scholarly journals Decreasing Serine Levels During Growth Transition Triggers Biofilm Formation inBacillus subtilis

2019 ◽  
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 ◽  
Translation Rate ◽  
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 recently 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 biofilm induction. How serine levels change in different growth stages, howB. subtilisregulates intracellular serine levels in response to metabolic status, 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. 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 to exponential phase. Interestingly, 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 various environmental and cellular signals. It was previously 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 revealed that serine levels decrease at the biofilm entry due to catabolite control and a shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared to exponential phase; three isoacceptors recognizing UCN serine codons are reduced much greater than the two recognizing AGC and AGU codons. This indicates a possible mechanism for selective ribosome pausing.

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

2019 ◽  
Vol 201 (15) ◽  
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.

Abstract 356: Adhesion of Staphylococcus Epidermidis to Fibrinogen Alters Clot Formation Kinetics and Ultimate Stiffness

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Carolyn Vitale ◽  
Tianhui Ma ◽  
Michael J Solomon ◽  
J. Scott VanEpps
Keyword(s):  
Stationary Phase ◽  
Deposition Rate ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Fibrin Clot ◽  
Exponential Phase ◽  
Automated Image Analysis ◽  
Clot Formation ◽  
Coagulation Proteins

Bacterial infection is known to increase the risk for thromboembolism. The mechanism underlying this correlation remains largely unknown. We recently showed that the common pathogen Staphylococcus epidermidis retards clot formation, increases clot elasticity and generates a heterogeneous clot structure that remodels over time. Here, we elucidate the mechanism of this process by evaluating the capacity for S. epidermidis to bind to fibrinogen as a function of its growth phase. We hypothesized that the effect of S. epidermidis on a fibrin clot is related to its propensity toward biofilm formation. Therefore, stationary phase (biofilm-like) S. epidermidis will have a more robust effect on clot kinetics and elasticity than exponential phase (planktonic). Furthermore, this difference is mediated by increased adhesion to fibrinogen. Rheometry was used to evaluate the formation and resultant elasticity of fibrin clots with exponential or stationary phase S. epidermidis . A functional in vitro model was developed to evaluate adhesion of S. epidermidis to a fibrinogen coated surface in a continuously flowing environment. Fluorescent labeled exponential and stationary phase S. epidermidis were visualized flowing through a parallel plate microfluidic chamber past immobilized fibrinogen. Images were obtained every 3 seconds for 30 min. Bacterial deposition rate and mean adhesion time were quantified by automated image analysis. A paired Student’s t-test was used for statistical analysis. Stationary phase S. epidermidis retards clot formation and increases resultant elasticity while exponential phase only slightly reduces elasticity. The bacterial deposition rate onto fibrinogen was significantly (p=0.03) greater for stationary phase (1741 ± 1513 cells/cm 2 · sec -1 ) vs exponential phase (676 ± 270 cells/cm 2 · sec -1 ). The average adhesion time however was similar for exponential and stationary phase cells. Coagulation proteins can provide a framework for bacterial adhesion, biofilm formation and infection. In turn infected thrombi with (biofilm-like) bacteria are stiffer which correlates to more frequent bacterial binding to fibrinogen. This provides a potential molecular mechanism for infection mediated thromboembolic events.

scholarly journals Effect of Escherichia coli Morphogene bolA on Biofilms

2004 ◽  
Vol 70 (9) ◽  
pp. 5682-5684 ◽  
Author(s):  
Helena L. A. Vieira ◽  
Patrick Freire ◽  
Cecília M. Arraiano
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Stationary Phase ◽  
Biofilm Formation ◽  
Stress Conditions ◽  
Biofilm Development

ABSTRACT Biofilm physiology is established under a low growth rate. The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development. In order to verify this hypothesis, we tested the effect of bolA on biofilm formation. Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.

scholarly journals An Autoregulatory Circuit Affecting Peptide Signaling in Bacillus subtilis

1999 ◽  
Vol 181 (17) ◽  
pp. 5193-5200 ◽  
Author(s):  
Beth A. Lazazzera ◽  
Iren G. Kurtser ◽  
Ryan S. McQuade ◽  
Alan D. Grossman
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Exponential Phase ◽  
Peptide Signaling ◽  
Expression Of Genes ◽  
Low Concentrations ◽  
Competence And Sporulation Factor

ABSTRACT The competence and sporulation factor (CSF) of Bacillus subtilis is an extracellular pentapeptide produced from the product of phrC. CSF has at least three activities: (i) at low concentrations, it stimulates expression of genes activated by the transcription factor ComA; at higher concentrations, it (ii) inhibits expression of those same genes and (iii) stimulates sporulation. Because the activities of CSF are concentration dependent, we measured the amount of extracellular CSF produced by cells. We found that by mid-exponential phase, CSF accumulated to concentrations (1 to 5 nM) that stimulate ComA-dependent gene expression. Upon entry into stationary phase, CSF reached 50 to 100 nM, concentrations that stimulate sporulation and inhibit ComA-dependent gene expression. Transcription of phrC was found to be controlled by two promoters: P1, which precedes rapC, the gene upstream ofphrC; and P2, which directs transcription ofphrC only. Both RapC and CSF were found to be part of autoregulatory loops that affect transcription from P1, which we show is activated by ComA∼P. RapC negatively regulates its own expression, presumably due to its ability to inhibit accumulation of ComA∼P. CSF positively regulates its own expression, presumably due to its ability to inhibit RapC activity. Transcription from P2, which is controlled by the alternate sigma factor ςH, increased as cells entered stationary phase, contributing to the increase in extracellular CSF at this time. In addition to controlling transcription ofphrC, ςH appears to control expression of at least one other gene required for production of CSF.

scholarly journals Protein Acetylation and Butyrylation Regulate the Phenotype and Metabolic Shifts of the Endospore-forming Clostridium acetobutylicum

2018 ◽  
Vol 17 (6) ◽  
pp. 1156-1169 ◽  
Author(s):  
Jun-Yu Xu ◽  
Zhen Xu ◽  
XinXin Liu ◽  
Minjia Tan ◽  
Bang-Ce Ye
Keyword(s):  
Stationary Phase ◽  
Clostridium Acetobutylicum ◽  
Dynamic Change ◽  
High Energy ◽  
Exponential Phase ◽  
Growth Stages ◽  
Lysine Acetylation ◽  
Functional Studies ◽  
Two Phases ◽  
Transitional Phase

Clostridium acetobutylicum is a strict anaerobic, endospore-forming bacterium, which is used for the production of the high energy biofuel butanol in metabolic engineering. The life cycle of C. acetobutylicum can be divided into two phases, with acetic and butyric acids being produced in the exponential phase (acidogenesis) and butanol formed in the stationary phase (solventogenesis). During the transitional phase from acidogenesis to solventogenesis and latter stationary phase, concentration peaks of the metabolic intermediates butyryl phosphate and acetyl phosphate are observed. As an acyl group donor, acyl-phosphate chemically acylates protein substrates. However, the regulatory mechanism of lysine acetylation and butyrylation involved in the phenotype and solventogenesis of C. acetobutylicum remains unknown. In our study, we conducted quantitative analysis of protein acetylome and butyrylome to explore the dynamic change of lysine acetylation and butyrylation in the exponential phase, transitional phase, and stationary phase of C. acetobutylicum. Total 458 lysine acetylation sites and 1078 lysine butyrylation sites were identified in 254 and 373 substrates, respectively. Bioinformatics analysis uncovered the similarities and differences between the two acylation modifications in C. acetobutylicum. Mutation analysis of butyrate kinase and the central transcriptional factor Spo0A was performed to characterize the unique role of lysine butyrylation in the metabolic pathway and sporulation process of C. acetobutylicum. Moreover, quantitative proteomic assays were performed to reveal the relationship between protein features (e.g. gene expression level and lysine acylation level) and metabolites in the three growth stages. This study expanded our knowledge of lysine acetylation and butyrylation in Clostridia and constituted a resource for functional studies on lysine acylation in bacteria.

scholarly journals Translocation of mycobacillin synthetase in Bacillus subtilis

1985 ◽  
Vol 225 (3) ◽  
pp. 639-643 ◽  
Author(s):  
N K Mukhopadhyay ◽  
S K Ghosh ◽  
S Majumder ◽  
S K Bose
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Exponential Phase ◽  
High Rate ◽  
Synthetase Activity ◽  
Late Exponential Phase ◽  
Extracellular Release ◽  
Particulate Form ◽  
Membrane Bound

The extracellular release of mycobacillin from Bacillus subtilis first occurred in the medium at the onset of stationary phase and continued at a high rate even after 6 days. Mycobacillin synthetase activity appeared earlier than late-exponential phase in the cytosol of producer cells and was not sedimentable even at 105 000 g. The activity then quickly reached the maximum late in the stationary phase. With further increase in the age of the culture, the activity gradually disappeared from the cytosol, to reappear concomitantly in the membrane in an insoluble particulate form, even in absence of protein synthesis. The membrane-bound synthetase activity was sedimentable at 10 000 g and was fairly active even after 5 days.

scholarly journals Quorum sensing in Bacillus subtilis slows down biofilm formation by enabling sporulation bet hedging

2019 ◽  
Author(s):  
Mihael Spacapan ◽  
Tjaša Danevčič ◽  
Polonca Štefanic ◽  
Ines Mandic-Mulec
Keyword(s):  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Bet Hedging ◽  
Wild Type ◽  
Master Regulator ◽  
Matrix Components ◽  
Sporulation Initiation ◽  
Biofilm Development ◽  
Biofilm Matrix

1.2ABSTRACTThe ComQXPA quorum sensing (QS) system of Bacillus subtilis, a Gram-positive, industrially relevant, endospore forming bacterium, promotes surfactin production. This lipopeptide increases transcription of several genes involved in biofilm matrix synthesis via the Spo0A-P master regulator. We hypothesized that the inactivation of the QS system will therefore result in decreased rates of floating biofilm formation. We find that this is not the case and that the QS deficient mutant forms pellicles with a faster rate and produces more biofilm matrix components than the wild type. As Spo0A-P is the master regulator of sporulation initiation we hypothesized that the ComQXPA dependent signaling promotes sporulation and consequently slows the growth rate of the wild type strain. Indeed, our results confirm that cells with the inactive QS initiate endospore formation in biofilms later and more synchronously than the wild type, as evidenced by spore frequencies and the PspoIIQ promoter activity. We argue, that the QS system acts as a switch that promotes stochastic sporulation initiation and consequently bet hedging behavior. By committing a subpopulation of cells to sporulation early during growth, wild type population grows slower and produces thinner biofilms but also assures better survival under stressful conditions.1.1IMPORTANCEBacillus subtilis is widely employed model organism to study biofilm formation and sporulation in Gram-positive bacteria. The ComQXPA quorum sensing (QS) system indirectly increases the transcription of genes involved in biofilm matrix formation, which predicts a positive role of this QS in biofilm development Here we show that QS mutants actually form more matrix components per pellicle than the wild type and that their pellicles are thicker and form with a faster rate. We explain this, by showing that cells with an inactive QS exhibit a delay in sporulation entry, which is also more synchronous relative to the wild type. We argue, that the ComQXPA QS system acts as a switch that contributes to the stochastic sporulation initiation and though this path promotes bet hedging behavior. This finding is important in terms of “quorum quenching” strategies aiming to down modulate biofilm development through inhibition of QS signaling and underscores the richness of QS regulated phenotypic outcomes among bacterial species.

scholarly journals A serine sensor for multicellularity in a bacterium

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Arvind R Subramaniam ◽  
Aaron DeLoughery ◽  
Niels Bradshaw ◽  
Yun Chen ◽  
Erin O’Shea ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Exponential Phase ◽  
Ribosome Profiling ◽  
Phase Growth ◽  
Translation Speed ◽  
Genome Wide ◽  
Ribosome Density ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus ◽  
Serine Codons

We report the discovery of a simple environmental sensing mechanism for biofilm formation in the bacterium Bacillus subtilis that operates without the involvement of a dedicated RNA or protein. Certain serine codons, the four TCN codons, in the gene for the biofilm repressor SinR caused a lowering of SinR levels under biofilm-inducing conditions. Synonymous substitutions of these TCN codons with AGC or AGT impaired biofilm formation and gene expression. Conversely, switching AGC or AGT to TCN codons upregulated biofilm formation. Genome-wide ribosome profiling showed that ribosome density was higher at UCN codons than at AGC or AGU during biofilm formation. Serine starvation recapitulated the effect of biofilm-inducing conditions on ribosome occupancy and SinR production. As serine is one of the first amino acids to be exhausted at the end of exponential phase growth, reduced translation speed at serine codons may be exploited by other microbes in adapting to stationary phase.

scholarly journals Ploidy of Bacillus subtilis, Bacillus megaterium, and three new isolates of Bacillus and Paenibacillus

2014 ◽  
Author(s):  
Benjamin Böttinger ◽  
Karolin Zerulla ◽  
Jörg Soppa
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Bacillus Megaterium ◽  
Ploidy Level ◽  
Methanogenic Archaea ◽  
Exponential Phase ◽  
Phylogenetic Groups ◽  
Circular Chromosome ◽  
Additional Group ◽  
Positive Genus

Bacteria were long assumed to be monoploid, maintaining one copy of a circular chromosome. In recent years it became obvious that the majority of species in several phylogenetic groups of prokaryotes are oligoploid or polyploid, e.g. in halophilic and methanogenic archaea, proteobacteria, and cyanobacteria. The present study aimed at investigating the distribution of ploidy in an additional group of prokaryotes, i.e. in the gram-positive genus Bacillus. First, the numbers of origins and termini of the two laboratory strains Bacillus subtilis and Bacillus megaterium were quantified using an optimized real time PCR approach. B. subtilis was found to be mero-oligoploid in exponential phase with, on average, 5.9 origins and 1.2 termini. In stationary phase the average numbers of origins per cell was considerably smaller. B. megaterium was found to be polyploid in exponential phase with about 12 copies of the origin and terminus. Again, the ploidy level was down-regulated in stationary phase. To verify that oligo-/polyploidy is not confined to strains with a long history of growth in the laboratory, three strains were newly isolated from soil, which were found to belong to the genera of Bacillus and Paenibacillus. All three strains were found to be oligoploid with a growth-phase dependent down-regulation of the ploidy level in stationary phase. Taken together, these results indicate that oligo-/polyploidy might be more widespread in Bacillus and related genera than assumed until now and that monoploidy is not typical.

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