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Microbial Cell Factories Jul 2017Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However,... (Review)
Review
Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.
Topics: Adenosine Triphosphate; Bacillus subtilis; Bacterial Proteins; Disulfides; Gene Expression Regulation, Bacterial; Lipoproteins; Membrane Proteins; Molecular Chaperones; Protein Folding; Protein Sorting Signals; Recombinant Proteins; Solubility; alpha-Amylases
PubMed: 28724440
DOI: 10.1186/s12934-017-0738-1 -
Current Opinion in Microbiology Apr 2016Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function... (Review)
Review
Bacillus subtilis provides a model for investigation of the bacterial cell envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the envelope. Although their individual regulons overlap, σ(W) is most closely associated with membrane-active agents, σ(X) with cationic antimicrobial peptide resistance, and σ(V) with resistance to lysozyme. Here, I highlight the role of the σ(M) regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of envelope synthesis and cell division, as well as stress-inducible alternative enzymes. Studies of these cell envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics.
Topics: Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Cell Membrane; Cell Wall; Gene Expression Regulation, Bacterial; Regulon; Sigma Factor
PubMed: 26901131
DOI: 10.1016/j.mib.2016.02.002 -
Journal of Bacteriology Feb 2020The MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we...
The MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we describe the global impact of and deletions at the protein level. Using a comprehensive proteomic approach, we showed that 33 and 55 proteins are differentially abundant in Δ and Δ cells, respectively, including proteins involved in metal acquisition, translation, central metabolism, and cell wall homeostasis. In addition, both mutants showed modifications in intracellular metal ion pools, with significant Mg accumulation in the Δ mutant. Phenotypic and morphological analyses of Δ and Δ mutants revealed their high sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress. Mutant strains had a modified cell wall thickness and accumulated lower levels of intracellular reactive oxygen species (ROS) than the wild-type strain. Remarkably, our results highlight an intimate connection between MntR, Zur, antibiotic sensitivity, and cell wall structure. Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In , Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of wild-type, Δ, and Δ strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of and genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.
Topics: Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Cell Wall; Gene Expression Regulation, Bacterial; Gene-Environment Interaction; Homeostasis; Metals; Mutation; Oxidation-Reduction; Proteomics; Repressor Proteins; Stress, Physiological
PubMed: 31818924
DOI: 10.1128/JB.00547-19 -
MBio Dec 2019and are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria,...
and are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent and share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between and Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same "hierarchy" of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history. High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, In this report, we investigate to what extent quantitative principles discovered in Gram-negative are applicable to Gram-positive We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes.
Topics: Bacillus subtilis; Bacterial Physiological Phenomena; Cell Cycle; Cell Division; DNA Replication; Escherichia coli; Replication Origin
PubMed: 31848269
DOI: 10.1128/mBio.02205-19 -
Applied and Environmental Microbiology Feb 2019Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To...
Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in , both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria. Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium , we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in , allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.
Topics: Bacillus subtilis; Bacterial Proteins; Biofilms; Biological Transport; Homeostasis; Iron; Siderophores
PubMed: 30446551
DOI: 10.1128/AEM.02439-18 -
Applied and Environmental Microbiology Jun 2023Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals....
Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals. Thus, its metabolic engineering attracts significant attention. Nevertheless, the limited availability of selective markers makes this process difficult and time-consuming, especially in the case of multistep biosynthetic pathways. Here, we employ CRISPR/Cas9 technology to build an easy cloning toolkit that addresses commonly encountered obstacles in the metabolic engineering of B. subtilis, including the chromosomal integration locus, promoter, terminator, and guide RNA (gRNA) target. Six promoters were characterized, and the promoter strengths ranged from 0.9- to 23-fold that of the commonly used strong promoter P. We characterized seven terminators in B. subtilis, and the termination efficiencies (TEs) of the seven terminators are all more than 90%. Six gRNA targets were designed upstream of the promoter and downstream of the terminator. Using a green fluorescent protein (GFP) reporter, we confirmed integration efficiency with the single-locus integration site is up to 100%. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important product, lycopene. By heterologous expression of the essential genes for lycopene synthesis on the B. subtilis genome, a total of 13 key genes involved in the lycopene biosynthetic pathway were manipulated. Moreover, our findings showed that the gene cluster -- could positively affect the production of lycopene, while the cluster --- had a negative effect on lycopene production. Hence, our multilocus integration strategy can facilitate the pathway assembly for production of complex chemicals and pharmaceuticals in B. subtilis. We present a toolkit that allows for rapid cloning procedures and one-step subcloning to move from plasmid-based expression to stable chromosome integration and expression in a production strain in less than a week. The utility of the customized tool was demonstrated by integrating the MEP (2C-methyl-d-erythritol-4-phosphate) pathway, part of the pentose phosphate pathway (PPP), and the hetero-lycopene biosynthesis genes by stable expression in the genome. The tool could be useful to engineer B. subtilis strains through diverse recombination events and ultimately improve its potential and scope of industrial application as biological chassis.
Topics: Lycopene; CRISPR-Cas Systems; Bacillus subtilis; Plasmids; Metabolic Engineering
PubMed: 37272803
DOI: 10.1128/aem.00230-23 -
Microbiology Spectrum Dec 2023WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in also chromosome segregation. How WhiA works in is unknown....
WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in also chromosome segregation. How WhiA works in is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.
Topics: Bacillus subtilis; DNA-Binding Proteins; Cell Division; Chromosomes; Fatty Acids; Bacterial Proteins
PubMed: 37916812
DOI: 10.1128/spectrum.01795-23 -
PloS One 2015There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to...
There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log10 reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.
Topics: Bacillus anthracis; Bacillus subtilis; Dose-Response Relationship, Radiation; Kinetics; Microbial Viability; Porosity; Species Specificity; Spores, Bacterial; Ultraviolet Rays
PubMed: 26372011
DOI: 10.1371/journal.pone.0138083 -
Journal of Bacteriology Jun 2021Biofilm dispersion is the final stage of biofilm development, during which biofilm cells actively escape from biofilms in response to deteriorating conditions within the...
Biofilm dispersion is the final stage of biofilm development, during which biofilm cells actively escape from biofilms in response to deteriorating conditions within the biofilm. Biofilm dispersion allows cells to spread to new locations and form new biofilms in better locations. However, dispersal mechanisms have been elucidated only in a limited number of bacteria. Here, we investigated biofilm dispersion in Bacillus subtilis. Biofilm dispersion was clearly observed when B. subtilis was grown under static conditions in modified LB medium containing glycerol and manganese. Biofilm dispersion was synergistically caused by two mechanisms: decreased expression of the operon encoding exopolysaccharide synthetases and the induction of sporulation. Indeed, constitutive expression of the operon in the sporulation-defective Δ mutant prevented biofilm dispersion. The addition of calcium to the medium prevented biofilm dispersion without significantly affecting the expression of the operon and sporulation genes. In synthetic medium, eliminating calcium did not prevent the expression of biofilm matrix genes and, thereby, biofilm formation, but it attenuated biofilm architecture. These results indicate that calcium structurally stabilizes biofilms and causes resistance to biofilm dispersion mechanisms. Sporulation-dependent biofilm dispersion required the operon, encoding dipicolinic acid (DPA) synthase. During sporulation, an enormous amount of DPA is synthesized and stored in spores as a chelate with calcium. We speculate that, during sporulation, calcium bound to biofilm matrix components may be transported to spores as a calcium-DPA complex, which weakens biofilm structure and leads to biofilm dispersion. Bacteria growing as biofilms are notoriously difficult to eradicate and sometimes pose serious threats to public health. Bacteria escape from biofilms by degrading them when biofilm conditions deteriorate. This process, called biofilm dispersion, has been studied as a promising strategy for safely controlling biofilms. However, the regulation and mechanism of biofilm dispersion has been elucidated only in a limited number of bacteria. Here, we identified two biofilm dispersion mechanisms in the Gram-positive, spore-forming bacterium Bacillus subtilis. The addition of calcium to the medium stabilized biofilms and caused resistance to dispersal mechanisms. Our findings provide new insights into biofilm dispersion and biofilm control.
Topics: Bacillus subtilis; Bacterial Proteins; Biofilms; Calcium; Gene Expression Regulation, Bacterial; Operon; Picolinic Acids; Spores, Bacterial
PubMed: 33927049
DOI: 10.1128/JB.00114-21 -
Isolation of Persister Cells of and Determination of Their Susceptibility to Antimicrobial Peptides.International Journal of Molecular... Sep 2021Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent...
Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant spores are not susceptible to the antimicrobial peptides.
Topics: Humans; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacillus subtilis; Cathelicidins; Cell Separation; Escherichia coli; Flow Cytometry; Microbial Sensitivity Tests; Polymerase Chain Reaction; Drug Resistance, Bacterial; Spores, Bacterial
PubMed: 34576222
DOI: 10.3390/ijms221810059