Molecular analysis of mutations in agrobacterium tumefaciens under selection pressure 1

MOLECULAR ANALYSIS OF MUTATIONS IN AGROBACTERIUM TUMEFACIENS UNDER SELECTION PRESSURE QIAN ZHUOLEI (B. Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS First of all, my deepest gratitude goes to my supervisor, Associate Professor Pan Shen Quan, not only for giving me the opportunity to undertake this interesting project but also for his patience, encouragement, practical and professional guidance throughout my Ph. D candidature. Secondly, I would like to express my heartfelt gratitude to A/P Leung Kai Yin for his guidance with the facilities and his advice on my research project. I also appreciate A/P Jin Shouguang, University of Florida, USA for giving me instructions in doing research and for the donation of plasmids pEX18Tc and pUCA19. I would also like to thank the following friends and members in my laboratory who have helped me in one way or another: Tan Lu Wee, Guo Minliang, Li Xiaobo, Zhang Li, Tu Haitao, Chang Limei, Hou Qingming, Jia Yonghui, Yang Kun, Wang Long, Lin Su, Tang Hock Chun, Alan John Lowton, Sun Deying and Seng Eng Khuan. I want to thank the friends from other laboratories who assisted me in many ways and spent happy time with me such as Sheng Donglai, Yu Hongbing, Li Mo, Tung Siew Lai, Wang Xiaoxing, Luo Min and Hu Yi etc. I would also like to give my sincere appreciation to Mr Ong Tang Kwee and Madam Ang Swee Eng for their technical aid in taking high-quality photographs for me. More over, I must thank my parents and my siblings, for their support in my career and life. Finally, I thank the National University of Singapore for awarding me a research scholarship to carry out this interesting project. i TABLE OF CONTENTS ACKNOWLEDGEMENTS I LIST OF PUBLICATIONS RELATED TO THIS STUDY .VI LIST OF FIGURES VII LIST OF TABLES . X LIST OF ABBREVIATIONS .XI SUMMARY XIII CHAPTER 1. LITERATURE REVIEW 1.1. Overview of the regulation of point mutation and insertion mutation in bacteria . 1.1.1.Regulation of point mutation in bacteria 1.1.1.1. Proof reading . 1.1.1.2. Mismatch repair 1.1.1.3. Oxidative DNA damage 1.1.1.4. Other regulators 11 1.1.2. Regulation of transposition in bacteria 12 1.1.2.1. Mechanism of transposition 13 1.1.2.2. Intrinsic control of transposition activity 16 1.1.2.3. Host-mediated regulation 17 1.1.2.4. Target preference . 20 1.1.2.5. Transposition can be induced by environmental stimulus 20 1.2. Some important features about Agrobacterium tumefaciens 22 1.2.1. Molecular basis of A. tumefaciens mediated transformation . 23 1.2.1.1. Virulence genes function . 25 1.2.1.2. Roles of chromosomal virulence genes of A. tumefaciens . 29 1.2.2. The genome of A. tumefaciens 32 1.3. Some important features of two-component systems . 33 1.3.1. The characteristics of sensor and regulator proteins . 34 1.3.2. Activities of the HPK and RR proteins 36 1.3.3. Two-component systems in A. tumefaciens . 37 1.3.3.2. VirA-VirG two component system . 38 ii 1.3.3.3. ChvG-ChvI two-component system . 39 1.4. Aims and significance of this study . 46 CHAPTER 2. MATERIALS AND METHODS . 47 2.1. Bacteria strains, plasmids, primers, media and antibiotics . 47 2.2. DNA manipulation . 47 2.2.1. Preparation of competent cells 47 2.2.2. Plasmid preparation . 54 2.2.3. Genomic DNA preparation from A. tumefaciencs . 55 2.2.4. DNA digestion and ligation . 56 2.2.5. Polymerase Chain Reaction (PCR) . 56 2.2.6. Chemical Transformation of E. coli 57 2.2.7. DNA electrophoresis analysis and purification . 57 2.3. Southern blot analysis 58 2.3.1. Labeling of probes with fluorescein 58 2.3.2. Membrane blots preparation 59 2.3.3. Hybridization and stringent wash 60 2.3.4. Blocking, antibody incubation and washing . 60 2.3.5. Signal generation and detection . 61 2.4. Transposon tagging 61 2.5. General protein techniques . 62 2.5.1. Buffers for protein manipulations . 62 2.5.2. SDS-PAGE gel electrophoresis . 62 2.5.3. Staining of SDS-PAGE separated proteins with standard coomassie blue 63 2.5.4. Western blot analysis . 63 2.6. RNA manipulations 65 2.6.1. RNA extraction from A. tumefaciens . 65 2.6.2. RT-PCR 66 CHAPTER 3. MUTATION SPECTRUM OF AGROBACTERIUM TUMEFACIENS TO DIFFERENT ANTIBIOTICS . 67 3.1. Introduction 67 3.1.1. The mechanism of bacterial resistance to tetracycline 67 3.1.1.1. The action of tetracycline 67 3.1.1.2. Mechanism of resistance to tetracycline . 68 3.1.2. The mechanisms of bacterial resistance to rifampicin 69 3.2. Materials and methods 71 iii 3.2.1. Transformation of A. tumefaciens by electroporation . 71 3.2.1.1. Preparation of electrocompetent A. tumefaciens cells 71 3.2.1.2. Transformation of electrocompetent A. tumefaciens cells with plasmid DNA by electroporation 71 3.2.2. Complementation 72 3.2.3. Determination of mutation frequency . 73 3.2.4. Creation of mutant library and localization of the insertion site . 73 3.2.5. DNA sequencing 75 3.3. Results . 76 3.3.1. Different mutation frequency of A. tumefaciens strains to tetracycline resistance on MG/L and AB . 76 3.3.2. Characterization of the mutations to tetracycline resistance of A. tumefaciens strains . 85 3.3.2.1. Different mutation modes of A6007 and A6340 . 85 3.3.2.2. The mutation modes of other A. tumefaciens strains 90 3.3.3. The mutation frequency of mutS mutant on tetracycline selective medium . 90 3.3.3.1. Generation of mutS deletion mutant . 94 3.3.3.2. The Tcr mutation frequency of A6007S and A6340GS . 95 3.3.4. Different mutation frequencies of A. tumefaciens strains to rifampicin resistance . 98 3.3.4.1. Mutation of A6007 and A6340 to rifampicin resistance . 100 3.3.4.2. The mutation of other A. tumafaciens strains to rifampicin resistance . 100 3.3.5. Characterization of the mutations in rpoB gene 103 P P 3.4. Discussion 106 CHAPTER 4. THE ROLE OF CHVG IN AGROBACTERIUM TUMEFACIENS 119 4.1. Introduction .119 4.2. Materials and methods .119 4.2.1. Membrane proteins preparation . 119 4.2.2. Two-dimensional PAGE gel electrophoresis . 120 4.2.2.1. Two-dimensional gel sample preparation . 120 4.2.2.2. Iso-electric focusing (IEF) 121 4.2.2.3. Second-dimensional PAGE . 121 4.2.2.4. Silver Stain 122 4.2.3. In-gel digestion 122 4.2.4. Semi-quantitative PCR 124 4.2.5. Inoculation of plant leaves with A. tumefaciens 124 4.3. Results . 125 4.3.1. Results of two-dimensional PAGE gel electrophoresis . 125 4.3.2. Result of MALDI-TOF 130 iv 4.3.3. Generation of Atu4026 knock-out mutant in A. tumefaciens 136 4.3.4. The function of AopB and Atu4026 140 4.3.5. The transposases expression inside A6340 under stressful conditions . 142 4.4. Discussion 143 CHAPTER 5. CONCLUSIONS AND FUTURE PROSPECTIVE 148 5.1. General conclusions . 148 5.2. Future prospective . 149 REFERENCES: 151 v LIST OF PUBLICATIONS RELATED TO THIS STUDY Qian Z., Li X., Tu H and Pan SQ. 2007. Coupling of point mutation and insertion mutation of Agrobacterium tumefaciens under selective pressure. (Manuscript in preparation). vi LIST OF FIGURES Fig. 1-1. Mismatch Repair by MutHLS. Fig. 1-2. The GO system. 10 Fig. 1-3. Mechanism of replicative transposition. 15 Fig. 1-4. A model for the Agrobacterium-mediated genetic transformation. 24 Fig. 1-5. Organization of a typical two-component regulatory system. 35 Fig. 1-6. Model of signal integration and activity by the ChvE/VirA-VirG signal transducing proteins. 40 Fig. 1-7. Hydropathy plot of ChvG and its predicted domains. 42 Fig. 1-8. Alignment of the putative amino acid sequence of ChvG and ExoS proteins. 45 Fig. 3-1. Secondary structure model of tetracycline-specific efflux pumps. 70 Fig. 3-2. Flow chart of the general DNA Walking ACP-PCRTM Technology. 74 Fig. 3-3. Mutation of different mutants to Tcr. 78-79 Fig. 3-4. Mutation of 483 and 483(pUCA19atu4029) on MG/L (left) and AB (right) Tc. 81 P P Fig. 3-5. Mutation loci of 483 (∆atu4029), 715 (∆lonD), Tcm3 (∆sdhA) and Tcm5 (∆atu2583). 83-84 Fig. 3-6. Hot spots of transposition and point mutations of A6007 and A6340 inside tetR. 87 Fig. 3-7. The two kinds of insertions detected inside tetR gene from the Tcr colonies. 88 PCR of the tetR and tetA fragment of six Agrobacterium strains. 91 P Fig. 3-8. Fig. 3-9. P Mechanism of mutS knockout in A. tumefaciens. 96 vii Fig. 3-10. PCR Result of mutS knockout. 97 Fig. 3-11. Mutation of A6007, A6340, A6007S and A6340GS to Tcr on MG/L. 99 Fig. 3-12. Mutation of A6007, A6340, and A6340(pUCA19chvG) to Rifr on MG/L and AB Rif. 101 Fig. 3-13. Mutation of 715 and 483 to Rifr on MG/L and AB Rif. 104 Fig. 3-14. Mutation of Tcm3 and Tcm5 to Rifr on MG/L and AB Rif. 105 Fig. 3-15. 107 P P P P P P P P Hot spots of the mutation occurred in the RpoB protein. Fig. 3-16. Coupling of point mutation and insertion mutation model. 112 Fig. 3-17. a, the reaction that is catalyzed by SdhA inside A. tumefaciens. b, TCA cycle. 114 Fig. 3-18. Non-phosphorylated glucose degradation pathway inside A. tumefaciens. 117 Fig. 4-1. Fig. 4-2. Fig. 4-3. Fig. 4-4. Fig. 4-5. Fig. 4-6. Fig. 4-7. Two-dimensional PAGE gel electrophoresis of A6007 whole cell proteins. 126 Two-dimensional PAGE gel electrophoresis of A6007 and A6340 whole cell proteins. 127 Two-dimensional PAGE gel electrophoresis of A6007 and A6340 whole cell proteins. 128 Western blot of A6007 and A6340 membrane proteins using AopB and Vbp1 as the first antibody respectively. 129 Two-dimensional PAGE gel electrophoresis of A6007 and A6340 membrane proteins. 131 a, The spectra of A1(upper), A2(lower).b, The spectra of A3(upper), A4(lower). 132-133 The MASS spectra of protein A4 which is the hypothetical protein Atu4026. 134 viii Fig. 4-8a. One dimensional SDS PAGE of A6007 and A6340 membrane proteins. 136 Fig. 4-8b. One dimensional SDS PAGE of A6007 and A6340 membrane proteins. 137 Fig. 4-9. 138 Generation of pEX18TcKmUFDF. Fig. 4-10. Result of knockout: colony and represent the successful Atu4026 knockout strain. 139 Fig. 4-11. Infection of A.tumefaciens cells on the leaves of Kalanchoe plants. 141 Fig. 4-12. Semi-quantitative PCR result of wild type and A6340 genes 16srRNA, orfA and orfB. 144 ix We were not surprised to find that most of the Tcr mutations from A6007S and P P A6340GS were point mutations (data not shown). This was probably because the deficiency of the MMR resulted in inefficienct DNA repair. We tried to determine the Tcr mutation frequency of A6007S and A6340GS on the P P minimal medium. However, only very weak colonies could be obtained after long time incubation on AB Tc plates. Further, we found that single colony of A6007S could be obtained on AB medium without any antibiotics (data not shown). Hence, the mutS mutant was not viable on the minimal medium although the growth was as well as wild type strain on the rich medium. 3.3.4. Different mutation frequencies of A. tumefaciens strains to rifampicin resistance The tetracycline system can monitor the occurrence of different mutagenic events including point mutations and insertion mutations. We would like to know whether the genes affected the mutation to tetracycline resistance can also affect the in other loci of A. tumefaciens genome. As it is known, bacteria gain rifampicin resistance just by a minor modification of the DNA dependent RNA polymerase gene rpoB. Therefore, the rifampicin system can only monitor the point mutation. Here, we firstly blast the A. tumefaciens RpoB according to the E. coli strain K12 RpoB amino acid sequence. The homology of these two RpoB proteins is 58% identical and 75% positive (data not shown). The E. coli RpoB is composed of 1283 amino acids. The rpoB gene of A. tumefaciens is Atu1956 (NCBI), located at the liner chromosome, 98 Figure 3-11: Mutation of A6007, A6340, A6007S and A6340GS to Tcr on MG/L. Approxiately 5×108 cells of A6007 (upper panel left) and A6340 (upper panel right) were plated on MG/L Tc respectively. Approxiately 5×108 cells of A6007S (lower panel left) and A6340GS (lower panel right) were plated on MG/L Tc respectively. P P P P P P 99 composed of 1378 amino acids. 3.3.4.1. Mutation of A6007 and A6340 to rifampicin resistance Similar with the mutation to tetracycline resistance, we examined the mutation to rifampicin resistance of A6007 and A6340 on rich medium MG/L. All the mutation frequency was calculated according to the triplication of the plating experiment. The result showed that the mutation to rifampicin resistance on MG/L medium of A6340 was more than 200 times lower than that of A6007 (Figure 3-12a Table 3-6). The mutation frequency to rifampicin resistance of A6007 was around 10-7 which was quite P P similar to that of tetracycline resistance. The chvG complemented strain restored the mutation frequency to a level a little bit higher than that of A6007 (Table 3-6). We also examined the mutation frequency on the minimal medium AB rifampicin. The results indicated that the A6340 still showed much lower frequency of mutation in comparison with that of A6007 (Figure 3-12b Table 3-6). This further indicates that an enzyme involved in the regulation of mutation in A. tumefaciens was controlled by ChvG. 3.3.4.2. The mutation of other A. tumafaciens strains to rifampicin resistance We carried on the examination of the mutation frequency to rifampicin resistance of other A. tumefaciens strains which showed lower mutation frequency to tetracycline resistance. These strains included 715, Tcm3 and Tcm5. We also checked the mutation frequency of the hyper-mutant 483 on the rifampicin medium. Different from A6340, 100 a b Figure 3-12: Mutation of A6007, A6340, and A6340(pUCA19chvG) to Rifr on MG/L and AB Rif. Approximately 5×108 cells of A6007, A6340, A6340(pUCA19chvG) were spread on MG/L and AB Rif respectively. a, A6007, A6340 and A6340(pUCA19chvG) on MG/L Rif (left to right). b, A6007, A6340 and A6340(pUCA19chvG) on AB Rif (left to right). P P P P 101 Table 3-6 Mutation frequency of all strains on rich medium MG/L Rif and minimal medium AB Rif. Strains A6007 A6340 A6340(pUCA19chvG) Tcm3 Tcm5 715 483 Genotype WT chvG– chvG–+chvG sdh– Atu2583– lonD– Atu4029– P P P P P P P MG/L Rif (10-9) AB Rif (10-9) 310.0 ± 67.4 170.7 ± 104.0 1.0 ± 1.0 11.3 ± 6.1 846.7 ± 102.0 481.3 ± 60.2 322.0 ± 197.5 321.3 ± 162.7 574.0 ± 253.3 420.7 ± 294.4 361.3 ± 185.6 312.3 ± 171.6 284.0 ± 54.1 126.7 ± 9.9 P P P P 102 all the other strains showed similar mutation frequency with wild type strain on both rich medium MG/L and minimal medium AB (Figure 3-13 and 3-14; Table 3-6). The mutation frequency for all of them was about 10-7. Hence, in the case of rpoB gene P P mutation, the mechanisms of mutation regulation seemed to be different from that of tetR mutation. This could be simply because they are different loci in the genome and regulated by different pathways. However, ChvG seemed to be at the upstream in the pathway of both Rifr and Tcr mutations. P P P P 3.3.5. Characterization of the mutations in rpoB gene Firstly, we did whole-cell PCR for the A6007 mutant colonies growing on the MG/L selective plates. Since the size of gene rpoB is as large as 4134 base pairs, it is very difficult to sequence the whole gene. According to Wolff et al., (2004), all the mutations to rpoB gene in E.coli strain occur between 443 and 2059. The length of E.coli RpoB is quite similar with A. tumefaciens RpoB and they share 58% homology. Therefore, we designed three pairs of primers from 341 to 2214 of the A. tumefaciens rpoB gene. We did PCR of these fragments and sequenced all of them as described in section 3.2.5. As expected, almost all the mutations happened within a small region. Thus we designed another pair of primers p1301 and pR1891 to PCR and sequencing. If the mutation site is outside the region, other primers may be used for 103 a b Figure 3-13: Mutation of 715 and 483 to Rifr on MG/L and AB Rif. Approximately 5×108 cells of 715 and 483 were plated on MG/L and AB Rif respectively. A, 715 on MG/L and AB Rif (left to right). b, 483 on MG/L and AB Rif (left to right). P P P P 104 a b Figure 3-14: Mutation of Tcm3 and Tcm5 to Rifr on MG/L and AB Rif. Approximately 5×108 cells of Tcm3 and Tcm5 were plated on MG/L and AB Rif respectively. a, Tcm3 on MG/L and AB Rif (left to right). b, Tcm5 on MG/L and AB Rif (left to right). P P P P 105 further PCR and sequencing (see 3.2.5.). As shown in Figure 3-15 and Table 3-7, totally 48 mutant colonies were sequenced. Almost all the mutation sites were within the RNA polymerase β subunit domain 2, and the region between domain and (1386-1672 of the rpoB gene). Only several mutation sites were located in the locus before domain as well as within the domain 6. This is consistent with the report on mutations in E.coli rpoB (Wolff, et al., 2004). It appeared that Ser522 is particularly critical as 14 out of the 48 mutations occured at this P P amino acid. In these instances the Ser was mutated to Pro, Trp or Leu (Table 3-7). 3.4. Discussion The objective of Chapter is to establish the relationship of insertion mutation and point mutation. Using A. tumefaciens as model system, we determined its point mutation and insertion mutation frequency to tetracycline resistance and demonstrated that point mutation and insertion mutation could be a coupling process since several Agrobacterium strains (A6007, 483, Tcm5) showed higher frequency of point mutation while the other strains (A6340, Tcm3 and 715) showed more insertion mutations. Furthermore, the study of mutation to rifampicin resistance indicated that chvG− strain P P A6340 also exhibited a lower frequency of mutation to the rifampicin resistance, thus suggesting that chvG could play a role in the upstream sequence of the mutational pathway. Here, we tested the tetracycline resistance of A. tumefaciens strains. This system had many advantages: the whole genome sequenced, known mechanism (elaborated in 106 Figure 3-15: Hot spots of the mutation occurred in the RpoB protein. 2,3,6 and represent RNA polymerase β subunit domain 2, 3, and respectively. It clearly showed that the domain3 contained most of the hot spots of mutation. 107 Table 3-7: Distribution of mutations in rpoB Site (bp) Amino acid change Base pair change 454 460 1386 1391 1409 1543 1556 1564 1565 1565 1577 1578 1583 1621 1622 1625 1627 1633 1672 2303 2303 2309 Val→Ile(152) Val→Phe(154) Arg→Arg(462) Arg→His(464) Met→Arg(470) Phe→Leu(515) Ser→Leu(519) Ser→Pro(522) Ser→Trp(522) Ser→Leu(522) Asp→Gly(526) Asp→Glu(526) Val→Ala(528) Ser→Ala(541) Ser→Leu(541) Ala→Gly(542) Leu→Val(543) Pro→Ser(545) Arg→Cys(558) Ser→Trp(768) Ser→Leu(768) Gln→Leu(770) GC→AT GC→TA TA→AT GC→AT TA→GC TA→CG CG→TA TA→CG CG→GC CG→TA AT→GC CG→GC TA→CG TA→GC CG→TA CG→GC CG→GC CG→TA CG→TA CG→GC CG→TA AT→TA TOTAL No. of occurrence: 1 1 1 1 1 48 108 section 1.2.2. and 3.1.1.2.) and a naturally high mutation frequency at a specific tetR gene. More importantly, different mutagenic events were involved in mutations to tetracycline resistance of A. tumefaciens. We classified these mutations into point mutations and insertions. Point mutations referred to all base substitutions, single base pair deletions and additions. The insertions in our case referred to the transposition of IS426 or IS50R (only in A6340) into the tetR gene. The two kinds of mutations could be related by some mechanisms. Different spectra of stationary-phase mutations have indeed been detected in Pseudomonas putida (Saumaa et al., 2002). Their system revealed that different mutants with base substitutions, deletions, and insertion of Tn4652 occurred with different growing time (Saumaa et al., 2002; Kasak et al., 1997). They also demonstrated that the mutations happened in growing bacterium were different from those appeared during stationary phase. However, there was no direct evidence to show that how the transposition, base substitutions and minor base pair deletions could be related. Thus, the relationship of point and insertion mutation was one of the most important issues in this project. Initially, we found that A. tumefaciens strain A6340 had an extremely low frequency of mutation to Tcr on MG/L rich medium compared with that of wild type strain A6007. P P On the other hand, A6340 exhibited a normal frequency of mutation on the minimal medium. This prompted us to find more mutant strains that had a similar mutation frequency to tetracycline as A6340. As a result, we identified that 715, Tcm3, Tcm5 also showed a very high susceptibility to tetracycline on rich medium and normal mutation frequency on minimal medium (Table 3-1). We identified a mutator strain 109 483 which showed dramatically higher mutation frequency on both rich and minimal medium as well. It seemed that medium may exert some effects on the mutation frequency. It was reported by Lampe et al. (1979) that medium could change the susceptibility of enterobacteria to ampicillin. More recently, Flores et al. (2005) analyzed the global transcriptional response of the PB11 (encode phosphotransferase system PTS) mutant when growing in minimal medium with glucose as carbon source. They found that the alternative sigma factor RpoS was up-regulated and several were detected to be transcribed by this sigma factor subunit. RpoS has been shown to promote mutation by several groups (Tsui et al., 1997; Broek et al., 2005). After calculating the mutation frequency to tetracycline resistance, we further characterized these mutants on the minimal medium by direct sequencing the tetR region. It was found that most of the mutants of A6340, 715 and Tcm3 belonged to insertion mutation. In contrast, a larger proportion of mutations of A6007, Tcm5 and 483 belonged to point mutation (Table 3-4). Therefore, we hypothesized that different mutation modes could be applied by the bacterium to overcome the stressful condition. More importantly, once the point mutation pathway was blocked in some mutants like A6340, the transposition pathway could be induced under certain condition. The model was shown in Figure 3-16. The implications of the model are: 1) ChvG may affect the point mutation and insertion mutation by different unknown factors; 2) we can not conclude now that if there is direct link between ChvG and the proteins that are involved in point mutation and insertion mutation like DinB and H-NS etc.; 3) as shown in the model, ChvG, LonD (inactivated in 715) and SdhA (inactivated in Tcm3) 110 stimulate the point mutation while they suppress the insertion mutation although the mutation frequency of the three mutant strains seems to be similar as A6007, thus the mechanism of mutation is different between A6340, 715, Tcm3 and wild type strain A6007. To our knowledge, this is the first description of coupling of point mutation and insertion mutation at the same time. As shown previously, ChvG-ChvI was a global regulator (Li et al., 2002) and it was likely that ChvG played a key role in responding to various physiological or environmental changes in A. tumefaciens. It could be helpful to determine if chvG suppressed the enzymes involved in transposition. Previous study showed that two-component system ColR-ColS regulated transposition of Tn4652 in Pseudomonas putida (Hõrak et al., 2004). A probable two-component regulator BarA was found to positively regulate RpoS which affected both point mutation and transposition (Mukhopadhyay et al., 2000). It is generally accepted that the activity of transpositions can be induced by environmental and population factors and in particular by stress in various organisms (Capy et al., 2000). It is reported that the transposable element IS426 displayed the de-repressed function (Farrand et al., 1990). The stress in our case might be the nutritional stress since the chvG mutant strain grew much slower and looked not as strong as A6007. As shown by Hall (1988), transposition could be induced during long time incubation. Apparently, the transposition of A6007 was induced after incubating of bacterium on the medium without any readily usable carbon source available for days (Li 2006 unpublished result). Moreover, Tcm3 which also showed higher 111 Figure 3-16 Coupling of point mutation and insertion mutation model. ChvG, LonD (deficient in strain 715) and SdhA (deficient in strain Tcm3) stimulate the point mutation while they also suppress the insertion mutation. The detailed explanation can be seen in the text. 112 transposition frequency on minimal medium (Table3-4), was found to be deficient in the gene sdhA encoding the enzyme succinate dehydrogenase which converts succinate to fumarate or vice versa in the TCA cycle (shown in Figure 3-17ab). Interestingly, consistent with our results, it is already reported that defect in AspA which catalyzed the reductive and oxidative branches of the TCA cycle in E. coli under anaerobic conditions also triggered the transposition to happen at an early stage with higher frequency (Twiss et al., 2005). AspA converts aspartate into fumarate under microaerophilic or anaerobic conditions, when the TCA cycle is split into reductive and oxidative branches. The author of the paper considered the transposition was responding to lack of a fermentable carbon source but not the aspartase itself played a negative role in transposition. This was similar to our conditions. The defect of SdhA in mutant strain Tcm3 may also cause the cell under nutritional stress and therefore stimulate the transposition. One thing that we must pay attention to was that obviously SdhA was in the middle of the succinate dehydrogenase operon (Figure 3-5b). It was quite possible the mini-Tn5 had a polar effect to SdhB which located immeadiately after SdhA. That could be a reason that the Tcm3 complement strain only partially restored the mutation to Tcr. P P An ATP-dependent protease LA2 (LonD) was disrupted in the strain 715. 715 showed much higher transposition frequency compared with that of wild type suggesting that protease may play a role during the transposition process. One possible reason could be the transposase was more stable in the protease mutant. Derbyshire et 113 [...]... and minimal medium AB Tc 64 Point mutation and insertion mutation frequency of A tumefaciens strains (AB Tc) 80 Point mutation and insertion mutation in tetR of A6007 and A6340 80 Mutation frequency of all strains on rich medium MG/L Rif and minimal medium AB Rif 82 Distribution of mutations in rpoB 86 Table 1- 2 Table 3 -1 Table 3-4 Table 3-5 Table 3-6 Table 3-7 x LIST OF ABBREVIATIONS A adenosine A... loss of any number of base pairs; insertions, or the addition of any number of base pairs; inversions, in which a piece of linear DNA molecule is excised and reinserted in reversed order; duplication, a form of insertion in which the added bases are the same as a sequence already in the genome, usually that immediately adjacent to the insert; and complex mutations, which may include any combination of. .. (Scheuermann and Echols, 19 84) Mutations in mutD, impair the proof reading activity of episilon, leading to a high rate of spontaneous mutation (Cox and Horner, 19 83) 2 The mutD phenotype demonstrates the biological importance of the epsilon subunit and its in vivo role in avoiding mutations (Echols et al., 19 83) 1. 1 .1. 2 Mismatch repair In addition to tactics that enhance the fidelity of replication, the... combination of these mutation In the following section, I will specifically review the definition and regulation of point mutation and insertion mutation 1. 1 Overview of the regulation of point mutation and insertion mutation in bacteria Point mutations are known as single base change, or at most the addition/deletion of a few nucleotides while much of the variation that occurs in bacteria (and other organisms)... structure like 1 insertions Specific sequences known as insertion sequences (IS) have evolved in a way as to have a specific ability to insert into other DNA sequences, thus generating insertion mutations (Dale, 19 98) The genes that have been involved in the point mutation process could be different from those in insertion mutation Here, this research will review the regulation of point mutation and insertion... called mutator cells (reviewed in Miller, 19 96) 1. 1 .1. 1 Proof reading Most DNA polymerases also possess exonuclease activity that may function in proof reading through the 3’ to 5’ exonuclease activity to remove the incorrectly paired bases (Echols and Goodman, 19 91) This mechanism of correcting errors considerably enhances the fidelity of replication The epsilon subunit of Escherichia coli DNA polymerase... point mutation is single base change, or at most the addition or deletion of a few nucleotides On the other hand, specific sequences known as insertion sequences (IS) may be inserted into other DNA sequences, thus generating insertion mutations Most of the studies in this field are focused on the mechanisms of either point mutations or insertion mutations Although different mutation types including... mutations than insertion mutations Since several Agrobacterium strains (A6007, 483, Tcm5) showed a higher frequency of point mutation while the other strains (A6340, Tcm3 and 715 ) showed more insertion mutations, we hypothesize that point mutation and insertion mutation are coupled: when a simple point mutation is suppressed, the insertion mutation could be induced under certain stressful circumstances...LIST OF TABLES Table 1- 1 Proteins required for DNA mismatch repair of E.coli and budding yeast S cerevisiae 7 Comparison of A tumefaciens ChvG with other bacterial sensor proteins 44 Mutation frequency at tetR of all strains on rich medium MG/L Tc and minimal medium AB Tc 48 Table 3-2 Genotype and encoded products of A tumefaciens strains 52-53 Table 3-3 Mutation frequency of different strains on... different mutation types including both point mutations and insertions existed in one mutational event (Saumaa et al., 2002; Mennecier et al., 2006), it is not clear how point and insertion mutations are coordinated in the event of selection pressure In this study, we developed a genetic assay to detect both types of mutations that can inactivate a gene It was found that A tumefaciens gave rise to spontaneous . mutation in bacteria 2 1. 1 .1. 1. Proof reading 2 1. 1 .1. 2. Mismatch repair 3 1. 1 .1. 3. Oxidative DNA damage 8 1. 1 .1. 4. Other regulators 11 1. 1.2. Regulation of transposition in bacteria 12 1. 1.2 .1. . LIST OF ABBREVIATIONS XI SUMMARY XIII CHAPTER 1. LITERATURE REVIEW 1 1. 1. Overview of the regulation of point mutation and insertion mutation in bacteria 1 1. 1 .1. Regulation of point mutation. Mechanism of transposition 13 1. 1.2.2. Intrinsic control of transposition activity 16 1. 1.2.3. Host-mediated regulation 17 1. 1.2.4. Target preference 20 1. 1.2.5. Transposition can be induced