Studies on specific and general defense strategies against reactive oxygen species in Bacillus subtilis [Elektronische Ressource] / vorgelegt von Jörg Mostertz

Sujets

• Biologie

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2004
• Nombre de lectures: 19
• Poids de l'ouvrage: 6 Mo

Extrait

Studies on Specific and General Defense Strategies
against Reactive Oxygen Species in Bacillus subtilis
I n a u g u r a l d i s s e r t a t i o n
zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald
vorgelegt von
Jörg Mostertz
geboren am 31.07.1971
in Berlin
Greifswald, 02.02.2004Dekan:
1. Gutachter:
2. Gutachter:
Tag der Promotion:Contents
Summary ...........................................................................................................................1
Zusammenfassung ...........................................................................................................2
Introduction.......................................................................................................................3
Experimental system ......................................................................................................3
Adaptive response networks...........................................................................................3
Oxidative stress............................................................................................................10
Scope of this thesis20
Transcriptome and Proteome Analysis of Bacillus subtilis Gene Expression in
Response to Superoxide and Peroxide Stress ..............................................................21
Introduction ..................................................................................................................21
Methods .......................................................................................................................24
Results and discussion .................................................................................................25
Patterns of Protein Carbonylation following Oxidative Stress in Wild-type and sigB
Mutant Bacillus subtilis Cells .........................................................................................43
Introduction ..................................................................................................................43
Methods45
Results.........................................................................................................................47
Discussion....................................................................................................................52
BScreening for Mutants of -Dependent Genes with Increased Sensitivity against
Hydrogen Peroxide in Bacillus subtilis..........................................................................57
Introduction57
Methods .......................................................................................................................59
Results.........................................................................................................................62
Discussion....................................................................................................................67
Functional Analysis of Thioredoxin in Bacillus subtilis................................................70
Introduction ..................................................................................................................70
Methods .......................................................................................................................71
Results and discussion .................................................................................................72
Conclusion ...................................................................................................................77
I
General Discussion.........................................................................................................79
Specific stress response in E. coli and B. subtilis ..........................................................80
General stress response in E. coli and 81
The role of Dps in E. coli and B. subtilis ........................................................................83
BThe general stress response of and its function in aerobic life...................................84
Conclusion ...................................................................................................................85
References ......................................................................................................................86
List of Publications .........................................................................................................99
II
SUMMARY
Summary
The present work consists of four parts, containing experimental data obtained from analysis of
Bacillus subtilis specific and general defense strategies against reactive oxygen species.
In the first part, the peroxide and superoxide stress stimulons of B. subtilis were analyzed by
means of transcriptomics and proteomics. Oxidative stress responsive genes were classified into
two groups: the gene expression pattern was either similar after both stresses or the genes
primarily responded to one stimulus. The high induction observed for members of the PerR-regulon
after both stimuli supported the assumption that activation of the peroxide specific PerR-regulon
1represented the primary stress response after superoxide and peroxide stress.
The second part focuses on protein carbonylation in B. subtilis wild-type and sigB mutant cells.
The introduction of carbonyl groups into amino acid side chains of proteins represents one possible
form of protein modification after attack by reactive oxygen species. Carbonyl groups are readily
detectable and the observed amounts can thus serve as an indicator for the severity of protein
damage. The results demonstrate clearly that B. subtilis proteins are susceptible to hydrogen
peroxide (H O ) mediated carbonylation damage. The application of low concentrations of H O2 2 2 2
prior to the exposure to otherwise lethal levels of peroxide reduced markedly the degree of protein
carbonylation, which also held true for glucose starved cells. Artificial preloading with general stress
proteins resulted in a lower level of protein carbonylation when cells were subjected to oxidative
stress, but no differences were detected between wild-type and sigB mutant cells.
In the third part, strains with mutations in genes encoding general stress proteins were screened
for decreased resistance after H O challenge. It was demonstrated that resistance to H O after2 2 2 2
transient heat treatment, likewise to conditions of glucose starvation, was at least partly mediated
B Bby the -dependent general stress response. The screening of mutants in -controlled genes
revealed an important role for the deoxyribonucleic acid (DNA)-binding protein Dps in the context of
B-mediated resistance to oxidative stress underlining previous reports. Therefore, the experimental
strategy opens a global view on the importance of DNA integrity in B. subtilis under conditions of
oxidative stress.
The fourth part includes analysis of a B. subtilis thioredoxin conditional mutant. The thiol-
disulfide oxidoreductase TrxA is an essential protein in B. subtilis that is suggested to be involved in
maintaining the cytoplasmic thiol-disulfide state even under conditions of oxidative stress. To
investigate the physiological role of TrxA, growth experiments and two-dimensional gel
electrophoresis were carried out with exponentially growing cells that were depleted of TrxA. The
observations indicate that TrxA is essentially involved in the re-reduction of phosphoadenosyl
phosphosulfate reductase CysH within the sulfate assimilation pathway of B. subtilis.

1
This part was done in cooperation with C. Scharf. Both authors contributed equally to it.
1
ZUSAMMENFASSUNG
Zusammenfassung
Die vorliegende Arbeit, bestehend aus vier Abschnitten, enthält experimentelle Daten aus Analysen
der spezifischen und generellen Schutzstrategien von Bacillus subtilis gegen reaktive Sauerstoffspezies.
In dem ersten Abschnitt ist die Analyse der Peroxid und Superoxid Stressstimulons von B. subtilis
anhand von Transkriptom- und Proteomdaten dargestellt. Gene, die auf oxidativen Stress reagieren,
konnten zwei Gruppen zugeordnet werden: entweder waren die Genexpressionsmuster nach Einwirkung
beider Stressoren ähnlich, oder die Genexpression wurde nur durch einen der beiden Stimuli verändert.
Die starke Induktion, die für Gene des PerR-Regulons nach beiden Stimuli beobachtet wurde, unterstützt
die These, dass die Aktivierung des Peroxid-spezifischen PerR-Regulons die primäre Stressantwort nach
2Superoxid- und nach Peroxidstress darstellt.
Der zweite Abschnitt enthält Daten zu Proteincarbonylierungen in Wildtyp- und sigB Mutantenzellen
von B. subtilis. Die Reaktion von Aminosäureseitenketten zu Carbonyl-haltigen Derivaten stellt eine der
möglichen Formen von Proteinmodifikationen nach Oxidation durch reaktive Sauerstoffspezies dar. Der
Nachweis von Proteincarbonylierungen ist methodisch gut zugänglich und ermittelte Mengen können als
Indikatoren für das Level der oxidativen Proteinschädigung herangezogen werden. Die Resultate
belegen, dass Proteine von B. subtilis nach Einwirkung von Hydrogenperoxid (H O )2 2
Carbonylierungsschäden aufweisen. Der Grad der Schädigung war weniger ausgeprägt, wenn Zellen v