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The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

ABSTRACT: As a result of its strong adaptation to wheat and rye sourdoughs, Lactobacillus sanfranciscensis has the smallest genome within the genus Lactobacillus. The concomitant absence of some important antioxidative enzymes and the inability to synthesize glutathione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1'-azobis(N,N-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initial concentration. In this study, RNA sequencing was used to reveal the transcriptional response of L. sanfranciscensis DSM 20451(T) (wild type [WT]) and its ?tcyB mutant with a nonfunctional cystine transporter after thiol stress caused by diamide. Along with the different expression of genes involved in amino acid starvation, pyrimidine synthesis, and energy production, our results show that thiol stress in the wild type can be compensated through activation of diverse chaperones and proteases whereas the ?tcyB mutant shifts its metabolism in the direction of survival. Only a small set of genes are significantly differentially expressed between the wild type and the mutant. In the WT, mainly genes which are associated with a heat shock response are upregulated whereas glutamine import and synthesis genes are downregulated. In the ?tcyB mutant, the whole opp operon was more highly expressed, as well as a protein which probably includes enzymes for methionine transport. The two proteins encoded by spxA and nrdH, which are involved in direct or indirect oxidative stress responses, are also upregulated in the mutant. This work emphasizes that even in the absence of definitive antioxidative enzymes, bacteria with a small genome and a high frequency of gene inactivation and elimination use small molecules such as the cysteine/cystine couple to overcome potential cell damage resulting from oxidative stress.

SUBMITTER: Stetina M

PROVIDER: S-EPMC4068661 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

Stetina Mandy M Behr Jürgen J Vogel Rudi F RF

Applied and environmental microbiology 20140502 14

As a result of its strong adaptation to wheat and rye sourdoughs, Lactobacillus sanfranciscensis has the smallest genome within the genus Lactobacillus. The concomitant absence of some important antioxidative enzymes and the inability to synthesize glutathione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1'-azobis(N,N-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initia ...[more]

PMID: 24795368

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Project description:The bacterium Bacillus subtilis is of high importance both as a model organism for Gram-positive bacteria and as an industrial workhorse in the production of biomolecules. In recent years, advancements have been made to engineer the bacterium even further toward industrial applications. In this study, we present a novel screening method for mutant libraries using diamide, an oxidizing agent that binds free thiols and creates disulfide bonds between them, thereby causing a so-called "disulfide stress" in bacteria. The method shows promise to selectively identify phenotypes in B. subtilis with improved tolerance toward oxidative and disulfide-associated stress. Phenotypes initially identified by transposon mutagenesis were recreated through targeted gene deletions. Among the resulting deletion mutants, the largest difference in diamide tolerance compared to the parental strain was observed for pfkA and ribT deletion strains. A proteomics analysis showed that diamide tolerance can be achieved through different routes involving increased expression of stress management proteins and reduced availability or activity of the RNA degradosome. We conclude that our screening method allows the facile identification of Bacillus strains with improved oxidative stress tolerance phenotypes. IMPORTANCE During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

Dataset Information

The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

Publications

The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

Similar Datasets

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