Molecular analysis of mutations in agrobacterium tumefaciens under selection pressure 1

Overview of the regulation of point mutation and insertion mutation in bacteria.... Hot spots of transposition and point mutations of A6007 and A6340 inside tetR.. Thus both the point m

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

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

1.1 Overview of the regulation of point mutation and insertion mutation in bacteria 1

1.1.1.Regulation of point 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 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

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

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 TcP r P 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

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

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

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)

LIST OF FIGURES

Fig 1-1 Mismatch Repair by MutHLS 4 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 TcP

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 TcP

r

colonies 88

Fig 3-8 PCR of the tetR and tetA fragment of six Agrobacterium

strains 91

Fig 3-9 Mechanism of mutS knockout in A tumefaciens 96

Fig 3-10 PCR Result of mutS knockout 97

Fig 3-11 Mutation of A6007, A6340, A6007S and A6340GS to

TcP r

on MG/L 99

Fig 3-12 Mutation of A6007, A6340, and A6340(pUCA19chvG) to

RifP r

on MG/L and AB Rif 101 Fig 3-13 Mutation of 715 and 483 to RifP

r

on MG/L and AB Rif 104 Fig 3-14 Mutation of Tcm3 and Tcm5 to RifP

r

on MG/L and AB Rif 105 Fig 3-15 Hot spots of the mutation occurred in the RpoB protein 107 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

Fig 4-2 Two-dimensional PAGE gel electrophoresis of A6007 and

A6340 whole cell proteins 127

Fig 4-3 Two-dimensional PAGE gel electrophoresis of A6007 and

A6340 whole cell proteins 128

Fig 4-4 Western blot of A6007 and A6340 membrane proteins using

AopB and Vbp1 as the first antibody respectively 129

Fig 4-5 Two-dimensional PAGE gel electrophoresis of A6007 and

A6340 membrane proteins 131

Fig 4-6 a, The spectra of A1(upper), A2(lower).b, The spectra of

A3(upper), A4(lower) 132-133 Fig 4-7 The MASS spectra of protein A4 which is the hypothetical

protein Atu4026 134

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 Generation of pEX18TcKmUFDF 138

Fig 4-10 Result of knockout: colony 1 and 8 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

LIST OF TABLES

Table 1-1 Proteins required for DNA mismatch repair of E.coli and

budding yeast S cerevisiae 7

Table 1-2 Comparison of A tumefaciens ChvG with other bacterial

sensor proteins 44

Table 3-1 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 rich medium MG/L Tc

and minimal medium AB Tc 64

Table 3-4 Point mutation and insertion mutation frequency of

A tumefaciens strains (AB Tc) 80

Table 3-5 Point mutation and insertion mutation in tetR of

A6007 and A6340 80

Table 3-6 Mutation frequency of all strains on rich medium MG/L

Rif and minimal medium AB Rif 82

Table 3-7 Distribution of mutations in rpoB 86

EDTA

TBP Tributylphosohine

Tc tetracycline TCA Tri Chloride Acetic Acid

Tn transposon

UV ultraviolet V/V volume per volume

X-gal 5-bromo-4-chloro-3-indolyl

β-D-galactopyranoside X-phos 5-bromo-4-chloro-3-indolyl

phosphate

Summary

Mutations may occur as point mutations and insertion mutations A 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 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

mutants resistant to tetracycline at a high frequency (Luo et al., 1999) Bioinformatics analysis revealed that the genome of C58 harbored two divergently transcribed genes,

tetA and tetR, encoding products that were very similar to proteins of the Tet(A) class

of tetracycline resistance systems (Luo et al., 1999) Thus both the point mutation and transposon insertion of tetR gene may give rise to the spontaneous mutation of bacteria

We found that the sensor protein ChvG mutant strain A6340 exhibited a

dramatically reduced mutation frequency to TcP

r

compared with that of wild type strain

A6007 on the rich medium In contrast, the mutation frequency of A6340 and A6007 was similar on the minimal medium This suggests that the acquisition of TcP

r

was not

due to the sensitivity of the chvG mutant to tetracycline Moreover, A6340 exhibited a

significantly higher insertion mutation frequency and lower point mutation frequency compared with A6007 on the minimal medium These indicate that the bacterial genotype may affect its mutation capacity A6007 and A6340 used different mutation modes although the apparent mutation frequency was comparable on the minimal medium Therefore ChvG may affect the mutation to TcP

r

; this effect was independent

of the DNA repair protein MutS, since the chvG and mutS double mutant did not show the phenotype of chvG or mutS

To identify the genes that affect the mutation frequency, A tumefaciens was

randomly mutagenized by mini-Tn5 The screening of the mutant libraries led to the identification of two other mutants 715 and Tcm3 that displayed higher frequency of insertion mutation and lower frequency of point mutation to TcP

r

on the minimal medium as compared to A6007 A gene encoding the putative ATP-dependent protease LA2 (LonD) was disrupted in the strain 715 We hypothesized that LonD protease

could degrade the transposase from IS426 in A tumefaciens Tcm3 contained an insertion at the gene sdhA encoding the enzyme succinate dehydrogenase, which

converts succinate to fumarate or vice versa in the TCA cycle The defect of SdhA in mutant strain Tcm3 may cause nutritional stress and therefore stimulate the

transposition On the other hand, we also identified some mutants that displayed similar mutation mode with A6007 such as 483 and Tcm5, both of which showed a

larger proportion of point 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 for a better survival of A tumefaciens

We also examined the mutation frequency of A tumefaciens strains on the

Rifampicin selective medium to see if ChvG regulated the mutation at other loci In contrast to the tetracycline system, point mutation but not insertion mutation at the

rpoB gene may generate the rifampicin resistance (RifP

r

) phenotype (Garibyan et al.,

2003) Thus the rifampicin system can only monitor the point mutation frequency We found that A6340 displayed dramatically lower frequency of point mutation on both of rich and minimal medium containing rifampicin as compared to the wild type

However, all the other strains Tcm3, Tcm5, 715 and 483 did not show any significant change in the frequency of mutation to rifampicin resistance The hot spots of mutation

within the rpoB gene were identified and the loci were quite consistent with the result obtained from E coli (Wolff et al., 2004) This experiment provided another piece of evidence that chvG played a direct or indirect role in mutations The result suggested

that the mechanism for mutation to RifP

The sensor protein ChvG appeared to affect the mutational pathway and it also

played a critical role during the tumorigenesis of A tumefaciens (Charles and Nester,

1993) Therefore it was important to for us to determine the ChvG-ChvI

two-component system regulatory pathway Two-dimensional gel electrophoresis was adopted to analyze the protein profiles for A6340 and A6007 Several new proteins, besides AopB were identified to be regulated by ChvG It would be of significance to determine if any of the proteins regulated by ChvG could play a role in the mutation process

In short, this study provides the first piece of evidence that point mutation is

coupled with insertion mutation in A tumefaciens This implies that bacteria may have

a regulatory system to monitor the success of mutational events

Chapter 1 Literature Review

Mutation is a sudden, random change in a HgeneH, or unit of hereditary material, that can alter an inheritable characteristic Although most mutations are not beneficial, since any change in the delicate balance of an organism having a high level of adaptation to its environment tends to be disruptive, the evolutionary success of bacteria relies on the beneficial mutations occurring to optimize their adaptability to constantly changing environmental conditions

Mutations are usually classified as base substitutions; deletions, or 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 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) is due to far more substantial alterations to the DNA structure like

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, 1998) 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 mutation

respectively by several genes

1.1.1 Regulation of point mutation in bacteria

Spontaneous mutations occur through errors in the replication of DNA It is essential that the newly synthesized DNA is a precise complementary copy of the template strand (Dale, 1998) Thus the cells develop the functions of proof reading and repair Usually, bacterial cells lacking in DNA repair show elevated spontaneous mutation rates and are called mutator cells (reviewed in Miller, 1996)

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, 1991) This mechanism of correcting errors considerably

enhances the fidelity of replication The epsilon subunit of Escherichia coli DNA polymerase III supplies the exonuclease activity and is encoded by the dnaQ gene (Scheuermann and Echols, 1984) Mutations in mutD, impair the proof reading activity

of episilon, leading to a high rate of spontaneous mutation (Cox and Horner, 1983)

The mutD phenotype demonstrates the biological importance of the epsilon subunit and its in vivo role in avoiding mutations (Echols et al., 1983)

1.1.1.2 Mismatch repair

In addition to tactics that enhance the fidelity of replication, the cell also possesses mechanism to correct damage that may occur in non-replicating DNA or replication errors that have escaped the proof reading process DNA mismatch repair (MMR) is one of several DNA repair pathways conserved from bacteria to humans (reviewed in Schofield and Hsieh, 2003) The MMR pathway targets base-base mismatches, e.g., G:

T, and insertion/ deletion loops (IDLs) that give rise to frameshifts The alterations in genes coding for DNA repair enzymes result in mutator phenotypes In addition, the mismatch repair system is involved in the maintenance of chromosomal structural integrity and in the control of horizontal gene transfer by preventing recombination between non-identical DNA sequences (Denamur and Matic, 2006)

In E coli, the mismatch repair pathway is initiated by the MutS, MutL and MutH

proteins as shown in Figure 1-1 MutS recognizes a mismatched base pair as well as insertions or deletions of one to four nucleotides MutL forms a complex with MutS

that activates the MutH endonuclease (Oliver et al., 2002) Nicking of the transiently

unmethylated DNA strand by MutH ensures that MMR targets repair to the newly

synthesized strand containing the error The DNA helicase II (uvrD gene product)

ensures the required separation of DNA strands

Figure 1-1 Mismatch Repair by MutHLS: recognition of mismatch (shown in red),

identifying the new DNA strand (using the hemimethylated GATC shown in blue) and cutting to encompass the unmethylated GATC and the misincorporated nucleotide (red G), excision of the DNA with the DNA helicase Uvr D (aided by exonuclease I and SSB), gap filling by DNA polymerase III and ligation

Adapted from HTUwww.personal.psu.edu/rch8/workmg/RepairDNACh7.htmUTH

The MutS and MutL of mismatch repair system have been highly conserved during

evolution The MutS (855 amino acids) and MutL (633 amino acids) of Pseudomonas aeruginosa strain PAO1 were found to be 63% and 50% identical to the corresponding

E coli proteins (Oliver et al., 2002) In eukaryotes the general features of MMR are conserved (Buermeyer et al., 1999) Table 1-1 shows the proteins required for DNA mismatch repair of E coli and budding yeast Saccharomyces cerevisiae Although the

proteins involved are different, the general mechanism is similar between eukaryotes and prokaryotes

Mutants lacking any of the specific components of the mismatch repair system, i.e

that have defects in either the mutH, mutL, mutS, or uvrD genes are strong mutators

that stimulate transitions (both G:C to A:T and A:T to G:C) at high rates (reviewed in

Miller, 1996; Boe et al., 2000; Yang et al., 2004) It is reported that general

stress-response sigma factor RpoS suppress the expression of mutS and therefore increases the frequency of mutation in Pseudomonas (Broek et al., 2005) and E coli (Tsui et al., 1997)

Interestingly, it was found that the mutant bacteria– defective in mismatch repair

from the natural isolates of E coli were present at an unexpected high frequency over 1% (Matic et al., 1997) Since E coli in humans is a commensal inhabitant of the

gastrointestinal tract as well as one of the most frequently isolated bacterial pathogens, the authors raised the possibility that there could be a link between mutator phenotype and pathogenicity More recently, it was found that no MutS expression could be

Adapted from Schofield and Hsieh, 2003

found in the natural isolates of the four strains including two E coli O157:H7 strains, a diarrhoeagenic E coli O55:H7 strain, and a uropathogenic strain (Li et al., 2003)

They suggested that because the bacteria in the natural habitat constantly experienced

a “feast or famine” existence, they may signal the need for a transient hyper-mutable and/or hyper-recombinagenic phenotype The hyper-mutable phenotype may produce the beneficial mutations and play a role in evolution

How can mismatch repair-deficient mutators outgrow nonmutator cells when they produce such vastly increased numbers of mutations that are predominantly

deleterious? It is shown that the mutator ourgrows the non-mutator strain only when the ratio of mutator: non-mutator population size is above a certain threshold

(Denamur and Matic, 2006) This threshold is determined by the ratio of the frequency

of mutants carrying beneficial alleles in mutator versus non-mutator population

1.1.1.3 Oxidative DNA damage

Reactive oxygen species can damage DNA and result in increased mutations that may lead to human cancers and contribute to aging A major oxidative damage lesion

is 8-oxo-guanine (8-GO), which forms 8-GO: A mismatches (Schofield and Hsieh, 2003)

E coli and Salmonella typhimurium have inducible enzyme systems that can

neutralize reactive oxygen species before they damage DNA The inducible enzymes include two superoxide dismutases that convert superoxides to hydrogen peroxide and

two catalases that convert the hydrogen peroxide to water The global regulons of these

functions are the oxyR, soxR and katF systems (Demple, 1991) Deletion of oxyR locus result in increased DNA damage and spontaneous mutation (Storz et al., 1987)

Some proteins are involved in the repair of DNA damage caused by mainly 8-GO reactive oxygen species MutM glycosylase removes 8-GO from 8-GO:C base pairs What happens more frequently is A across from the GO lesion since replicative

polymerase insert A 5-fold to 200-fold more frequently than C across from the GO

lesion (Shibutani et al., 1991) MutY glycosylase removes A from 8-GO:A (Lu et al.,

2001), allowing DNA polymerase I-mediated repair synthesis to restore a C The

action of mutY and mutM can be concluded in the following model shown in Figure

1-2 b 8-GO can also be introduced into the DNA if the dGTP precursor is oxidized If the 8-oxodGTP is incorporated across from A, then A:T convert to C:G MutT, a base excision repair enzyme, hydrolyzes dGOTP, thus preventing the conversion

(Figure1-2c)

Double mutants lacking both MutM and MutY activities have extraordinarily high

mutation rates and the mutations in the mutY , mutM strains are mainly G:C to A:T transversions (Michaels et al., 1992) The transversion from C to A was elevated

significantly in the Pseudomonas putida MutY-defective strain (Saumaa et al., 2002)

Figure 1-2: The GO system A 2’-deoxy-7,8-dihydro-8-oxoguanosine (8-GO) B Oxidative

damage can lead to GO lesions in DNA which are repaired by the concerted action of the MutM and MutY proteins MutM removes 8-GO from 8-GO:C base pairs; MutY removes A

from 8-GO:A C MutT is active on 8-oxodGTP and hydrolyze it to 8-oxodGMP, effectively

removing the triphosphate from the deoxynucleotide pool

Adapted from Miller, 1996

1.1.1.4 Other regulators

Besides the genes involved in the DNA repair can regulate the point mutation of

bacteria, some other genes can also affect the point mutation DNA polymerase IV is a poorly processive error-prone DNA polymerase and a member of the large, elaborated DinB/UmuDC superfamily of DNA polymerases in bacteria, archaea, and eukaryotes

(Friedberg et al., 2000) When the expression of the SOS-inducible polymerase dinB is increased in E coli, both -1 frameshift at mononuleotide repeats and base substitution mutations increase, although not to the same extent (Kim et al., 1997) It has been reported that pol IV was required for most adaptive point mutation at lac in the E coli,

but not for mutations in growing cells, survival of UV or oxidative damage, or

adaptive amplification (McKenzie et al., 2001) The deletion of the dinB gene resulted

in about 75% decrease in spontaneous frameshift and base substitution mutations in

strain (Strauss et al., 2000)

Similar with DinB, the stationary-phase sigma factor RpoS was also found to promote the point mutation frequency It was found that RpoS negatively controlled

the MutS, thus stimulating the whole mutation frequency in E coli and Pseudomonas (Tsui et al., 1997; Broek et al., 2005) In Pseudomonas putida, the occurrence of 2- to

3- bp deletions required the stationary-phase sigma factor RpoS, which indicated that

some mutagenic pathway was positively controlled by RpoS (Saumaa et al., 2002)

There are many papers describing genes that are shown to regulate the point

mutations in bacteria For example, overproduction of the multidrug resistance

transcription regulator, EmrR, results in a large increase in frameshift and base

substitution in E coli (Yang et al., 2004) Two-component system comA and comK

involved in the regulation of differentiation in post-exponential growth were found to regulate stationary-phase mutagenesis (Sung and Yasbin, 2002)

Although it is no surprise that mutation increases when exonucleolytic proofreading

is decreased, at first glance it is surprising that mutation can increase if the activity of certain repair proteins is increased (Frosina, 2000) For example, increased expression

of 3-methydadenine DNA glycosylase II (which excised damaged, but to some extent undamaged, bases from DNA) increased the mutation rate as measured by increased

spontaneous mutation to rifampicin resistance in E coli (Caporale, 2003; Berdal et al.,

1998)

1.1.2 Regulation of transposition in bacteria

Different from point mutation, transposition is the large-scale alteration in the DNA structure Movable element is important in the generation of the natural diversity of micro-organisms Bacteria commonly carry several such sequences, often as multiple copies, and a substantial proportion of spontaneous mutations may be due to

inactivation of genes by insertion of a copy of an IS element (Dale, 1998)

Transposons are essentially similar to IS elements in that they have the ability to move (transpose) from one site to another; they differ in carrying one or more

identifiable genetic markers like antibiotic resistance These transposons have played a key role in the evolution and spread of antibiotic resistance Many studies demonstrate the significant impact of mobile elements on genome organization and evolution In bacteria, transposons facilitate formation of DNA inversion, deletions, and

chromosome fusions (Shapiro, 1979) The mechanism of transposition will be

discussed in the following section

1.1.2.1 Mechanism of transposition

In considering mechanisms of transposition, it is not necessary to distinguish

between insertion sequences and transpositions, as the same mechanisms apply to both types of transposable elements (Dale, 1998) Transposition happens by two kinds of mechanisms: replicative mechanism, a copy of the element is inserted at a different site while the original copy is retained; non-replicative transposition, also called conservative transposition, the insertion sequences do not replicate when they

transpose

The replicative transposition mechanism is shown in Figure 1-3 The stages are as follows:

a Single-strand breaks (nicks) in the DNA are produced at each 3’ end of the

transposon (in opposite strands) The recipient DNA is also nicked, at two places

on either side of a short target sequence The staggered nature of the nicks is the ultimate cause of the duplication of the target sequence

b The free ends of the transposon are joined to the free ends generated by the nicks

in the recipient DNA

c The free 3’ ends of the recipient DNA act as primers for the synthesis of DNA strands, using host replication or repair enzymes This synthesis will proceed through the transposon, separating the two strands until it reaches the existing complementary strand, to which the new strand will be joined by the action of DNA ligase

d Since there are two copies of the transposon in the same direction, recombination between the two copies gives rise to the end-products of transposition: donor and recipient each contain a copy of transposon

Some of the transposable elements follow the non-replicative mechanisms A model for non-replicative transposition is the “cut and paste” model In that case, the transposition is completely excised from the donor molecule before being attached to the target site; then the free transposon DNA joins to cut ends of target; repair

synthesis fills in gaps around the transposon

The process of transposition must be carefully controlled to prevent the

accumulation of potentially deleterious insertions in the host genome The

transposable elements have evolved numerous shrewd strategies to exercise this

control The mechanisms act at various steps of transposition process: transcription of

the transposase gene; its translation; transposase stability and activity; and DNA

binding and catalysis (reviewed in Nagy and Chandler, 2004) The detailed regulation

of transposition will be discussed in the following section

1.1.2.2 Intrinsic control of transposition activity

Intrinsic control includes all regulatory mechanisms inherent to the transposable element itself Many endogenous promoters which drive transposase expression are inefficient thus limiting transposase levels at the transcription level For example, the

in vivo activity of the endogenous promoter of IS21 is undetectable (Reimmann et al., 1989) IS50 produces two proteins: the full-length transposase and a shorter derivative

truncated at its N-terminal end which acts as an inhibitor of tranposase activity These two proteins are expressed from two distinct promoters but are translated in the same phase to control the transposase level (Johnson and Reznikoff, 1984) It is found that

the regulation of the transposase of Tn4652 tnpA is mediated by the

transposon-encoded protein TnpC (Hõrak and Kivisaar, 1999)

A less conventional mechanism has been demonstrated with IS1 and with elements related to IS3 In these cases, a key protein required for transposition is translated from

two different reading frames on the mRNA: the ribosomes start reading the mRNA in one frame and then at a defined point shift back one base and continue reading in a different frame This ensures that very little available functional enzyme (Dale, 1998)

1.1.2.3 Host-mediated regulation

The involvement of host factors adds another level of regulation of transposition besides the inherent regulation by itself The host imposes this level by limiting the availability of the proteins required to modulate the timing and efficiency of

transposition In vitro studies have shown that transposition is orchestrated in a large

DNA-protein complex, the transpososome, in which all of the protein and DNA

substrates are assembled so as to ensure a productive event

There is no general rule and each transposable element may have its own set of host factors and indeed use a given host factor in different ways Their intervention can occur at several levels including transposase expression and transpososome assembly,

or in the later stages that necessitate DNA repair (reviewed in Nagy and Chandler, 2004)

Firstly, I would like to talk about the regulation of transposition by DNA

architectural proteins Nucleiod proteins are small, abundant, DNA-binding proteins that profoundly affect the local and global structure of the chromosome, and play a

major role in gene regulation The Escherichia coli proteins H-NS is recognized as an

important component among the major nucleiod-associated proteins H-NS, stands for histone-like nucleiod structuring protein, is also known to regulate stress response pathways and virulence genes in bacteria People found that mutation in H-NS resulted

in the reduction of transposition of IS1 to at lease 100-fold compared with the

the hns-null mutant (Rouquette et al., 2004) In the same year, Swingle et al (2004)

demonstrated that H-NS was required for efficient transposition of all three elements-

IS903, Tn10 and Tn552 in a papillation assay, suggesting a general role for H-NS in

bacterial transposition One year later, a thorough study about H-NS was conducted by

the Wardle group, showing that H-NS promotes Tn10 transposition by binding directly

to the transposition complex or transpososome (Wardle et al., 2005) They presented evidence that, upon binding, H-NS induced the unfolding of the Tn10 transpososome

and helped to maintain the transpososome in an unfolding state

Similar with H-NS, IHF (integration host factor) is another nucleiod-associated

protein involved in transposition IHF plays a key role in the assembly of the Tn10 transpososome; it binds to the outside end (OE) of Tn10 immediately adjacent to the

primary transposase binding site and bends the DNA to stabilize the transposase-OE

interaction (Sakai et al., 1995 and 2000)

For phage Mu, binding of IHF to a site approximately 1 kb from the left end

stimulates repressor binding, presumably by severely bending the DNA, and regulates

a divergent promoter which is involved both in repressor and transposase expression

(reviewed in Nagy and Chandler, 2004) Similarly, the tnpA, transposase of Tn4652, is found to be modulated by IHF in P putida (Hõrak and Kivisaar, 1998) The presence

of an IHF-binding site upstream of the tnpA promoter enhances the promoter activity Further, it was proved that transposition of Tn4652 in stationary phase P putida was essentially limited by the amount of IHF (Ilves et al., 2004) No transposition of

Tn4652 occurred in a P putida ihfA-defective strain Moreover, overexpression of IHF

resulted in significant enhancement of transposition compared with wild-type strain

Other factors that may regulate the transposition include DNA methylase, enzymes involved in DNA repair, SOS systems etc DNA adenine methylation (DAM) has been shown to affect the activity of the transposon ends directly Evidence was presented

that the damP

–

mutants have increased IS10 activity in E coli (Roberts et al., 1985)

Fully methlylated ends were less active than hemimethylated ends Since the sites would be transiently hemimethylated following passage of a replication fork, they

suggested that IS10 transposition may preferentially occur immediately after passage

of a chromosomal replication fork

It should not be difficult to imagine that the enzymes for DNA repair and DNA gyrase are needed for the transposition from its own process shown in Figure 1-3 Many transposable elements generate small direct repeats of target DNA flanking the insertion These are the result of staggered attack of the target sequence by the two transposition ends These must undergo repair to avoid damage to the genome

(reviewed in Nagy and Chandler, 2004) It was shown that both the polymerase

activity and 5’ to 3’ exonuclease activity of DNA polymerase I were required for

transposition of Tn5 (Sasakama et al., 1981)

In an extensive screen of the mutant library for the host factors that regulated

transposition, Coros et al (2005) provided genetic evidence that GTP was required for transposition of IS903 and Tn552 in E coli

1.1.2.4 Target preference

Transposable elements have influenced the genetic and physical composition of all

modern organisms The choice of a target can also have critical implications both for the transposon and for the host It seems that the target choice of transposon is quite promiscuous on a genome scale However, data suggest the integration of some

transposons is far from random Computational and genetic analysis of multiple IS903

insertion sites predicted a preferred target consisting of a 21 bp palindromic pattern centered on the 9 bp target duplication generated during transposition The 5 bp

flanking sequences were the most important sequences required for site-specific

insertion of IS903 (Hu et al., 2001)

It is reported that insertion hot- and cold- spot genes were found to have more than

1,000-fold variation in utilization frequency for the Mu phage DNA to E coli The

author suggested the local chromosome structure is more important than DNA

sequence in determining Mu target-site selection (Manna et al., 2004)

1.1.2.5 Transposition can be induced by environmental stimulus

In the natural world, individuals, populations and species all have to cope with environmental change Individual organisms have to adapt physiologically through responses that are immediate and reversible These adaptations may lead to genetic changes and to evolution of inherited characteristics Many of the genetic changes are

due to increased activity of transposable elements (reviewed by Capy et al., 2000)

The DNA architectural proteins may vary with the bacterial growth phase,

supercoiling, temperature and the various other systems involved in DNA metabolism All these conditions may vary according to the physiology of bacteria which must compromise with environmental changes Therefore, it is not hard to imagine the transposition activity may be induced by some stimulus People reported that the

activities of transposition and excision proteins of IS103 could well respond to the

physiological conditions that exist within a colony that had depleted its environmental

nutrients (Hall, 1988) In the paper reported by Kasak et al (1997), they described a

system that PheP

mutants appear only after starvation:

no insertions of Tn4652 were observed among the preexisting PheP

strain (Ilves et al., 2001) The rpoS gene, codes for alternative transcriptional sigma

factor and is upregulated during the limited nutrient period, controls expression of

multiple stationary-phase genes (Hengge-Aronis, 1999) More recently, Twiss et al (2005) reported that transposition was modulated by a diverse set of host factors in E coli and was stimulated by nutritional stress They identified a mutant strain deficient

in gene aspA encoded an enzyme involved in the oxidative branches of the TCA cycle

showed higher transposition activity They suggested it was the first direct genetic link between transposition and nutritional stress

1.2 Some important features about Agrobacterium tumefaciens

The model system used in this study is Agrobacterium tumefaciens Thus it is necessary to introduce some important features about this bacterium A tumefaciens is

a Gram-negative, soil-borne plant pathogen that can cause crown gall disease, a

tumorous disease oat infection sites, on a wide range of plant species (Van et al., 1974; Waston et al., 1975; Hooykaas et al., 1994) A tumefaciens can transfer a DNA

segment, called the transferred DNA (T-DNA), from its tumor-inducing (Ti) plasmid into the plant cell nucleus, where the T-DNA integrates into the plant genome The

T-DNA transfer from A tumefaciens to plant cells is characteristic of the type VI

secretion system that exports DNA and protein substrates (Christie and Vogel, 2000) T-DNA carries genes that are involved in the synthesis of plant growth regulators Integration of the T-DNA into the host cell genome and the subsequent expression of the T-DNA encoded genes results in the formation of neoplastic growths, known as crown gall tumors which synthesize opines and serve to provide the major carbon and

nitrogen source for A tumefaciens (reviewed in Tzfira et al., 2000; Kado, 2000; Gelvin, 2000) 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 hosts range of A tumefaciens includes not only the dicotyledonous plants (reviewed in Hooykaas et al., 1994), such as fruit trees and vines, but also the monocotyledonous plants (Hiei et al., 1997; Komari et al.,

1998) Furthermore, it has been found that A tumefaciens can also transform fungus, yeast and even mammalian cells as well (Bundock et al., 1995; Relic et al., 1998) When suitably modified, we could make A tumefaciens the most effective vector for

gene transfer in plant biotechnology Interestingly, based on the transformation

technology developed in the A tumefaciens, people tried to transform plants by other plant associated symbiotic bacteria like Sinorhizobium meliloti, thus providing a versatile “open source” platform for plant technology (Broothaerts et al., 2005)

1.2.1 Molecular basis of A tumefaciens mediated transformation

A tumefaciens is the only known natural vector for inter-kingdom gene transfer The

transferred DNA (T-DNA) is referred to as the T-region when located on the Ti

plasmid T-region on native Ti plasmid is approximately 10-30 kbp in size (Barker et al., 1983) T-regions are defined by T-DNA border sequences These borders are 25 bp

perfect direct repeats and highly homologous in sequence (Peralta and Ream, 1985; De

Vos et al., 1981) The process of T-DNA transfer consists of several important steps as shown in Figure 1-4: bacterium chemotaxis and attachment, vir genes induction,

T-DNA processing, T-DNA transfer and nuclear targeting, T-DNA integration into the plant genome and transferred gene expression Initially, in coordination with the

monosaccharide transporter ChvE and in the presence of the appropriate phenolic and sugar molecules, VirA autophosphorylates and subsequently transphosphorylates the

VirG protein (Jin et al., 1990) The activated VirG helps to increase the level of

transcription of the vir genes Then the vir genes products are directly involved in the