The analysis of plasmid rearrangements observed in the soil bacterium OR168 after the introduction of transposon Tn5 : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

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The Analysis of Plasmid

Rearrangements Observed in the Soil Bacterium OR168 After the

Introduction of Transposon Tn5

A thesis presented in fulfilment of the requirements for the

degree of

Master of Science

in

Genetics at Massey University, Palmerston North.

David John Gerard Harte 1994

·ABSTRACT

Transposon Tn5 mutagenesis has been used extensively in Escherichia coli and various other Gram-negative bacteria to produce both random and site directed mutants. The popularity of Tn5 as a mutagen stems from its apparent random insertion into the genome, leading to non-leaky polar mutations. It also confers on many bacteria resistance to aminoglycosides, providing a strong selectable marker.

The site of insertion can be mapped by Southern DNA hybridisation against a specific Tn5 probe.

Tn5-containing derivatives of the Rhizobia-like soil isolate, OR168, were produced using the broad host-range suicide plasmid vector pSUP 1011. After the transfer of pSUPlOll to OR168 via heterogeneric bacterial conjugation, stable OR168::Tn5 exconjugants were selectively isolated at frequencies of approximately 10-⁴ per recipient. None of the 53 OR168::Tn5 exconjugants screened showed the parental plasmid profile. Visible alterations to the plasmid profile were common with respect to the native plasmid profile. These events generally showed large deletions from, or additions to, the native replicons of OR168. The alterations also included a low incidence of a decrease in plasmid number. Analysis of the exconjugant population shows that the insertion of Tn5 into the genome of OR 168 may not be strictly random. It was shown that 66% of OR168::Tn5 exconjugants screened contain a plasmid-borne Tn5 element, with 90% of those involving Tn5 insertion in the same episome. There is evidence that events other than classical conservative transposition have occurred after the introduction of pSUPlOl 1 into the OR168 genome.

Screening of the isolated ORl 68: :Tn5 population for pSUP 1011 vector sequences revealed the presence of the pSUPlOl 1-derived RP4-mob fragment in 33 of 35 OR168::Tn5 exconjugants containing a plasmid-borne Tn5 element. Analysis also revealed the acquisition of Tn5 alone, presumably by conservative transposition, occurred only twice in the 35 events involving a plasmid target. This suggests that another site within the RP4 fragment can act as a surrogate transposase recognition site. Alternatively, the insertion of the RP4-mob::Tn5 sequence into a plasmid target may involve a site specific recombination process peculiar to the ORl 68 isolate.

No mechanism was elucidated for the formation of many of the alterations in plasmid mobility. Restriction fragment lengths in the immediate vicinity of the anomalous RP4-mob::Tn5 insertion are identical in different plasmids. This may indicate sequence duplication among the OR168 plasmids. Such duplication may precipitate, through homologous recombination processes, the plasmid instability observed.

CONTENTS

ABSTRACT

List of Figures List of Tables Abbreviations

1. INTRODUCTION

1.1 Alteration in Genome Organisation and Gene

1.2

1.3

1.4

1.5

1.6

Transfer in Bacteria

Genetic Recombination

1.2.1 General Recombination 1.2.2 Site Specific Recombination 1.2.3 Illegitimate Recombination

Mechanisms for Intergeneric Gene Transfer 1.3.1 Bacterial Conjugation

1.3.2 Transduction 1.3.3 Transformation

Bacterial Plasmids

1.4.1 Incompatibility Group P Plasmids

1.4.2 Native Plasmids of Rhizobia

Transposons

1.5.1 Transposon Tn5

1.5.2 Transposition in Prokaryotes

1.5.3 DNA Rearrangements Associated with Tn5 1.5.4 Tn5 Mutagenesis and Plasmid Marking

Aims

VI VI Vll

1

1

2 2 4 4

5 6 7 8

9 9 10

11 11 14 18 18

20

11

Ill

2. MATERIALS AND METHODS 24

2.1 Chemicals and Reagents 24

2.2 Bacterial Strains and Plasmid Vectors 25

2.2.1 Characterisation of Bacterial Strains Used 25

2.2.2 Growth and Maintenance of Bacterial Cultures 27

2.3 Conjugation of Bacteria: ORI 68 x PN302 Crosses 30

2.3.1 Preparation of Parental Strains 30

2.3.2 Conjugation Procedure 30

2.3.3 Recovery of Cross Progeny 30

2.3.4 Estimation of Frequency of Transposition Event 31 2.3.5 Estimation of Frequency of Spontaneous Mutations 31

2.3.6 Isolation of Exconjugants 31

2.4 Analytical Minigel Electrophoresis 32

2.5 Horizontal Eckhardt Gel Electrophoresis 33

2.5.1 Reagents and Solutions 33

2.5.2 Agarose Gel Preparation 34

2.5.3 Sample Preparation and Electrophoresis 34

2.6 Gel Staining and Photography 35

2.7 DNA Isolation and Purification Methods 35

2.7.l Rapid Isolation of Plasmid DNA 35

2.7.2 Phenol-Chloroform Extraction of DNA 36 2.7.3 Ethanol Precipitation and Washing of DNA 37 2.7.4 Measurement of DNA Concentration and Purity 37

2.8 Southern Blotting Analysis of DNA 38

2.8.1 Solutions for Vacuum Blotting 38

2.8.2 Preparation of the Blotting System 38

2.8.3 Blotting Procedure 39

2.9 Nucleic Acid Slot-Blots 40

2.10 Extraction, Digestion and Electrophoresis of Genomic DNA 41

2.10.1 Isolation of Total Genomic DNA 41

2.10.2 RE Digestion and Electrophoretic fragment Separation 42

IV

2.11 Preparation of Probe DNA 43

2.11.1 Large-Scale Preparation of Plasmid DNA 44 2.11.2 Small-Scale Preparation of Plasmid DNA 45

2.12 Probe DNA Labelling, Hybridisation and Detection 47

2.12.1 Labelling Probe DNA 48

2.12.2 Hybridisation of Labelled DNA to Target DNA 48 2.12.3 Detection of Probe-Target DNA Hybrids 50

2.13 Screening of Exconjugants for Antibiotic Resistance Markers 51 2.13.1 Determination of the Bactericidal Concentration

of Chloramphenicol 52

2.13.2 Screening of Exconjugants for Antibiotic

Resistance Markers 52

3. RESULTS 53

3.1 Creation and Isolation of OR168::Tn5 Derivatives 53

3.2 Determination of Plasmid Sizes for ORI68 and

Selected Exconjugants 57

3.3 Comparison of Plasmid Profiles 62

3.3.1 Eckhardt Gel Electrophoresis Results 62

3.3.2 Examination of Variability in CCC Band Size 66

3.3.3 The Megaplasmid Band 68

3.4 Screening of Exconjugants for Antibiotic

Resistance Markers 87

3.5 Determining the Role of Tn5 in the Observed Plasmid

Rearrangements 88

3.5.1 Determining the Site of Tn5 Insertion 88 3.5.2 Determining the Number of Tn5 Elements in

the Exconjugant Genomes 93

3.6 Slot-Blot Analysis of Exconjugants 98

V

3.7 Further Investigation of Tn5 Insertion Site and Related

Changes in Plasmid Mobility 104

3.7.1 Detailed Analyses of Exconjugant Total Genomic

DNA Restriction Patterns 105

3.8 Investigation of the RP4-mob::Tn5 Insertion Site on pOR168d 119

3.9 Plasmid Rearrangements Revisited 128

3.10 Transmissible Plasmids in OR168::Tn5 Exconjugant 130 3.10.1 Attempted Recovery of ORI 68::RP4-oriT:: Tn5 Plasmid 130

4. GENERAL DISCUSSION AND CONCLUSIONS 132

4.1 Discussion 132

4.1.1 The Relationship Between the Site of Tn5 Insertion

and Plasmid Rearrangement 132

4.1.2 Insertion of pSUP 1011 Sequences in Plasmid pORl 68d 133 4.1.3 Reports of Anomalous Tn5 Activity and their Relevance

to Observations Made in this Study 136 4.1.4 Suggestions for the Mechanism Causing the

Observed Plasmid Rearrangements 139

I I

4.2 Areas Requiring Further Investigation 141

4.3 Conclusions 143

5. ACKNOWLEDGEMENTS 144

6. BIBLIOGRAPHY 145

LIST OF FIGURES 1.5.1

1.5.2

Structure of Tn5

Models for transposition mechanisms

3.2.1 Eckhardt plasmid profiles of reference strains with known

3.2.2

plasmid sizes compared with plasmid profile of OR168 and exconjugants with plasmids of unknown size Semi-log10 plot of plasmid size against RM for determining

plasmid sizes of exconjugants and OR168 3.3.1 Plasmid profiles and location of Tn5 in OR168::Tn5

to 3.3.9 exconjugants obtained via heterogeneric

conjugation between OR168 and E. coli PN302.

3.3.10 Summary of the comparative plasmid profiles seen after the conjugative transfer of Tn5 into OR168

3.3.11 Semi-log₁₀ plot of plasmid size against RM for determining plasmid sizes of exconjugants from Eckhardt gels 3.5.1 Summary of comparative plasmid profiles of exconjugants

with plasmid-borne Tn5 elements

3.5.2. Electrophoresis and detection of Tn5 in digested total genomic

12 13

60

61

70-79

80

81

96

DNA from OR168::Tn5 exconjugants 97

3.7.1 Electrophoresis and detection of Tn5 in digested total genomic

to 3.7.9 DNA from OR168::Tn5 exconjugants and the OR168 parent 108-116 3.8.1 Electrophoresis and detection of pSUPlOl 1-derived DNA

sequences in digested total genomic DNA from the

exconjugants DHl 13, DH201 and DH216 122-125 3.8.2 Restriction mapping of the insertion site of the donor sequences in

plasmid 'd' of the exconjugants DHl 13, DH201, and DH216 127

LIST OF TABLES 2.1

3.1.1 3.1.2

List of bacterial strains and plasmids used

Isolation of progeny from ORI 68 x PN302 crosses Incidence of putative transposition events from crosses 3.1.3 Spontaneous marker mutations in parental strains 3.2.1 Plasmid size determinations

3.3.1 Sizing of plasmid bands visible on gels shown in Figures to 3.3.9 3.3.la to 3.3.9a

3.6.1 Summary of hybridisation results of Eckhardt gels and slot-blots 3.7.1 Summary of sizes of restriction fragments hybridising to Tn5-Probe

to 3.7.3 on Southern blots shown in Figures 3.7.1 to 3.7.9 3.8.1 Summary of total genomic DNA fragment sizes hybridising to

probes derived from pKan2, pSUP202, and pSUPlOl 1

29 56 56 56 59

82-86 103

117-118

126

vi

ABBREVIATIONS

AMPPD AMP-D AP Ap ATP BCIP bp CCC Cm CsCl CTAB DIG DM DNA dsDNA EDTA EtBr IE IncF lncP IS kb Km

LB

NBT

Nm nt

oc

OE oriT

RM Sp SSC ssDNA TBE Tc TE tra Tris Tn TY

3-(2 '-Spiroadamantane )-4-methoxy-4-(3 "-phosphoryloxy) -phenyl-1,2-dioxetane

Unstable dephosphorylated intermediate of AMPPD degradation Alkaline phosphatase

Ampicillin

Adenine triphosphate

5-Bromo-4-chloro-2-indolylphosphate Base pair

Covalently closed circular Chloramphenicol

Cesium chloride

Hexadecyltrimethyl ammonium bromide Digoxigenin

Distance migrated Deoxyribonucleic acid Double-stranded DNA

(Ethylenedinitrilo) tetra-acetic acid Ethidium bromide

Inside end (of IS50 and Tn5) Incompatibility group F plasmid Incompatibility group P plasmid Insertion sequence

Kilobase pairs Kanamycin

Luria-Bertani medium

4-Nitro blue tetrazolium chloride Neomycin

Nucleotide Open circular

Outside end (of IS50 and Tn5) Origin of transfer

Relative electrophoretic mobility Spectinomycin

Standard sodium citrate single-stranded DNA Tris-borate-EDT A Tetracycline Tris-EDT A buffer Transfer genes

2-Amino-2-(hydroxymethyl)- 1,3-propanediol acetate Transposon

Tryptone Yeast extract medium

Vll