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Role of Alarmones in The Protection of Escherichia coli Against Stress
Souad MOUMENE
[Thesis].The University of Manchester, School of Pharmacy and Pharmaceutical Sciences;2012.
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Abstract
Escherichia coli has evolved in environments which may commonly be acidic and thus developed adaptive mechanisms to minimise acid-induced damage. It has previously been observed that adapted bacteria to moderately acidic conditions can grow in media considerably below their optimum growth pH. To explain this phenomenon, a hypothesis which suggested that diffusible molecules (alarmones) may serve as early warning systems of acidic conditions was proposed. Alarmones are thought to be produced upon exposure to mildly-acidic conditions. They then diffuse in the environment and elicit a protective response against acid in recipient cells. The protective activity of those putative alarmones against lethal acid was investigated. The main aim of this project is to determine the mode of action of those alarmones at the molecular level. Preliminary experiments confirmed acid resistance conferred by alarmones to populations of E. coli C600. The stability of those alarmones at different temperatures and following proteinase K treatment was investigated. Moreover, investigations into whether alarmones are autoinducer-2 (AI-2) molecules and whether alarmones increase the percentage of persisters in an E. coli population were undertaken. Subsequently, microarray analyses of both alarmone-induced and non-induced cultures were performed to reveal E. coli genes induced by alarmones. Moreover, proteomic studies using two-dimensional gel electrophoresis were conducted to reveal proteins induced by alarmones. Supernatants from alarmone-induced cultures conferred statistically significant protection (p<0.01) on recipient cultures against lethal acid (pH3). Alarmones were inactivated by heat (60oC) and by proteinase K. The autoinducer-2 (AI-2) assay revealed that alarmones are not AI-2 molecules. In addition, alarmones did not increase the percentage of persisters. In order to elucidate potential mechanisms for alarmone-mediated protection, the genomic expression and protein induction of alarmone-induced cells using microarray analysis and two-dimensional gel electrophoresis, respectively, were performed. Two-dimensional gel electrophoresis of transduced cultures indicated that around 13 proteins were induced in the alarmone-protected populations of E. coli C600. Mass-spectrometric analysis revealed that these alarmone-inducible proteins include the acid stress chaperone HdeB and the DNA-binding transcriptional dual regulator, H-NS which plays an important role in stress adaptation. Microarray analyses of transduced cultures indicated that 671 open reading frames (ORFs) were significantly differentially expressed between alarmone-protected and control populations (p<0.05). 508 ORFs were upregulated in the induced cells including 10 genes related to acid-resistance and 36 different genes related to multidrug efflux system proteins whereas 163 ORFs including the autoinducer-2 system were downregulated. E. coli releases diffusible signalling compounds which mediate adaptation to acid stress of recipient cells. Microarray and proteomic data show that two acid fitness island genes (hdeB and hdeD) and three genes that encode the antiporters of the three amino-acid-dependent acid resistance mechanisms (gadC, adiC and cadB) were among the upregulated genes. This work confirms that there is communication between bacterial cells in general and warning messages amongst E. coli C600 cells in particular in the presence of stress.
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Doctor of Philosophy
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262
Table of contents:
Abstract……………………………………………………………………………......8Declaration………………………………………………………………….……......9Copyright
Statement…………………………………………………………..…10Research Contributions…………………………………………………………11Acknowledgements………………………………………………………………12Dedication………………………………………………………………………...…13Abbreviations………………………………………………………………………14List
of Tables……………………………………………………………………....16List of Figures……………………………………………………………………...17List
of Appendices…………………………………………………………….…..20Chapter 1 General Introduction…………………………..……...……...…..221.1
Introduction……………………………………………………………………..221.2 Effects of environmental variables on
microbial growth……….……231.2.1 Temperature……………………………………………………………….....……231.2.2 pH………………………………...…………………………………………….…251.2.3
Water-availability…………………………………………………………....……261.2.4 Oxygen………………………………...………………………………………….261.2.5
Presence of toxic agents………………………………...……………………….…271.3 Bacterial adaptation to changes
in environmental factors…………..281.3.1 General stress response in Escherichia coli…………............................................281.3.2
The sigma factor “RpoS (σs)” and σs-controlled-genes………………………..…291.3.2.1 Role of
rpoS in thermotolerance………………………………………………...301.3.2.2 Role of rpoS in acid tolerance………………………………………………..…311.3.2.3
Role of rpoS in oxidative stress…………………………………………………331.3.2.4 Role of rpoS in ethanol
tolerance………………………………………….……331.3.2.5 Role of rpoS in tolerance to high osmolarity……………………………………331.3.3
Specific stress responses in Escherichia coli……………………………………..341.3.3.1 E. coli stress
response to heat……………………………………………….341.3.3.2 E. coli stress response to cold-shock………………………………………..341.3.3.3
E. coli stress response to oxidative stress…………………………………...351.3.3.4 E. coli stress
response to oxygen deprivation………………………….……351.3.3.5 E. coli stress response to osmotic
stress…………………………………….361.3.3.6 E. coli stress response to nutrient limitation………………………………...361.3.3.7
E. coli stress response to acid stress………………………………………...371.3.3.7.1 Control of cytoplasmic
pH……………………………………………38 1.3.3.7.2 pH homeostasis in Gram-negative bacteria………………………...…381.3.3.7.3
pH homeostasis in Gram-positive bacteria……………………………391.3.3.7.4 Acid tolerance, acid
resistance and acid habituation in Escherichia coli….…………………………………………………………………………...391.3.3.7.4.1
RpoS-dependent acid resistance system………………...…421.3.3.7.4.2 Glutamate/glutamine-dependent
acid resistance system…..421.3.3.7.4.3 Arginine-dependent acid resistance system………………..451.3.3.7.4.4
Lysine-dependent acid resistance system………………….451.3.3.7.5 Acid resistance in the
virulent strain E. coli O157:H7………………..461.4 Alarmones……………………………………………………………………….471.4.1
The biological role of alarmones………………………………………………….481.4.2 Findings related to habituation
in acidic pH………………………………………491.4.3 Properties of extracellular sensing components
and extracellular inducing components……………………………………………………………………………...501.4.4 Suggested
mechanism of action for alarmones…………………………………...521.4.5 Difference between extracellular
sensing components and extracellular inducing components……………………………………………………………………………...54
1.4.6 Biological basis of protection conferred by alarmones……………………...……55
1.4.7 Stress cross-tolerance……………………………………………………………..56 1.4.8 Inhibition
of the synthesis of alarmones……………………………………….....571.5 Aims and Objectives………………………………………………………..…58Chapter
2 Materials and Methods……………………………………….602.1 Bacterial strains………………………………………………………………..60
2.2 Growth of bacteria…………………………………………………………… 602.3 Chemicals………………………………………………………………………..612.4
Spectrophotometric assessment of bacterial growth………………….612.5 Determination of viable
counts……………………………………………..612.6 Measured versus calculated optical densities…………………………...632.7
Growth curves of bacteria at different pH values……………………..642.8 Effect of acidic pH
on bacterial growth and viability………………...652.9 Production of bacterial alarmones………………………………………...652.9.1
Production of bacterial alarmones by acid stress………………………………….652.9.1.1 Production
of bacterial alarmones by acid stress (pH5) ………………………..652.9.1.2 Production of bacterial
alarmones by acid stress (pH4) ………………………..652.9.2 Production of bacterial alarmones
by heat stress………………………………….662.10 Measurement of potential protection conferred by
alarmones.......662.10.1 Protection of bacterial alarmones produced by acid stress………………………662.10.1.1
Method involving serial dilutions……………………………………………...662.10.1.2 Method involving plating
out of the samples directly…………………………672.10.2 Protection of bacterial alarmones produced
by heat stress………………………672.11 Stability studies……………………………………………………………….682.11.1 Temperature……………………………………………………………………...682.11.2
Proteinase K treatment…………………………………………………………...692.12 Extraction of alarmones into
organic and aqueous phases………...692.13 Autoinducer-2 assay…………………………………………………………702.14
Investigation of potential link between alarmones and persisters……………………………………………………………………………....702.15
Growth rate effect of alarmones………………………………………….712.16 Proteomic analyses of alarmone-induced
and non-induced Escherichia coli protein extracts…………………………………………..……..712.16.1 Protein
extraction………………………………………………………………...712.16.2 Protein extraction: an alternative method……………………………………..…712.16.3
Quantification of extracted proteins………………………………………….…..722.16.4 Isoelectric focusing
(IEF): the first dimension of two-dimensional gel electrophoresis………………………………………………………………………..…732.16.4.1
Sample application and IPG strips rehydration………………………..732.16.4.2 Focusing of the
IPG strips……………………………………………..742.16.4.3 Equilibration of the IPG strips…………………………………………742.16.5
SDS-PAGE electrophoresis: the second dimension of two-dimensional gel electrophoresis………………………………………………………………………..…742.16.5.1
Casting of gels…………………………………………………………742.16.5.2 Electrophoresis………………………………………………………...752.16.5.3
Visualisation of gels………………………………………………...…752.17 Microarray analysis………………………………………………….….752.17.1
Preparation of cells for RNA extraction…………………………………………752.17.2 Isolation of total
RNA……………………………………………………….…..752.17.3 Determination of RNA quantity and quality…………………………………….762.17.4
Ethanol precipitation of RNA……………………………………………………782.17.5 Microarray procedure……………………………………………………………792.17.5.1
cDNA preparation……………………………………………………..802.17.5.1.1 Reagents and materials required…………………………...802.17.5.1.2
Reagent preparation………………………………………..802.17.5.1.3 Random priming cDNA synthesis…………………………802.17.5.2
RNA degradation………………………………………………………812.17.5.3 Purification and quantification of
cDNA synthesis products………….812.17.5.4 cDNA fragmentation…………………………………………………..812.17.5.5
Terminal labelling…………………………………………………..…822.17.5.6 E. coli target hybridisation…………………………………………….822.17.5.6.1
Reagents and materials…………………………….………822.17.5.6.2 Miscellaneous reagents…………………………………….832.17.5.6.3
Miscellaneous supplies……………………………….……832.17.5.6.4 Reagent preparation……………………………………..…832.17.5.6.5
Hybridisation procedure……………………………...……832.17.5.7 Washing, staining and scanning of
the array slide………………………….…842.17.5.8 Analysis of microarray data……………………………………………………84Chapter
3 Bacterial response to unfavourable heat and acid conditions…………………………………………………………………………...853.1
Introduction…………………………………………………………………..…853.2 Acid and Heat as Stress Agents……………………………………….……853.2.1
Acid…………………………………………………………………………….…853.2.2 Heat…………………………………………………………………………..……863.3
Alarmones/Extracellular Signals………………………………………..…863.4 Methods…………………………………………………………………………..863.4.1
Growth curves of different bacterial strains………………………………………863.4.2 Determination of
bacterial susceptibility towards acid and heat stress…………...863.4.2.1 Death kinetics
of E. coli C600 exposed to acidic pH……………………..873.4.2.2 Death kinetics of E. coli
DH5α exposed to acidic pH………………….....873.4.2.3 Death kinetics of E. coli wild type
exposed to acidic pH…………………873.4.2.4 Death kinetics of Staphylococcus epidermidis
exposed to acidic pH…….873.4.2.5 Death kinetics of E. coli C600 exposed to heat………………………...…87
3.4.3 Effect of acidic pH on bacterial growth and viability…………………………….883.4.4 Production
of bacterial alarmones by acid stress…………………………………883.4.5 Production of bacterial
alarmones by heat stress…………………………………883.4.6 Measurement of potential protection conferred
by alarmones……………………883.4.6.1 Protection conferred by bacterial alarmones produced
by acid stress: serial dilution method……………………………………………………………………883.4.6.2 Protection
conferred by bacterial alarmones produced by acid stress: method involving plating
out of the samples directly…………………………….893.4.6.3 Protection conferred by bacterial
alarmones produced by heat stress…….893.4.7 Stability studies of potential alarmones…………………………………...………893.4.8
Extraction of alarmones into organic and aqueous phases……………..…………893.4.9 Growth rate
effect of alarmones…………………………………………………..893.4.10 Autoinducer-2 assay……………………………………………………………..893.4.11
Investigations of any link between alarmones and persisters……………………893.5 Results………………………………………………………………………….…893.5.1
Kill curves of different bacterial strains………………………………………..…893.5.2 D-value estimation
for E. coli C600………………………………………………923.5.3 Growth curves of different bacterial strains………………………………………953.5.3.1
Growth curves of E. coli C600 in different pHs………………………….953.5.3.2 Effect of acidic
pH on E. coli C600 growth and viability………………..96 3.5.3.3 Growth curves of E. coli
DH5α in different pHs…………………………963.5.3.4 Growth curves of E. coli w/t in different
pHs………………………….....973.5.3.5 Growth curves of Staphylococcus epidermidis in different
pHs……….....983.5.3.6 Effect of acidic pH on Staphylococcus epidermidis growth and
viability………………….………………………………………………………...99 3.5.4 Protective effect of alarmones………………………………………………...…1003.5.4.1 Acid
tolerance induction in E. coli C600 and E. coli (w/t) by medium filtrates from pH
5 culture…………………………………………………...1003.5.4.2 Acid tolerance induction in E. coli C600
by medium filtrates from pH 4 culture………………………………………………………………………..1023.5.4.2.1 Stability
of putative alarmones………………………………...1033.5.4.2.2 Extraction of alarmones into hydrophilic
and hydrophobic layers……………………………………………………………………..1043.5.4.2.3 Growth rate of alarmone-induced
and control populations of E. coli C600………………………………………………………………...1053.5.4.2.4 Acid
tolerance induction in E. coli DH5α…………………….1063.5.4.2.5 Are alarmones produced by
Staphylococcus epidermidis?........1083.5.4.2.6 Effect of E. coli C600 alarmones on
Staphylococcus epidermidis…………………………………………………...……….….1093.5.4.2.7 Are alarmones autoinducer-2?
………………………………...109 3.5.4.2.8 Investigation of any link between alarmones and persisters…...1113.5.4.3
Possible heat tolerance induction in E. coli C600 through E. coli C600 culture exposed
to 55°C………………………………………………………….1123.6 Discussion………………………………………………………………………1153.7 Conclusion…………………………………………………………………..….120Chapter
4 Proteomic Analyses of Alarmone-induced and Non-induced Escherichia coli C600……………………………………………1214.1
Introduction……………………………………………………………………1214.2 Methods…………………………………………………………………………1234.2.1
Protein extraction and solubilisation………………………………………………1234.2.2 Protein separation
by two-dimensional gel electrophoresis………………..….…1234.2.3 Protein detection…………………………………………………………………1234.2.4
Data analysis…………………………………………………………………..…1244.3 Results…………………………………………………………………………...1244.3.1
Differentially-expressed proteins identified using the isoelectric focusing pH range
of 3-10……………………………………..…………………………………………..1254.3.2 Differentially-expressed proteins
identified using the isoelectric focusing pH range of 3-6……………………………..……………………………………………………129
4.3.3 Differentially-expressed proteins identified using the isoelectric focusing
pH range of 5-8………………………………………….……………………………………….1354.4 Discussion………………………………………………………………………1414.5
Conclusion……………………………………..……………………………….147Chapter 5: DNA microarrays of alarmone-induced
and non-induced populations of Escherichia coli C600……………………...1485.1 Introduction………………………..…………………………………..………1485.2
Affymetrix GeneChip® Arrays technology…………..…………………1495.3 Data collection and normalisation…………..……………………………1495.4
Determination of differentially-expressed genes…………..………….1505.4.1 Supervised methods…………..……………………………………..…………...1525.4.2
Data filtering…………..……………………………………..…………………..152 5.5 Resources for meta-analysis…………..…………………………………...1525.6
DNA microarray experimental methods…………..……………………1525.7 DNA microarray results…………..…………………………………..…….1535.7.1
ORFs relating to acid resistance……………………..………………………..…1565.7.2 ORFs relating to hydrogenase
activity……………………………………..……1575.7.3 ORFs relating to multidrug efflux pump proteins……………………..……...…1575.7.3.1
ORFs relating to the ATP-binding cassette (ABC) transporters……...…1585.7.3.2 ORFs
relating to the major facilitator superfamily (MFS) …………...…1595.7.3.3 ORFs relating
to membrane fusion protein family………………………1605.7.3.4 ORFs relating to the resistance
nodulation cell division family…………1615.7.4 ORFs relating to biofilm formation……………………..……………………….1615.7.5
ORFs relating to outer and inner membrane proteins……………………..……..1615.7.6 ORFs relating
to DNA replication and cell division……………………..……....1625.7.7 ORFs relating to phosphate
metabolism and transport……………………..……1635.7.8 ORFs relating to nitrate and/or nitrite
metabolism and transport………………..1645.7.9 ORFs relating to amino-acids……………………..…………………………..…1655.7.9.1
Arginine……………………..…………………………………..……….1655.7.9.2 Alanine and glycine……………………..……………………………….1655.7.9.3
Cysteine……………………..…………………………………..……….1655.7.9.4 Histidine……………………..…………………………………..………1665.7.9.5
Methionine……………………..…………………………………..…….166 5.7.9.6 Proline……………………..…………………………………..…………166
5.7.9.7 Serine……………………..…………………………………..………….1665.7.9.8 Threonine……………………..……………………………………….…1665.7.9.9
Tryptophan……………………..…………………………………..……1665.7.9.10 Leucine/isoleucine/valine……………………..…………………….…..1675.7.10
ORFs relating to cytochrome oxidase enzymes………………..………………1675.7.11 ORFs relating to
autoinducer-2 (AI-2) ………………..……………………….1685.7.12 ORFs relating to non-coding RNA (ncRNA)
………………..………………...1685.7.13 ORFs relating to lipopolysaccharide biosynthesis………………..……………1685.7.14
ORFs relating to ribose transport………………..……………………………..1695.7.15 ORFs relating to NADH
dehydrogenase I………………..…………………….1695.7.16 Miscellaneous ORFs………………..…………………………………………..1695.8
Discussion…………………..…………………………………………………..1735.9 Conclusion…………………..………………………………………………….190Chapter
6 General discussion and suggestions for future work……………………………………………………………………………….…1916.1
General discussion…………………………………………………………………1916.2 Conclusion…………………………………………………………………………1996.3
Suggested future work………………………………………………………….…..200Appendices..............................................................................................................201References..............................................................................................................213
Abstract:
Escherichia coli has evolved in environments which may commonly be acidic and thus
developed adaptive mechanisms to minimise acid-induced damage. It has previously been
observed that adapted bacteria to moderately acidic conditions can grow in media considerably
below their optimum growth pH. To explain this phenomenon, a hypothesis which suggested
that diffusible molecules (alarmones) may serve as early warning systems of acidic
conditions was proposed. Alarmones are thought to be produced upon exposure to mildly-acidic
conditions. They then diffuse in the environment and elicit a protective response
against acid in recipient cells. The protective activity of those putative alarmones
against lethal acid was investigated. The main aim of this project is to determine
the mode of action of those alarmones at the molecular level. Preliminary experiments
confirmed acid resistance conferred by alarmones to populations of E. coli C600. The
stability of those alarmones at different temperatures and following proteinase K
treatment was investigated. Moreover, investigations into whether alarmones are autoinducer-2
(AI-2) molecules and whether alarmones increase the percentage of persisters in an
E. coli population were undertaken. Subsequently, microarray analyses of both alarmone-induced
and non-induced cultures were performed to reveal E. coli genes induced by alarmones.
Moreover, proteomic studies using two-dimensional gel electrophoresis were conducted
to reveal proteins induced by alarmones. Supernatants from alarmone-induced cultures
conferred statistically significant protection (p<0.01) on recipient cultures against
lethal acid (pH3). Alarmones were inactivated by heat (60oC) and by proteinase K.
The autoinducer-2 (AI-2) assay revealed that alarmones are not AI-2 molecules. In
addition, alarmones did not increase the percentage of persisters. In order to elucidate
potential mechanisms for alarmone-mediated protection, the genomic expression and
protein induction of alarmone-induced cells using microarray analysis and two-dimensional
gel electrophoresis, respectively, were performed. Two-dimensional gel electrophoresis
of transduced cultures indicated that around 13 proteins were induced in the alarmone-protected
populations of E. coli C600. Mass-spectrometric analysis revealed that these alarmone-inducible
proteins include the acid stress chaperone HdeB and the DNA-binding transcriptional
dual regulator, H-NS which plays an important role in stress adaptation. Microarray
analyses of transduced cultures indicated that 671 open reading frames (ORFs) were
significantly differentially expressed between alarmone-protected and control populations
(p<0.05). 508 ORFs were upregulated in the induced cells including 10 genes related
to acid-resistance and 36 different genes related to multidrug efflux system proteins
whereas 163 ORFs including the autoinducer-2 system were downregulated. E. coli releases
diffusible signalling compounds which mediate adaptation to acid stress of recipient
cells. Microarray and proteomic data show that two acid fitness island genes (hdeB
and hdeD) and three genes that encode the antiporters of the three amino-acid-dependent
acid resistance mechanisms (gadC, adiC and cadB) were among the upregulated genes.
This work confirms that there is communication between bacterial cells in general
and warning messages amongst E. coli C600 cells in particular in the presence of stress.
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Created by:
Moumene, Souad
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17th June, 2012, 20:41:40
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Moumene, Souad
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26th June, 2012, 18:09:09