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Diamide-based screening method for isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries

ABSTRACT: The bacterium Bacillus subtilis is of high importance both as a model organism for Gram-positive bacteria and as an industrial work horse in the production of biomolecules. In recent years, advancements have been made to engineer the bacterium even further towards industrial applications. In this study, we present a novel screening method for mutant libraries using diamide, an oxidising 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 towards 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 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

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Bacillus Subtilis Subsp. Subtilis

SUBMITTER: Sandra Maass

LAB HEAD: Dörte Becher

PROVIDER: PXD041512 | Pride | 2023-09-25

REPOSITORIES: Pride

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Project description:Genome wide transcriptional changes induced by various types of oxidative stresses as well as salt stress were studied in a DatfA mutant and the appropriate control A. nidulans strains. Although a significant number of stereotypically regulated genes was identified (Core Oxidative Stress Response or COSR genes) when the global transcriptional effects of five different oxidative stress conditions were compared the number of co-regulated genes decreased to 13 when NaCl stress was included into the analyses. The appearance of only a few co-regulated genes and the great number of genes regulated merely by one certain type of stress do not support the existence of a S. cerevisiae-type Environmental Stress Response in A. nidulans. Deletion of atfA, a true functional ortholog of fission yeastâs âall-purposeâ stress response transcription factor, increased the oxidative stress sensitivity of A. nidulans and affected the transcription of several genes under both unstressed and stressed conditions. The number of genes under AtfA control was quite stress-type dependent; e.g. deletion of atfA altered the transcription of a wide spectrum of genes under menadione sodium bisulfite stress but had only a minor effect on the transcriptome profiles when A. nidulans cultures were exposed to H2O2, tBOOH, NaCl and, especially, to diamide stress. These observations suggest that the function of AtfA in the regulation of various stress responses is much smaller than we thought before or other transcription factors can take over a number of AtfAâs functions when the atfA gene is deleted. It is noteworthy that both oxidative and salt stress induced the transcription of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of further clusters. Surprisingly, certain clusters were down-regulated by the stress conditions tested and the majority of them were not stress-responsive at all. Therefore, stress dependent regulation seems to be a frequent but far not a general feature of the regulation of secondary metabolism in A. nidulans. 14 samples, 7 with the control strain and 7 with an DatfA strain (each series contains samples from untreated as well as menadione, low concentration hidrogen-peroxide, high concentration hidrogen-peroxide, tert-butylhydroperoxide, diamide and NaCl treated cultures)

Project description:Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 deletion mutants, on induction/repression dynamics of 200 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We follow up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during diamide stress, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and show that the M-bM-^@M-^\activatingM-bM-^@M-^] mark H3K4me3 functions largely as a repressor in yeast. We set to track how histone modifications change during transcriptional reprogramming. We perfomed a time course experiment following treatment of mid-log growing yeast (BY4741) with diamide. Samples were collected at t=0,4,8,15,30,60 and fixed by crosslinking. We extracted mononucleosomes by MNase digestion and then applied ChIP for selected histone modifications. The IPed material was hybridized to genomic tiling arrays against the input material.

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Diamide-based screening method for isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries

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