Characterization of the putative lipase motif of agrobacterium virulence protein virj

CHARACTERIZATION OF THE PUTATIVE LIPASE MOTIF OF AGROBACTERIUM VIRULENCE PROTEIN VIRJ ZHANG LI (B.Sc., PKU.) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS First of all, I would like to give my deepest gratitude to my supervisor, Associate Professor Pan Shen Quan, for giving me the opportunity to undertake this interesting project. I would like to thank him for his encouragement, guidance and expert advice throughout my M. Sc candidature. I am also grateful to Assistant Professor Yang Hong Yuan and Markus Wenk, not only for their kind advice for my research work but also for giving me the opportunity to conduct my LPS and lipid study in their labs. I also thank Mr. Ho Zi Zong and Ms. Anne K. Bendt for their kind instructions and help on the equipments under their care. In addition, I truly appreciate the following friends and members of my laboratory for their assistance, without any complaint both physically and spiritually during the course of my M.Sc study: Ms. Tan Lu Wee, Ms. Chang Limei, Mr. Hou Qingming, Mr. Guo Minliang, Mr. Tang Hock Chun, Mr. Li Xiaobo, Ms. Qian Zhuolei, Mr. Sun Deying and Mr. Alan Lowton. Apart from these, I would like to thank the National University of Singapore for supporting me with a research scholarship throughout my M. Sc candidature. During my two and a half years here in Singapore, I have enjoyed a home life. I have always felt warm and happy even though I am a foreigner. I really owe thanks to all my teachers and my friends for their kind help. Their contributions to the completion of this thesis are deeply appreciated. 1 TABLE OF CONTENT ACKNOWLEDGEMENTS...........................................................................................0 TABLE OF CONTENT.................................................................................................2 SUMMARY...................................................................................................................5 LIST OF FIGURES .......................................................................................................7 LIST OF ABBREVIATIONS........................................................................................9 LITERATURE REVIEW ............................................................................................10 1.1. MOLECULAR MECHANISM OF A. TUMEFACIENS ...................................................12 1.1.1. vir gene induction ......................................................................................13 1.1.2. T-complex formation .................................................................................15 1.1.3. T-complex delivery....................................................................................18 1.1.4. Nuclear localization of T-DNA .................................................................24 1.1.5. T-DNA integration.....................................................................................26 1.1.6. Functions of chromosomal virulence genes...............................................28 1.2. ACVB AND VIRJ ..................................................................................................29 1.3. AIMS OF THIS PROJECT .......................................................................................31 MATERIALS AND METHODS.................................................................................33 2.1. PLASMIDS, STRAINS AND MEDIA .........................................................................33 2.1.1. Strains, plasmids and primers ....................................................................33 2.1.2. Media, antibiotics and other stock solutions..............................................36 2.1.3. Antibiotics and other stock solutions.........................................................37 2.1.4. Growth conditions and strain storage ........................................................38 2.1.5. Overexpression of protein in E. coli ..........................................................38 2.1.5. Virulence gene induction of A. tumefaciens ..............................................38 2.2. DNA MANIPULATIONS .......................................................................................39 2.2.1. Preparation of competent cell ....................................................................39 2.2.2. Plasmid DNA preparation..........................................................................39 2.2.3. DNA digestion and ligation .......................................................................40 2.2.4. Polymerase chain reaction (PCR) ..............................................................40 2.2.5. DNA electrophoresis and purification .......................................................41 2 2.2.6. Transformation of E. coli...........................................................................42 2.2.7. Transformation of A. tumefaciens..............................................................43 2.3. PROTEIN TECHNIQUES ........................................................................................44 2.3.1. Buffers for protein manipulations..............................................................44 2.3.2. Pull down assay..........................................................................................45 2.3.3. Purification of VirB7-His ..........................................................................47 2.3.4. SDS-PAGE analysis...................................................................................48 2.3.5. Silver staining ............................................................................................50 2.3.6. Western blot analysis .................................................................................50 2.4. LIPOPOLYSACCHARIDES METHODOLOGY ...........................................................51 2.4.1. LPS preparation by hot phenol ..................................................................51 2.4.2. TLC analysis of LPS..................................................................................52 2.4.3. LPS electrophoresis and staining ...............................................................53 2.5. ANALYSIS OF WHOLE CELL LIPIDS ......................................................................53 2.5.1. Preparation of whole cell lipids .................................................................53 2.5.2. TLC analysis of lipids................................................................................53 2.5.3. Electrospray ionization (ESI) ion-trap MS analysis of lipids ....................54 2.6. PLANT TUMORIGENESIS ASSAY...........................................................................54 2.7. SUBCELLULAR FRACTIONATION OF A. TUMEFACIENS ..........................................55 RESULTS ....................................................................................................................57 3.1. FUNCTIONAL ASSAY OF VIRJ-HIS.......................................................................57 3.1.1. Construction of VirJ-His expression vectors .............................................57 3.1.2. A. tumefaciens strain B119 is sensitive to carbenicillin ............................62 3.1.3. Functional test of VirJ-His in A. tumefaciens............................................62 3.2. ANALYSIS OF LIPOPOLYSACCHARIDES (LPS).....................................................65 3.2.1. Thin-layer chromatography (TLC) analysis of LPS ..................................66 3.2.2. LPS analysis by electrophoresis.................................................................67 3.3. ANALYSIS OF WHOLE CELL LIPIDS ......................................................................70 3.4. PULL-DOWN ASSAY OF VIRJ-HIS ........................................................................78 3.5. POSTTRANSLATIONAL MODIFICATION OF VIRB7................................................81 3.5.1. Functional test of VirB7-His in A. tumefaciens .........................................82 3.5.2. Purification of VirB7-His ..........................................................................82 3.5.3. Mass Spectrometry (MS) analysis VirB7-His ...........................................88 3 3.6. ANALYSIS OF CELL MEMBRANE PROTEINS ..........................................................92 DISCUSSION ..............................................................................................................95 4.1. VIRJ DOES NOT AFFECT THE STRUCTURAL INTEGRATION OF LIPOPOLYSACCHARIDES IN A. TUMEFACIENS ..............................................................95 4.2. VIRJ DOES NOT AFFECT THE POSTTRANSLATIONAL MODIFICATION OF VIRB7 ....96 4.3. VIRJ MAY AFFECT THE VIRULENCE OF A. TUMEFACIENS BY IMPAIRING THE TDNA TRANSFER PROCESS .........................................................................................98 4.4. FUTURE STUDY...................................................................................................99 REFERENCES ..........................................................................................................101 4 SUMMARY Agrobacterium tumefaciens transfers a specific fragment of DNA (T-DNA) of its tumor-inducing (Ti) plasmid into plant cells. In the octopine strains of A. tumefaciens, a gene named virJ located on the Ti plasmid can functionally complement a chromosomal gene (acvB) mutant. But this virJ gene is not found in the nopaline strains of A. tumefaciens. While mutation of one of these two genes has no effect on the bacterial virulence, mutation of both of these two genes has been shown to abolish the ability of A. tumefaciens to cause tumors on plants. Analysis of protein sequences of AcvB and VirJ has suggested that they both contain a putative lipase or acyltransferase motif. Mutation of the lipase motif would lead to the malfunctioning of the proteins, indicating that the lipase motif plays a key role in the function of these proteins. Previous studies have shown that inactivation of the lipase motif affected the stability of some components of the VirB channel, including VirB7, VirB8, VirB9 and VirB10 (Pan, 1999). In acvB and virJ double mutant strain, the dimerization of VirB7 was not affected but VirB7 was more prone to be digested by protease K (Lu Baifang, 2000; unpublished data). To understand the biochemical function of VirJ, genetic and biochemical experiments were carried out to further characterize VirJ. Experimental data from this study have shown that mutation of virJ has no effect on the LPS or lipid profile of A. tumefaciens, based on the results from thin-layer chromatography, SDS-PAGE analysis and electrospray ionization (ESI) ion-trap mass spectrometry (MS) analysis. Protein-protein interaction study via pull-down analysis has indicated that VirJ could interact with VirD2, VirE2, VirD4 and AopB but not with VirB7, VirB8, VirB10 and 5 VirB11. Molecular weight of VirB7 purified from B119 and A208 background did not show any difference, suggesting that VirJ may not be involved in the posttranslational modification of VirB7. 6 LIST OF FIGURES Fig. 1.1. Agrobacterium-plant cell interaction 11 Fig. 1.2. A model depicting the subcellular locations and interactions of 23 the VirB and VirD4 subunits of the A. tumefaciens VirB/D4 T4SS Fig. 3.1. Construction of plasmids pHisJ1 59 Fig. 3.2. Sequencing result of virJ-his in pHisJ1 60 Fig. 3.3. Amino acid sequences of the lipase motif of VirJ-His encoded 61 by pHisJ1, pHisJ2 and pHisJ3 Fig. 3.4. Plant tumorigenesis assay 64 Fig. 3.5. Thin-layer chromatography analysis (TLC) of LPS from A. 68 tumefaciens Fig. 3.6. LPS analysis using SDS-PAGE (15%) 69 Fig. 3.7. Thin-layer chromatography of whole cell lipids from A. 72 tumefaciens Fig. 3.8. ESI-MS analysis of lipids extracted from A. tumefaciens strain 73 B119 Fig. 3.9. ESI-MS analysis of lipids extracted from A. tumefaciens strain 74 B119(pHisJ1) Fig. 3.10. ESI-MS analysis of lipids extracted from A. tumefaciens strain 75 B119(pHisJ2) Fig. 3.11. ESI-MS analysis of lipids extracted from A. tumefaciens strain 76 B119(pHisJ3) Fig. 3.12. ESI-MS analysis of lipids extracted from different A. 77 tumefaciens strains (overlaid) Fig. 3.13. Pull-down assay of VirJ-His from A. tumefaciens 80 Fig. 3.14. Purification of VirB7-His from TG1(pB78HSW) cultured in 84 LB Fig. 3.15. Purification of VirB7-His from B721(pB78HSW) and 86 B119(pB78HSW) 7 Fig. 3.16. Purification of VirB7-His from B721(pB78HSW) and 87 B119(pB78HSW) Fig. 3.17. Analysis of the amino acid sequence of VirB7-His 89 Fig. 3.18. MALDI-TOF TOF MS analysis of VirB7-His isolated from B721(pB78HSW) MALDI-TOF TOF MS analysis of VirB7-His isolated from B119(pB78HSW) Analysis of the cell membrane proteins from A. tumefaciens 90 Fig. 3.19. Fig. 3.20. 91 94 strains B119(pHisJ1) and B119(pHisJ2) 8 LIST OF ABBREVIATIONS A adenosine μm micrometre Amp ampicillin min minute(s) AS acetosyringone ml milliliter(s) bp base pair(s) mM millimole C- terminal carboxyl terminal n nano- C cytidine nm manometer Cb Carbenicillin N- terminal amino terminal Chl chloramphenical PAGE polyacrylamide gel DMSO dimethylsulfoxide DNA deoxyribonucleic acid R resistant/resistance dNTP deooxyribonucleoside RNase ribonuclease triphosphate rpm revolutions per minute double-stranded DNA SDS sodium dodecyl sulfate dithiothreitol sec second(s) Ethylenediaminetetra ssDNA single-stranded DNA acetic acid 1× TAE 40 mM Tris-acetate, 1 dsDNA DTT EDTA g electrophoresis grams or gravitational mM EDTA force, according to the TBS Tris-buffered saline intended meaning Tc tetracycline isopropyl-β-D- V/V volume per volume thiogalactoside wt wild type kb kilobase(s) or 1000 bp MCS multiple cloning site(s) kD kilodalton(s) M molar Km kanamycin MES 2-[N-morpholino] lacZ β-galactosidase gene mg milligram(s) LPS lipopolysaccharides μ micro- TLC thin-layer μg microgram(s) μl microliter(s) IPTG ethanesulfonic aci chromatography MS mass spectrometry 9 LITERATURE REVIEW Agrobacterium tumefaciens is a Gram-negative, non-sporing, motile, rod-shaped bacterium, closely related to Rhizobium which forms nitrogen-fixing nodules on clover and other leguminous plants. The study of A. tumefaciens has lasted almost one century ever since Smith and Townsend found that this bacterium can cause tumors on plants (Smith et al, 1907). During the tumor inducing process, A. tumefaciens attaches to plant cells and transfer a DNA segment, called the transferred DNA (TDNA), from its tumor-inducing (Ti) plasmid into the plant cell nucleus. Upon the integration of the T-DNA into the plant genome, the expression of the genes on the TDNA will lead to the accumulation of the plant hormones, causing the plant cells to proliferate limitlessly and finally form tumors (Van et al, 1974; Waston et al, 1975; Hooykaas et al, 1994). The tumors synthesize opines, which are the sole carbon and nitrogen source of A. tumefaciens. Based on the type of opines they use, A. tumefaciens are usually classified into octopine and nopaline strains (Sheng and Citovsky, 1996). As the research of A. tumefaciens progresses, more knowledge about this pathogen has been accumulated. It is now known that the host range of A. tumefaciens includes not only the dicotyledonous plants (Hooykaas et al, 1994), such as fruit trees and vines, but also the monocotyledonous plants (Hiei et al, 1997; Komari et al, 1998). In 1996, it was further found that A. tumefaciens can transform yeast cells by homologous recombination (Bunkock et al, 1996). In 1998, the ability of A. tumefaciens to transform mammalian cells (Hela cells) was also demonstrated (Relic et al, 1998; Talya Kunik et al, 2001). The development of A. tumefaciens as a 10 Fig. 1.1. Agrobacterium-plant cell interaction. Critical steps that occur to or within the bacterium and those within the plant cell are highlighted, along with genes and/or proteins known to mediate these events: 1. Attachment of Agrobacterium to host cell surface receptors; 2. Recognition of plant signals by bacterial VirA/VirG sensor-transducer system; 3. Activation of bacterial vir genes; 4. Processing and production of transferable T-strand; 5. Export of T-DNA into plant cell via VirB/D4 channel; 6. Intracytoplasmic transport of T-complex; 7. Nuclear import of T-complex; 8. T-DNA integration. IM, bacterial inner membrane; NPC, nuclear pore complex; OM, bacterial outer membrane; PP, bacterial periplasm. (Cited from Tzfira and Citovsky, 2002) 11 plant genetic vector has been one of the most important technical developments in the past 25 years. 1.1. Molecular mechanism of A. tumefaciens A. tumefaciens is the only known natural vector for inter-kingdom gene transfer. There are three genetic components of A. tumefaciens that are required for plant cell transformation. The first is the T-DNA, which is actually transported from the bacterium into the plant cell. The T-DNA is a discrete segment of DNA located on the 200 kb Ti plasmid of A. tumefaciens and is delineated by two 25 bp imperfect direct repeats known as the T-DNA borders (De Vos et al, 1981). The T-borders are highly homologous in sequence (Yadav et al, 1982; Jouanin et al, 1989). The right border repeat of the T-DNA is required for the effective transformation of the plant cells and functions in a unidirectional manner (Miranda et al, 1992). The 35 kb virulence (vir) regions which are composed of eight major loci (virA, virB, virC, virD, virE, virG, virJ and virH) are the second component that is required for the T-DNA production and delivery (Winans, 1992; Kado et al, 1991 and 1994; Pan et al, 1995). All of the vir operons are induced by plant phenolic compounds, such as acetosyringone (AS) and specific monosacchairdes, as a regulon via the VirA/VirG two-component system. The protein products of these genes, termed virulence (Vir) proteins, can generate a copy of the T-DNA and mediate its transfer into the host cell. The third component is a set of chromosomal virulence (chv) genes, some of which are involved in bacterial chemotaxis toward and attachment to a wounded plant cell (Sheng and Citovsky, 1996). The last two genetic components play important roles in the T-DNA processing and movement from A. tumefaciens into the plant cell nucleus. This review describes the characteristics and functions of Vir proteins and several Chv proteins, which are involved in T-DNA transfer from A. tumefaciens into plant cells. 12 1.1.1. vir gene induction There are about 25 vir genes located on the Ti plasmid that are required for the tumorigenesis (Stachel and Nester, 1986). All vir operons are transcriptionally induced during infection in response to a family of related phenolic compounds or a family of sugars, some of which are released from the plant wounds. The environmental signal of central importance in vir gene induction is extracellular pH ranging from 5.0 to 5.8 (Winans, 1992). Two Ti plasmid encoded proteins, named VirA and VirG, are required for the induction of vir genes (Rogowsky et al, 1987; Stachel et al, 1986; Winana et al, 1988). VirA and VirG are the members of a gene family of the two-component regulatory systems, involving a sensor and a response regulator that regulate the induction of the vir genes in response to the plant wound signal compounds (Leroux et al, 1987; Melchers et al, 1986 and 1987; Morel et al, 1989; Powell et al, 1987; and Winans et al, 1986). Induction by monosaccharides requires another protein named ChvE which is chromosomally encoded (Huang et al, 1990). ChvE is a periplasmic glucosegalactose-binding protein that can enhance the induction by phenolic compounds or sugars (Cangelosi et al, 1990; Lee et al, 1992). VirA is a sensor protein that acts directly or indirectly as the receptor for plant phenolic compounds. This protein is an inner membrane protein which belongs to the histidine protein kinase family (Leroux et al, 1987). VirA has four domains which include a periplasmic domain, a linker domain, a kinase domain and a receiver domain. The periplasmic domain is required for tumor induction because of its binding with a variety of monosaccharides and also the periplasmic sugar-binding 13 protein, ChvE (Cangelosi et al, 1990; 1991). The linker domain of VirA functions as a receptor for the phenolic compounds and acidity. The kinase domain and the receiver domain are crucial for the signal transduction (Jin et al, 1990a; 1990b; 1990c). Physical and genetic evidences have indicated that VirA exists as a homodimer in its native conformation and the homodimer is the functional state in the plant-bacterium signal transduction (Pan et al, 1993). When VirA senses the phenolic compounds released from the wounded plant cells, it will get autophosphorylated at His-474 (Lee et al, 1995; 1996; Ninfa et al, 1988; 1991; 1993) and further transfer the phosphate group to Asp-52 of VirG. VirG is a member of the response regulator class of proteins, whose N-terminal halves are the targets of phosphorylation and the C-terminal halves generally have promoter-binding properties (Albright et al, 1989; Miller et al, 1989; Tempe et al, 1982). When VirG is phophorylated by VirA, the phospho-VirG activates the transcription of the remainder of the vir genes by binding particularly to the vir-box, which is a conserved regulator element found upstream of most of the vir genes. Non-phosphorylatable mutant VirA and VirG proteins have been found to lose their ability to induce the expression of vir genes (Jin et al, 1990a; 1990b; 1990c). On the other hands, multiple copies of VirG in A. tumefaciens have been demonstrated to greatly enhance the vir gene expression and the transient transformation frequency of some plants tissues (Liu et al, 1992). Having multiple copies of VirG has also enabled a higher level of vir gene induction by acetosyringone (AS), even at alkaline pH (Liu et al, 1993). 14 A. tumefaciens also possesses a putative chromosomally encoded twocomponent signal transduction system, known as the ChvG-ChvI system. Once the bacteria are in close proximity or contact with the plant or animal cells during infection, it is quite likely that they will encounter an acidic pH environment. Sensing the acidity appears to be important for A. tumefaciens to cope with the environment in plants and to cause tumors on these plants. ChvG is proposed to be a histidine protein kinase that might act as the sensor to directly or indirectly sense the extracellular acidity, while ChvI is suggested to be the response regulator. Mutation of these two proteins would abolish the tomorigenecity of A. tumefaciens (Trevor et al, 1993). 1.1.2. T-complex formation Proteins responsible for the production of the T-complex are encoded by the virC, virD and virE operons. Upon the activation and expression of these vir genes, a single-stranded T-DNA would be generated. T-complex is formed when one molecule of T-DNA is associated covalently with one molecule of VirD2 and a large number of VirE2 molecules. The T-border sequences which define the borders of the T-DNA are the target sites for the VirD1/VirD2 endonuclease and serve as the covalent sites for VirD2 (Howard et al, 1989; Pansegrau et al, 1993; Wang et al, 1984; 1987; Albright et al, 1987; Yanofsky et al, 1986). An “overdrive” sequence near the T-DNA right border not only helps to establish the functional polarity of right and left borders but also enhances the transmission of the T-DNA into the plant cells, but the exact operating mechanism is still unclear (Jen et al, 1986; Hansen et al, 1992). VirD1 and VirD2 encoded by the virD operon are essential for the production of the T-complex. VirD1 functions as a topoisomerase. It recognizes and binds with the 15 T-DNA to promote the binding of VirD2 and catalyzes the conversion of the supercoiled DNA to the relaxed DNA (Ghai et al, 1989). virD2 encodes an endonuclease (Yanofsky et al, 1986) which nicks the T-DNA border sequence in a site- and strand-specific manner and covalently attaches to the 5' end of the nicked DNA via the tyrosine 29 residue (Pansegrau, 1993; Jasper, 1994; Zupan et al, 2000; Gelvin, 2000; Vogel et al, 1992). The nicked DNA is then displaced by replication of the bottom strand to release the single-stranded T-DNA. In vivo study has shown that the expression of VirD1 and VirD2 is sufficient for the generation of T-DNA both in E. coli and in A. tumefaciens. However, in vitro study shows that VirD2 alone is enough for mediating the precise cleavage of the T-border sequences, while VirD1 is essential for the cleavage of double-stranded DNA substrates. Another two proteins termed as VirC1 and VirC2, may interact with VirD1 and VirD2 during the nicking reaction. VirC1 was found to increase the efficiency of TDNA production by interaction with the overdrive sequence near the right T-border on the Ti plasmid when VirD2 and VirE2 proteins were limited (De Vos and Zambryski, 1989). After T-strand was generated, VirE2 proteins were found to coat the T-DNA immediately to protect it from degradation by the proteases within A. tumefaciens and the plant cells (Gietl et al, 1980; Christie et al, 1988; Rossi et al, 1996). VirE2 is a protein that has high affinity for the single-stranded DNA (ssDNA). In vitro, VirE2 can bind with single stranded DNA without sequence specificity (Citovsky et al, 1989; Sen et al, 1989). During this process, VirE1 was also found to be required (McBride and Knauf, 1988; Winans et al, 1987). As a small, acidic protein with an amphipathic 16 α-helix at its C-terminus which functions as a specific molecular chaperone for VirE2, VirE1 can help to regulate the translation of VirE2 and prevent VirE2 from self aggregation by interacting with the N-terminus of VirE2. In other words, VirE1 enhances the stability of VirE2 and maintains VirE2 in an export-competent state (Deng et al, 1999) before VirD2 pilots the T-complex into the plant cell nucleus, where the transferred T-DNA is integrated into the plant genome. Although VirE2 can bind with the T-DNA, it is still not clear whether the binding occurred inside A. tumefaciens or within the plant cells. Two models have been proposed for the transfer of VirE2. In the first model, it is suggested that VirE2 is transferred together with the T-DNA in the form of T-complex through the VirB/D4 channel. This is based on the observation that VirE2 is one of the most abundant virulence proteins in A. tumefaciens and it can bind ssDNA in a strong and cooperative manner in vitro. Besides, the T-strand and VirE2 could be coimmunoprecipitated from the extracts of vir-induced A. tumefaciens. But more and more evidence suggests that VirE2 may actually be transferred into the plant cell independent of the T-DNA and that the T-complex is formed within the plant cell cytoplasm. First of all, complementation study has shown that VirE2 mutant strain could be rescued by coinfection with strains that expressed VirE2 but lacked the T-DNA (Christie et al, 1988; Otten et al, 1984). Secondly, the transfer of VirE2 requires the expression of VirE1, but the transfer of the T-DNA does not, indicating that VirE1 is the export chaperone of VirE2. Conversely, transfer of the TDNA requires VirC1 and VirC2, but the transfer of VirE2 does not require these two proteins (Deng et al, 1999; Suzuki et al, 1988; Christie et al, 1988). Recent 17 biophysical report has further suggested that VirE2 itself could form channels on the artificial membranes through which the T-DNA might be transferred (Dumas, 2001; Myriam et al, 2005). Studies have shown that VirD2, VirE2 and VirF could be exported across the cell membranes independent of VirB (Chen et al, 2000). These pieces of evidence imply that VirE2 is transported through the VirB/VirD4 channel or an alternative route and subsequently inserted into the plant plasma membrane, allowing the transport of the T-strand (a ss-T-DNA-VirD2 complex) (Dumas et al, 2001). 1.1.3. T-complex delivery After the T-complex is formed, it is ready to be delivered into the plant cells. The T-complex transfer apparatus is a type IV secretion system, which is encoded by the virB operon and virD4 (Zupan et al, 1998; Deng and Nester, 1998). To date, the systems that secrete various substrates through the bacteria cell wall are classified into four different types (Christie, 1997). Type I secretion pathway is typified by Escherichia coli hemolysin export, which requires three accessory proteins: a transport ATPase in the inner membrane, an outer membrane accessory protein and a protein spanning the periplasm (Fath et al, 1993). Type II is typified by pullulanase export in Klebsiella oxytoca which is sec-dependent. The substrate is first transferred into the periplasm in a sec-dependent manner before it is secreted across the outer membrane via a specialized apparatus (Hobbs et al, 1993). Type III secretion pathway is a sec-independent pathway typified by Yop export in the human pathogen Yersinia pestis. The substrates are translocated directly cross the cell membranes into the host cells (Hueck, 1998). And type IV systems primarily transfer DNA-protein complexes 18 from donor to recipient during conjugation (Salmond, 1994; reviewed by Zupan et al, 1998). virB operon is the largest operon of the vir region, which encodes 11 genes. Except for virB1, all the other genes are essential for the tumorigenecity of A. tumefaiciens (Berger et al, 1994; Christie, 1997), but virB1 can increase the efficiency of the T-complex transmembrane assembly (Fullner, 1998; Lai and Kado, 1998). All virB gene products and VirD4 are localized to the cell membrane, where they form a transmembrane channel to translocate the T-DNA and the associated virulence proteins from A. tumefaciens to the recipient cells (Thorstenson, et al, 1993). Recent study has shown that the secretion apparatus is assembled at the cell pole. Neither the assembly nor the polar localization of the VirB proteins require ATP utilization by ATPase encoded by virB4 and virB11 (Judd et al, 2005). Analysis of the sequence VirB1 indicates that the periplasmic protein VirB1 carries a lysozyme and lytic transglycosylase motif in its amino-terminal half, suggesting that VirB1 may locally lyze the peptidoglycan layer of the cell wall and prepare the sites for the assembly of the transporter (Llosa et al, 2000; Baron et al, 1997). This activity of transglycosylase is important for the biogenesis of the T-pilus but not for the transfer of the substrates (Liu et al, 2003). VirB1 undergoes C-terminal processing after it is exported to the periplasm and a smaller protein VirB1*, which is the C-terminal 73 amino acids of VirB1, is found to be secreted and loosely associated with the outer membrane. VirB1* can also form a complex with VirB9, indicating its role in the assembly of the transporter (Baron et al, 1997). 19 VirB2 is the major component of the T-pili that are generated when A. tumefaciens cells are induced with the plant phenolic compounds (Lai et al, 1998; Sagulenko et al, 2001; Schmidt-Eisenlohr et al, 1999). VirB2 is processed by the removal of the 47-amino-acid signal peptide. The remaining 74-amino-acid peptide is linked by a peptide bond between the amino- and carboxyl-terminal residue to generate a cyclic peptide (the T pilin) (Eisenbrandt et al, 1999). The T pilin subunits are transported across the cell membrane and assembled into the exocellular, flexuous T-pili which protrude from the cell wall (Lai et al, 2000; 2001). The signal peptidase cleavage sequence is crucial for the virulence, since mutations near the cleavage sites would result in severe attenuation of the virulence (Lai et al, 2001). VirB3 and VirB4 may promote the formation of the virulent pilus. VirB3 is localized preferentially to the outer membrane in a VirB4-dependent manner (Jones, 1994), while VirB4 is an ATPase which is essential for the virulence of A. tumefaciens (Christie et al, 1989). Mutation in the Walker A nucleotide triphosphate binding motif of VirB4 would affect the localization of VirB3 and the mutant strain would exhibit attenuated virulence on plants (Berger and Christie, 1993). Another virulence protein VirB5 has been found to cofractionate with the T-pilus components and appears to be a minor component of the T- pilus (Schmidt-Eisenlohr et al, 1999). VirB6, which is associated with the inner membrane, is found to be required for the stabilization of VirB5 and VirB3 and the formation of VirB7 homodimers. Deletion of VirB6 would lead to reduced expression of VirB7 monomers. The formation of VirB7-VirB9 heterodimers and VirB7 homodimers were also abolished, which could not be restored by VirB7 expression in trans (Siegfried et al, 2000). 20 VirB7 is a lipoprotein which is crucial for the assembly of the membrane spanning transporter and the pilus. The matured VirB7 protein is anchored to the outer membrane by its lipid moiety (Fernandez et al, 1996; Baron et al, 1997). Coimmunoprecipitation results have indicated that VirB7 could form homodimers and also heterodimers with VirB9 via disulfide bridges. Deletion of virB7 gene would lead to the reduced expression levels of VirB4, VirB5, VirB8, VirB9, VirB10 and VirB11, indicating that the synthesis and the stability of the majority of VirB proteins are dependent on VirB7 (Fernandez et al, 1996). In addition, deletion of virB9 gene would reduce the expression of VirB4, VirB5, Virb8, VirB10 and VirB11, but not VirB7 (Fernandez et al, 1996). It is hypothesized that the association of VirB with VirB7-VirB9 complex stabilizes their accumulation during the assembly of the transporters. Study of VirB7 has revealed a signal sequence which is ended with Ala-LeuSer-Gly-Cys at the amino terminus of VirB7. This sequence is in consensus with the signal peptidase II cleavage site, indicating that VirB7 is a lipoprotein (Hayashi, 1990). Studies of lipoproteins in E. coli have led to a proposed modification pathway of these proteins in bacteria. Briefly, bacterial lipoproteins are first modified by adding a thioether-linked diglyceride to the invariant Cys residue in the signal sequence. Then two fatty acids are added to the diglyceride via ester likage. Signal peptidase II cleaves before the modified Cys residue and is followed by acylation at the Cys residue by amide linkage to the palmitic acid, generating a 41-amino-acid polypeptide with a modified Cys residue at the amino terminus. It has been 21 demonstrated that only the matured VirB7 is stable and competent to function as a virulence factor (Fernandez et al, 1996). VirB11 is another ATPase with a Walker A motif in its carboxyl terminus and it is located in the inner membrane independent of other VirB proteins. Analysis of the Walker A motif mutant strains has indicated that the membrane interaction is modulated by ATP binding or hydrolysis. Therefore VirB11 may function as a chaperone to facilitate the movement of the T-complex substrate to cross the cytoplasmic membrane by supplying energy (Lai and Kado, 2000). The final component of the transporters is VirD4, the third ATPase that is required for the virulence. VirD4 is an inner membrane protein and is required for the formation of the T-pilus (Fullner, 1996). It is proposed that VirD4 functions as a coupling protein for the transfer of the virulence factors to the other members of the secretion apparatus by an energy dependent manner. Although the transfer of T-DNA from A. tumefaciens into the plant cells are mediated by the VirB channel, recent studies have shown that the T-DNA associated proteins are exported independently of VirB (Chen et al, 2000). It is possible that VirD2, VirE2, and another protein VirF are transported across the cell membranes by a specific pathway different from that transports the T-DNA. 22 Fig. 1.2. A model depicting the subcellular locations and interactions of the VirB and VirD4 subunits of the A. tumefaciens VirB/D4 T4SS. The VirD4 coupling protein assembles as a homohexameric, F1-ATPase-like structure juxtaposed to the VirB channel complex. VirB11, a hexameric ATPase structurally similar to the members of the AAA ATPase superfamily, is positioned at the cytoplasmic face of the channel entrance, possibly directing substrate transfer through a VirB6/VirB8 inner membrane (IM) channel. The VirB2 pilin and VirB9 comprise the channel subunits to mediate substrate transfer to and across the outer membrane (OM). VirB10 regulates substrate transfer by linking IM and OM VirB subcomplexes. (Cited from Cascales and Christie, 2004) 23 1.1.4. Nuclear localization of T-DNA Once inside the cytoplasm of the host cells, the T-complex will enter the host cell nucleus where it can integrate into the host genome. Since T-DNA itself does not contain any specific sequence, any foreign DNA fragment placed between the T-DNA borders can be transported into the plant cells and subsequently integrated into the plant genome. This suggested that the associated protein components must have played some roles in the nuclear localization of the T-complex. The T-complex is a large structure about 13nm in diameter (Citovsky et al, 1997; Abu-Arish et al, 2004) which is too large to enter the nucleus by diffusion but is within the size limits of the active nuclear import. Indeed, both VirD2 and VirE2 have nucleus localization sequences (NLS) (Herrera-Estrella et al, 1990; Citovsky et al, 1992, 1994; Howard et al, 1992; Tinland et al, 1992). VirD2 has two NLSs, one monopartite NLS at the N-terminus and one bipartite at the C-terminus. The C-terminal NLS is responsible for the transfer of the T-DNA into the cell nucleus but not the one at the N-terminus, since mutation in the Nterminal NLS showed no effect on the T-DNA transfer (Shurvinton et al, 1992; Rossi et al, 1993; Narasimhulu et al, 1996; Mysore et al, 1998) but the VirD2 mutant strain lacking the C-terminal NLS was unable to mediate the plant nuclear targeting of the T-complex (Rossi et al, 1993; Ziemienowicz et al, 2000 and 2001). The N-terminal half of VirD2 may be involved in the integration process of the T-DNA in the plant nucleus (Koukolikova-Nicola et al, 1993; Shurvinton et al, 1992). Studies have showed that VirD2 alone was sufficient to import short ssDNA, but VirE2 was required to import long ssDNA additionally (Ziemienowicz et al, 2000; 24 2001). VirE2 contains two separate bipartite NLS in the center, one located in residues 212-252 and the other located in residues 288-317 (Gietl et al, 1987; Christie et al, 1988; Citovsky et al, 1988; Das, 1988). These sequences have some overlap with the ssDNA binding sequence, indicating that the NLSs of VirE2 might also be involved in binding the single stranded T-DNA (Citovsky et al, 1992; Citovsky et al, 1994). Deletion of NLS1 in VirE2 would reduce its ssDNA binding ability while deletion of NLS2 would completely abolish the ssDNA binding and nuclear localization activities. These imply that the NLS of these two proteins might play different roles in nuclear localization of the T-complex. Recent studies have found that the NLSs of octopine VirE2 might differ from that of the nopaline-type Ti plasmids in that they are not functional in the nuclear import of proteins in Xenopus oocytes, Drosophila embryos (Guralnick et al, 1996) and yeast cells (Rhee et al, 2000). With a slight modification of the NLS of VirE2, VirE2 was found to be functional in targeting DNA into the nuclei of the animal cells (Guralnick et al, 1996). This suggests that nuclear targeting signals in plant and animal cells might differ slightly (Gelvin, 2000). Another protein VirE3, which is exported into the host cells during transformation, has just recently been shown to be involved in the nuclear targeting of the T-complex. It can facilitate the nuclear import of VirE2 via the karyopherin αmediated pathway and thus allowing the subsequent T-DNA expression (Lacroix et al, 2004). It has been proposed that VirE3 might function as an ‘adaptor’ molecule between VirE2 and karyopherin α which can ‘piggy-back’ VirE2 into the host cell nucleus (Lacroix et al, 2004). 25 Besides the above virulence proteins, some plant factors may also be involved in the process of nucleus localization of the T-DNA. Several such plant factors that can interact with VirD2 and VirE2 have been identified. VirD2 was found to have interaction with three members of the cyclophilin chaperone family of Arabidopsis, named RocA, Roc4 and CypA (Deng et al, 1998), as well as a type 2C serine/threonine protein phosphatase (PP2C) (Gelvin, 2000). It is proposed that dephosphorylation of the NLS of VirD2 by PP2C has a negative effect on the localization of the T-DNA into the nucleus. Finally, VirD2 was also found to interact with a member of the Arabidopsis karyopherin α family, AtkAPα (Ballas and Citovsky, 1997). Members of this protein family mediate nuclear import of NLScontaining proteins (Nakielny and Dreyfuss, 1999). Just like VirD2, VirE2 also interacts with some plant host factors. Two of these are VIP1 and VIP2 (Tzfira and Citovsky, 2000; Tzfira et al, 2001). It is proposed that VIP1 binds with VirE2 and target it into the nucleus of the host cell by a “piggy-back” mechanism. During this process, VIP1 can interact with a cellular karyppherin αe.g.AtkAPα which mediates the nuclear import of the T-DNA and associated virulence proteins. Therefore, VIP1 might be an adaptor between VirE2 and the conventional nuclear import machinery of the host cells (Doyle et al, 2002). 1.1.5. T-DNA integration T-DNA integration is the final step of the transformation process. However, less is known about how the T-DNA is integrated into the plant genome. It is possible that some plant factors may play some roles in this process. 26 VirD2 might play a dual role in the integration process by ensuring both fidelity and efficiency (Tinland et al, 1995; Rossi et al, 1996; Mysore et al, 1998). VirD2 not only guide the T-DNA into the plant nucleus, but also help in the integration of the TDNA into the plant genome. Studies have shown that VirD2-T-DNA complex has both ligase and polymerase activities. Besides VirD2, some plant proteins may also take part in this process. One of these proteins is VIP2, an Arabidopsis protein that bind with VirE2 (Tzfira and Citovsky, 2000). VIP2 can interact with VIP1 in the yeast two-hybrid system (Tzfira and Citovsky, 2000). It is possible that VIP2 form a complex together with VIP1 and VirE2 and then mediates the intranuclear transport of VirE2 and its cognate T-strand to the chromosomal regions where the host DNA is more exposed. Genetic experiments have shown that some Arabidopsis rat (resistant to Agrobacterium transformation) mutants exhibited lower rates of stable transformation when compared with the wild type plants, indicating that some host factors are involved in the transformation process. It has been shown that in rat5 a histone H2A gene is disrupted and the T-DNA integration step of transformation is blocked. Complementation analysis and overexpression studies have indicated that histone H2A plays a role in A. tumefaciens transformation. It is possible that histone H2A plays an important role in the illegitimate recombination of the T-DNA into the plant genome (Mysore et al, 2000). Recently, another virulence protein, VirF, which is also transferred into the host cells, is found to be functional within the plant cell. However virF is only located on 27 the octopine-type Ti plasmids (Melchers et al, 1990; Schrammeijer et al, 1998). Based on the fact that octopine- and nopaline-strain of A. tumefaciens share a range of hosts but differ in the virulence towards other hosts, it is supposed that VirF is a host range factor of A. tumefaciens (Regensburg-Tuink and Hooykas, 1993). VirF is secreted to the plant cell via the VirB-VirD4 transport system, where it can interact with the other plant factors (Vergunst et al, 2000). One such plant factors is the Arabidopsis SKp-1 like (ASK) proteins, which binds with VirF in a yeast two-hybrid assay (Schrammerijer et al, 2001). SKp-1 is a subunit of the SCF (Skp1/Cdc53-cullin/F-box) complexes. It is possible that the F-box of VirF is recognized by the Skp proteins and therefore recruited to the SCF complex where it is proteolyzed by the ubiquitindependent degradation pathway (Del Pozo and Estelle, 2000). Since the proteolysis process regulates the plant cell into S phase, it is suggested that VirF might stimulate the plant cells to divide and become more susceptible to the integration of the T-DNA (Xiao and Jang, 2000). 1.1.6. Functions of chromosomal virulence genes Besides the virulence genes on the Ti plasmid, some genes on the chromosome are also required for the virulence of A. tumefaciens (Gelvin, 2000). But unlike the virulence genes on the Ti plasmid, the functions of these chromosomal genes have not been well studied. The products of gene pscA, chvB and chvA are propsed to function in the biosynthesis, modification and export of specific extracellular polysaccharides. It is suggested that these genes may encode proteins that are associated with the synthesis and transportation of β-1,2-glycan during the attachment phase of A. tumefaciens 28 (Thomashow et al, 1987; Douglas et al, 1982; Cangelosi et al, 1987). Four gene products, ChvD, ChvE, MiaA and Ros, regulate the expression of the vir genes in addition to the VirA/VirG system. In particular, ChvE can interact with the periplasmic domain of VirA and transfer the environmental signal to the bacteria (Winans et al, 1988; Huang et al, 1990; Gray et al, 1992; Close et al, 1985). Gene product of chvG and chvI provide an additional two-component system which is required for the virulence (Charles and Nester, 1993; Mantis and Winans, 1993). The functions of the chromosomal genes att and acvB are still not clear but acvB is necessary for infection (Matthyse, 1987; Wirawan et al, 1993). Recently, another two chromosomal genes that are also associated with the virulence of A. tumefaciens have been identified. These are katA and aopB (Xu and Pan, 2000; Jia et al, 2002). katA encodes a catalase that is involved in the detoxification of hydrogen peroxide. Mutation of this gene will attenuate the bacterial ability to cause tumors on plants but does not affect its viability (Xu et al, 2001). aopB is the homologue of a Rhizobium gene encoding an outer membrane protein. The expression of this gene requires the wild type ChvG/ChvI two-component system. But the detailed function of this protein is still unknown. 1.2. acvB and virJ In 1993, AcvB, a 47 kD protein, was identified as another chromosomal factor and the mutation caused by random insertion of a Tn5 transposon could affect the virulence of its parental strain A208. The mutant strain (B119) exhibited similar growth rates in both rich medium and minimal medium when compared to that of the parental strain. Although the growth was not affected, the virulence was abolished. 29 The strain was avirulent on Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. Attachment assay has showed that no significant difference in the attachment ability was observed. Sequence analysis of AcvB has indicated that the Nterminus of the protein contains a characteristic signal sequence (Wirawan et al, 1993). The matured AcvB is localized to the periplasm of bacteria and its expression in A. tumefaciens is independent of the induction by acetosyringone (AS). T-DNA generation is not affected in B119, indicating that AcvB is not involved in this process. Since AcvB can bind with the single stranded DNA in a sequence independent manner, it is possible that AcvB functions in the periplasm of A. tumefaciens by binding with the T-strand (Kang et al, 1994). virJ is located on the vir region of octopine-type Ti plasmid between virA and virB but it is not found on the nopaline-type Ti plasmid, pTiC58 (Pan et al, 1995; Kalogeraki and Winans, 1995). VirJ shares about 50% identity with AcvB in aminoacid sequence which could be found in both octopine-type and nopaline-type strains. The homologous region lies in the C-terminal half of AcvB. The expression of VirJ is under the control of the virA/virG two-component system regulated by acetosyringone which has no effect on acvB. The functions of VirJ and AcvB are still not clear. Expression of VirJ can restore the virulence of acvB mutant strain, indicating that they express the same factor required for the virulence, or play similar roles. The strains lacking both acvB and virJ had an impaired ability in T-DNA transfer, suggesting that these two proteins function in T-DNA transfer process (Pan et al, 1995). Subcellular fractionation experiments showed that VirJ mainly exist in the periplasm of A. tumefaciens. Pull 30 down assay showed that VirJ could interact with both the transport apparatus and type IV secretion substrates in A. tumefaciens, indicating that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ (Pantoja et al, 2002). Therefore, a two-step secretion pathway model has been proposed. In this model, the secretion substrates VirD2, VirE2 and VirF are first translocated into the periplasm through a specific transporter. In the periplasm, the substrates can form complexes with VirJ, which is transferred into the periplasm via a sec-like pathway. VirJ associates with VirD4 and the VirB pilus independently of one another and mediate the transfer of the substrates across the outer membrane via the VirB channel (Pantoja et al, 2002). 1.3. Aims of this project Sequence analysis of VirJ and AcvB has revealed that they both contain a lipase or acyltransferase motif that is required for the function of VirJ and AcvB (Pan, 1999; unpublished data). Studies have shown that mutation of the lipase motif would abolish the virulence of A. tumefaciens, but mutation outside the lipase motif has no effect on the bacteria. This indicates that the lipase motif of these two proteins play an important role during the tumorigenesis process. Cell fractionation and EDTA treatment studies showed that the mutation of the lipase motif caused an accumulation of VirB9 in the periplasm and also affected the stability of VirB7, VirB8, VirB9 and VirB10 but not other components of the VirB channel. The effect on VirB4 and VirE2 were minimal. However, the specific functions of VirJ still remain unclear. The aim of this project is to understand the biochemical function of VirJ. One specific aim is to study how the lipase motif of VirJ affects the virulence of A. 31 tumefaciens. For this purpose, first lipopolysaccharides (LPS) and whole cell lipids pattern were analysed. Other specific aims are to study the interaction of VirJ with other virulence proteins; to examine whether the lipase motif of VirJ would affect the posttranslational modification of the lipoprotein VirB7; and finally to determine the pattern of the bacterial cell membrane proteins. 32 MATERIALS AND METHODS 2.1. Plasmids, strains and media 2.1.1. Strains, plasmids and primers Bacterial strain, a Relevant characteristic(s) or sequences Source or reference DH5α endA1, hsdR17, supE44, thi-1 recA1 gyrA96, relA1, D(argF-lacZYA)U169, φ80dlacZ ΔM15. Host strain of plasmid replication. Bethesda Research Laboratorie s MT607 Pro-82, thi-1, hsdR17, supE44, end44, endA1, recA56. Host strain of triparental mating. Finan et al, 1986 MT616 MT607 (pKR600), mobilizer. Helper strain of triparental mating Finan et al, 1986 supE, HsdΔ5, thi, Δ(lac-proAB) F’[traD36, proAB+, q lacI , lacZΔM15]. Host strain of plasmid US Biochemisc als A348 Wild type strain, octopine-type A136(pTiA6NC) Laboratory collection A208 Wild type strain, nopaline-type, A136(pTiT37) Matsumoto et al, 1986 B119 Derivative of A208, Tn5 insertion at acvB, Km B721 A136 (pTiA6NC), octopine type deletion of virB7 and acvB. Used to confirm the function of pB78HSW plasmid or primer Strains Escherichia coli TG1 Agrobacterium tumefaciens R Wirawan et al, 1986 Labotory collection 33 Plasmids pUCA19 Cloning vector, repA, lacZ’, Ori, Plac, Amp R Jing s. g. pHisJ1 pUCA19 encoding a 1kb BamHI and EcoRI fragment containing the promoter of virJ in A. tumefaciens and R virJ-his. Cb This study pHisJ2 pUCA19 encoding a 1kb BamHI and EcoRI fragment containing the promoter of virJ in A. tumefaciens and R virJ-his with Ser127 replaced by threonine. Cb This study pHisJ3 pUCA19 encoding a 1kb BamHI and EcoRI fragment containing the promoter of virJ in A. tumefaciens and R virJ-his with Ser136 replaced by threonine. Cb pB78HSW pSW172 encoding virB8 and virB7-his, Tc R This study This study Primers HisJ-5 5’-CGCGGATCCGCTGCAGCCTTTCTGGTTCT-3’ This study HisJ-3 5’CCGGAATTCTTAATGATGATGATGATGATGAA GAGGTGCAGGACCTGAA-3’ This study P1 5’TTTACTTATAGGATATACTTTCGGCGCTGACGT -3’ This study P2 5’ACGTCAGCGCCGAAAGTATATCCTATAAGTAA A-3’ This study P3 5’GACGTCATGCCGGCAACCTTCAATAGGCTTAC G-3’ This study 34 P4 a 5’CGTAAGCCTATTGAAGGTTGCCGGCATGACGT C-3’ This study VirJ-1 5’-GAATGACCGCGCCAATGG-3’ This study VirJ-2 5’-CCGCTGAAGCCTATCACC-3’ This study Amp, ampicillin; Km, kanamycin; Tc, tetracycline 35 2.1.2. Media, antibiotics and other stock solutions Media or solutions Preparation a,b Reference LB (Luria broth) Tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; pH 7.5 Sambrook et al, 1989 SOB Tryptone, 20 g; yeast extract, 5 g; NaCl, 0.5 g; 10 ml of 250 mM KCl; pH 7.0,sterilize by autoclaving and add 5ml of filter-sterilized 2 M MgCl2 Sambrook et al, 1989 TB 10 mM PIPS, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl MG/L LB, 500 ml; mannitol, 10 g; sodium glutamate, 2.32 g; KH2PO4, 0.5 g; NaCl, 0.2 g; MgSO4.7H2O, 0.2 g; biotin, 2 μg; pH 7.0 Cangelosi et al, 1991 AB (Minimal medium) 20 × AB salts, 50 ml; 20 × AB buffer, 50 ml; 0.5% glucose 900 ml (autoclaved separately and mixed together before use) Cangelosi et al, 1991 IB (Induction Medium) 20 × AB salts, 50 ml; 20 × AB buffer, 1 ml; 0.5 M MES (pH 5.5), 8 ml; 30% glucose, 60 ml (autoclaved separately and mix together before use) Cangelosi et al, 1991 20 × AB salts NH4Cl, 20 g; MgSO4. 7H2O, 6 g; KCl, 3 g; CaCl2, 0.2 g; FeSO4.7H2O, 50 mg Cangelosi et al, 1991 K2HPO4, 60 g; NaH2PO4, 23 g; pH 7.0 Cangelosi et al, 1991 MES, 97.6 g; pH 5.5 Cangelosi et al, 1991 20 × AB buffer 0.5 M MES a Preparation for 1 liter, and sterilized by autoclaving; b For solid media, 1.5% agar was added. 36 2.1.3. Antibiotics and other stock solutions Antibiotics or soulutions Preparations Stock Concentration (mg/ml) Working Con. Working Con. in A. in E. coli tumefaciens (μg/ml) (μg/ml) Kanamycin (Km) Dissolved in dH2O, filter sterilized 100 50 100 Carbenicillin (Cb) Same as above 100 100 100 Tetracycline (Tc) Dissolved in absolute ethanol 5 10 5 Chloramphenical (Chl) Same as above 34 17 -- Isopropyl-β-Dthiogalactoside (IPTG) Dissolved in dH2O, filter sterilized 24 24 24 Acetosyringone Dissolved in dimethyl sulfoxide 100 mM -- 100 μm 37 2.1.4. Growth conditions and strain storage E. coli strains were grown at 37°C in LB (Sambrook et al, 1989) and A. tumefaciens strains were grown at 28°C in MG/L or IB supplemented with the appropriate antibiotics when necessary. Strains from single colonies were cultured on agar plates at 28°C or 37°C until single colonies appeared. Single colonies were picked up and mixed with MG/L or LB containing 50% glycerol. Strains were stored at -80°C. 2.1.5. Overexpression of protein in E. coli E. coli carrying the expression vector was inoculated into liquid LB with the appropriate antibiotics and cultured at 37°C overnight. The cell culture was diluted with fresh medium to the cell density of OD600= 0.5. IPTG was added to the culture to the final concentration of 0.3 mM. The culture was then incubated at 37°C for another 4 hours before the cells were harvested for subsequent protein purification. 2.1.5. Virulence gene induction of A. tumefaciens To induce the expression of virulence proteins in A .tumefaciens, cells were first cultured overnight on MG/L plate until single colonies appeared (normally 2-3 days) at 28°C. Colonies were inoculated into MG/L liquid medium with constant shaking at 225 rpm. Cells were then collected by centrifugation at 4000 rpm for 10 min and washed with IB twice. Cells were resuspended in IB and OD600 was adjusted to 0.3. Acetosyringone (AS) was added to the final concentration of 100 μM. Virulence gene expression was induced at 28°C for 18 hours with shaking. 38 2.2. DNA manipulations 2.2.1. Preparation of competent cell E. coli strain DH5α was routinely used as the host for cloning experiments, unless otherwise specified. E. coli cells were streaked from frozen stock and cultured overnight on LB plates at 37°C. Then, several single colonies were picked and inoculated into100 ml of SOB medium in a 1-liter conical flask. The cells were cultured at room temperature (about 19°C) with vigorous shaking (250 rpm) to an OD600 of 0.5 to 0.7. The cells were chilled on ice for 10 min before collected by centrifugation at 2600 rpm for 5 min at 4°C. The cell pellets were resuspended in 30 ml of ice-cold TB buffer (10 mM PIPS, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, pH 6.7; all components except MnCl2 were dissolved and autoclaved; 1M MnCl2 solution was filter-sterilized and added to make TB buffer; stored at 4°C) and then incubated on ice for 10 min. Cells were collected by centrifugation as above and resuspended in 5 ml of ice-cold TB buffer. Thereafter, DMSO was added to the final concentration of 7% and the cells were aliquoted into pre-cooled sterile Eppendorf tubes at 100 μl each. The competent cells were kept at -80°C until needed. 2.2.2. Plasmid DNA preparation Plasmid DNA was prepared following the method described previously with some modifications (Sambrook et al, 1989). Briefly, E. coli cells from 2 ml of overnight culture were collected by centrifugation at 12000 rpm for 1 min. The cell pellet was resuspended in 100 μl of ice-cold solution I (50 mM glucose, 25 mM TrisHCl, 10 mM EDTA, pH 8.0) thoroughly by vigorous vortex. Then, 200 μl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and mixed by gentle inverting 39 rapidly for 4-6 times. The solutions should become viscous and slightly clear. After adding 150 μl of Solution III (3 M potassium, 5 M acetate) to the solution, the mixture was inverted for 4-6 times to disperse Solution III through the viscous bacterial lysate. The lysate was extracted with equal volume of chloroform once by centrifugation at 14000 rpm for 5 min. The supernatant was then transferred to a clean Eppendorf tube. To precipitate the plasmid DNA, 2 volumes of cold ethanol was added and the mixture were centrifuged as above. The DNA pellet was washed once with 70% ethanol and dried in a vacuum concentrator. The extracted plasmid DNA was dissolved in 20 μl of sterile water and stored at -20°C. 2.2.3. DNA digestion and ligation DNA digestion and ligation were conducted following the instructions of the manufacturers of digestion and ligation enzymes. The restriction enzymes were purchased from Promega. Digestion reaction systems were comprised of buffer, enzyme, DNA and water, and were incubated at 37°C for an appropriate period. Digested vectors and fragments used for ligation were cleaned. Ligation was performed at room temperature for 4 hours or overnight. 2.2.4. Polymerase chain reaction (PCR) Polymerase chain reaction was carried out using a PCR machine in a thin wall PCR tube. The PCR solutions (25 μl) usually included the following: 10 × PCR buffer (without MgCl2) 2.5 μl 25 mM MgCl2 1.5 μl Primer 1 (10 pmol/μl) 1 μl 40 Primer 2 (10 pmol/μl) 1 μl dNTPs (10 mM each) 0.5 μl Template DNA 20-100 ng Taq DNA polymerase 0.5 μl (1 unit) Add distilled water to a final volume of 25 μl The PCR was run using the following program: 1 cycle 94°C for 5 min 30-35 cycles 94°C for 30 seconds Annealing at (Tm-5)°C for 60-90 seconds Extension at 72°C for 1 min per kb 1 cycle 72°C for 10 min Hold 16°C 2.2.5. DNA electrophoresis and purification DNA fragments were separated in 1% TAE (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0)-agrose gel along with a standard DNA marker (Fermentas). Digested DNA vectors and fragments from genomic DNA or PCR products for ligation and transformation were purified following the instructions provided by the manufacturer. Briefly, DNA was separated in a 1% agrose gel. The gel slice containing the desired DNA band was excised and transferred into a pre-weighted Eppendorf tube before 3 volumes (100 mg gel ≈ 100 μl) of buffer QG were added. The sample was incubated in a 55°C waterbath for 5-10 min to dissolve the gel slice 41 completely. For DNA fragments larger than 4 kb or smaller than 500 bp, 1 gel volume of isopropanol was added. The mixture was then transferred into a QIAquick spin column in a 2 ml collection tube. The binding of the DNA to the column was fulfilled by centrifugation for 1 min at 14000 rpm. The column was then washed once with 750 μl of buffer PE with one additional centrifugation to remove residual ethanol. The column was placed into a clean 1.5 ml tube. To elute DNA, 30 μl of sterile water was applied to the centre of the column membrane and the column was centrifuged at 14000 rpm for 1 min. The eluted liquid containing the purified DNA was stored at 20°C. 2.2.6. Transformation of E. coli Plasmids or ligation products were introduced into E. coli by transformation for amplification or screening (Sambrook et al, 1989). Frozen competent cells (100 μl) were thawed on ice. Plasmid (50-100 ng in 1-2μl) or ligation product (20 μl) was added and the contents of the tubes were mixed by swirling gently. The tubes were then incubated on ice for 30 min. The mixture of cells and DNA was heat-shocked at 42°C for 90 seconds. After chilling the cells on ice for 2 min, 750 μl of fresh LB medium was added. The cultures were incubated at 37°C for 1 hour with shaking. The cells were collected and spread onto LB agar plates containing the appropriate antibiotic(s). Colonies usually appear after incubation at 37°C for12-16 hours. 42 2.2.7. Transformation of A. tumefaciens Introduction of the broad-range plasmids into Agrobacterium tumefaciens was carried out by triparental mating (Ditta et al, 1980) or electroporation (Cangelosi et al, 1991). Triparental mating from E. coli into A. tumefaciens was conducted by mixing equal proportions of the helper strain MT616, the donor strain and the recipient strain cells together on MG/L agar plates and incubating the plates overnight at 28°C. A small amount of the mating mixture was picked and streaked onto AB agar plates containing the appropriate antibiotics. The A. tumefaciens exconjugates usually appeared after 2-3 days of incubation. After purified on AB plates for several times, single colonies of A. tumefaciens were streaked on MG/L agar plate with the appropriate antibiotic(s). For triparental mating from A. tumefaciens into E. coli, a similar procedure was taken with some modifications and adjustments. The mating mixture of the helper strain MT616, the donor strain and the recipient strain MT607 was incubated on LB plates overnight at 28°C and streaked onto LB plates containing the appropriate antibiotics. The plates were cultured overnight at 37°C, since A. tumefaciens could not grow at 37°C. Electroporation was also used in this study for the introduction of plasmids into A. tumefaciens. Electrocompetent A. tumefaciens cells were prepared as follows. Cells cultured overnight at 28°C were scrapped with a sterile wooden stick and then transferred into an autoclaved Eppendorf tube. The cells were washed three times with ice-cold 15% glycerol before the cell pellet was resuspended in 50-100 μl of icecold 15% glycerol and then the plasmid DNA (50-100 ng in 1-2 μl) was added. The 43 mixture of cells and DNA was transferred into a chilled BioRad electroporation cuvette and kept on ice for 10 min. Gene Pulser II Electroporation System (BioRad) was set to the 25-μF capacitor, voltage of 2.5 kV and controller unit of 400 Ω. The outside of the cuvette was wiped with C-fold towel to get rid of the moisture before the cuvette was slide into the shocking chamber base. The cells were usually pulsed for 8-10 milli-seconds. Thereafter, 1 ml of MG/L medium was added and the mixture was transferred into a 15-ml culture tube. After culturing at 28°C for 1h, the cells were collected and spread onto MG/L plates containing the selectable antibiotics. Colonies usually appeared on the third day after electroporation. 2.3. Protein techniques 2.3.1. Buffers for protein manipulations The buffers used for protein manipulations in this study are listed below: Name 10 × Tris-buffered saline (10 × TBS) Components (for 1 L) pH adjustment 0.2 M Tris base Adjust pH to 7.6 1.37 M Sodium chloride 38 ml 1M Hydrochloric acid 1 × TBST 10 × Tank buffer 0.1% Tween-20 (v/v) in 1 × TBS 0.25 M Tris No need to check 1.92 M glycine pH 0.1% SDS 10 × Transfer buffer 48 mM Tris Adjust pH to 8.3 38 mM glycin 0.37 g SDS 44 20% methanol 4 × separating gel buffer 1.5 M Tris-HCl Adjust pH to 8.8 4 × stacking gel buffer 0.5 M Tris-HCl Adjust pH to 6.8 1 × SDS gel-loading buffer 50 mM Tris-HCl (pH 6.8) 100 mM dithiothreitol 2% SDS 0.1% bromophenol blue 20% glycerol Staining solution 0.25 g Coomassie Brilliant blue R (Gibco) 400 ml methanol 70 ml acetic acid Destaining solution I 400 ml methanol 70 ml acetic acid Destaining solution II 70 ml Acetic acid 50 ml Methanol 2.3.2. Pull down assay Pull-down assay is an in vitro method used to determine the physical interaction between two or more proteins. The minimal requirement for a pull-down assay is the availability of a purified and tagged protein (the bait) which will be used to capture and ‘pull-down’ the protein-binding partner (the prey). In this study, pull down assay was conducted using Immobilized Metal Affinity Chromatography (IMAC). In order to test the interactions of VirJ with other proteins, 45 native VirJ-His was isolated from A. tumefaciens. The protocol used to isolate native VirJ-His and its interacting proteins is outlined as below. A. tumefaciens was first inoculated into MG/L and then induced with AS in IB. Bacteria were collected by centrifugation at 4000 rmp for 10 min and were washed once with PBS (pH 7.6). The pellet was then resuspended in 2 ml of the resuspension buffer (50 mM NaH2PO4, 300 mM NaCl, pH 7.4). Lysozyme was added to the final concentration of 4 mg/ml. After vortexing vigorously, the sample was shaked on ice for half an hour to break up the cells before 10 ml of binding buffer (50 mM NaH2PO4, 0.5% Triton X-100, 300 mM NaCl, 2 mM PMSF, pH 7.4) was added to dilute the sample. The lyzed cells were centrifuged at 12000 rpm for 15 min at 4ºC to pellet any debris and unbroken cells. The supernatant was kept as the total cell lysate. A small amount of the cell lysate was taken out for protein analysis control. The rest was ready for binding assay and was transferred into a new tube. The cell lysate could be stored at -80ºC if needed. Resin (Talon, ClonTech) was prepared proportional to the quantity of the bacteria and the expression level. In general, about 250 μl resins had the capacity to bind VirJ-His from 1.2 x 1011 cells. The resin was centrifuged at 700 g for 2 min and the supernatant was discarded before the resin was equilibrated by adding 5 volumes of cell resuspension buffer and left for several minutes. After that, the resin was centrifuged at 700 g for 2 min and the supernatant was discarded carefully. The resin would be ready for binding with His-tagged proteins after the prior washing steps. Sample was added to the resin and the VirJ-His proteins were bound with the resin by shaking at 4ºC gently for one hour before the sample was centrifuged at 700 g for 2 min. Thereafter, the supernatant was poured out without disturbing the resin pellet. After setting aside a small amount of the supernatant for 46 purification performance analysis, the resin pellet was washed with 10 volumes of binding buffer for three times and was then transferred into the column without introducing bubbles. VirJ-His were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM immidazole, pH 7.4). The resin was washed in 10 volumes of dH2O and then was resuspended in 30% ethanol for storage at 4ºC. 2.3.3. Purification of VirB7-His Purification of VirB7-His was conducted using Immobilized Metal Affinity Chromatography (IMAC). For the purification of low expression level VirB7 either from E. coli or from A. tumefaciens, the protocol provided by the manufacturer was modified to attain better purification results. For isolation and purification of VirB7-His from E. coli, TG1(pB78HSW) was inoculated into LB liquid with tetracycline overnight and OD600 was adjusted to 0.5 by diluting with fresh LB. IPTG was added into the medium to the final concentration of 0.3 mM. After 4 hours of induction, cells were collected and washed once with PBS (pH 7.6). In total, one litre of cell culture was collected. Cells were then resuspended in the resuspension buffer (50 mM NaH2PO4, 300 mM NaCl, 0.5% Triton X-100 and 8 M Urea, 2 mM PMSF, pH 7.4). The resuspension was passed through French Press twice. The lyzed cells were passed three times through the 18gauge syringe needle to reduce the viscosity of the samples. Cell lysate was centrifuged at 12000 g for 15 min and the supernatant was ready for binding with resin column. 47 After shaking for 1 hour at room temperature, the resins incubated with clarified lysate prepared as above were washed with cell resuspension buffer twice and then washed with resuspension buffer without Triton X-100 three times. The resins were then transferred into a resin column and the protein was finally eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 8 M Urea, 50 mM MES, pH 3.8). Fractions of a bed volume were collected and VirB7 was always eluted at the second elution fraction. For purification of VirB7-His from A. tumefaciens, the method was similar with that from E. coli except that A. tumefaciens cells were induced with AS in IB. In total, 2 litres of cells from IB were collected for the purification purpose. 2.3.4. SDS-PAGE analysis Protein profiles were analyzed using SDS-PAGE (Laemmli, 1970) based on the molecular weight. The electrophoresis apparatus used was the Mini-Protean III Electrophoresis Cell (BioRad). The apparatus was assembled according to the instructions provided by the manufacturer. The monomer stock solution of acrylamide/bis-acrylamide (30.8%T/2.7%C) was prepared as described in Molecular Cloning (Sambrook et al, 1989) and stored in the dark at 4°C. APS (10%) solution was freshly prepared before each use. Separating gel buffer (4 ×, 1.5 M Tris-HCl, pH 8.8) and stacking gel buffer (4 ×, 0.5 M Tris-HCl, pH 6.8) were stored at room temperature. Tank buffer was prepared as a 10 × stock solution (0.25 M Tris-HCl, 1.92 M glycine, 1% SDS, pH 8.3) and stored at room temperature. Gel loading buffer (2 ×, 100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue and 20% glycerol, 48 0.2 M DTT) was prepared without DTT and stored at room temperature. DTT was added from a 2 M stock solution and stored at -20°C before use. Polyacrylamide gels were prepared following the instructions of Hoefer Scientific Instruments (Protein electrophoresis-applications guide, 1994). In this study, 12% PAGE gel was used for the analysis of proteins, unless otherwise specified. After careful washing of the glass plates with 70% ethanol and drying, the plates were assembled. 10 ml of 12% separating gel was prepared by gently mixing 3.3 ml of H2O, 4 ml of monomer solution, 2.5 ml of separating gel buffer and 0.1 ml of 10% SDS. After thorough mixing, 0.1 ml of 10% APS and 3.5 μl of TEMED were added. Immediately, a suitable amount of the separating gel solution was pipetted and transferred into the region in-between the plate sandwiches with 1.5 cm left for stacking gel. The separating gel was over-laid with dH2O and left for about half an hour for the gel to polymerize. After that, 2 ml of stacking gel was prepared by mixing 1.4 ml of H2O, 0.33 ml of monomer, 250 μl of stacking gel buffer and 20 μl of 10% SDS together followed by carefully mixing with 20 μl of 10% APS and 2 μl of TEMED. The mixture was then pored on top of the separating gel. The comb was inserted and allowed for polymerize before running. Samples were mixed with the loading buffer and boiled for 5 min to allow proteins to denature. After the gel was completely polymerized, the wells were washed with dH2O after which samples were loaded and the gels were run in tank buffer at a constant current of 8 mA per plate until loading dye reached the bottom of the gel. 49 2.3.5. Silver staining Silvering staining was used when the protein concentration was low and could not be detected with standard Coomassie blue method. Soaking and washing were all carried out in a clean glass tray and all buffers were freshly prepared. Briefly, after electrophoresis, the gel was fixed in fixing solution (40% methanol, 13.5% formalin) for 10 min followed by washing in water twice for 5 min each. Then the gel was soaked in 0.02% Na2S2O3 and washed in water twice for 20 sec each. Next, the gel was soaked in 0.1% AgNO3 for another 10 min and finally the gel was rinsed with water and again with a small volume of developing solution (3% sodium carbonate, 0.05% formalin, 0.000016% Na2S2O3). The gel was developed in the developing solution until band intensities were adequate (normally 1-3 min). Staining process was stopped by adding 2.3 M citric acid and continued shaking for 10 min. Development would continue a little after adding citric acid. The gel was washed in water for 10 min and soaked in water for 30 min or longer before drying. 2.3.6. Western blot analysis The sample was mixed with equal volume of 2 × loading dye buffer (Laemmli, 1970) and boiled in a water bath for 5 min. After cooling, the sample was loaded into a SDS- polyacrylamide gel and separated at a constant current of 8 mA per plate. The protein was transferred onto the Immun-BlotTM PVDF membranes (Bio-Rad) from the gel in a mini gel transfer system for 4 hours at 200 mA or for overnight at 100 mA (for transfer of VirB7-His, the transfer condition was modified to 70 mA for one hour). After that the non-specific binding sites were blocked by immersing the membrane in 10 % non-fat milk (Nestle) in TBST for at least 2 h at room temperature on an orbital shaker. The membrane was then washed in TBST buffer for 3×10 min 50 and incubated in the diluted primary antibody for 1 h at room temperature. The membrane was washed three times as above before being incubated in the diluted secondary antibody for 1 h at room temperature. After washing thoroughly as above, the membrane was subjected to signal detection with the enhanced chemiluminescence (ECL) Western blot detection system according to the recommendations of the manufacturer (Amersham). 2.4. Lipopolysaccharides methodology The method used to isolate LPS was based on the hot phenol-water method developed by Apicella et al (1997) with some modifications. Because LPS can be dissolved in water and phenol, while proteins would become denatured in phenol, it is possible to isolate LPS from bacteria using hot phenol. 2.4.1. LPS preparation by hot phenol 10 ml of AS induced A. tumefaciens culture (OD600=0.3) was pelleted and washed twice with PBS (pH 7.6) containing 0.15 mM CaCl2 and 0.5 mM MgCl2 at 4ºC. Cell pellet was resuspended and washed in 300 μl of dH2O and transferred into glass tubes. Equal volume of 95% phenol (pre-heated to 68ºC) was added to the suspension and incubated at 68ºC for 15 min with vigorous agitation. The suspension was chilled on ice and then centrifuged at 8500 g for 15 min at 4ºC. The supernatant was taken out carefully and another 300 μl of water was added to the phenol phase to retract LPS from phenol again. After centrifugation, the supernatant was combined and dialyzed against water for 2 days to remove the trace odor of phenol. After dialysis, DNase and RNase were added to the final concentration of 200 μg/ml and 50 μg/ml respectively and incubated at 37ºC for 2 hours. LPS was precipitated by adding 51 10 volumes of 95% ethanol with 0.5 M sodium acetate. Samples were stored at -20ºC overnight before they were centrifuged at 2000 g for 10 min at 4ºC. The supernatant of each sample was discarded and the pellet was dissolved in 100 μl dH2O. 2.4.2. TLC analysis of LPS Thin-layer chromatography (TLC) is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. This method can be used to distinguish LPS from different strains as well as LPS from the wild type and the mutant strains. The method used to analyze LPS using TLC in this study is based on the protocol provided by Buttke and Ingram (1975). In general, LPS was diluted with chloroform: methanol (2:1, v/v) and vortexed for 4 sec for three times. 100 μl of sample was loaded to the pre-coated silica plate using syringe by an AutoSpotter at 65ºC at speed 2. For chromatography of whole LPS, isobutyric acid: concentrated NH4OH: water was used at the ratio (vol/vol/vol) of 57: 4: 39. TLC plate was separated by placing the plate into the TLC developing tank, which was filled with 100 ml of the separation solvent for 1 hour before use. When development was finished (normally would last about 7-8 hours), contaminants such as salts which would affect the detection of LPS were removed by washing the TLC plates sequentially with propanol-water (1:1, v/v) twice and then with methanol-water (1:1, v/v) once (Lee et al, 2004). LPS was visualized by charring at 150ºC for 10 min after spraying with 10% sulphuric acid in ethanol. 52 2.4.3. LPS electrophoresis and staining The electrophoresis method used to analyze LPS was the same as applied to the analysis of proteins except that SDS was omitted from the separating gel (Apicella et al, 1997). After electrophoresis, LPS was visualized by staining with silver. 2.5. Analysis of whole cell lipids 2.5.1. Preparation of whole cell lipids A. tumefaciens cells were collected and transferred into the FEP tubes, which would not release any chemicals that interfered with the analysis of lipids. 6 ml of chloroform: methanol (2:1, v/v) was added to the tubes and the samples were shaked at 250 rpm in the cold room overnight. Samples were topped up with dH2O and centrifuged at 4000 rpm for 25 min at room temperature. After centrifugation, the sample would be separated into three layers, with an aqua phase on top, an organic phase at the bottom and a protein pellet in between. The organic phase was transferred into a glass tube carefully and the sample was dried with N2 fluid. After drying, the pellet was resuspended into 300 μl of chloroform: methanol (1:1, v/v) by vigorous vortexing. After the sample was aliquoted, 100 μl of the sample was vacuum dried for TLC analysis, 100 μl for Electrospray ionization (ESI) ion-trap MS analysis and the rest was stored at -20ºC. 2.5.2. TLC analysis of lipids The method used to separate the whole cell lipids was similar with that used to analyze LPS, except that the developing solvent was different. For the separation of polar lipids, the developing solvent contained chloroform: methanol: water at a 53 volume ratio of 60: 12: 1. To analyze the non-polar lipids, the developing solvent was freshly prepared by mixing hexane, ethyl ether and formic acid at a volume ratio of 45: 5: 1. TLC development would finish in about 2-3 hours. After that, the TLC plate was stained with iodine until the band intensities were adequate. 2.5.3. Electrospray ionization (ESI) ion-trap MS analysis of lipids Electrospray ionization (ESI) allows production of molecular ions directly from samples in solution. It can be used for small and large molecular-weight biopolymers (peptides, proteins, carbohydrates, and DNA fragments) and lipids. The sample must be soluble, stable in solution, polar, and relatively clean (free of non-volatile buffers, detergents, salts, etc.). ESI was performed in the negative-ion mode using an LCA Deca ion-trap mass spectrometer, equipped with a capillary LC system. Carrier solvent (chloroform: methanol, 1:1) was used at a flow rate of 10 μl/min to deliver 2 μl injected samples. The operational parameters for the ESI source and ion transfer optics were as follows: Capillary (V) 3000; Sample Cone (V) 50; Extraction Cone (V) 1.0; Source Temperature (ºC) 80; Desolvation (L/hr) 350; MCP Detector (V) 2500. The run time for each sample was 4 min. Nitrogen was used as sheath gas. Spectra were scanned in the range of m/z 300-2000. 2.6. Plant tumorigenesis assay The tumorigenic ability of A. tumefaciens was tested on Kalanchoe daigremontiana leaves. In general, A. tumefaciens were cultured overnight in MG/L supplied with the appropriate antibiotics (Young and Nester, 1988). The bacterial 54 density was adjusted to OD600=0.5 with MG/L. Thereafter, fully expanded leaves of Kalanchoe was sterilized with 70% ethanol and wounded with a sterile needle of about 1 cm long before 5 μl of bacterial suspension was deposited into the wound. Each strain was inoculated three times at least on four independent plants. After growing for 3-5 weeks in the plant growth room, tumor formation was scored. 2.7. Subcellular fractionation of A. tumefaciens Fractionation of A. tumefaciens was conducted as described by de Maagd and Lugtenberg (1986) with some modifications. Induced A. tumefaciens cells were collected and washed once with the induction medium (Pan et al, 1993) and twice with 50 mM Tris-HCl, pH 8.0, by centrifugation at room temperature. The cell pellet was resuspended in 50 mM Tris-HCl (pH 8.0), 20% sucrose, 2 mM EDTA, and 0.2 mg/ml lysozyme. The cell suspension was incubated at room temperature for 30 min and centrifuged at 3000g for 15 min at 4ºC. All the remaining steps were carried out at 4ºC. The resulting supernatant was further centrifuged at 20000 g for 15 min and the supernatant was saved as the periplasmic fraction. The combined cell pellet was resuspended in 50 mM Tris-HCl, pH 8.5, 20% sucrose, 0.2 mM DTT, 2 mM PMSF, 0.2 mg/ml DNase I and 0.2 mg/ml RNase A. The cell suspension was passed through a French press minicell three times and then incubated on ice for 30 min. The cell lysate was diluted with 2 volumes of 50 mM Tris-HCl (pH 8.5) and centrifuged at 1000 g for 20 min. The supernatant was then centrifuged at 40000 rpm for 3 hours. The supernatant was saved as the cytosolic fraction. The pellet was resuspended in 2.5 ml of 5 mM EDTA (pH 8.0), 0.2 mM DTT and 20% sucrose by sonication. The suspension was layered onto a gradient containing a top layer of 6 ml of 5 mM EDTA (pH 8.0), 53% sucrose over a bottom layer of 2 ml of 5 mM EDTA (pH 8.0) and 70% sucrose. The gradient was centrifuged at 18000 rpm for 18 hr. 55 The upper and lower bands were apparent and well separated from each other, and they were collected as the inner and outer membrane fractions, respectively. After fractionation, 1 mM phenylmethylsulfonyl fluoride was added to each of the subcellular fractions. The proteins in each of the subcellular fractions were concentrated by Centriprep-10 (Amicon). The Laemmli sample buffer (Laemmli, 1970) was added to each of the subcellular fractions and boiled for 5 min. All the samples were derived from the same amount of cell pellet used for fractionation. 56 RESULTS 3.1. Functional assay of VirJ-His In 1995, two groups of researchers independently found that a protein VirJ that is encoded by the Ti plasmid can complement the acvB mutant strain (Pan et al, 1995; Kalogeraki and Winans, 1995). This indicates that VirJ and AcvB play the same or similar roles in A. tumefaciens. virJ is only found on the Ti plasmid of octopine but not nopaline strains and acvB is a chromosomal gene. Therefore, nopaline strains encode only AcvB while octopine strains have both AcvB and VirJ (Pan et al, 1995). In virJ and acvB double mutant strain, the stabilities of VirB8, VirB9, VirB10 and VirB11 were affected, but not that of VirB4 and VirE2. Further analysis of AcvB and VirJ revealed that they both contained a lipase or acyltransferase motif which is of ten amino acids and is conserved among lipases and acyltransferases (Pan, 1999; Person et al, 1989). Previous studies have shown that mutation in this motif would make VirJ unable to complement acvB mutant, suggesting that this motif plays a key role in the function of VirJ. 3.1.1. Construction of VirJ-His expression vectors In order to study the function of the lipase motif of VirJ within A. tumefaciens, plasmids that express VirJ with a 6xHis-tag fused to the C-terminus (VirJ-His(6)) were constructed. Briefly, the 1 kb fragment of virJ and its promoter sequence was amplified with primers HisJ-5 and HisJ-3 from the wild type strain A348 by PCR. After the PCR product was purified, two restriction enzymes BamHI and EcoRI were used to digest both the insert and the vector pUCA19 at 37ºC overnight. The digested fragments were purified and were ligated by T4 DNA ligase. The ligation product was 57 then transformed into DH5α for selection. The resulting plasmid was defined as pHisJ1 that contained the promoter of virJ in A. tumefaciens and virJ-his (Figure 3.1). The sequence of virJ was confirmed by DNA sequencing to ensure there was no point mutation that could have arisen during PCR (Figure 3.2). To test the function of the lipase motif of VirJ, one amino acid within the lipase motif and one outside it were mutated using site-directed mutagenesis scheme separately. For this purpose, two sets of primers were designed, one pair named P1 and P2 was designed for generating point mutation within the lipase motif, and the other pair named P3 and P4 was used to generate point mutation of a site outside the lipase motif. Two rounds of PCR were performed. Firstly pHisJ1 was used as the template and was amplified with different combinations of primers, including HisJ-5 and P2, HisJ-3 and P1. After the PCR products were purified, the second round of PCR was performed with primers HisJ-5 and HisJ-3 using the PCR products of the first round as the template. Then the PCR product was purified, digested and ligated with vector pUCA19 that had been digested with BamHI and EcoRI. The ligation product was transformed into DH5α for selection. The correct plasmid was named pHisJ2 which contained the promoter sequence of virJ in A. tumefaciens and virJ-his with Ser127 replaced by threonine. The same method was used to generate pHisJ3 with the promoter of virJ in A. tumefaciens and virJ-his with Ser136 replaced by threonine using primers P3 and P4 (Figure 3.1). The sequences of these two plasmids were also verified by sequencing (Figure 3.3). 58 MCS Plac BamHI virJ promoter EcoRI virJ his tag + lac Z’ Figure 3.1. Construction of plasmids pHisJ1. virJ and its promoter sequence was amplified from A348 with primers HisJ-5 and HisJ-3 by PCR. The PCR products were digested with BamHI and EcoRI and were ligated with pUCA19, which is a broad-host-range plasmid. Primers P1 and P2, which contained the appropriate mutation, where used to replace Ser127 with threonine in pHisJ2. P3 and P4, with an appropriate mutation within them, were used to replace Ser136 with threonine in pHisJ3. 59 1 ATGGCGATAAAATTGGTATTGATACTCGTATTTACACTGTTTCTCGCGGCAGACGCTGCC 61 TATGCGAATGACCGCGCCAATGGTGTCATGTGGTCAAACGGGGGCGAAGCTGGAGTGAGA 121 CTTCCTCTTCGGGTTTTCAATGCCAAGCCAGCCAAGAACACGGTGGCGATCATTTATTCC 181 GGAGACGCTGGATGGCAAAATATCGATGAGGTGATTGGTACCTATCTGCAGACGGAAGGG 241 ATTCCTGTCATTGGCGTCAGTTCACTTCGGTATTTCTGGTCGGAGCGGTCTCCAAGCGAA 301 ACTGCTAAGGATCTTGGTCACATAATCGATGTCTACACCAAGCATTTCGGTGTGCAGAAT 361 GTTTTACTTATAGGATATTCTTTCGGCGCTGACGTCATGCCGGCAAGCTTCAATAGGCTT 421 ACGCTTGAGCAAAAAAATCGGGTTAAGCAAATCTCTCTCTTGGCATTGTCACATCAAGTC 481 GACTATGTCGTCTCATTTAGGGGCTGGCTCCAACTCGAAACCGAAGGTAAGGGCGGCAAT 541 CCTCTGGATGATCTCAGATTCATTGACCCTGCAATCGTCCAATGCATGTACGGGCGCGAA 601 GACCGTAATAATGCTTGCCCATCTCTCCGACAGACCGGCGCAGAGGTGATAGGCTTCAGC 661 GGAGGCCATCACTTTGGTAATGATTTCAAAAAACTGTCTACGCGCGTCGTCTCAGGCCTC 721 GTGGCACGCCTAAGTCATCAGTATTCTTCAGGTCCTGCACCGCTTCATCATCATCATCAT 781 CATTAA Figure 3.2. Sequencing result of virJ-his in pHisJ1 (only the sequence of virJ-his was shown). The yellow box indicates the location of lipase motif and the red box showes the location of 6xhis-tag 60 121 pHisJ1: 127 130 136 …… vqnVLLIGYSFGAdvmpasfn …… 127 pHisJ2: …… vqnVLLIGYTFGAdvmpasfn …… tctÆact 136 pHisJ3: …… vqnVLLIGYSFGAdvmpatfn …… agcÆatc Figure 3.3. Amino acid sequences of the lipase motif of VirJ-His encoded by pHisJ1, pHisJ2 and pHisJ3. The amino acid sequences of the lipase motif are shown in red. The mutated amino acids are labeled in green. In pHisJ2, T379 was changed to A, therefore Ser127 was replaced by threonine. In pHisJ3, G407 was changed to C and therefore Ser136 was replaced by threonine. 61 3.1.2. A. tumefaciens strain B119 is sensitive to carbenicillin Next the three plasmids were transformed into A. tumefaciens strain B119 by electroporation. After electroporation, 100 μl of the cell culture was spread evenly on the MG/L plates containing 100 μg/ml carbenicillin. Colonies were supposed to appear in 2-3 days. However, no colony appeared even after 5 days. Since B119 is an acvB mutant strain and AcvB is normally secreted to the cell periplasm in the wild type, it is possible that mutation of acvB affects the cell membrane and therefore affects its tolerance to carbenicillin. In order to find out the appropriate concentration of the antibiotic used to culture B119 harbouring the carbenicillin resistant plasmid, the cell culture was spread onto MG/L plates with different concentrations of carbenicillin, from 1 μg/ml, 5 μg/ml, 10 μg/ml to 50 μg/ml. Colonies appeared on the MG/L plates with 1 μg/ml and 5 μg/ml of carbenicillin in 3 days, but not on other plates. B119 alone was streaked on MG/L plate with 1 μg/ml and 5 μg/ml of carbenicillin to verify the tolerance of B119 to carbenicillin. As a result, B119 could grow on the MG/L plate with 1 μg/ml carbenicillin but not on the MG/L plate with 5 μg/ml of carbenicillin. Therefore, MG/L plate with 5 μg/ml of carbenicillin was suitable for growing B119 harbouring carbenicillin resistant plasmids. 3.1.3. Functional test of VirJ-His in A. tumefaciens In order to determine whether VirJ-His can functionally complement the acvB mutant strain (B119) and verify the effect of the lipase motif on the virulence of A. tumefaciens, B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) were inoculated on the healthy leaves of K. daigremontiana and the tumors were scored after three weeks. Tumorigenesis assay showed that VirJ-His could restore the virulence of B119, indicating that it played the same role as the wild type VirJ. While VirJ with a point 62 mutation within the lipase motif could not restore the virulence of B119, VirJ with a mutation outside the lipase motif could still restore the virulence of B119 (Figure. 3.4). 63 A348 A208 B119 B119 B119(pHisJ2) B721 B119(pHisJ1) B119(pHisJ3) B119(pB78HSW) B721(pB78HSW) Figure 3.4. Plant tumorigenesis assay. A. tumefaciens was grown on MG/L with appropriate antibiotics at 28ºC overnight. Cells were scraped off the plate and resuspended in MG/L to OD600=0.5. Fully expanded leaves of K. daigremontiana were wounded with sterilized needles. 5 μl of A. tumefaciens cell suspensions were deposited into the wounds. Plants were grown at room temperature (about 25ºC) for three weeks before the tumors were tested. 64 3.2. Analysis of Lipopolysaccharides (LPS) The cell membranes of Gram-negative bacteria, including human pathogens such as E. coli and Salmonella enterica, consist of an asymmetric bilayer, the outer leaflet of which consists predominantly of lipopolysaccharides (LPS) with proteins taking up much of the remaining surface. This may be important for the growth and survival of the bacteria in harsh environments including those within eukaryotic hosts. Lipopolysaccharides derived from different groups of Gram-negative bacteria have a common basic structure, a lipid core (lipid A) attached to a polysaccharide moiety. Lipid A provides the anchor that secures the molecule within the membrane. It is believed to mediate most of the biological functions of LPS and can alone reproduce its toxicity. A 2-keto-3-deoxyoctonate links the lipid A region to the oligosaccharide region called the O-antigen (Rietschel et al, 1992; 1994). This Oantigen is the only variable region in the LPS molecule and is responsible for the heterogeneity of the response between LPS from different strains of bacteria. LPS that lacks the O-antigenic side chain often are referred to as rough owing to their colonial morphology, whereas bacteria which have this LPS component are referred to as smooth (Darveau and Hancock, 1983) Lipid A is a unique and distinctive phosphoglycolipid, the structure of which is highly conserved among species. However, variations in the fine structure can arise from the type of hexosamine present, the degree of phosphorylation, the presence of phosphate substituents, and importantly in the nature, chain length, number, and the position of the acyl groups. 65 In A. tumefaciens, chvA and chvB were found to affect the structure of LPS and the mutation of these two genes would lead to the abolishment of the tumorigenic ability of A. tumefaciens (Cangelosi et al, 1989). In this project, we first wanted to know whether mutation of the lipase motif of VirJ would affect the LPS of A. tumefaciens, since VirJ is a putative lipase or acyltransferase, and the formation of LPS requires the involvement of lipase or acyltransferase (Proce et al, 1994). If the mutation of the lipase motif changes the structure of LPS, the structure of the membrane of the cells would be changed and it is possible that it will affect the virulence of the bacteria. It is also possible that mutation of LPS may affect the attachment of A. tumefaciens to the plant cells. In order to test these, LPS from different A. tumefaciens strains were isolated and analyzed. 3.2.1. Thin-layer chromatography (TLC) analysis of LPS Thin-layer chromatography is a particularly frequent approach to obtain information about the various phospholipid components of the lipid extract under investigation. It is one of the best choices for rapid screening of samples because it is cheap, portable and has a minimal procedure of cleanup, little method development time and many samples can be analyzed in parallel (Lee et al, 2004). In 1975, TLC was developed as a new procedure to distinguish between LPSs of different strains and also between the wild type organism and the mutated form, as the mutants are unable to synthesize complete LPS (Buttke and Ingram, 1975). 66 In this study, LPS from A. tumefaciens strains B119, B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) were isolated using hot phenol-water method and were separated on a TLC plate. Differential size of LPS was expected to be separated due to the different motility of LPS in the organic solvent. However, after charring the TLC plate in the oven, only smears could be observed. No differences could be observed on the TLC plate (Figure 3.5). This is reasonable since LPSs of bacteria are a mixture of LPS with different length of O-antigenic chains. This indicated that the resolution of TLC is not sensitive enough to detect the differences of LPS in our experimental samples and a more accurate method may be required to separate LPS isolated from A. tumefaciens. 3.2.2. LPS analysis by electrophoresis LPS prepared by hot phenol-water method was separated by 15% polyacrylamide gel electrophoresis and stained with silver. Upon gel electrophoresis the lipopolysaccharides form a "ladder" of components which gives better resolution than the TLC method. LPS samples were dissolved into three major bands and several minor bands in the gel. The band patterns of LPS from B119 were the same with those from B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) (Figure 3.6). No detectable difference in the LPS pattern was observed from these four strains. Therefore, it is proposed that VirJ does not function in the formation of lipopolysaccharides of A. tumefaciens. 67 1 2 3 4 Figure 3.5. Thin-layer chromatography analysis (TLC) of LPS from A .tuemfaciens. A. tumefaciens strains were inoculated into MG/L followed by induced with AS in IB medium. LPS extracted with hot phenol-water method was spotted at the bottom of the TLC plate and was separated in the developing solvent containing isobutyric acid: concentrated NH4OH: water at 57: 4: 39 (vol/ vol/ vol). LPS was visualized by charring after spraying with 10% sulphuric acid in ethanol. Lane 1: LPS from B119, Lane 2: LPS from B119(pHisJ1), Lane 3: LPS from B119(pHisJ2) and Lane 4: LPS from B119(pHisJ3). The arrow indicates the smears of LPS. 68 250 kD 150 kD 100 kD 75 kD 50 kD 37 kD 25 kD 20 kD M 1 2 3 4 Figure 3.6. LPS analysis using SDS-PAGE (15%). A. tumefaciens was first cultured in MG/L and then induced in IB with AS for 18 hours. LPS were isolated from the bacteria cells using hot phenol-water method. LPS were subjected to 15% SDS-PAGE and stained with silver. M: Marker; lane 1: LPS from B119, Lane 2: LPS from B119(pHisJ1), Lane 3: LPS from B119(pHisJ2) and Lane 4: LPS from B119(pHisJ3). 69 3.3. Analysis of whole cell lipids VirJ is a putative lipase or acyltransferase which is secreted to the periplasm of A. tumefaciens. Since lipids are the major components of cell membranes, it is possible that VirJ could affect the lipid profile of the cell membranes, then making cell membranes unstable, which may in turn affect the stability of the cell membrane proteins, especially the components of the type IV secretion apparatus. However, the specific substrate of VirJ has not been identified. Previously, no lipase activity was detected with an in vivo agar plate assay even if MBP-VirJ was overexpressed by IPTG induction (Pan, 1999). Besides, no lipase activity was detected from purified MBP-VirJ fusion protein in vitro using either an esterase substrate p-nitrophenyl acetate, which can be hydrolyzed by many lipases, or a lipase substrate kit (Sigma). Therefore, whether VirJ possesses any lipase, acyltransferase or other enzymatic activity remains to be demonstrated. For this purpose, whole cell lipids were prepared by extraction with chloroformmethanol. 500 ml of A. tumefaciens induced with AS in the induction medium were collected for the preparation of the whole cell lipids. Lipids were analysed using TLC and electrospray ionization-mass spectrometry (ESI-MS) separately with the aim to find out the lipids that are different in the wild type and the mutant strains. Two separating systems of TLC were performed in order to separate the polar and non-polar lipids. The developing solvent contained chloroform: methanol: water at a volume ratio of 60: 12: 1 was used to separate polar lipids and the developing solvent containing hexane, ethyl ether and formic acid at a volume ratio of 45: 5: 1 70 was used to separate non-polar lipids. After running, TLC plates were stained with iodine and bands could be observed (Figure 3.7). From the TLC analysis, the polar lipids of A. tumefaciens were separated into different species of lipids, including phospholidyl etherolamine, phospholidic acid, phosphoglycerol and cardiolipin. The non-polar lipids were identified as triacyl glycerol. No significant difference could be observed from the lipid patterns analyzed by TLC. The shift in the motility of the lipids in chloroform: methanol: water system was presumably due to the fact that the samples loaded in the middle of the TLC plate always run slower compared with those loaded near the margin. From the TLC results, VirJ does not seem to affect the major components of the lipids in A. tumefaciens. It is likely that VirJ is only a protein which encodes a small lipase or acyltransferase motif that does not influence the biosynthesis of major lipids. However, it is still possible that VirJ could affect one kind of lipids and the effect could not be detected by TLC because of the resolution. To get better resolution of the lipid profile in A. tumefaciens, lipids isolated from B119, B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) were subjected to ESI-MS analysis. Signals were screened within the range of 300m/z to 2000m/z and all peaks were identified within the range from 700m/z to 900m/z. Signal patterns analyzed from B119, B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) were overlaid to search for the difference (from Figure 3.8 to Figure 3.12). Although some difference in the concentration of certain types of lipids were detected, but these differences were not related with the mutation. Therefore, it can be concluded that VirJ probably does not affect the lipid profile of A. tumefaciens. 71 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Figure 3.7. Thin-layer chromatography of whole cell lipids from A. tumefaciens. A. tumefaciens cells were cultured in MG/L followed by induction with AS in the induction medium. Whole cells lipids were extracted by treating the cells with chloroform: methanol (2:1, v/v). Lipids were dried with N2 fluid and were resuspended in chloroform: methanol (1:1, v/v). Lipids were visualized by staining with iodine after running. Left: lipid analyzed using chloroform: methanol: water system (volume ratio of 60: 12: 1). Right: lipid analyzed using hexane, ethyl ether and formic acid system (volume ratio of 45: 5: 1). Lane 1: phospholidyl etherolamine; Lane 2: phospholidic acid; Lane 3 and Lane 9: lipids from B119; Lane 4 and Lane 10: lipids from B119(pHisJ1); Lane 5 and Lane 11: lipids from B119(pHisJ2); Lane 6 and Lane 12: lipids from B119(pHisJ3); Lane 7: phosphoglycerol; Lane 8: cardiolipin; Lane 13: mono-di;triacyl glycerl mix; Lane 14: triacyl glycerol mix. 72 Figure 3.8. ESI-MS analysis of lipids extracted from A. tumefaciens strain B119 73 Figure 3.9. ESI-MS analysis of lipids extracted from A. tumefaciens strain B119(pHisJ1) 74 Figure 3.10.ESI-MS analysis of lipids extracted from A. tumefaciens strain B119(pHisJ2) 75 Figure 3.11.ESI-MS analysis of lipids extracted from A. tumefaciens strain B119(pHisJ3) 76 Figure 3.12. ESI-MS analysis of lipids extracted from different A. tumefaciens strains (overlaid) 77 3.4. Pull-down assay of VirJ-His The analysis of lipids from B119, B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) has showed that VirJ does not affect the lipids profile of A. tumefaciens. Since VirJ is secreted to the periplasm of A. tumefaciens, it is possible that VirJ may function by affecting the membrane proteins, especially those components of the type IV secretion pathways. Previously, Pantoja et al (2002) have found that VirJ could interact with both the transport apparatus and the type IV secretion substrates in A. tumefaciens. They proposed that VirJ functions as a mediator which helps to localize those different virulence factors to the specific locations. In Pan’s study (1999), cell fractionation and EDTA treatment studies showed that mutation of the lipase motif affected the stability of VirB7, VirB8, VirB9 and VirB10 but not other components of the VirB channel. Of all the virulence proteins, VirB7 is the only lipoprotein and is pivotal for the formation of the type IV secretion apparatus. Therefore, it is interesting to know whether mutation of the lipase motif would affect its interaction with these virulence proteins, especially VirB7. For this purpose, a pull-down assay was conducted. In this assay, VirJ-His was used as the bait and was isolated from A. tumefaciens stain B119, B119(pHisJ1), B119(pHisJ2) and B119(pHisJ3) that were induced with acetosyringone in the induction medium separately using Immobilized Metal Affinity Chromatography (IMAC). All procedures were very mild in which the cells were first treated with lysozyme to break the cell wall and then was treated with a buffer containing 0.5% Triton which can release the membrane proteins. During this procedure, the 78 interaction of VirJ with other proteins may not be destroyed and therefore the proteins that can interact with VirJ were also pulled-down together with VirJ. Proteins were eluted from the column with immidazole. Elution fractions were combined and were subject to 17.5% SDS-PAGE followed by western blotting with anti-VirB7 antibody or 12% SDS-PAGE followed by western blotting with anti-AopB, VirB8, VirB10 and VirB11 antibodies or 10% SDS-PAGE followed by western blotting with anti-VirD2, VirE2 and VirD4 antibodies to detect the interaction of VirJ-His with these proteins. However, our results differed from that of Pantoja et al (2002). In our experiements, no interactions of VirJ with VirB7, VirB8, VirB10 and VirB11 were detected and only the interaction with VirD2, VirE2, VirD4 and AopB were found in our pull down assay as shown in Figure 3.13. No difference was found between the wild type and the mutant VirJ during the interaction with VirE2, VirD2, VirD4 and AopB. The interactions with other virulence factors were not studied due to the lack of the appropriate antibodies. The difference of our pull down assay with that of Pantoja et al (2002) is that VirJ-His was used in our study instead of 3xFlag-VirJ. It is possible that the flag tag interacted with the components of VirB channel but not VirJ. 79 75 kD VirD2 75 kD VirE2 75 kD VirD4 25 kD AopB 7 kD VirB7 VirB8 25 kD 35 kD VirB10 37 kD VirB11 1 2 3 4 5 Figure 3.13. Pull-down assay of VirJ-His from A. tumefaciens. VirJ-His was used as the bait and was isolated from A. tumefaciens strains using Immobilized Metal Affinity Chromatography (IMAC). Proteins were analysed by SDS-PAGE and immunoblotting. Lane 1: A208 whole-cell homogenate sample; Lane 2: B119; Lane 3: B119(pHisJ1); Lane 4: B119(pHisJ2); Lane 5: B119(pHisJ3). Molecular weights are indicated on the left and specific antisera used for detection are indicated on the right. 80 3.5. Posttranslational modification of VirB7 VirB7 is one component of the VirB channel and is playing a key role in the assembly and stabilization of the T-DNA transfer apparatus. No detectable accumulations of VirB7, VirB8 and VirB9 could be observed in virB7 deletion strain, and VirB4, VirB5, VirB10 and VirB11 existed at lower levels in ΔB7 when compared with wild type A348. Studies have shown that VirB7 form homodimers and heterodimers with VirB9, and the homodimers are crucial for the formation of VirB channel. Failure in the accumulation of stable VirB7 would lead to an anchorless VirB channel and result in the instability of the VirB channel proteins (Fernandez et al, 1996). VirB7 is the only lipoprotein found in the VirB channel which is exposed at the periplasmic surface of the outer membrane. Posttranslational modification of VirB7 was completed in two steps: first, a thioether-linked diglyceride was added to the invariant Cys residue in the signal sequence; then it is further acylated at the Cys residue by amide linkage to the palmitic acid after it is cleaved by SPII before the modified Cys, generating a 41-amino-acid polypeptide with a modified Cys residue at the amino terminus. This process is mediated by lipase and acyltransferase. Similar phenotypes were observed by comparing the phenotypes of ΔB7 strain and acvB, virJ double mutant strain. Since both AcvB and VirJ possess a lipase or acyltransferase motif, and VirB7 is a lipoprotein which is crucial for the assembly of VirB channel, we asked the question whether AcvB and VirJ are involved in the acylation process of VirB7 or whether VirJ/AcvB helps to localize VirB7 to the outer membrane. 81 For this purpose, VirB7-His was studied both in the wild type and acvB/virJ mutant strains. These experiments were aimed to compare the molecular weight of VirB7 purified from different strains to give some clues as to whether VirJ is involved in the posttranslational modification process of VirB7, since failure in the lipid modification process would lead to reduced molecular weight of VirB7. 3.5.1. Functional test of VirB7-His in A. tumefaciens In order to test if VirB7-His could functionally complement virB7 mutant strain, plasmid pB78HSW which encodes VirB7 with a 6xHis-tag fused to the C-terminus (VirB7-His(6)) was transferred into B721, which is acvB and virB7 double mutant strain by triparental mating from TG1(pB78HSW). The resulting strain B721(pB78HSW) was confirmed by mating the plasmid back to E. coli MT607 with the helper strain MT616. The same plasmid was also transferred into B119. The function of the VirB7-His was confirmed by tumorigenesis assay. Plant inoculation experiment showed that mutation of virB7 abolished the virulence of strain B721, which could be restored by VirB7-His encoded by the plasmid pB78HSW. This indicates that VirB7-His functions as the wild type VirB7 (Figure 3.4.). 3.5.2. Purification of VirB7-His The method of isolation and purification of VirB7-His from E. coli was not established. In this study, VirB7-His was driven by the lacZ promoter on the vector pSW172. Therefore, E. coli was first induced with 0.3 mM IPTG at 37 ºC for 4 hours to induce the overexpression of VirB7. However, after induction, no overexpression 82 was observed on 17.5% SDS-PAGE stained by standard Coomassie Blue method. Western blotting assay showed that VirB7-His was expressed at a very low level. This could be because that VirB7 is a lipoprotein and is located on the cell membrane. Therefore, one liter of TG1(pB78HSW) induced with IPTG in LB was collected (OD600=0.5) and VirB7 was purified using the method mentioned in the Materials and Method section. All fractions after elution were mixed with loading dye and boiled for 5 min and were subjected to 17.5% SDS-PAGE followed by western blotting with anti-His tag antibody. Experimental results showed that TG1(pB78HSW) expressed a very low level of VirB7-His (exposed half an hour during western blotting assay). The density of the band detected by western blotting assay in the flow through fraction was similar to that of the crude cell extract of TG1(pB78HSW), suggesting that in E. coli VirB7-His could not bind with the resin well or VirB7 was unstable in TG1 cells. In elution fraction 2, VirB7 was detected although the concentration was extremely low (Figure 3.14). 83 7 kD 1 2 3 4 5 6 7 8 Figure 3.14. Purification of VirB7-His from TG1(pB78HSW) cultured in LB. TG1(pB78HSW) was induced with IPTG. VirB7-His was purified from E. coli with Immobilized Metal Affinity Chromatography (IMAC). VirB7 was detected by immonoblotting with anti His-tag antibody. Lane1: crude cell extract of TG1(pB78HSW); Lane 2: the supernatant after binding with Talon resin; Lane 3 to Lane 8: 1st to 6th elution fractions. 84 The same method was used to isolate and purify VirB7-His from B721(pB78HSW) and B119(pB78HSW). In B119(pB78HSW), there coexisted two virB7 genes, one is the wild type virB7 located on the Ti plasmid driven by the virB promoter, the other one is the VirB7-His on pB78HSW driven by the lacZ promoter. In B721(pB78HSW), only VirB7-His existed. Therefore, before the purification was conducted, the expression level of VirB7-His was compared in these two strains using anti-His tag antibody. Western blotting assay showed that these two strains expressed VirB7-His at the same level, and no difference in the size of these VirB7 proteins could be found on 17.5% PAGE. Next, VirB7-His was purified from B721(pB78HSW) and B119(pB78HSW). Western analysis showed that almost all of the VirB7-His was bound with the resin when compared with the samples before and after resin binding. The elution peak was observed in the second elution fraction (Figure 3.15). To evaluate the quality and efficiency of the purification, the second elution fraction was concentrated using vivaspin protein concentrator (3.5 kD) and was subjected to 17.5% SDS-PAGE followed by Coomassie Blue staining (Figure 3.16). From the gel, the same level of VirB7-His was extracted from both strains although many contaminated proteins existed. This may be because that VirB7-His was such a small protein which expressed at a low level that many other larger proteins could compete with VirB7 in binding with the resin. 85 7 kD B721(pB78HSW) 7 kD B119(pB78HSW) 1 2 3 4 5 6 7 8 Figure 3.15. Purification of VirB7-His from B721(pB78HSW) and B119(pB78HSW). Cells were cultured in MG/L and induced with AS in IB. VirB7 was purified using Metal Affinity Colomn and was detected with anti His-tag antibody. Upper panel: purification of VirB7 from B721/pB78HSW; Lower panel: purification of VirB7 from B119/pB78HSW. Lane1: crude cell extract of TG1/pB78HSW; Lane 2: the supernatant after binding with Talon resin; Lane 3 to Lane 8: 1st to 6th elution fractions. 86 150 kD 75 kD 50 kD 37 kD 25 kD 15 kD 7 kD 1 2 3 4 5 6 7 Figure 3.16. Purification of VirB7-His from B721(pB78HSW) and B119(pB78HSW). Bacterial cells were cultured in MG/L and induced with AS in IB.VirB7-His was purified with Metal Affinity Column and was eluted with MES at pH 3.8. Protein samples were subjected to 17.5% SDS-PAGE and stained with Coomassie Blue. Lane1: Marker; Lane 2: elution fraction 2 from B721(pB78HSW); Lane 3: flow of elution fraction 2 from B721(pB8HSW) during concentration; Lane 4: concentrated elution fraction 2 from B721(pB78HSW); Lane 5: elution fraction 2 from B119(pB78HSW); Lane 6: flow of elution fraction 2 from B119(pB8HSW) during concentration; Lane 7: concentrated elution fraction 2 from B119(pB78HSW). The arrows indicates VirB7-His. 87 3.5.3. Mass Spectrometry (MS) analysis VirB7-His In order to measure the accurate molecular weight of VirB7-His isolated from B119(pB78HSW) and B721(pB78HSW), the bands at about 7 kD were excised from the gel and sent to Protein and Proteomic Centre (PPC, NUS) for MALDI-TOF TOF MS analysis. Protein samples were first digested with trypsin followed by cleaning before they were analysed. Normally trypsin will cleave at the site after amino acid K and R. Analysis of the sequence of VirB7 showed that VirB7 should be digested into several fragments ranging from 2 amino acids to 23 amino acids (Figure 3.17.). However, after MS analysis, two fragments could not be found in the samples. One was polypeptides of amino acid 14 to amino acid 25; another one was of amino acid 52 to the N-terminus of the 6xHis tag. The C-terminal 14 amino acids were cleaved by the SPII during posttranslational modification within the cell. By comparing the molecular weight of the fragments detected by MS, no difference could be detected between VirB7-His from the wild type and virB7 mutant background (Figure 3.18. and Figure 3.19.) 88 VirB7-His precursor 1 11 21 31 41 51 MKYCLLCLVVALSGCQTNDTIASCKGPIFPLNVGRWQPTPSDLQLRNSGGRYDGAHHHHHH VirB7-His 15 26 36 47 52 61 CQTNDTIASCKGPIFPLNVGRWQPTPSDLQLRNSGGRYDGAHHHHHH (Missing) CQTNDTIASCK GPIFPLNVGR M.W.1069.61 WQPTPSDLQLR M.W.1340.71 WQPTPSDLQLRNSGGR M.W.1811.94 GPIFPLNVGRWQPTPSDLQLR M.W.2391.35 (Missing) NSGGRYDGAHHHHHH Figure 3.17. Analysis of the amino acid sequence of VirB7-His. Matured VirB7 is a 41-amino-acid polypeptide which undergoes lipid modification. First, a thioetherlinked diglyceride was added to the invariant Cys-15 residue in the signal sequence; then it is further acylated by amide linkage to the palmitic acid after it is cleaved by SPII before the modified Cys. The N-terminal signal peptides are labelled in red. Blue boxes indicate the peptides that could be detected during MALDI-TOF MS MS analysis. Two yellow boxes indicate the missing fragments. 89 Figure 3.18. MALDI-TOF TOF MS anaylsis of VirB7-His isolated from B721(pB78HSW). 90 Figure 3.19. MALDI-TOF TOF MS analysis of VirB7-His isolated from B119(pB78HSW). 91 3.6. Analysis of cell membrane proteins Since VirJ does not affect the lipid profile of A. tumefaciens, we next wanted to know whether cell membrane proteins were affected. Pan et al (1999) showed that mutation of the lipase motif of VirJ could cause the instability of VirB7, VirB8, VirB9 and VirB10 but not other components of the VirB channel, which span both the inner and the outer membrane of A. tumefaciens. In order to confirm this, 500 ml of acetosyringone induced B119(pHisJ1) which expressed wild type VirJ, and B119(pHisJ2) which expressed VirJ with a site mutation within the lipase motif, were collected for the preparation of the cell membrane proteins. The cell membrane proteins were isolated by ultracentrifugation and the inner and outer membranes of the cells were further separated by sucrose gradient centrifugation. After sucrose gradient centrifugation, two yellow bands representing the inner membrane and the outer membrane fractions respectively could be observed in the centrifuge tubes. Cell membranes were carefully pipetted out and were transferred into the clean tubes. 2x loading dye was added to the protein samples and the samples were boiled for 5 min. Samples were subjected to 12% SDS-PAGE and bands were visualized by silver staining. Previously, the instabilities of VirB7, VirB8, VirB9 and VirB10 were observed by western blotting assay by comparison of the intensities of the signals (Pan, 1999). Due to the lack of the appropriate antibodies against these VirB proteins, membrane proteins were analyzed using SDS-PAGE and silver staining method. By comparing the inner membrane proteins and the outer membrane proteins of B119(pHisJ1) and B119(pHisJ2), no obvious difference was identified. However, this does not mean that 92 VirJ does not affect the structure of the VirB channel. VirB proteins are membrane proteins and the expression level might not be high enough to be detected using silver staining. It is recommended to use 2-D gel electrophoresis to analyse the membrane proteins of B119 harbouring wild type VirJ and VirJ with mutation within the lipase motif or outside the lipase motif for the identification of differences in the future. 93 75 kD 50 kD 37 kD 25 kD 20 kD 15 kD 10 kD 1 2 3 4 5 Figure 3.20. Analysis of the cell membrane proteins from A. tumefaciens strain B119(pHisJ1) and B119(pHisJ2). Cell membrane proteins were extracted from A. tumefaciens strain B119(pHisJ1) and B119(pHisJ2) by ultracentrifugation and were further separated into the inner membranes and outer membranes by sucrose gradient centrifugation. Membrane proteins were subjected to 12% SDS-PAGE and were stained with silver. Lane 1: Marker; Lane 2: Inner membrane of B119(pHisJ1); Lane 3: Inner membrane of B119(pHisJ2); Lane 4: Outer membrane of B119(pHisJ1); Lane 5: Outer membrane of B119(pHisJ2). 94 DISCUSSION 4.1. VirJ does not affect the structural integration of lipopolysaccharides in A. tumefaciens A. tumefaciens would induce tumors on the wounded plants when they sense the signal compounds released from the wounded sites. The development of pathogenesis is a complex process and is conditioned by the recognition and absorption of the bacterium on the host. Besides, in order to transfer its T-DNA into the plant cell, the bacterium has to be adsorbed in the wounded area. This is a process that is modulated by the components of the external membrane of the bacterium including both the proteins and lipopolysaccharides (LPS) (Pueppke and Benny, 1984). In the latter case, the interaction is based on the recognition of a portion of the lipopolysaccharides by the receptor proteins situated on the plant cell wall. Further studies showed that the Oantigenic part of LPS is recognized by the plant, and mutation of the O-antigen chain will lead to avirulence of the bacterium (Matthysse 1984; New et al, 1986). In our study, we are interested to know whether VirJ is involved in the formation of LPS of A. tumefaciens since VirJ encodes a putative lipase motif and LPS contain a lipid portion. However, it is unlikely that mutation of the lipase motif of VirJ would affect the virulence of A. tumefaciens by affecting the structure of LPS. First, analysis of LPS extracted from different A. tumefaciens strains did not exhibit any difference on PAGE, which is a standard method to analyse LPS. Second, if VirJ is involved in the formation of LPS, it is more likely that mutation of the lipase motif would affect the lipid A portion but not other parts of LPS. However, it is the Oantigen part but not the lipid A portion that is involved in the attachment of A. tumefaciens to the plant cells. Third, previous studies have shown that mutation of 95 VirJ or AcvB does not affect the attachment of the bacterium (Wirawan et al, 1993). Therefore, we concluded that VirJ affect the virulence of A. tumefaciens in a way other than participating in the formation of LPS. 4.2. VirJ does not affect the posttranslational modification of VirB7 Since VirJ does not affect the formation of LPS, we wanted to know if it is involved in the posttranslational modification process of VirB7. Several clues led to the hypothesis that VirJ may be involved in this process. First of all, both VirJ and AcvB contain a lipase or acyltransferase motif and VirB7 is the only lipoproteins of the VirB channel. Secondly, posttranslational modification of VirB7 requires the activity of lipase and acyltransferase. Thirdly, mutation of virJ and virB7 would lead to the similar phenotypes of A. tumefaciens: mutation of virB7 lead to the instabilities of VirB4, VirB5, VirB8, VirB9, VirB10 and VirB11 and mutation of virJ lead to the instabilities of VirB7, VirB8, VirB9 and VirB10. In addition, VirB7 is of significant importance for the assembly of the VirB channel. Therefore, we hypothesized that VirJ could affect the lipid modification of VirB7 and consequently affect the structure of VirB channel. However, based on the experimental results, it is indicated that VirJ may actually not be involved in the modification process of VirB7. The first line of evidence comes from the analysis of VirB7 purified from B119(pB78HSW) and B721(pB78HSW). As mentioned above, VirB7 is a lipoprotein and the lipid portion plays a key role in anchoring VirB7 to the outer membrane of the cells and therefore provides the anchor for the formation of the VirB channel. Mutation of the lipid portion would make VirB7 loosely attached to the surface of the bacteria and easily to be degraded by protease. In this study, we hypothesized that in 96 B119(pB78HSW), VirB7 could not be correctly acylated due to the lack of functional VirJ and AcvB. And in B721(pB78HSW) VirB7 would be acylated and anchored to the outer surface of the cell. Thus it would be more difficult to purify VirB7 from B119(pB78HSW) in which VirB7 is readily to be degraded, or the concentration of VirB7 would be much lower compared with that of the wild type. However, VirB7His was purified successfully from both B119(pB78HSW) and B721(pB78HSW) and the final production were comparable in these two strains. This indicates that in B119 (pB78HSW), VirB7 was expressed at a normal level and was properly anchored. Another line of evidence comes from the analysis of VirB7 on PAGE. When VirB7 is properly acylated, its molecular weight should be at least 0.2 kD larger than VirB7 which is not acylated depending on how many molecules of palmatic acids are attached to VirB7. However, VirB7-His purified from the wild type and the mutant strains exhibited the same mobility on PAGE. No difference in the molecular weight could be observed. Previous studies showed that no difference in the retention time could be identified when VirB7 isolated from different strains were analyzed using HPLC, which separate molecules due to the polarity differences (Lu Baifang, 2000, unpublished). Because the attachment of the lipid portion to VirB7 would greatly change its polarity, it is possible that VirJ is not involved in the lipid modification process of VirB7. Thirdly, MALDI-TOF TOF MS analysis of VirB7 purified from B119(pB78HSW) and B721(pB78HSW) also gave some hints that VirJ does not affect the acylation of VirB7 although two fragments were not detected during analysis. One fragment from amino acid 52 to the N-terminus of the 6xHis tag was 97 missing because it was too small to be identified by the machine. And the missing of the other fragment which includes the Cys that was modified with lipid was possibly due to the fact that the lipid portion made it difficult to be extracted from the gel. In conclusion, it is more likely that VirJ and AcvB are not involved in the posttranslational modification process of VirB7. The stability of VirB7 in B119 was similar when compared to that of the wild type. VirB7 purified from B119(pB78HSW) and B721(pB78HSW) exhibited similar characteristics and therefore it is more likely that VirB7 was acylated in B119. 4.3. VirJ may affect the virulence of A. tumefaciens by impairing the TDNA transfer process Pervious studies have demonstrated that either AcvB or VirJ is required for the tumorigenecity of A. tumefaciens. The acvB and virJ double mutant strain showed no difference in the attachment to the plant cells. T-DNA production was not impaired in the mutant when compared with the wild type (Wirawan et al, 1993; Kang et al, 1994; Pan et al, 1995). Further study has showed that B119 which lacks both AcvB and VirJ could not deliver T-DNA into plant cell, suggesting that AcvB and VirJ encode a factor which is required for T-DNA transfer. Our study has showed that VirJ can interact with both VirD2 and VirE2, which associate with the T-DNA in a form of the T-complex. No interaction of VirJ with VirB proteins were observed in our pull-down assay and the result was different with that of Pantoja et al (2002). The only difference in these two experiments was that in our pull-down assay, we used VirB7-His to pull down those proteins that could 98 interact with VirJ, but Pantoja et al used 3xFlag as a tag. Whether VirJ interacts with the VirB channel needs further investigations. Kang et al (1994) have shown preliminary results which indicated that AcvB has single stranded DNA binding activity. Since VirJ is the homologue of AcvB, it is possible that VirJ can also bind with single stranded DNA and help in the T-DNA transfer process. Till now, little is known about the function of AcvB and VirJ. Previous studies showed that acvB mutant blocked the T-DNA transfer at some step other than vir gene induction, and AcvB is not required for the transfer of the conjugal DNA or for the viability in the wound. In addition, the acvB mutant could not be complemented by coinoculation (Winans et al, 1995). Recently, Chen et al (2000) found that VirD2, VirE2 and VirF could be secreted to the supernatant of cell culture. In 2001, Dumas found that VirE2 could integrate in black lipid membranes (BLM) and to form large anion-selective channels that transfer single-stranded DNA across the membrane in vitro. Duckely et al (2005) found that VirE2 and VirE1 can associate with all kinds of lipids. However, the transfer of VirD2 and VirE2 is sec-independent, while the transfer of VirJ is sec-dependent, suggesting that VirJ may not function in assisting the translocation of VirE2 and VirD2 across the cell membrane. 4.4. Future study In order to examine the effect of VirJ on the cell membrane proteins, 2-D gel can be used to screen for the difference in the wild type strain and virJ mutant strain. This will give a better resolution than the one-D gel. 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Microbiol. 1: 649-655 112 [...]... that mutation of the lipase motif would abolish the virulence of A tumefaciens, but mutation outside the lipase motif has no effect on the bacteria This indicates that the lipase motif of these two proteins play an important role during the tumorigenesis process Cell fractionation and EDTA treatment studies showed that the mutation of the lipase motif caused an accumulation of VirB9 in the periplasm... periplasm and also affected the stability of VirB7, VirB8, VirB9 and VirB10 but not other components of the VirB channel The effect on VirB4 and VirE2 were minimal However, the specific functions of VirJ still remain unclear The aim of this project is to understand the biochemical function of VirJ One specific aim is to study how the lipase motif of VirJ affects the virulence of A 31 tumefaciens For this... nopaline-type strains The homologous region lies in the C-terminal half of AcvB The expression of VirJ is under the control of the virA/virG two-component system regulated by acetosyringone which has no effect on acvB The functions of VirJ and AcvB are still not clear Expression of VirJ can restore the virulence of acvB mutant strain, indicating that they express the same factor required for the virulence, or... lipopolysaccharides (LPS) and whole cell lipids pattern were analysed Other specific aims are to study the interaction of VirJ with other virulence proteins; to examine whether the lipase motif of VirJ would affect the posttranslational modification of the lipoprotein VirB7; and finally to determine the pattern of the bacterial cell membrane proteins 32 MATERIALS AND METHODS 2.1 Plasmids, strains and media... 1.1.6 Functions of chromosomal virulence genes Besides the virulence genes on the Ti plasmid, some genes on the chromosome are also required for the virulence of A tumefaciens (Gelvin, 2000) But unlike the virulence genes on the Ti plasmid, the functions of these chromosomal genes have not been well studied The products of gene pscA, chvB and chvA are propsed to function in the biosynthesis, modification... facilitate the movement of the T-complex substrate to cross the cytoplasmic membrane by supplying energy (Lai and Kado, 2000) The final component of the transporters is VirD4, the third ATPase that is required for the virulence VirD4 is an inner membrane protein and is required for the formation of the T-pilus (Fullner, 1996) It is proposed that VirD4 functions as a coupling protein for the transfer of the virulence. .. into the periplasm via a sec-like pathway VirJ associates with VirD4 and the VirB pilus independently of one another and mediate the transfer of the substrates across the outer membrane via the VirB channel (Pantoja et al, 2002) 1.3 Aims of this project Sequence analysis of VirJ and AcvB has revealed that they both contain a lipase or acyltransferase motif that is required for the function of VirJ. .. indicating that the synthesis and the stability of the majority of VirB proteins are dependent on VirB7 (Fernandez et al, 1996) In addition, deletion of virB9 gene would reduce the expression of VirB4, VirB5, Virb8, VirB10 and VirB11, but not VirB7 (Fernandez et al, 1996) It is hypothesized that the association of VirB with VirB7-VirB9 complex stabilizes their accumulation during the assembly of the transporters... VirA, the phospho-VirG activates the transcription of the remainder of the vir genes by binding particularly to the vir-box, which is a conserved regulator element found upstream of most of the vir genes Non-phosphorylatable mutant VirA and VirG proteins have been found to lose their ability to induce the expression of vir genes (Jin et al, 1990a; 1990b; 1990c) On the other hands, multiple copies of VirG... while deletion of NLS2 would completely abolish the ssDNA binding and nuclear localization activities These imply that the NLS of these two proteins might play different roles in nuclear localization of the T-complex Recent studies have found that the NLSs of octopine VirE2 might differ from that of the nopaline-type Ti plasmids in that they are not functional in the nuclear import of proteins in Xenopus ... aims are to study the interaction of VirJ with other virulence proteins; to examine whether the lipase motif of VirJ would affect the posttranslational modification of the lipoprotein VirB7; and... acyltransferase motif Mutation of the lipase motif would lead to the malfunctioning of the proteins, indicating that the lipase motif plays a key role in the function of these proteins Previous studies... abolish the virulence of A tumefaciens, but mutation outside the lipase motif has no effect on the bacteria This indicates that the lipase motif of these two proteins play an important role during the