Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus subtilis

Galactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. In Bacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since the galE mutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to a galE null mutant in the presence of galactose, we identified mutations in sinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role in B. subtilis biofilm formation and that the expressions of both the gal and eps genes are interrelated. Finally, we propose that B. subtilis and other members of the Bacillus genus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources.Molecular and Cellular Biolog

A serine sensor for multicellularity in a bacterium

We report the discovery of a simple environmental sensing mechanism for biofilm formation in the bacterium Bacillus subtilis that operates without the involvement of a dedicated RNA or protein. Certain serine codons, the four TCN codons, in the gene for the biofilm repressor SinR caused a lowering of SinR levels under biofilm-inducing conditions. Synonymous substitutions of these TCN codons with AGC or AGT impaired biofilm formation and gene expression. Conversely, switching AGC or AGT to TCN codons upregulated biofilm formation. Genome-wide ribosome profiling showed that ribosome density was higher at UCN codons than at AGC or AGU during biofilm formation. Serine starvation recapitulated the effect of biofilm-inducing conditions on ribosome occupancy and SinR production. As serine is one of the first amino acids to be exhausted at the end of exponential phase growth, reduced translation speed at serine codons may be exploited by other microbes in adapting to stationary phase. DOI: http://dx.doi.org/10.7554/eLife.01501.00

Characterization of Subtilin L-Q11, a Novel Class I Bacteriocin Synthesized by Bacillus subtilis L-Q11 Isolated From Orchard Soil

Bacteriocins are peptides or proteins synthesized by bacterial ribosomes that show killing or inhibitory activities against different groups of bacteria. Bacteriocins are considered potential alternatives to traditional antibiotics, preservatives in pharmaceutical and food industries. A strain L-Q11 isolated from orchard soil was phylogenetically characterized as Bacillus subtilis based on 16S rRNA gene sequencing analysis. A novel class I bacteriocin (Subtilin L-Q11), was identified and purified from L-Q11 cell-free supernatant in a four-step procedure, including salt precipitation, cation exchange, gel filtration, and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass (3,552.9 Da) of this novel bacteriocin was determined by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The purified Subtilin L-Q11 exhibited optimal features in pH tolerance, thermostability, and sensitivity to proteases. Further, Subtilin L-Q11 showed inhibitory activities against a number of bacteria including some human pathogens and food spoilage bacteria, in particular Staphylococcus aureus. All these important features make this novel bacteriocin a potential candidate for the development of a new antibacterial drug or food preservative in the future

Insights into the diversity and survival strategies of soil bacterial isolates from the Atacama Desert

The Atacama Desert, the driest, with the highest radiation, and one of the most ancient deserts in the world, is a hostile environment for life. We have a collection of 74 unique bacterial isolates after cultivation and confirmation by 16S rRNA gene sequencing. Pigmentation, biofilm formation, antimicrobial production against Escherichia coli MG1655 and Staphylococcus aureus HG003, and antibiotic resistance were assessed on these isolates. We found that approximately a third of the colonies produced pigments, 80% of isolates formed biofilms, many isolates produce growth inhibiting activities against E. coli and/or S. aureus, and many were resistant to antibiotics. The functional characterization of these isolates gives us insight into the adaptive bacterial strategies in harsh environments and enables us to learn about their possible use in agriculture, healthcare, or biotechnology

Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity

TraR of Agrobacterium tumefaciens is a member of the LuxR family of quorum-sensing transcription factors and regulates genes required for conjugation and vegetative replication of the tumor-inducing (Ti) plasmid in the presence of the autoinducer 3-oxooctanoyl-homoserine lactone (OOHL). In the absence of OOHL, TraR is rapidly destroyed by proteolysis, suggesting that this ligand is required for TraR folding. To date, no TraR variant has been found that is active in the absence of OOHL. In this study, we conducted whole-cell and plasmid mutagenesis experiments to search for constitutive mutations of traR and identified two constitutive alleles. Surprisingly, neither contained a point mutation within the traR gene, but rather, both encoded fusion proteins between TraR and the N-terminal domain of an aminoglycoside N-acetyltransferase, encoded by a plasmid-borne antibiotic resistance gene present in the original strain. Data from Western immunoblot assays, pulse-chase assays, and immunoprecipitation assays show that these fusion proteins are far more stable to proteolysis than native apo-TraR. We also constructed a library of traR alleles encoding random amino-terminal fusions and selected for constitutive TraR activity. Five independent fusion proteins were identified by this approach. These fusion proteins accumulated to far higher levels than wild-type TraR in the absence of OOHL. One of these fusions was overexpressed in Escherichia coli and showed detectable tra box binding in the absence of OOHL. These data suggest that the native amino terminus of TraR may signal proteolysis and that fusing it to other proteins might sequester it from intracellular proteases

A bacterial volatile signal for biofilm formation

Bacteria constantly monitor the environment they reside in and respond to potential changes in the environment through a variety of signal sensing and transduction mechanisms in a timely fashion. Those signaling mechanisms often involve application of small, diffusible chemical molecules. Volatiles are a group of small air-transmittable chemicals that are produced universally by all kingdoms of organisms. Past studies have shown that volatiles can function as cell-cell communication signals not only within species, but also cross-species. However, little is known about how the volatile-mediated signaling mechanism works. In our recent study (Chen, et al. mBio (2015), 6: e00392-15), we demonstrated that the soil bacterium Bacillus subtilis uses acetic acid as a volatile signal to coordinate the timing of biofilm formation within physically separated cells in the community. We also showed that the bacterium possesses an intertwined gene network to produce, secrete, sense, and respond to acetic acid, in stimulating biofilm formation. Interestingly, many of those genes are highly conserved in other bacterial species, raising the possibility that acetic acid may act as a volatile signal for cross-species communication

The Chaperone GroESL Enhances the Accumulation of Soluble, Active TraR Protein, a Quorum-Sensing Transcription Factor from Agrobacterium tumefaciens▿

TraR of Agrobacterium tumefaciens is a LuxR-type quorum-sensing transcription factor that regulates genes required for replication and conjugation of the tumor-inducing (Ti) plasmid. TraR requires its cognate autoinducer N-3-oxooctanoyl-homoserine lactone (OOHL) for resistance of proteolysis in wild-type bacteria and for correct protein folding and solubility when overexpressed in E. coli. In this study, we ask whether GroESL might also play a role in TraR folding, as this molecular chaperone assists many proteins in attaining their native tertiary structure. Expression of E. coli GroESL in a strain expressing TraR increases the solubility of TraR and increases transcriptional activity of a TraR-dependent promoter. Both solubility and activity still require OOHL. We also studied the folding of TraR in the closely related bacterium Sinorhizobium meliloti. A mutation in one groEL gene slightly decreased the expression of a TraR-dependent promoter, strongly decreased the accumulation of TraR in Western immunoblot assays, and also strongly influenced the fate of pulse-labeled TraR

Bacillus subtilis Cell Differentiation, Biofilm Formation and Environmental Prevalence

Bacillus subtilis is a soil-dwelling, spore-forming Gram-positive bacterium capable of cell differentiation. For decades, B. subtilis has been used as a model organism to study development of specialized cell types. In this minireview, we discuss cell differentiation in B. subtilis, covering both past research and recent progresses, and the role of cell differentiation in biofilm formation and prevalence of this bacterium in the environment. We review B. subtilis as a classic model for studies of endospore formation, and highlight more recent investigations on cell fate determination and generation of multiple cell types during biofilm formation. We present mechanistic details of how cell fate determination and mutually exclusive cell differentiation are regulated during biofilm formation