Project description:Bacillus subtilis is a Gram-positive bacterium that serves as an important experimental system. B. subtilis NCIB 3610 is an undomesticated strain that exhibits phenotypes lost from the more common domesticated laboratory strains. Here, we announce the complete genome sequence of DK1042, a genetically competent derivative of NCIB 3610.
Project description:Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain,B. subtilis B-1, which forms biofilms containing mainly ?-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of ?-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms.
Project description:Surface superhydrophobicity makes bacterial biofilms very difficult to fight, and it is a combination of their matrix composition and complex surface roughness which synergistically protects these biomaterials from wetting. Although trying to eradicate biofilms with aqueous (antibiotic) solutions is common practice, this can be a futile approach if the biofilms have superhydrophobic properties. To date, there are not many options available to reduce the liquid repellency of biofilms or to prevent this material property from developing. Here, we present a solution to this challenge. We demonstrate how the addition of metal ions such as copper and zinc during or after biofilm formation can render the surface of otherwise superhydrophobic B. subtilis NCIB 3610 biofilms completely wettable. As a result of this procedure, these smoother, hydrophilic biofilms are more susceptible to aqueous antibiotics solutions. Our strategy proposes a scalable and widely applicable step in a multi-faceted approach to eradicate biofilms.
Project description:Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.
Project description:Bacillus subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis which form smooth, essentially featureless colonies, undomesticated strains such as NCIB 3610 form architecturally complex biofilms. NCIB 3610 also contains an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyperrugose biofilm phenotype. Here we explored the role of rapP phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartyl phosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap phr gene cassettes routinely form regulatory pairs; i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro. Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to posttranslational modification in vivo and directly inhibit RapP activity or, more likely, PhrH upregulates the expression of a peptide that, in turn, directly binds to RapP and inhibits its Spo0F phosphatase activity.
Project description:Cooperation is beneficial to group behaviors like multicellularity, but is vulnerable to exploitation by cheaters. Here we analyze mechanisms that protect against exploitation of extracellular surfactin in swarms of Bacillus subtilis. Unexpectedly, the reference strain NCIB 3610 displays inherent resistance to surfactin-non-producing cheaters, while a different wild isolate is susceptible. We trace this interstrain difference down to a single amino acid change in the plasmid-borne regulator RapP, which is necessary and sufficient for cheater mitigation. This allele, prevalent in many Bacillus species, optimizes transcription of the surfactin operon to the minimum needed for full cooperation. When combined with a strain lacking rapP, NCIB 3610 acts as a cheater itself-except it does not harm the population at high proportions since it still produces enough surfactin. This strategy of minimal production is thus a doubly advantageous mechanism to limit exploitation of public goods, and is readily evolved from existing regulatory networks.
Project description:The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion--the first step in colonization and biofilm formation--is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future.