A Bumpy Pathway to Stationary-Phase Survival in Bacillus subtilis

ABSTRACT Bacillus subtilis yabE encodes a predicted resuscitation-promoting factor/stationary-phase survival (Rpf/Sps) family autolysin. Here, we demonstrate that yabE is negatively regulated by a cis-acting antisense RNA which, in turn, is regulated by two extracytoplasmic function σ factors: σX and σM.

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Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽

10.1128/mbio.00143-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Yanyu Zhu ◽

Mainak Mustafi ◽

James C. Weisshaar

Keyword(s):

Escherichia Coli ◽

Fluorescence Microscopy ◽

Rna Polymerase ◽

Stationary Phase ◽

Exponential Growth ◽

Mean Square Displacement ◽

Quiescent State ◽

Chromosomal Dna ◽

Content Type ◽

E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

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Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

mSphere ◽

10.1128/msphere.00464-20 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 1

Author(s):

Bhagyashree Swarge ◽

Chahida Nafid ◽

Norbert Vischer ◽

Gertjan Kramer ◽

Peter Setlow ◽

...

Keyword(s):

Protein Synthesis ◽

Bacillus Subtilis ◽

Fluorescence Intensity ◽

Spore Germination ◽

Malic Enzyme ◽

Present Report ◽

Quiescent State ◽

Content Type ◽

Gfp Fluorescence ◽

Spore Maturation

ABSTRACT Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore’s inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695–707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination. IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

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The Presence of Conjugative Plasmid pLS20 Affects Global Transcription of Its Bacillus subtilis Host and Confers Beneficial Stress Resistance to Cells

Applied and Environmental Microbiology ◽

10.1128/aem.03154-13 ◽

2013 ◽

Vol 80 (4) ◽

pp. 1349-1358 ◽

Cited By ~ 16

Author(s):

Thomas C. Rösch ◽

Wladislaw Golman ◽

Laura Hucklesby ◽

Jose E. Gonzalez-Pastor ◽

Peter L. Graumann

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Host Cell ◽

Stress Resistance ◽

Transcriptional Profiling ◽

Conjugative Plasmid ◽

Receptor Protein ◽

Content Type ◽

Phage Receptor ◽

In Cells

ABSTRACTConjugation activity of plasmid pLS20 fromBacillus subtilissubsp.nattois induced when cells are diluted into fresh medium and diminishes as cells enter into stationary-phase growth. Transcriptional profiling shows that during mid-exponential growth, more than 5% of the host genes are affected in the presence of the plasmid, in contrast to the minor changes seen in freshly diluted and stationary-phase cells. Changes occurred in many metabolic pathways, although pLS20 does not confer any detectable burden on its host cell, as well as in membrane and cell wall-associated processes, in the large motility operon, and in several other cellular processes. In agreement with these changes, we found considerable alterations in motility and enzyme activity and increased resistance against several different forms of stress in cells containing the plasmid, revealing that the presence of pLS20 has a broad impact on the physiology of its host cell and increases its stress resistance in multiple aspects. Additionally, we found that the lack of chromosomal geneyueB, known to encode a phage receptor protein, which is upregulated in cells containing pLS20, strongly reduced conjugation efficiency, revealing that pLS20 not only increases fitness of its host but also employs host proteins for efficient transfer into a new cell.

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Escherichia coli Has a Unique Transcriptional Program in Long-Term Stationary Phase Allowing Identification of Genes Important for Survival

mSystems ◽

10.1128/msystems.00364-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Karin E. Kram ◽

Autumn L. Henderson ◽

Steven E. Finkel

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Experimental Evolution ◽

Natural Environments ◽

Complex Environments ◽

Transcriptional Program ◽

Complex Environment ◽

Content Type ◽

Stressful Environment

ABSTRACT Microbes live in complex and constantly changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we began to unravel the cells’ responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold shock. By competing cells lacking these genes with wild-type cells, we show that these genes are also important for survival during LTSP. These data can help identify gene products that may play a role in survival in this complex environment and lead to identification of novel functions of proteins. IMPORTANCE Experimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses that cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study, we began to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more-natural conditions.

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Pseudomonas-Saccharomyces Interactions: Influence of Fungal Metabolism on Bacterial Physiology and Survival

Journal of Bacteriology ◽

10.1128/jb.187.3.940-948.2005 ◽

2005 ◽

Vol 187 (3) ◽

pp. 940-948 ◽

Cited By ~ 39

Author(s):

Julia D. Romano ◽

Roberto Kolter

Keyword(s):

Stationary Phase ◽

Molecular Basis ◽

Synthetic Medium ◽

Grape Juice ◽

Natural Environments ◽

Bacterial Physiology ◽

Beneficial Effects ◽

Stationary Phase Survival ◽

Yeast Mutants ◽

Deletion Library

ABSTRACT Fungal-bacterial interactions are ubiquitous, yet their molecular basis is only poorly understood. In this study, a novel beneficial interaction between a strain of Pseudomonas putida and the fungus Saccharomyces cerevisiae was identified. When the bacteria were incubated alone in grape juice or in synthetic medium containing various concentrations of glucose, they lost viability rapidly during stationary phase. However, when the bacteria were incubated in these media in the presence of the fungus, their stationary phase survival improved dramatically. On agar plates containing glucose, the beneficial effects of the fungus were manifested in robust bacterial growth and exopolysaccharide production that led to visible mucoidy. In contrast, bacteria grew poorly and were nonmucoid in such media in the absence of the fungus. By using the available S. cerevisiae deletion library, yeast mutants that were unable to mediate this beneficial interaction were identified. These mutants revealed that the beneficial effect on bacterial physiology and survival was mediated by the ability of the fungus to metabolize the available glucose and consequent effects on the medium's pH. In natural environments where the concentration of glucose is high, it is likely that the presence of fungi has had profound beneficial effects on the physiology and survival of certain P. putida strains throughout their natural history.

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Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis

Journal of Bacteriology ◽

10.1128/jb.00807-19 ◽

2020 ◽

Vol 202 (9) ◽

Author(s):

Hilda C. Leyva-Sánchez ◽

Norberto Villegas-Negrete ◽

Karen Abundiz-Yañez ◽

Ronald E. Yasbin ◽

Eduardo A. Robleto ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Excision Repair ◽

Dna Lesions ◽

Ap Endonuclease ◽

Content Type ◽

Error Prone Repair ◽

Base Excision ◽

Oxidized Guanine

ABSTRACT We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in Bacillus subtilis YB955 (hisC952 metB5 leuC427). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed B. subtilis YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, mfd and greA were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of mfd or greA abrogated the production of stress-associated amino acid revertants in the GO and nfo exoA nth strains, respectively. These results suggest that in nutritionally stressed B. subtilis cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase ΔytkD ΔmutM ΔmutY (referred to here as ΔGO) and Δnfo ΔexoA Δnth (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of mfd or greA increased the production of His+, Met+, or Leu+ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of B. subtilis. IMPORTANCE In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed B. subtilis strain YB955 (hisC952 metB5 leuC427) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing B. subtilis to escape from growth-limiting conditions.

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Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Malaysian Journal of Microbiology ◽

10.21161/mjm.1461801 ◽

2018 ◽

Author(s):

Quay, D. H. X. ◽

Qureshi, A. ◽

Bhakta, S. ◽

Keep, N. H.

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Gene Encoding ◽

Stationary Phase Survival

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A Decrease in Serine Levels during Growth Transition Triggers Biofilm Formation inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00155-19 ◽

2019 ◽

Vol 201 (15) ◽

Cited By ~ 1

Author(s):

Jennifer Greenwich ◽

Alicyn Reverdy ◽

Kevin Gozzi ◽

Grace Di Cecco ◽

Tommy Tashjian ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Biofilm Formation ◽

Exponential Phase ◽

Growth Stages ◽

Content Type ◽

Biofilm Development ◽

Ribosome Pausing ◽

Trna Isoacceptors ◽

Serine Codons

ABSTRACTBiofilm development inBacillus subtilisis regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate ofsinR, which encodes a master repressor for biofilm matrix genes and ultimately triggers biofilm induction. How serine levels change in different growth stages, howB. subtilisregulates intracellular serine levels, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here, we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. The deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared with exponential phase. However, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development inB. subtilis.IMPORTANCEInBacillus subtilis, biofilm formation is triggered in response to environmental and cellular signals. It was proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we reveal that serine levels decrease at the biofilm entry due to catabolite control and a serine shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared with exponential phase; three isoacceptors recognizing UCN serine codons are reduced much more than the two recognizing AGC and AGU codons. This finding indicates a possible mechanism for selective ribosome pausing.

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Adaptive, or Stationary-Phase, Mutagenesis, a Component of Bacterial Differentiation in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.184.20.5641-5653.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5641-5653 ◽

Cited By ~ 64

Author(s):

Huang-Mo Sung ◽

Ronald E. Yasbin

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Ecological Niche ◽

Reca Protein ◽

Exponential Phase ◽

The Other ◽

Positive Bacterium ◽

Bacterial Populations ◽

Bacterial Differentiation ◽

Small Subpopulation

ABSTRACT Adaptive (stationary-phase) mutagenesis occurs in the gram-positive bacterium Bacillus subtilis. Furthermore, taking advantage of B. subtilis as a paradigm for the study of prokaryotic differentiation and development, we have shown that this type of mutagenesis is subject to regulation involving at least two of the genes that are involved in the regulation of post-exponential phase prokaryotic differentiation, i.e., comA and comK. On the other hand, a functional RecA protein was not required for this type of mutagenesis. The results seem to suggest that a small subpopulation(s) of the culture is involved in adaptive mutagenesis and that this subpopulation(s) is hypermutable. The existence of such a hypermutable subpopulation(s) raises important considerations with respect to evolution, the development of specific mutations, the nature of bacterial populations, and the level of communication among bacteria in an ecological niche.

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