Poly-gamma-glutamic acid secretion protects Bacillus subtilis from zinc and copper intoxication

AbstractZinc and copper are essential micronutrients that serve as a cofactors for numerous enzymes. However, when present at elevated concentrations, zinc and copper are highly toxic to bacteria. To combat the effects of zinc and copper excess, bacteria have evolved a wide array of defense mechanisms. Here, we show that the Gram positive soil bacterium, Bacillus subtilis, produces the extracellular polymeric substance, poly-gamma-glutamate (γ-PGA) as a protective mechanism in response to zinc and copper excess. Furthermore, we provide evidence that zinc and copper dependent γ-PGA production is independent of the DegS-DegQ two component regulatory system and likely occurs at a post-transcriptional level. These data provide new insight into bacterial metal resistance mechanisms and contribute to our understanding of the regulation of bacterial γ-PGA biosynthesis.ImportanceZinc and copper are potent antimicrobial compounds. As such, bacteria have evolved a diverse range of tools to prevent metal intoxication. Here, we show that the Gram-positive model organism, Bacillus subtilis, produces poly-gamma-glutamic acid (γ-PGA) as a protective mechanism against zinc and copper intoxication and that zinc and copper dependent γ-PGA production occurs by a yet undefined mechanism independent of known γ-PGA regulation pathways.

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Transcriptional Control of the Sulfur-RegulatedcysH Operon, Containing Genes Involved inl-Cysteine Biosynthesis in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.182.20.5885-5892.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5885-5892 ◽

Cited By ~ 31

Author(s):

Maria Cecilia Mansilla ◽

Daniela Albanesi ◽

Diego de Mendoza

Keyword(s):

Bacillus Subtilis ◽

Transcription Initiation ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Sulfur Metabolism ◽

Transcriptional Level ◽

Cysteine Biosynthesis ◽

Gram Positive ◽

Sulfur Starvation ◽

High Level

ABSTRACT The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria. In this study we describe the expression pattern of the B. subtilis cysHoperon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the cysH, cysP,ylnB, ylnC, ylnD, ylnE, and ylnF genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. ThecysH operon contains a 5′ leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of B. subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the −35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that O-acetyl-l-serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the cysHoperon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.

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Biofilm hydrophobicity in environmental isolates of Bacillus subtilis

Microbiology ◽

10.1099/mic.0.001082 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Margarita Kalamara ◽

James C. Abbott ◽

Cait E. MacPhee ◽

Nicola R. Stanley-Wall

Keyword(s):

Extracellular Matrix ◽

Bacillus Subtilis ◽

Glutamic Acid ◽

Biofilm Formation ◽

Type Species ◽

Diverse Range ◽

Content Type ◽

Link Type ◽

The Matrix ◽

Highly Hydrophobic

Biofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our novel isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason for the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.

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Biofilm hydrophobicity in environmental isolates of Bacillus subtilis

10.1101/2021.04.29.441976 ◽

2021 ◽

Author(s):

Margarita Kalamara ◽

James C. Abbott ◽

Cait E. MacPhee ◽

Nicola. R. Stanley-Wall

Keyword(s):

Extracellular Matrix ◽

Bacillus Subtilis ◽

Glutamic Acid ◽

Biofilm Formation ◽

Environmental Isolates ◽

Diverse Range ◽

European Nucleotide Archive ◽

Universal Feature ◽

The Matrix ◽

Highly Hydrophobic

AbstractBiofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our new isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason behind the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.RepositoriesRaw sequence reads and annotated assemblies have been submitted to the European Nucleotide Archive under accession PRJEB43128.

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CONFIGURATION OF THE CELLULAR GLUTAMIC ACID OF BACILLUS SUBTILIS

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)50980-7 ◽

1951 ◽

Vol 191 (1) ◽

pp. 305-307

Author(s):

Lloyd T. Jenkins ◽

Leon S. Ciereszko

Keyword(s):

Bacillus Subtilis ◽

Glutamic Acid

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High-level exogenous glutamic acid-independent production of poly-(γ-glutamic acid) with organic acid addition in a new isolated Bacillus subtilis C10

Bioresource Technology ◽

10.1016/j.biortech.2011.11.085 ◽

2012 ◽

Vol 116 ◽

pp. 241-246 ◽

Cited By ~ 40

Author(s):

Huili Zhang ◽

Jianzhong Zhu ◽

Xiangcheng Zhu ◽

Jin Cai ◽

Anyi Zhang ◽

...

Keyword(s):

Bacillus Subtilis ◽

Glutamic Acid ◽

Organic Acid ◽

Acid Addition ◽

High Level

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Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

Nature Communications ◽

10.1038/s41467-019-13408-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Naomi Shimokawa-Chiba ◽

Claudia Müller ◽

Keigo Fujiwara ◽

Bertrand Beckert ◽

Koreaki Ito ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stop Codon ◽

Release Factor ◽

Positive Bacterium ◽

Gram Positive ◽

E Coli ◽

Cryo Electron Microscopy ◽

Dead End ◽

Bacterium Bacillus Subtilis ◽

Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

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Inducible Transport of Citrate in a Gram-positive Bacterium, Bacillus subtilis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)62427-5 ◽

1971 ◽

Vol 246 (4) ◽

pp. 1032-1040

Author(s):

Klaus Willecke ◽

Arthur B. Pardee

Keyword(s):

Bacillus Subtilis ◽

Positive Bacterium ◽

Gram Positive ◽

Bacterium Bacillus Subtilis ◽

Bacterium Bacillus

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Physiology of D-Glutamic Acid-requiring Mutants of Bacillus subtilis

Nature ◽

10.1038/186818a0 ◽

1960 ◽

Vol 186 (4727) ◽

pp. 818-818

Author(s):

HARUO MOMOSE ◽

YÔNOSUKE IKEDA

Keyword(s):

Bacillus Subtilis ◽

Glutamic Acid

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Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽

10.1128/msphere.00655-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Robert S. Brzozowski ◽

Brooke R. Tomlinson ◽

Michael D. Sacco ◽

Judy J. Chen ◽

Anika N. Ali ◽

...

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Cell Size ◽

Unknown Function ◽

Suppressor Mutation ◽

Gram Positive ◽

Content Type ◽

Suppressor Mutations ◽

Extragenic Suppressor ◽

Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

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