Exploitation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> as a robust workhorse for production of heterologous proteins and beyond

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.     

Morphologies and phenotypes in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> biofilms

In this study, we explored Bacillus subtilis biofilm growth under various conditions such as the use of substrates with different stiffnesses and nutrient levels using a well-developed optical imaging technique to spatially and temporally track biofilm growth. We also developed a quantitative method to characterize B. subtilis biofilm morphologies under various growth conditions. To determine biofilm rim irregularities, we used the dimensionless P2A ratio, defined as P²/4πA, where P is the perimeter and A is the area of the biofilm. To estimate biofilm thickness from transmission images, we developed a calibration procedure based on Beer- Lambert’s law and cross sectioning. Furthermore, to determine the distributions of different B. subtilis cell phenotypes during biofilm growth, we used a triple-fluorescence-labeled B. subtilis strain that expressed motility, matrix production, and sporulation. Based on this work, we are able to tune biofilm growth by changing its growing environment.     

A Disulfide Bond-Containing Alkaline Phosphatase Triggers a BdbC-Dependent Secretion Stress Response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for α-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

Biosynthesis of secreted ribonucleases of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> and <Emphasis Type="Italic">Bacillus circulans</Emphasis> under nitrogen starvation

The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein.     

Heterologous expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> gene of glutamyl endopeptidase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5–5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.     

d-Amino Acids Do Not Inhibit Biofilm Formation in Staphylococcus aureus

Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation.     

FlgM proteins from different bacteria exhibit different structural characteristics

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85 °C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.     

The biofilm formation defect of a Bacillus subtilis flotillin‐defective mutant involves the protease FtsH

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix‐producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft‐specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal‐localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.     

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria ^1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors ^7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells ^11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm ^18-21.Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation ^11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis^15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin ²⁵. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers ¹⁵.We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B.subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.     

The majority of the matrix protein TapA is dispensable for Bacillus subtilis colony biofilm architecture

Biofilm formation is a co-operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease-resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA-fibre formation but is dispensable for in vitro TasA-fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.     

Artificial septal targeting of Bacillus subtilis cell division proteins in Escherichia coli: an interspecies approach to the study of protein-protein interactions in multiprotein complexes

Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.     

d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.     

Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS_Sm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions.     

Predation by Myxococcus xanthus Induces Bacillus subtilis To Form Spore-Filled Megastructures

Biofilm formation is a common mechanism for surviving environmental stress and can be triggered by both intraspecies and interspecies interactions. Prolonged predator-prey interactions between the soil bacterium Myxococcus xanthus and Bacillus subtilis were found to induce the formation of a new type of B. subtilis biofilm, termed megastructures. Megastructures are tree-like brachiations that are as large as 500 μm in diameter, are raised above the surface between 150 and 200 μm, and are filled with viable endospores embedded within a dense matrix. Megastructure formation did not depend on TasA, EpsE, SinI, RemA, or surfactin production and thus is genetically distinguishable from colony biofilm formation on MSgg medium. As B. subtilis endospores are not susceptible to predation by M. xanthus, megastructures appear to provide an alternative mechanism for survival. In addition, M. xanthus fruiting bodies were found immediately adjacent to the megastructures in nearly all instances, suggesting that M. xanthus is unable to acquire sufficient nutrients from cells housed within the megastructures. Lastly, a B. subtilis mutant lacking the ability to defend itself via bacillaene production formed megastructures more rapidly than the parent. Together, the results indicate that production of the megastructure facilitates B. subtilis escape into dormancy via sporulation.