Characterisation and functional analysis of the putative agr system in Clostridium acetobutylicum

267 Pages·2017·5.57 MB·English

by  Christine Middleton

Characterisation and functional analysis of
the putative agr system in Clostridium
acetobutylicum

JAMIE SCOTT, BSc. (Hons), MSc.

Thesis submitted to the University of Nottingham
For the degree of Doctor of Philosophy

December 2012
Declaration

Unless otherwise acknowledged, the work presented in this thesis is my
own. No part has been submitted for another degree in the University of
Nottingham or any other institute of learning.

Jamie Scott

December 2012
Abstract
Clostridium acetobutylicum is an industrially important Gram positive
organism which is capable of producing economically important chemicals
in the Acetone, Butanol and Ethanol (ABE) process. An orthologue of the
accessory gene regulator (agr) locus of Staphylococcus aureus has been
found to be present in the genome of Clostridium acetobutylicum. In
S.aureus, agr encodes a quorum sensing (QS) system that controls the
expression of virulence in this species. Analysis of the agr region in
C.acetobutylicum was conducted using reverse transcriptase PCR which
showed the agrB and agrD genes to be linked. This was also the case with
the agrC and agrA genes but there was no conclusive evidence to suggest
that all 4 genes resided on the same operon. The use of cat-based
reporter vectors which incorporate chloramphenicol acetyl transferase
were used to look at the expression profiles of the agrB and agrC putative
operons. The agrC construct showed activity consistent with the expected
pattern of expression but this was not repeated with the agrB construct.
Antisense RNA vectors were constructed with the intention of disrupting
the agr genes but had no observable effect. This work was superseded by
a newly available method to knockout the agrA gene by allelic exchange
and the use of the ClosTron system to obtain gene inactivation in agrB,
agrC and agrA. Gas chromatography analysis of these mutants showed
little or no difference in product formation and a sporulation assay was
developed which revealed that these mutants were inhibited in spore
production. Finally, microarray analysis has been used to look at the effect
of agrB inactivation on the gene expression of C.acetobutylicum. The
expression of known sporulation genes was found to be differentially
regulated. This study presents some of the first evidence to support the
hypothesis that agr may be a major regulator in C.acetobutylicum and may
act in a cell density dependent manner via a diffusible signal molecule.

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Acknowledgements
I would like to thank my supervisors Professor Nigel Minton and Dr Alan
Cockayne for their help and support throughout my PhD.
Many thanks to the Clostridial research group both past and present who
have helped me along the way. Special thanks to Dr Glen Carter who
taught me all his tricks in Molecular Biology and Dr John Heap who
developed many of the techniques used in my work. I would also like to
thank Victoria Wright for her time and hard work in producing the
microarray data in this study and Professor Klaus Winzer for his help and
advice.

I would like to thank the research group of Professor Philippe Soucaille at
what is now National Institute for Applied Sciences, Toulouse, France who
provided some of the plasmids and techniques used in my work. My
thanks also to the research group of Professor Hubert Bahl in Rostock
Germany who also provided advice on handling Clostridium
acetobutylicum.

Many thanks to the staff of NHS Great Yarmouth and Waveney who have
provided help and sympathy during the time I have spent writing my thesis.

Finally I would like to thank my family and friends who have provided so
much support to me over the years. Thanks to my Grandparents in
particular my Grandfather Derrick Lewis who was such a good role model
and my Grandmother Mary Lewis whose faith in me was unwavering.
Special thanks to my parents Glen Carter and Steve Carter for their hard
work and support.

Last but not least I would like to thank my wife Lisa Scott for her patience
support and love through the time of writing my thesis. Without her help
and the support of those mentioned I would not have been able to
complete this PhD.

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Table of Contents
ABSTRACT ................................................................................................. I
ACKNOWLEDGEMENTS .......................................................................... II
TABLE OF FIGURES .............................................................................. VII
LIST OF TABLES .................................................................................... XI
LIST OF ABBREVIATIONS ..................................................................... XI
1.0 INTRODUCTION .................................................................................. 1
1.1 THE GENUS CLOSTRIDIUM ............................................................... 1
1.2 CLOSTRIDIUM ACETOBUTYLICUM .................................................. 1
1.3 ACIDOGENESIS .................................................................................. 2
1.4 SOLVENTOGENESIS .......................................................................... 2
1.4.1 ACETONE ......................................................................................... 4
1.4.2 BUTANOL ......................................................................................... 4
1.5 GENES AND OPERONS INVOLVED WITH SOLVENTOGENESIS ... 4
1.5.1 EXPRESSION PROFILES OF METABOLIC GENES ..................................... 6
1.6 REGULATION OF SOLVENTOGENESIS ........................................... 6
1.6.1 SPO0A ............................................................................................ 7
1.6.2 ABR AND SINR ................................................................................. 7
1.6.3 SOLR .............................................................................................. 8
1.7 METABOLIC ENGINEERING OF C.ACETOBUTYLICUM .................. 9
1.7.1 REPORTER STUDIES ....................................................................... 10
1.7.2 ANTISENSE RNA ............................................................................ 11
1.7.3 MICROARRAY ................................................................................. 13
1.8 SPORULATION ................................................................................. 14
1.8.1 GENETIC CONTROL OF SPORULATION................................................ 16
1.8.2 ROLE OF SPO0A ............................................................................ 16
1.8.3 AXIAL FILAMENT FORMATION AND ASYMMETRIC DIVISION .................... 18
1.8.4 SIGMA FACTORS INVOLVED IN SPORULATION ..................................... 19
1.8.5 ROLE OF ΣF .................................................................................... 19
1.8.6 ROLE OF ΣE .................................................................................... 20
1.8.7 ENGULFMENT (ΣG) .......................................................................... 22
1.8.8 ROLE OF ΣK .................................................................................... 22
1.9 SIGMA FACTORS IN C.ACETOBUTYLICUM ................................... 23
1.11 QUORUM SENSING ........................................................................ 24
1.12 QUORUM SENSING IN GRAM-NEGATIVE BACTERIA ................. 25
1.13 QUORUM SENSING IN GRAM-POSITIVE BACTERIA .................. 28
1.14 THE AGR SYSTEM OF STAPHYLOCOCCUS AUREUS ................ 29
1.15 THE ROLE OF AGR LIKE SYSTEMS IN OTHER SPECIES ........... 33
1.16 QUORUM SENSING IN CLOSTRIDIUM .......................................... 36
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1.16.1 THE AGR HOMOLOGUE IN C.ACETOBUTYLICUM................................. 38
1.17 GENETIC MANIPULATION OF CLOSTRIDIUM ............................. 39
1.17.1 GENERATION OF RANDOM MUTANTS ............................................... 39
1.17.2 DIRECTED GENE INACTIVATION USING HOST MEDIATED RECOMBINATION
.............................................................................................................. 41
1.17.3 ANTISENSE RNA .......................................................................... 47
1.17.4 CLOSTRON ................................................................................... 47
1.18 DEVELOPMENT OF THE CLOSTRIDIAL RAM ......................................... 50
2.0 MATERIALS AND METHODS ........................................................... 54
2.1 BACTERIAL STRAINS AND PLASMIDS .......................................... 54
2.2 CHEMICAL REAGENTS .................................................................... 57
2.3 GROWTH MEDIA ............................................................................... 58
2.3.1 LURIA-BERTANI MEDIUM .................................................................. 58
2.3.2 2 X YT MEDIUM ............................................................................. 58
2.3.3 2 X YTG MEDIUM ........................................................................... 58
2.3.4 CLOSTRIDIAL BASAL MEDIUM CBM .................................................. 58
2.3.5 REINFORCED CLOSTRIDIAL MEDIUM RCM ........................................ 59
2.3.6 CLOSTRIDIAL BASAL SPORULATION MEDIUM CBSM .......................... 59
2.3.7 SYNTHETIC MEDIUM ........................................................................ 60
2.4 SUPPLEMENTS .................................................................................. 60
2.5 GROWTH CONDITIONS ....................................................................... 60
2.6 DNA MANIPULATIONS ..................................................................... 61
2.6.1 PLASMID PREPARATION ................................................................... 61
2.6.2 RESTRICTION DIGESTS .................................................................... 61
2.6.3 BLUNT-ENDING OF DNA FRAGMENTS USING KLENOW POLYMERASE .. 62
2.6.4 BLUNT-ENDING OF DNA FRAGMENTS USING T4 DNA POLYMERASE .. 62
2.6.5 DEPHOSPHORYLATION OF LINEARISED DNA FRAGMENTS .................. 62
2.6.6 LIGATION OF DNA FRAGMENTS ........................................................ 62
2.6.7 PCR AND RESTRICTION DIGEST CLEAN UP ...................................... 63
2.6.8 EXTRACTION OF DNA FRAGMENTS FROM AGAROSE .......................... 63
2.7 AGAROSE GEL ELECTROPHORESIS ............................................ 63
2.8 POLYMERASE CHAIN REACTION .................................................. 64
2.8.2 GENERAL PCR PARAMETERS .......................................................... 67
2.8.3 COLONY PCR ................................................................................ 68
2.9 TRANSFER OF PLASMID DNA INTO BACTERIAL CELLS ............ 68
2.9.1 DIALYSIS OF DNA LIGATION REACTIONS........................................... 68
2.9.2 PREPARATION OF ELECTROCOMPETENT E. COLI ................................ 68
2.9.3 ELECTROPORATION OF PLASMID DNA INTO E.COLI ........................... 69
2.9.4 PREPARATION OF CHEMICALLY COMPETENT E. COLI.......................... 69
2.9.5 TRANSFORMATION OF PLASMID DNA INTO CHEMICALLY COMPETENT E.
COLI ....................................................................................................... 70
2.9.6 METHYLATION OF PLASMID DNA FOR TRANSFER INTO
C.ACETOBUTYLICUM ................................................................................ 70
2.9.7 CONJUGATION OF PLASMID DNA INTO C. ACETOBUTYLICUM .............. 70
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2.9.8 ELECTROPORATION OF PLASMID DNA INTO C. ACETOBUTYLICUM: USING
C. BEIJERINCKII METHOD .......................................................................... 71
2.9.9 ELECTROPORATION OF PLASMID DNA INTO C. ACETOBUTYLICUM USING
THE NEESON AND TITBALL METHOD ........................................................... 72
2.9.10 ELECTROPORATION INTO C.ACETOBUTYLICUM: USING THE
MERMELSTEIN AND PAPOUTSAKIS METHOD ................................................ 72
2.10 EXCISION OF MLS CASSETTE USING FLP RECOMBINASE ...... 73
2.11 T/A CLONING .................................................................................. 74
2.12 BLUE/ WHITE SELECTION ............................................................. 74
2.13 RNA ANALYSIS ............................................................................... 74
2.13.1 PREPARATION OF TOTAL RNA FROM C. ACETOBUTYLICUM ............... 75
2.13.2 REMOVAL OF CONTAMINATING GENOMIC DNA FROM TOTAL RNA .... 75
2.13.3 DNASE I TREATED RNA SAMPLE CLEAN UP ................................... 76
2.13.4 ONE-STEP RT-PCR...................................................................... 76
2.14 DNA MICROARRAY ........................................................................ 77
2.14.1 PREPARATION OF RNA FROM C.ACETOBUTYLICUM .......................... 77
2.14.2 MICROARRAY METHOD .................................................................. 77
2.15 CHLORAMPHENICOL ACETYL TRANSFERASE ASSAY ............ 79
2.16 CLOSTRON SYSTEM ...................................................................... 80
2.17 GAS CHROMATOGRAPHY ANALYSIS ......................................... 80
2.18 SPORULATION ASSAYS ................................................................ 81
2.18.1 LIQUID BASED ASSAY ..................................................................... 81
2.18.2 PLATE BASED ASSAY ..................................................................... 82
3.0 IDENTIFICATION AND ANALYSIS OF A PUTATIVE AGR SYSTEM
IN C.ACETOBUTYLICUM ........................................................................ 83
3.1 INTRODUCTION ................................................................................ 83
3.2 RESULTS ........................................................................................... 85
3.2.1 EXPLOITATION OF THE C. ACETOBUTYLICUM GENOME ........................ 85
3.2.2 TRANSCRIPTIONAL ANALYSIS OF THE AGR OPERON ........................... 87

3.2.3 GENE TRANSFER TECHNIQUES ......................................................... 89
3.2.3.1 Conjugation of C acetobutylicum 824 .................................... 89
3.2.3.2 Inhibition of restriction modification system using ARD and its
effect on conjugation efficiency ......................................................... 91
3.2.3.3 Development of a consistent electroporation protocol ........... 94
3.2.4 CONSTRUCTION OF REPORTER VECTORS .......................................... 94
3.2.4.1 GUS based vectors ............................................................... 94
3.2.5 DEVELOPMENT OF CATP BASED EXPRESSION VECTORS ...................... 96
3.3 DISCUSSION ................................................................................... 100
4.0 GENE KNOCKOUT IN C.ACETOBUTYLICUM AND ITS EFFECTS
ON SOLVENTOGENISIS AND SPORULATION ................................... 102
4.1 INTRODUCTION .............................................................................. 102
4.2 RESULTS ......................................................................................... 102
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4.2.1 DEVELOPMENT OF ANTISENSE VECTORS ......................................... 102
4.2.2 WHOLE GENE KNOCKOUT USING ALLELIC EXCHANGE ........................ 105
4.2.3 CLOSTRON INSERTIONAL MUTATION ............................................... 110
4.2.3.1 The use of pMTL007 for agr gene knockout in
C.acetobutylicum ............................................................................. 110
4.2.3.2 Complementation of mutants ............................................... 111
4.2.4 THE EFFECT OF GENE KNOCKOUT WITH RAM ON C.ACETOBUTYLICUM
PRODUCT FORMATION ............................................................................ 113
4.2.4.1 Effect of agr knockout on ethanol production ...................... 114
4.2.4.2 Effect of agr knockout on acetone production ..................... 115
4.2.4.3 Effect of agr knockout on acetate production ...................... 116
4.2.4.4 Effect of agr knockout on butyrate production ..................... 117
4.2.4.5 Effect of agr knockout on butanol production ...................... 117
4.2.5 REMOVAL OF THE SELECTIVE MARKER USING PMTL007C-E2 ........... 124
4.2.6 EFFECT OF ERMB REMOVAL ON AGR KNOCKOUT MUTANTS ............... 126
4.2.6.1 Ethanol production............................................................... 127
4.2.6.2 Acetone production.............................................................. 128
4.2.6.3 Acetate production............................................................... 129
4.2.6.4 Butyrate production ............................................................. 130
4.2.6.5 Butanol production............................................................... 132
4.2.7 SPORULATION .............................................................................. 133
4.2.7.1 Effect of agr knockouts on sporulation................................. 134
4.3 DISCUSSION .................................................................................. 137
4.3.1Sporulation .............................................................................. 141
4.3.2 Complementation ................................................................... 142
5.0 THE EFFECT OF AGRB KNOCKOUT ON GENE EXPRESSION IN C.
ACETOBUTYLICUM. ............................................................................. 143
5.1 INTRODUCTION .............................................................................. 143
5.2 RESULTS ......................................................................................... 144
5.2.1 GENES INVOLVED IN SOLVENTOGENESIS ........................................ 148
5.2.2 GENES INVOLVED IN SPORULATION ................................................. 151
5.2.2.1 Sporulation in B.subtilis ....................................................... 151
5.2.2.2 Initiation of Sporulation ........................................................ 151
5.2.2.3 Stage I - Axial filament formation ......................................... 153
5.2.2.4 Stage II – Asymmetric division ............................................ 155
5.2.2.5 SpoIIIE ................................................................................. 156
5.2.2.6 Sigma factors ...................................................................... 157
5.2.2.7 Stage III - Engulfment of the prespore ................................. 158
5.2.2.8 Sigma Factor G (σG) ............................................................ 160
5.2.2.9 σG Regulon .......................................................................... 161
5.2.2.9 Sigma factor K (σK) .............................................................. 163
5.2.2.10 Uncharacterised sigma factors .......................................... 164
5.2.2.11. pSOL-1 genes .................................................................. 165
5.2.3 CELL MOTILITY, CHEMOTAXIS AND SIGNAL TRANSDUCTION MECHANISMS
............................................................................................................ 167
5.2.3.1 COG class N – Cell motility and secretion ........................... 167
5.2.3.2 COG Group T – Signal transduction mechanisms ............... 169
5.2.4 CARBOHYDRATE TRANSPORT AND METABOLISM ............................... 172
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5.3 DISCUSSION ................................................................................... 174
5.3.1 THE EFFECT OF THE AGRB DELETION ON SPORULATION .................... 174
5.3.2 THE EFFECT OF THE AGR DELETION ON SOLVENTOGENESIS .............. 177
5.3.3 THE EFFECT OF THE AGR KNOCKOUT ON MOTILITY GENES ................. 180
5.3.4 DOES AGR ACT IN A CELL DENSITY DEPENDENT MANNER? ................. 180
5.4.5 LESS STRINGENT STATISTICAL ANALYSIS REVEALS SUPPORTING
EVIDENCE THAT THE AGR SYSTEM REGULATES SPORULATION AND
SOLVENTOGENESIS ................................................................................ 182
6.0 GENERAL DISCUSSION ................................................................. 183
6.1 REGULATION OF THE AGR HOMOLOGUES IN
C.ACETOBUTYLICUM .......................................................................... 183
6.2 GENERATION OF AGR KNOCKOUT MUTANTS ........................... 184
6.3 PHENOTYPIC EFFECTS OF AGR MUTATION .............................. 187
6.4 FUTURE STUDIES .......................................................................... 188
BIBLIOGRAPHY .................................................................................... 192
APPENDIX I – Z SCORES OF THE THREE AGR MUTANTS WITH THE
RAM FOR THE LEVELS OF PRODUCT FORMATION ........................ 222
APPENDIX II – Z SCORES OF THE FOUR AGR MUTANTS
CONTAINING NO SELECTIVE MARKER FOR THE LEVELS OF
PRODUCT FORMATION ....................................................................... 223
APPENDIX III - CLUSTERS OF ORTHOLOGOUS GROUPS OF
PROTEINS (COGS) ............................................................................... 224
APPENDIX IV - PUBLICATIONS ........................................................... 248

Table of Figures
Chapter 1

Figure 1.1 The biosynthetic pathways involved in acidogenesis and
solventogenesis in C.acetobutylicum………………………..3
Figure 1.2 The sol operon in C.acetobutylicum…………………………5
Figure 1.3 Illustration of the sinI/R operon of B.subtilis………………...8
Figure 1.4 The B.subtilis sporulation pathway showing the 8 stages of
sporulation……………………………………………………..15
Figure 1.5 Phosphorylation cascade leading to the activation of Spo0A
in B.subtilis……………………………………………………..17
Figure 1.6 A simplified representation of the role of sigma factors and
other regulatory proteins involved in the regulation of
sporulation in B.subtilis……………………………………….21
Figure 1.7 The V.fischeri lux paradigm regulatory cascade ….………27
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Figure 1.8 The agr locus and signalling system………………………..32
Figure 1.9 The organisation of the com gene cluster and the pathways
involved in an agr like quorum sensing response of
B.subtilis……………………………………………………......36
Figure 1.10 A single crossover event leading to an insertional
mutation…………………………………………………….…...42

Figure 1.11 Double crossover event in which two recombinational
events can lead to a stabilised mutant……………………...43

Figure 1.12 The process of insertional deletion using the Targetron
and ClosTron systems………………………………………..51

Chapter 3
Figure 3.1 The arrangement of the putative agr operon in
C.acetobutylicum and its comparison with the S.aureus agr
operon……………………………………………………….....86
Figure 3.2 RT-PCR analysis of the agr locus in C.acetobutylicum…..88
Figure 3.3 PCR products using primersCDcond1 and CDcond4 specific
to pMTL9361…………………………...................................90
Figure 3.4 An agarose gel showing evidence that 3 of the
thiamphenicol resistant colonies which have been proven to
harbour pMTL9361 are C.acetobutylicum 824…………….91
Figure 3.5 Construction of ard based plasmids pJSARD916 and
pJSARD5592……………………………………………….....93
Figure 3.6 Method used for construction of pJS01…………………….95
Figure 3.7 Method used for construction of pJS04 and pJS05……….98
Figure 3.8 Three graphs showing replicate CAT reporter activity of the
two reporter vectors…………………………………………..99

Chapter 4
Figure 4.1 Construction of the two antisense vectors pJS06 and pJS07
from pSOS95…………………………………………………104
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