Molecular basis of agrobacterium mediated gene transfer into mammalian cells 4

Chapter 4. ChvG regulates acidic pH-inducible genes on the chromosomes and Ti plasmid 4.1. Introduction The interaction between bacteria and their hosts is complicated. On one hand, bacteria must survive in harsh extracellular milieu in which they will have to confront the bactericidal substances while sensing potential host signals. On the other hand, they have to invade host cells and resist various cellular strategies aimed to eliminate them. Thus, they can survive in various rapid and unexpected environment, such as fluctuating nutrient and toxin levels, acidity, temperature, cell density, and water availability and have little ability to change their environment. Therefore, they must monitor and respond to environmental signals in a rapid and accurate manner. The penalty for losing touch with their immediate environment is often death. The prototypical two-component system is a major signaling mechanism that mediates the response to various environmental stimuli in bacteria (Nixon et al., 1986; Ronson et al., 1987; Winans et al., 1986; Parkinson. 1993; Tokishita and Mizuno, 1994). In this sensory-response system, bacteria make use of signaling pathways that involve phosphorylation of key effector proteins by a histidine protein kinase (HK). These pathways help bacteria to adjust their metabolism and structure in response to environmental signals in a turning on/off manner. In addition, expression of virulence factors in both the plant and mammalian pathogens are also controlled by the twocomponent systems in order to adapt to the various host environments in which they are exposed during the various stages of infection (Uhl and Miller, 1996). 119 4.1.1. General overview of two-component systems in prokaryotic cells The first bacterial two-component signal transduction system was found by Ninfa and Magasanik (1986) from the NR system, a regulatory system that controls gene expression in response to nitrogen-source availability in E. coli. Then, sequence analysis showed that there are numerous other bacterial sensory systems containing the similar sequences of NR system (Nixon et al., 1986). Subsequent work confirmed that these systems operated via a signaling mechanism analogous to that utilized by the NR system. To date, two-component systems are widely found in both Gram-positive and Gram-negative pathogenic bacteria. For example, surveys of representative completed genomes have identified 62 twocomponent proteins in Escherichia coli, 27 in Streptococcus pneumoniae, 70 in Bacillus subtilis, 24 in Methanobacterium thermoautotrophicum. (Mizuno, 1997; Mizuno, 1997; Smith et al., 1997; Fabret et al., 1999: Throup et al., 2000). These systems not only regulate basic housekeeping functions but also control expression of toxins and other proteins important for pathogenesis. However, not all prokaryotes utilize two-component systems as extensively as E. coli. The number of twocomponent systems differs greatly in different species, ranging from as in Mycoplasma genitalium to 80 as in Synechocystis sp., in which these proteins account for about 2.5% of the genome (Mizuno, 1997). Although two-component systems have not been identified in animals, worm and fly genomes, it is indeed present in fungi, slime molds, and plants though far less numerous than in bacteria. There appear to be significant differences in the way two-component systems are used in different species. Generally, in most prokaryotic systems, the output response is affected directly by the response regulator (RR), which functions as a transcription 120 factor. In eukaryotic systems, two-component proteins are found at the beginning of signaling pathways where they interface with more conventional eukaryotic signaling strategies such as mitogen-activated protein (MAP) kinase and cyclic nucleotide cascades (Loomis et al., 1997; Wurgler-Murphy and Saito, 1997). The typical two-component system consists of a sensor kinase (HPK), which perceives environmental signal with its N-terminal input domain, and a response regulator protein (RR), which mediates cell response with its C-terminal output domain by regulating expression of specific genes (Fig. 4.1 a) (Ann et al., 2001). Signaling is achieved by phosphotransfer from a highly conserved His residues in the sensor’s transmitter domain, which is autophosphorylated in the presence of the appropriate stimulus, to an Asp residue in the N-terminal receiver domain of the regulator protein. The phosphorylation of receiver domain may result in a conformational change of the response regulator, which elicits the specific response. The output response of the system is determined by the level of phosphorylated RR. The genes encoding the sensor and regulator are often cotranscribed as a single transcript. Two kinds of phosphorylation pathways are found in Eubacteria, Archaea and Eukarya: His/Asp and Ser/Thr/Tyr. Both phosphorylation pathways (His/Asp and Ser/Thr/Tyr) can function in both prokaryotes and eukaryotes. His-Asp phosphotransfer systems account for the majority of signaling pathways in eubacteria but are quite rare in eukaryotes, in which kinase cascades involving Ser/Thr and Tyr phosphorylation predominate (Loomis et al., 1997; Wurgler-Murphy et al., 1997; Zhang, 1996). 121 Fig. 4.1. Two-component phosphotransfer schemes. (a) A typical two-component phosphotransfer system consists of a dimeric transmembrane sensor HK and a cytoplasmic RR. A monomer of a representative HK is shown with transmembrane segments indicated by TM1 and TM2. Conserved sequence motifs N, G1, F and G2, are located in the ATP-binding domain. HKs catalyze ATP-dependent autophosphorylation of a specific conserved His residue (H). The activities of HKs are modulated by environmental signals. The phosphoryl group (P) is then transferred to a specific Asp residue (D) located within the conserved regulatory domain of an RR. Phosphorylation of the RR typically activates an associated (or downstream) effector domain, which ultimately elicits a specific cellular response. (b)A multi-component phosphorelay system often begins with a hybrid HK that has an additional RR regulatory domain at the C-terminus. More than one His¯Asp phosphoryl transfer reaction takes place and the scheme usually involves a His-containing phosphotransfer (HPt) protein that serves as a His-phosphorylated intermediate. Abbreviations: HK, histidine protein kinase; RR, response regulator protein (cited from Ann et al., 2001). 122 The two-component systems are involved in the sensing of a wide variety of abiotic (e.g. pH, temperature or osmolarity) or biotic signals. Some signals are produced by a host, while others are synthesized by the bacterial populations themselves. In the latter case, the signals may be produced in coordination with the cell density of a population and thereby ensure regulatory mechanisms commonly known as quorum sensing. For a number of two-component systems, the signals are not yet known. The two-component systems of pathogen also play an important role in sensing and responding to the stress signals released by the host. After a successful infection, the host immune response will eventually ensues the arrival of nutrophils and macrophages. To survive this response, bacteria need to develop the ability to resist phagocytosis and/or to survive in the in vivo environment (such as the stress condition in the phagosome, including acidification and a wide rage of lethal enzymes). Such sensing and reponse could be mediated by a two-component system involved in the stress response. etaRS in E. faecalis was one of such examples, which was identified as a two-component system to be involved in stress response (low pH and high ethanol concentration) and bacterial virulence (Teng et al., 2002). 4.1.2. Structure and activities of sensor histidine protein kinase (HPK) In the typical two-component systems, sensor histidine protein kinases (HPKs) are usually transmembrane proteins that monitor external stimuli with variable extracellular domains and transmit this information to the RR by a phosphorylation event. The sizes of the member of the sensor HPK family range from 40 kDa to 200 kDa. HPKs function as homodimers, in which one HPK monomer catalyzes the phosphorylation of the conserved His residue in the second monomer. Archetypal constructs of HPK contain two typical modulars: a diverse sensing domain which can 123 be more than 500 residues long and a highly conserved kinase core region, also called the transmitter domain. This 350-residue long region exhibits sequence that is generally conserved in the histidine protein kinase superfamily (Nixon et al., 1986; Ronson et al., 1987; Parkinson and Kofoid, 1992; Parkinson, 1993). 4.1.2.1. The kinase core module The kinase core is about 350 amino acids in length and is responsible for binding ATP/ADP and directing kinase transphosphorylation. Five conserved amino acid motifs define the core region and have been termed the H, N , G1, F and G2 boxs (Parkinson and Kofoid, 1992). The H box contains the conserved His residue that is the site of phosphorylation. In most HPKs, the H box is part of the dimerization domain and functions as an intermediate in the phosphotransfer pathway, accepting a phosphoryl group from upstream donor (a HK bound ATP) and transferring it to the downstream RR domain. Therefore, residue surrounding the conserved His is expected to be involved in phosphotransfer catalysis as well as protein–protein recognition. The N, G1, F and G2 boxes comprise the nucleotide binding cleft. These motifs are usually contiguous, but the spacing between them is somewhat varied. In archetypal HPKs, the conserved His is located within the dimerization domain, adjacent to the nucleotide binding domain. However, not all HKs have the same domain organization. In CheA, the HPK of the bacterial chemotaxis system, the H box is located in the P1 domain, two domains N-terminal to the ATP-binding domain, P4 (Hess et al., 1988). Other systems derived from typical HPK use His-containing phosphotransfer domains (HPt), distinct from the His-containing dimerization domains, which contain an invariant His capable of carrying out phosphoryl transfer (Fig. 4.1 b). In some 124 cascades, this HPt domain is distinct from the kinase and constitutes an isolated module. There are many features that distinguish HPKs from the larger family of Ser/Thr/Tyr kinases (STTK). First, unlike typical protein kinase reaction in which a kinase catalyzes direct transfer of a phosphate from ATP to the substrate, each HPK must first be autophosphorylated, and then the phosphoryl group from HPK-P is passed to the specific RR. The second difference is that there is a one-to-one relationship between HK and RR in most two-component pathways, while one protein kinase phosphorylates multiple targets in classic protein kinase amplification cascades. Thirdly, the site of HPK autophosphorylation is a His residue and the site of RR phosphorylation is an Asp residue (Bourret et al., 1990). The energetic and chemical stabilities of phospho-His and phospho-Asp differ significantly from those of "more traditional" phospho-amino acids (phospho-Tyr, phospho-Ser, and phosphoThr). In addition to directing the forward phosphorylation reaction, some HPKs also have a phosphatase activity, enabling them to catalyze dephosphorylation on their cognate RRs. Through these opposing actions, the HPK regulates the phosphorylation level of the downstream RR, controlling the flow of information through the signaling pathway. This dephosphorylation is commonly present in phosphotransfer pathways that need to be shut down quickly (Hsing and Silhavy, 1997). 4.1.2.2. Sensing domain The sensing domains of HPKs share little primary sequence similarity, reflecting many different specific ligand/stimulus to which HPKs are responsive to. Based on localization, HPKs are divided into two classes: soluble and membrane bound. In 125 membrane bound HPKs that contain extracellular domains, the sensor domains are periplasmic and could detect the environmental signals. While in soluble HPKs such as chemotaxis kinase CheA, the sensor domains could be regulated by intracellular stimuli and/or interactions with cytoplasmic domains of other proteins. In many cases (including that of EnvZ), deletion of sensor modules results in a partially or completely active form of the kinase (Parkinson and Kofoid, 1992). This indicates that the sensor domain may also serve as kinase inhibitor and suggests that a common mechanism of kinase regulation could be the removal of this inhibition. 4.1.3. Structure and activities of response regulator proteins (RRs) In prokaryotic systems, RRs are typically found at the ends of phosphotransfer pathways and function as phosphorylation-activated switches to affect the adaptive response. Most RRs have a two-domain structure with a conserved N-terminal regulatory domain and a variable C-terminal effector domain (Bourret et al., 1990; Brissette et al., 1991; Stewart, 1993; Lukat et al., 1991) The RRs’ regulatory domain containing the Asp-phosphorylation site is about 125-residues and has three activities (Volz, 1993). First of all, they can catalyze phosphoryl transfer from the phosphorylated HPKs to one of their own Asp residues. Secondly, they can catalyze autodephosphorylation and thus limit the lifetime of the activated state. Finally, they also regulate the activities of their associated effector domains in a switches on/off manner. The effector domains are diverse with respect to both structure and function. In order to elicit the output response, most of them have a DNA-binding module and function to activate and/or repress transcription of specific genes. However, some RRs control more diverse responses such as the regulation of motility, activation of 126 mitogen-activated protein (MAP) kinase cascades, and modulation of cyclic nucleotide levels. Based on homology of their DNA-binding domains, the RRs can be divided into three major subfamilies (Makino et al., 1988; Stewart, 1993; Weiss and Magasanik, 1988). These subfamilies are designated OmpR (with a modified winged-helix fold), NarL (with a four-helix bundle) and NtrC (with an ATPase and a helical DNA binding domain) after their representative members. Due to the specific DNA sequences that effector domains recognized, the arrangement of binding sites and individual mechanisms of activation of transcriptional machinery differ from each other even within the same subfamiily. How a conserved regulatory domain can regulate so many varied effector domain activities has been a central question in the two-component system studies. The genetic, biochemical and biophysical studies of many different RRs support that RR regulatory domains function as generic on/off switch modules. These domains can exist in equilibrium between two predominant conformations, corresponding to the inactive and active states. Phosphorylation induces conformational changes that shift the equilibrium towards the active conformer. It provides a very simple and adaptable mechanism for the regulation of RRs activity. The different molecular surfaces of the regulatory domain in the two conformations can facilitate specific protein-protein interactions and thus different output responses can be achieved. 4.1.4. Two-component systems identified in A. tumefaciens The ability to respond to environmental stimuli is especially important for αproteobacteria that are associated pericellularly or intracellularly with animals and plants either as pathogens or as endosymbionts. Many two-component sensory transduction systems operate in this group of bacteria and they often control complex 127 regulatory networks and are critically important for the establishment of a relation between these bacteria and their hosts, no matter whether the latter are animals or plants (Cheng and Walker, 1998; Sola-Landa et al., 1998). For example, S. meliloti ChvI/Exos regulates the production of polysaccharides and B. abortus BvrR/BvrS controls cell invasion and intracellular survival which are likely to be involved in regulating the synthesis of OM components essential in the interaction with eukaryotic host cells. As a member of α-proteobacteria, Agrobacterium tumefaciens also contains many two-component pathways. BLAST analyses of the A. tumefaciens genome database have revealed that there are at least 25 putative two-component regulatory gene pairs (Goodner et al 2001). The two well studied of these are VirA/VirG and ChvI/ChvG, which play important roles in A. tumefaciens mediated tumorigenesis (Charles et al., 1992; Charles and Nester, 1993; Parkinson and Kofoid, 1992; Winans, 1992; Winans et al., 1994). 4.1.4.1. VirA/VirG is the first two-component system identified in A. tumefaciens The Ti plasmid encoded VirA/VirG system is the best studied two-component pathway in Agrobacterium. As a transmembrane protein, VirA works as the sensor part of a two-component system, while VirG functions as the cytoplasmic transcriptional regulator (Winans et al., 1986, 1989,1994). This two-component system controls the expression of the Ti-plasmid-harbored vir genes that are required for causing crown gall tumors on plants. VirA is an 92 kDa membrane-bound histidine protein kinase (Chang et al., 1992; Leroux et al., 1987; Winans et al., 1989) and exists as a homodimer in its native 128 8kb 6kb Fig. 4.3. Southern blot analysis of homologous recombinants. The total DNA of Agrobacterium strains were extracted and digested with ClaI (lanes 1, 3-5) or PstI (lanes 2, 6-8), respectively. gfpuv (from mini-Tn5) was used as probe. Lane 1, A6007; lane 2, A6340; lane 3, CG9; lane 4, A6007-CG9; lane 5, A6340-CG9; lane 6, AG6; lane 7, A6007-AG6; lane 8, A6340-AG6. 139 4.3.2. Effects of chvG on the regulation of acid-inducible genes located on the chromosomes Previously, our lab identified two chromosomal genes (aopB and katA) that are involved in A. tumefaciens mediated tumor induction and are inducible by acidic pH (Xu and Pan, 2000; Jia et al., 2002); aopB is located on the circular chromosome and katA on the linear chromosome (Wood et al , 2001). To determine whether ChvG is involved in the regulation of these two genes, the expression of aopB::gfp and katA::gfp fusions in the presence and absence of ChvG was measured. a plasmid containing the aopB::gfp fusion was constructed by cloning a 9.0 kb SphI DNA fragment from the genomic DNA of CGI1 (Jia et al, 2002) into pSW172 to generate pJYH15. This 9.0 kb SphI DNA fragment contained the mini-Tn5 insertion at aopB and the sequences flanking the mini-Tn5 insertion. pJYH15 was introduced into the chvG- mutant strain A6340 and the corresponding chvG+ strain A6007 and then the aopB::gfp expression was measured by green fluorescence (Table 4.1) and immunoblotting (Fig. 4.4). The result showed that the aopB::gfp expression in the chvG+ strain A6007 was induced about 8-fold by the change from pH 7.0 to pH 5.5. This is consistent with the previous observation that aopB was induced by acidic pH. However, in the chvG- mutant A6340, the aopB::gfp expression was undectectable on IB buffered at pH 7.0 or pH 5.5. This suggests that ChvG is absolutely required for the expression of aopB. To study the effect of chvG on katA expression, katA::gfp expression in the absence of any functional katA gene was examined, as the KatA protein represses katA::gfp expression (Xu et al, 2001). Different genetic backgrounds for the katA::gfp fusion construct were created by introducing the total DNA from AG6, 140 Table 4.1. The effects of chvG on the expression of acid-inducible genes located on the two chromosomesa gfp reporter fusion presence of chvG A6007-CG9 16S RNA::gfp A6340-CG9 Strains I rb Fold of acidic inductionc IB pH 7.0 IB pH 5.5 + 1210 1160 0.96 16S RNA::gfp - 1172 1075 0.92 A6007(pJYH15) aopB::gfp + 60 502 8.40 A6340(pJYH15) aopB::gfp - --- A6007-AG6 katA::gfp + 154 1406 9.13 A6340-AG6 katA::gfp - 1387 1328 0.96 A6340-AG6(pLP36) katA::gfp + 1395 1505 1.08 A6340AG6(pTC147) katA::gfp + (+chvI) 326 1383 4.24 A6340-AG6(pTC201) katA::gfp +d (+chvI) 360 924 2.57 a The results presented in the table represent three independent experiments. b The relative fluorescence intensities (Ir) of Agrobacterium tumefaciens strains containing the gfp reporter fusions grown on IB pH 7.0 or IB pH 5.5 were measured as described in the Materials and Methods. c The fold of acidic induction for each gene was determined by dividing the Ir of the bacteria grown on IB pH 5.5 by that on IB pH 7.0. d The plasmid pTC201 contains chvG and chvI from S. meliloti 141 16SRNA-gfp aopB-gfp katA-gfp 10 11 12 13 14 15 16 17 18 GFP 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 _ A 63 _ A 60 + C 7- G + A 63 _ _ -C 40 G A 60 J (p 07 + YH A + 5) 63 pJ 0( _ YH ) 15 _ 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 + + _ _ + + + + pH chvG ) ) 6 36 47 G G P A A C pL 40 07 pT 6( 6( 63 60 G G A A -A A 40 03 63 A A Fig. 4.4. The effects of ChvG on the expression of acid-inducible genes aopB and katA as determined by Western analysis. A. tumefaciens cells containing rrn::gfp, aopB::gfp or katA::gfp fusion were grown on IB agar plates buffered at pH 7.0 or pH 5.5 as described in the Materials and Methods. The gene expression levels in the bacterial cells were analyzed in the absence (-) [A6340, A6340-CG9, A6340(pJYH15) and A6340-AG6] or presence (+) [A6007-CG9, A6007(pJYH15), A6007-AG6, A6340-AG6(pLP36) and A6340-AG6(pTC147)] of a functional chvG. The same amount of bacterial cells was collected for each strain, and the total protein was electrophoresed on SDS/12% polyarylamide gel. The GFP protein was visualized by immunoblotting using the GFP antibody. 142 which is a katA- mutant containing the katA::gfp fusion, into A6007 and A6340 and selecting for homologous recombinants (see above). This resulted in two recombinants A6007-AG6 and A6340-AG6, which lack a functional katA gene but contain the katA::gfp fusion in the A6007 and A6340 background, respectively. katA::gfp expression in these strains was measured. (Table 4.1 and Fig. 4.4) and found that the katA::gfp expression in the chvG+ background was induced about 9fold by the change from neutral to acidic pH, consistent with the previous observation that katA was inducible by acidic pH. In the chvG- background, the katA::gfp was still expressed, but at the same level at pH 7.0 and pH 5.5. These data suggest that chvG is required for the responsiveness of katA gene expression to low pH, but not for expression of the katA gene. In addition, the katA::gfp expression level in the chvGcells grown at pH 7.0 or pH 5.5 was comparable to that in the chvG+ cells grown at pH 5.5. These data suggest that katA expression is repressed at neutral pH and chvG derepresses katA expression at a low pH. Thus, chvG appears to regulate aopB and katA genes differently. Studies with regard to complementation the chvG- mutation with the plamid pLP36 containing only the chvG gene was conducted. Although the ChvG protein was detected in strain A6340 (pLP36) by Western analysis (Li et al., 2002), the responsiveness of katA expression to a pH change was not restored (Table 4.1 and Fig. 4.4). When the plasmid pTC147 containing both chvG and chvI (Charles and Nester, 1993) was introduced into A6340-AG6, the ability to respond to the pH change was partially restored (Table 4.1 and Fig. 4.4). Although both pLP36 and pTC147 restored the ability of bacteria to grow on acidic media, it was not apparent why the chromosomal chvG mutation could not be fully complemented by a plasmid-harbored chvG gene,. Notably, the expression of both B. abortus bvrS and bvrR (homologous 143 to chvG and chvI respectively) is required to complement a bvrR mutation (SolaLanda et al, 1998). 4.3.3. Effect of chvG on the expression of vir genes The possibility that ChvG might affect the expression of vir genes encoded on the Ti plasmid was explored since the maximal expression of these genes requires an acidic pH environment in addition to plant phenolic compounds, such as acetosyringone (AS) (Winans, 1992). The requirement for the low pH is a function of VirG, which requires both an acidic pH and plant signal molecules for maximal activity. The plasmid pSM243cd (containing a virB::lacZ fusion) and pSM358cd (containing a virE::lacZ fusion) (Stachel and Zambryski, 1986) were introduced into A6007 and A6340. A6340 is hypersensitive to carbenicillin (Cb), even when the plasmid containing a Cb resistance gene was introduced into A6340. Thus, low concentration of Cb (5 µg/ml) was used to select for A6340(pSM243cd) and A6340(pSM358cd), which were subsequently verified by triparental mating to be Cb resistant. The expression of virB::lacZ or virE::lacZ in the bacteria grown on IB buffered at pH 7.0 or pH 5.5was measured by including 100 µM of AS in the media buffered at both pH 7.0 and pH 5.5 to induce vir gene expression. Thus, any difference in the vir gene expression between pH 7.0 and pH 5.5 should be due to the change in pH. As shown in Table 4.2, the virB::lacZ and virE::lacZ gene expression in the chvG+ strain A6007 were induced about 15-fold and 10-fold, respectively, by acid (pH 5.5). However, the virB::lacZ and virE::lacZ expression in the chvG- mutant A6340, although measurable, was much lower than the activity in the chvG+ strain A6007. This is consistent with the previous observation that ChvI was required for 144 Table 4.2. The effect of chvG on the expression of vir genes located on the Ti plasmida lacZ reporter fusions Presence of the chromosomal chvG A6007 NAd A6340 Strains β-galactosidase activityb Induction foldc IB+AS pH 7.0 IB+AS pH 5.5 + NA NA - NA A6007(pSM243cd) virB::lacZ + 200 2959 14.8 A6340(pSM243cd) virB::lacZ - 15 27 1.8 A6007(pSM358cd) virE::lacZ + 713 6777 9.5 A6340(pSM358cd) virE::lacZ - 83 192 2.3 A6007(pSHM4) chvH::lacZ + 86 80 0.9 A6340(pSHM4) chvH::lacZ - 93 82 0.9 a The results presented in the table represent three independent experiments. b The β-galactosidase activity of Agrobacterium tumefaciens strains containing the chvH::lacZ, virB::lacZ or virE::lacZ fusion on IB pH 7.0 or IB pH 5.5 in the presence of 100 µM acetosyringone (AS) were measured as described in the Materials and Methods. c The induction fold was calculated by dividing the gene expression level of the bacteria grown on IB pH 5.5 by that on IB 7.0. d NA, not applicable. 145 the expression of virB and virG (Mantis, 1993). In addition, the induction of both virB::lacZ and virE::lacZ expression by acidic pH was marginal in the chvG- mutant. Thus, ChvG plays an important role in the expression of virB and virE, although it is not essential for the expression of virB and virE while it is for aopB. These data suggested that ChvG also regulates the acidic responsiveness of the vir genes. 4.3.4. ChvG did not affect genes that were not acid-inducible In order to determine whether the effects of chvG on katA and aopB gene expression were specific under the tested conditions, the expression of a 16S ribosomal RNA gene (designated as rrn) in CG9 was studied. This strain contains an rrn::gfp fusion with the mini-Tn5 transposon inserted onto an rrn gene encoding 16S ribosomal RNA. Different genetic backgrounds for the rrn::gfp fusion were created by introducing the total DNA of CG9 into A6007 and A6340 and selecting for homologous recombinants in the same way when A6007-AG6 and A6340-AG6 were created. The resulting strains A6007-CG9 and A6340-CG9 contained the rrn::gfp fusion in the A6007 and A6340 background, respectively. We examined the GFP expression in these strains and found that the 16S ribosomal RNA expression was constant in chvG+ and chvG- backgrounds (Table 4.1 and Fig. 4.4), suggesting that the 16S ribosomal RNA gene is not acid-inducible and not affected by ChvG. This suggests that the effects of ChvG on aopB and katA are specific to acid-inducible genes. This is consistent with the previous observation that a lac::lacZ fusion was not significantly affected by a chvI mutation (Mantis, 1993). To determine whether the effects of chvG on the virB and virE gene expression were specific under the tested conditions, the expression of a chromosomal gene chvH that is also involved in virulence (Peng et al, 2001) was also examined, by using the 146 reporter construct chvH::lacZ. This construct was harbored on a plasmid pSMH4, which carries the 507 bp chvH sequence upstream of the start codon and 12 bp of chvH coding sequence fused in frame with the lacZ coding sequence. The results showed that the chvH::lacZ expression was not affected either by ChvG or acidic pH. This supports earlier observation that ChvG is involved in the induction of Ti-plasmid harbored virB and virE by an acidic pH. It is likely that ChvG regulates all vir genes harbored on the Ti-plasmid, as they are regulated as one regulon through the virA/virG two-component system (Winans, 1992). 4.3.5. Complementation of chvG mutation with S. meliloti exoS gene Agrobacterium is closely related to Rhizobium in many aspects during their interactions with plants. Mutations in ndvB of Rhizobium or chvB in Agrobacterium could be complemented by the mutual counterpart; and the wild type cgs gene from Brucella abortus could restore the synthesis of cyclic β-(1–2) glucan of both ndvB and chvB mutants (De Iannino et al., 2000). Since the ChvG protein has a high homology (81% identity and 89% similarity) with the S. meliloti counterpart ExoS (Li et al., 2002), whether A. tumefaciencs ChvG and S. meliloti ExoS are functionally related or not was determined by carrying out two sets of experiments since the chvG mutation is pleiotropic. The plasmid pTC201 containing S. meliloti exoS and chvI was introducted into chvG-katA double mutant strains, A6340-AG6 by triparental mating. Initial tests whether exoS could restore the ability of A6340-AG6 to grow on acidic media, as the chvG- mutant (A6340) was severely impaired in its growth at pH 5.5 but not at pH 7.0 showed that A6340-AG6 (pTC201) could grow on IB medium buffered at pH5.5 (data not shown). 147 The level of GFP expression by measuring green fluorescence intensity was also compared. As expected, in the presence of S. meliloti exoS, the katA::gfp expression in A6340-AG6 was induced about 2.5-fold by the acidic pH, although the induction is not as high as wild type A6007-AG6 (Table 4.1). This suggested that exoS gene from S. meliloti could partially complement the function of chvG gene to regulate the responsiveness of katA gene to acid pH. These results suggest that ExoS can partially complement the function of chvG in coping with the environment, such as acidic pH, and also regulating low-pH inducible genes, such as the responsiveness of katA to external microenvironment. 4.4. Discussion To efficiently infect host including plant and animal cells, A. tumefaciens may coordinate the expression of the genes responsible for the virulence with the genes involved in metabolism and/or genes necessary to overcome host defense mechanisms. This studies have demonstrated that the two-component regulatory system chvG/chvI plays a role in controlling the expression of vir genes and acidic pH-inducible chromosomal genes in A. tumefaciens. The Ti-plasmid-harbored vir genes are directly responsible for the virulence to cause tumors on plants; it appears that at least some of the chromosomal acidic-pH inducible genes like katA (Xu and Pan, 2000) are involved in defending against plant defense response. Expression of these two classes of genes appears to be coordinated by ChvG, as it is involved in the acidic pHinduction of both classes of the genes. The vir genes are coordinately induced in response to three environmental signals: phenolic compounds, monosaccharides and acidic pH (Charles and Nester, 1993). It is known that the membrane-bound sensor protein VirA senses the phenolic 148 compounds; the periplasmic protein ChvE senses the monosaccharides with the participation of the VirA periplasmic domain. Our studies presented here provide evidence that ChvG might sense acidic pH as it plays an important role in the acidic pH-induction of the vir genes. This is consistent with the previous observation that a chvI null mutation abolished the low pH induction of virG gene expression, which is mediated by at least two independent regulatory pathways; the chvI mutation also attenuated the vir gene expression (Mantis and Winans, 1993). ChvG plays an important role in regulating chromosomal aopB and katA, both of which are acidic pH-inducible and involved in the Agrobacterium-plant interaction. At the same time, ChvG is not required for the expression of other chromosomal virulence genes such as chvH. Charles et al (1993) found that the chvG mutants A6340 and A7678 were much more sensitive to detergents and antibiotics than the wild type A. tumefaciens strains, which suggested that the permeability of the cell envelope could be affected by chvG/chvI. It is supposed that chvG/chvI may regulate the expression of some envelope proteins. Indeed, ChvG was found to be required for the expression of aopB, which encodes an outer membrane protein exposed on the bacterial cell surface (Jia et al., 2002). Consistent with this, the B. abortus bvrS/bvrR (homologous to A. tumefaciens chvG/chvI) has been found to regulate some outer membrane proteins in B. abortus (Guzman-Verri et al, 2002). These suggest that ChvG might be also required for the bacterial fitness. chvG is responsible for the acidic pH-inducibility of the genes that are located on Ti plasmid as well as the circular and linear chromosomes. The functions encoded by these genes may include: (a) virulence to cause tumors on plants; (b) DNA delivery into mammalian cells; (c) response to against host defense; and (d) bacterial 149 fitness. The ways that ChvG regulated the acidic pH-inducible genes also appeared to be different. Since ChvG is a membrane-bound protein that is a putative sensor kinase, we hypothesize that ChvG is a global sensor protein that is directly or indirectly responsible for sensing the pH changes. Perhaps the pH sensing occured extracellularly, as only the growth media were adjusted to be acidic and thus the acidic pH should be extracellular. In addition, ChvG possesses a periplasmic domain that might be involved in the pH sensing. The global pH sensing by ChvG might affect the expression of different regulatory genes, which then regulate the expression of acidic pH-inducible genes through different cascades of events. This presumably could explain the apparent differences in ChvG-regulated expression of different genes. ChvG is absolutely essential for the expression of and consequently the induction of aopB by acidic pH. This suggests that ChvG/ChvI might more directly affect the aopB promoter. It remains unknown whether ChvI directly interacts with the aopB promoter. ChvG is required for the responsiveness of katA gene expression to the pH change, but not for the expression of the katA gene. ChvG appears to play a role in the acidic pHinduction of katA by repressing the katA expression at neutral pH. This suggests that ChvG/ChvI might negatively regulate genes like katA at neutral pH, while working as a positive regulatory system at acidic pH. ChvG plays an important role in the induction of Ti-plasmid harbored vir gene by an acidic pH; but it is not absolutely essential for the vir gene expression. It appears that ChvG might be indirectly involved in the vir gene expression, as virG is known to directly regulate all the vir genes (Winans 1992). 150 It would be of interest to understand how ChvG senses the acidity at the molecular level and determine whether ChvG regulates the expression of all the acidic pH-inducible genes in A. tumefaciens. Nevertheless, some of the ChvG-regulated genes may be responsible for the bacterial ability to cope with its hosts. This is supported by the following observations: (1) ChvG and ChvI are required for the bacterial growth on acidic media (Charles and Nester, 1993); (2) the plant tissue environment is acidic with minimal nutrition (Li et al, 1999); and (3) the ChvGregulated katA is involved in the detoxification of H2O2 released during Agrobacterium-plant interaction (Xu and Pan, 2000). (4) ChvG is involved in both plant and mammalian cells gene transfer. Other acidic pH-inducible genes like vir are directly responsible for the bacterial virulence. These indicate that ChvG-mediated sensing of the acidity is directly related to the bacterial ability to cope with its hosts, suggesting that ChvG is a global sensor for initiating the infection process. Notably, the entire ChvG length is homologous to the corresponding protein sensors in some members in the α-subdivision of proteobacteria, such as Sinorhizobium, Mesorhizobium, Brucella, Bartonella and Caulobacter. Some αproteobacteria like Rickettsia conorii and R. prowazekii lack a ChvG homolog. Since R. conorii and R. prowazekii are obligate intracellular bacteria, it will remain unclear whether ChvG homologs are present in all free-living α-proteobacteria until more bacterial genomes are determined. The homology between A. tumefaciens ChvG and S. meliloti ExoS is the strongest. Since S. meliloti is also a plant-associated bacterium, it would be of significance to determine if S. meliloti ExoS is responsible for sensing the acidity of the plant environment during its symbiosis with the plants. The S. meliloti ExoS has been shown to play a role in regulating the production of succinoglycan, which is important for the establishment of symbiosis. Our results 151 suggested that ExoS could partially complement the function of chvG in vivo. Thus, we propose that ExoS might also be involved in regulating the expression of acidic pH-inducible genes in S. meliloti. S. meliloti and A. tumefaciens might employ similar strategies in coping with their microenvironment by sensing the external signals such as pH. However, more work need to be conducted to verify these presumptions. Brucella and Bartonella species are human pathogens that cause brucellosis and bartonellosis, respectively. The BvrS protein, a ChvG homolog in B. abortus, has been shown to be important for the bacterial ability to inhibit lysosome fusion and replicate inside animal cells (Sola-Landa et al., 1998). The ChvG homologs in the Brucella and Bartonella species are homologous to the A. tumefaciens ChvG to a significant degree even in the putative periplasmic domains, which might be directly involved in sensing external signal(s). Once bacteria like Brucella and Bartonella are internalized into animal cells, it is possible that they may encounter an acidic pH environment within the vesicles containing them. Therefore, it would be of interest to determine if these ChvG homologs are involved in sensing acidic pH or other signal(s). The ChvG homolog (CC0238) in C. crescentus is also homologous to a limited degree (40% identities; 55% positives) to the periplasmic domain of A. tumefaciens ChvG. C. crescentus lives in a dilute aquatic environment; it would be of significance to determine if this ChvG homolog senses the aquatic pH. However, studies of this homolog might be complicated by the fact that the C. crescentus genome possesses another ChvG homolog (CC2670), which is homologous (32% identities; 50% positives) only to the C-terminal kinase domain of A. tumefaciens ChvG. Various 152 bacteria other than α-proteobacteria also possess ChvG homologs; however, the homology is limited only to the C-terminal kinase domains and not in the periplasmic domains (Table 4.2), suggesting that they might be involved in sensing signal(s) other than pH. Nevertheless, it should be of significance to determine how ChvG senses pH and whether it senses other signal(s). 153 Table 4.3. Comparison of Agrobacterium tumefaciens ChvG with other bacterial sensor proteins Standard BLAST results with A. tumefaciens ChvG in the databases α-Proteobacteria Sinorhizobium meliloti ExoS Mesorhizobium loti ExoS Brucella melitensis ChvG Brucella abortus BvrS A putative Bartonella bacilliformis kinase A putative Caulobacter crescentus kinase Homology based on BLAST Identities Positives 78% 66% 58% 58% 53% 40% 86% 77% 70% 71% 69% 55% 22-33% 46-52% 24% 20% 41% 42% Various other bacteria, Gram-negative or positive Standard BLAST hits BLAST sequences results with A. tumefaciens ChvG Sinorhizobium meliloti ActS Salmonella typhurium PhoQ 154 [...]... program to identify the genes regulated by ChvG/ChvI system, which might be involved in A tumefaciens-dependent plant and/or mammalian cell gene transfer, could help us to understand the tumorigensis mechanism of Agrobacterium 4. 2 Materials and methods 4. 2.1 Construction of homologous recombinants by electroporation The introduction of specific mutation genes into different genetic Agrobacterium background... AG6, 140 Table 4. 1 The effects of chvG on the expression of acid-inducible genes located on the two chromosomesa gfp reporter fusion presence of chvG A6007-CG9 16S RNA::gfp A6 340 -CG9 Strains I rb Fold of acidic inductionc IB pH 7.0 IB pH 5.5 + 1210 1160 0.96 16S RNA::gfp - 1172 1075 0.92 A6007(pJYH15) aopB::gfp + 60 502 8 .40 A6 340 (pJYH15) aopB::gfp - 0 0 - A6007-AG6 katA::gfp + 1 54 140 6 9.13 A6 340 -AG6... inducible genes 1 34 residing on A tumefaciens chromosomes However, it is not clear whether any of these genes participate in the tumorigenesis process or how they are regulated The ChvG/ChvI two-component system plays an important role in signaltransduction of Agrobacterium- mediated plant tumorigensis In addition, ChvG was found to be involved in the Agrobacterium- mediated mammalian cell gene transfer Therefore,... G 14 P3 A A C pL 40 07 pT 6( 6( 63 60 G G A A A 0-A 34 40 6 63 A A Fig 4. 4 The effects of ChvG on the expression of acid-inducible genes aopB and katA as determined by Western analysis A tumefaciens cells containing rrn::gfp, aopB::gfp or katA::gfp fusion were grown on IB agar plates buffered at pH 7.0 or pH 5.5 as described in the Materials and Methods The gene expression levels in the bacterial cells. .. 2 3 4 5 6 7 8 8kb 6kb Fig 4. 3 Southern blot analysis of homologous recombinants The total DNA of Agrobacterium strains were extracted and digested with ClaI (lanes 1, 3-5) or PstI (lanes 2, 6-8), respectively gfpuv (from mini-Tn5) was used as probe Lane 1, A6007; lane 2, A6 340 ; lane 3, CG9; lane 4, A6007-CG9; lane 5, A6 340 -CG9; lane 6, AG6; lane 7, A6007-AG6; lane 8, A6 340 -AG6 139 4. 3.2 Effects of chvG... by Southern blot analysis 4. 2.2 Low pH induction of Agrobacterium genes A tumefaciens cells containing the appropriate fusions were grown on AB agar plates at 28 °C for 2 days and then transferred to agar plates of IB buffered at pH 5.5 or pH 7.0 For the bacterial cells containing the vir gene fusions, 100 µM acetosyringone (AS) was added into the IB media to induce the vir genes expression After grown... min and the supernatant was transferred to a cuvette to measure the absorbance at 42 0 nm (A420) The units of β Galactosidase alkaline phosphatase activity were calculated using the following formula: 137 A420 × 1000 × 6 t (min) × A600 4. 3 Results 4. 3.1 Construction of specific genetic background strains via homologous recombination To study the effect of chvG on chromosomal gene expression, A tumefaciens... absence (-) [A6 340 , A6 340 -CG9, A6 340 (pJYH15) and A6 340 -AG6] or presence (+) [A6007-CG9, A6007(pJYH15), A6007-AG6, A6 340 -AG6(pLP36) and A6 340 -AG6(pTC 147 )] of a functional chvG The same amount of bacterial cells was collected for each strain, and the total protein was electrophoresed on SDS/12% polyarylamide gel The GFP protein was visualized by immunoblotting using the GFP antibody 142 which is a katA-... activity The plasmid pSM 243 cd (containing a virB::lacZ fusion) and pSM358cd (containing a virE::lacZ fusion) (Stachel and Zambryski, 1986) were introduced into A6007 and A6 340 A6 340 is hypersensitive to carbenicillin (Cb), even when the plasmid containing a Cb resistance gene was introduced into A6 340 Thus, low concentration of Cb (5 µg/ml) was used to select for A6 340 (pSM 243 cd) and A6 340 (pSM358cd), which... chvG gene was conducted Although the ChvG protein was detected in strain A6 340 (pLP36) by Western analysis (Li et al., 2002), the responsiveness of katA expression to a pH change was not restored (Table 4. 1 and Fig 4. 4) When the plasmid pTC 147 containing both chvG and chvI (Charles and Nester, 1993) was introduced into A6 340 -AG6, the ability to respond to the pH change was partially restored (Table 4. 1 . mechanism of Agrobacterium. 4. 2. Materials and methods 4. 2.1. Construction of homologous recombinants by electroporation The introduction of specific mutation genes into different genetic Agrobacterium. role in signal- transduction of Agrobacterium- mediated plant tumorigensis. In addition, ChvG was found to be involved in the Agrobacterium- mediated mammalian cell gene transfer. Therefore, a screening. 4. 2.2. Low pH induction of Agrobacterium genes A. tumefaciens cells containing the appropriate fusions were grown on AB agar plates at 28 °C for 2 days and then transferred to agar plates of