Quantitative analysis of asynchronous transcription-translation and transcription processivity in Bacillus subtilis under various growth conditions.
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ABSTRACT:
SUBMITTER: Zhu M
PROVIDER: S-EPMC8586808 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.
| S-EPMC7483943 | biostudies-literature
Project description:The general assumption is that when bacteria run out of nutrients they become dormant or form spores. Here we show, using a new technique, that under deep starvation conditions non-sporulating Bacillus subtilis cells do not become dormant but continue to grow. B. subtilis can form (endo)spores and this has been regarded as the principal mechanism through which it survives long periods of nutrient depletion. However, in this study we demonstrate that non-sporulating B. subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics and oxidative stress. Interestingly, these cells appeared to be metabolically active, and transcriptome analyses indicated that their gene-expression profile differs substantially from both stationary phase cells, and exponentially growing cells. Surprisingly, using an inhibitor for cell division, we discovered that these coccoid-like B. subtilis cells are not dormant but actually grow and divide, albeit with a doubling time of ~4 days. It emerged that secreted proteases, allowing acquisition of nutrients from lysed brethren, are essential for this growth mode. In fact, nutrient levels comparable to 10,000 times diluted LB (Lysogeny broth) appeared to be sufficient to sustain this growth. The very slow growth provides an alternative strategy for B. subtilis to survive nutrient depletion and environmental stresses. We propose to call this the oligotrophic growth state. This state might be common among bacterial species to survive deep starvation conditions.
2018-08-01 | GSE102140 | GEO
Project description:Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427, under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu(+) reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.
| S-EPMC2897666 | biostudies-literature
Project description:The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis' intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis' chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable ?-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein ?-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis' metabolism and will facilitate future work to develop this organism for synthetic biology.
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Project description:In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified.We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His6-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not.Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.
| S-EPMC5080769 | biostudies-literature
Project description:The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.
| S-EPMC10024249 | biostudies-literature
Project description:Bacillus subtilis’ sigma factor F is the first forespore specific transcription factor and controls genes required for the early stages of prespore development. The role of sigF is well studied under conditions that induce sporulation. Here, the impact of a sigF disruption on the transcriptome of exponentially growing cultures is studied by micro-array analysis. This is relevant because in various experiments, such as industrial fermentation, prolonged experimental evolution or zero-growth studies, sporulation is an undesirable trait that should be avoided, e.g by a sigF mutation. Under conditions that typically don’t induce sporulation, the transcriptome showed minor signs of sporulation initiation. The number of genes differentially expressed and the magnitude of expression were, as expected, quite small in comparison with sporulation conditions. The genes mildly down-regulated were mostly involved in anabolism and the genes mildly up-regulated were mostly involved in catabolism, in particular fatty acid degradation genes. Probably this is related to the arrest at sporulation stage II occurring in the sigF mutant where additional energy is required for further growth of the polar compartments formed.
2015-09-30 | GSE64918 | GEO
Project description:Using biological control agents (BCAs) is an essential component of integrated pest and diseases management. Despite much research on biocontrol of plant diseases, success in field crops has been limited with most successes being achieved in greenhouse cultivation. This lack of success is often attributed to the complex ecological processes involved in biocontrol. We used next generation sequencing (NGS) technology to study environmental fate of Bacillus subtilis, a widely used BCA, focusing on its dispersal aspect in open field and under protection. The dispersal of B. subtilis was very limited, particularly under protection. The reduction in the BCA population size was relatively small within 8 days; indeed, no overall reduction in the relative abundance was observed under the protected condition. These results suggested that limited dispersal is probably the main reason for its variable (and often low) control efficacy under field conditions. Thus to increase biocontrol efficacy, it is necessary to frequently apply this BCA with the application interval depending on the growth rate of target host tissues. Phyllosphere microbiota differed significantly between plants grown in open field and under protection but were not greatly affected by the introduced BCA.
| S-EPMC4776175 | biostudies-literature
Project description:Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l-valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l-glutamate supply. The synthesis of one mole of l-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l-glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l-valine synthesis under oxygen deprivation may be due to solving these problems. Increased l-valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l-valine production in E. coli under microaerobic conditions. We have created l-valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 ?ilvBN ?ilvIH ?ilvGME::PL -ilvBN N17K DA) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l-valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l-valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l-valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE-possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l-valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l-valine by means of "valine fermentation" was shown in E. coli. This methodology has the advantages of being an economical and environmentally friendly process.
| S-EPMC6449708 | biostudies-literature
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes.
2015-01-26 | GSE65272 | GEO