Molecular mechanisms involved in Bacillus subtilis biofilm formation

Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil‐dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programmes of cellular specialization and cell–cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis and thereby placing a special emphasis on summarizing the most recent discoveries in the field.     

Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. Recently, we reported regarding genes spaB, spaT, and spaC (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58:132-142, 1992) which are involved in the biosynthesis of subtilin, and genes spaR and spaK (C. Klein, C. Kaletta, and K.-D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), which regulate subtilin biosynthesis via a histidine kinase/response regulator system. Further sequence analysis revealed the presence of three additional open reading frames, spaI, spaF, and spaG, downstream of the structural gene spaS. The spaI gene encodes a hydrophilic 19.3-kDa lipoprotein containing a consensus signal sequence, indicating that this protein might be membrane anchored. A similar gene, nisI, has been identified in the nisin producer. SpaF shows strong homology to members of the family of ABC transporters. spaG encodes a hydrophobic protein which might form the active transporter together with SpaF. Gene disruption mutants in all three genes were still able to produce subtilin; however, these mutants were more sensitive to subtilin than the wild-type strain. These results show that these genes are involved in the immunity mechanism of the producer strain. A similar involvement of an ABC transporter in the self-protection mechanism has been described for the McbE and McbF transporter, which confers immunity against microcin B17 in Escherichia coli. Mutants containing mutations in the genes spaR and spaK, which are responsible for regulation of subtilin biosynthesis, also became more sensitive to subtilin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thinking about Bacillus subtilis as a multicellular organism

Initial attempts to use colony morphogenesis as a tool to investigate bacterial multicellularity were limited by the fact that laboratory strains often have lost many of their developmental properties. Recent advances in elucidating the molecular mechanisms underlying colony morphogenesis have been made possible through the use of undomesticated strains. In particular, Bacillus subtilis has proven to be a remarkable model system to study colony morphogenesis because of its well-characterized developmental features. Genetic screens that analyze mutants defective in colony morphology have led to the discovery of an intricate regulatory network that controls the production of an extracellular matrix. This matrix is essential for the development of complex colony architecture characterized by aerial projections that serve as preferential sites for sporulation. While much progress has been made, the challenge for future studies will be to determine the underlying mechanisms that regulate development such that differentiation occurs in a spatially and temporally organized manner.     

Proteins involved in formation of the outermost layer of Bacillus subtilis spores

To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.     

Genes controlling xylan utilization by Bacillus subtilis.

Eight mutants of Bacillus subtilis deficient in xylan utilization were isolated and characterized genetically and biochemically. Each mutant was obtained independently after nitrosoguanidine mutagenesis. All of the analyzed mutations were shown to be linked. Reciprocal transformation crosses revealed the existence of two genes controlling xylan utilization which have been designated xynA and xynB. Available data have indicated that these two genes code for two xylan-degrading enzymes existing in the wild-type strains, an extracellular beta-xylanase (xynA) and a cell-associated beta-xylosidase (xynB).     

Genes involved in SkfA killing factor production protect a Bacillus subtilis lipase against proteolysis

Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.     

Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I

Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the meso-diaminopimelate (LD-A2pm) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and lssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A2pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group lssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyl-LL-A2pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase 1. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapG. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase 1, not in the carboxylase, and to belong to the dapG locus, dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A2pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapG genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.     

Regulation of endospore formation in Bacillus subtilis

Spore formation in bacteria poses a number of biological problems of fundamental significance. Asymmetric cell division at the onset of sporulation is a powerful model for studying basic cell-cycle problems, including chromosome segregation and septum formation. Sporulation is one of the best understood examples of cellular development and differentiation. Fascinating problems posed by sporulation include the temporal and spatial control of gene expression, intercellular communication and various aspects of cell morphogenesis.     

Autoregulation of alpha-amylase formation in Bacillus subtilis

The phenomenon of self-regulation of alpha-amylase formation in Bacillus subtilis is discovered. The dependence of regulatory effect upon the phase of culture development in shown.     

Bistability and biofilm formation in Bacillus subtilis

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

Creating New Genes by Plasmid Recombination in Escherichia coli and Bacillus subtilis

Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.