New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. The Comparison of Proteomic Responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli. Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. coli enzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.

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RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase—α, β, β′. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β’ subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

Bacillus subtilis develops genetic competence for the uptake of foreign DNA when cells enter stationary phase and a high cell density is reached. These signals are integrated by the competence transcription factor ComK, which is subject to transcriptional, post-transcriptional and post-translational regulation. Many proteins are involved in the development of competence, both to control ComK activity and to mediate DNA uptake. However, for many proteins, the precise function they play in competence development is unknown. In this study, we assessed whether proteins required for genetic transformation play a role in the activation of ComK or rather act downstream of competence gene expression. While these possibilities could be distinguished for most of the tested factors, we assume that two proteins, PNPase and the transcription factor YtrA, are required both for full ComK activity and for the downstream processes of DNA uptake and integration. Further analyses of the role of the transcription factor YtrA for the competence development revealed that the overexpression of the YtrBCDEF ABC transporter in the ytrA mutant causes the loss of genetic competence. Moreover, overexpression of this ABC transporter also affects biofilm formation. Since the ytrGABCDEF operon is naturally induced by cell wall-targeting antibiotics, we tested the cell wall properties upon overexpression of the ABC transporter and observed an increased thickness of the cell wall. The composition and properties of the cell wall are important for competence development and biofilm formation, suggesting that the observed phenotypes are the result of the increased cell wall thickness as an outcome of YtrBCDEF overexpression.

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase -α, β, β′. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed massive decreases in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.If the continued activity of a protein may become harmful to the bacteria, it is important not only to prevent expression of the corresponding gene but also to take two important measures: (i) switch off the protein's activity and (ii) degrade the mRNA to exclude further production of the protein. The inactivation or even degradation of proteins is well documented in the model bacteria. For example, in both E. coli and B. subtilis the uptake of toxic ammonium is limited by a regulatory interaction of the ammonium transporter with GlnK, a regulatory protein of the PII family (1,2). Similarly, the uptake of potentially toxic potassium can be prevented by inhibition of potassium transporters at high environmental potassium concentrations, either by the second messenger cyclic di-AMP or by interaction with a dedicated modified signal transduction protein, PtsN (3,4,5). To prevent the accumulation of potentially harmful mRNAs, bacteria rely on a very fast mRNA turnover. Indeed, in E. coli and B. subtilis more than 80% of all transcripts have average half-lives of less than 8 minutes, as compared to about 30 minutes and 10 hours in yeast or human cells, respectively (6,7,8,9). Thus, the mRNA turnover is much faster than the generation time. The high mRNA turnover rate in bacteria contributes to the fast adaptation even in rapidly growing cells. The rapid mRNA turnover is therefore a major factor to resolve the apparent growth speed-adaptation trade-off. 4RNases are the key elements to achieve the rapid mRNA turnover in bacteria. Theses enzymes can degrade bulk mRNA in a rather unspecific manner, just depending on the accessibility of the RNA molecules as well as perform highly specific cleavages that serve to process an RNA molecule to its mature form. In all organisms, RNA degradation involves an interplay ...

The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA supercoiling in exponential phase stimulates transcription from rRNA promoters, and DNA relaxation in stationary phase contributes to cessation of their activity. Novobiocin treatment of B. subtilis cells relaxes DNA and decreases rRNA promoter activity despite an increase in the GTP level, a known positive regulator of B. subtilis rRNA promoters. Comparative analyses of steps during transcription initiation then reveal differences between rRNA promoters and a control promoter, Pveg, whose activity is less affected by changes in supercoiling. Additional data then show that DNA relaxation decreases transcription also from promoters dependent on alternative sigma factors σB, σD, σE, σF, and σH with the exception of σN where the trend is the opposite. To summarize, this study identifies DNA topology as a factor important (i) for the expression of rRNA in B. subtilis in response to nutrient availability in the environment, and (ii) for transcription activities of B. subtilis RNAP holoenzymes containing alternative sigma factors.

Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. Identification of the late ISC interactome revealed unexpected involvement of the aerobic marker protein BolA and specific interaction of IscA with the outer mitosomal membrane. Although we confirmed that the synthesis of the Fe-S cluster remained the only metabolic role of mitosomes, we also showed that mitosomes lack client proteins that require the [4Fe-4S] cluster. Instead, by knocking out the bolA gene from the G. intestinalis genome, we showed that, unlike aerobic mitochondria, the late ISC mitosomal pathway is involved in the assembly of cytosolic [4Fe-4S] clusters. Thus, this work reveals an unexpected link between the formation of mitochondrial and cytosolic [4Fe- 4S] clusters. This may either be a consequence of mitochondrial adaptation to life without oxygen, or it represents a general metabolic coupling that has not been previously observed in the complex mitochondrial metabolism of aerobes.