The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).

Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.

Ecological sociobiology is an emerging field that aims to frame social evolution in terms of ecological adaptation. Here we explore the ecological context for evolution of quorum sensing diversity in bacteria, where social communication is limited to members of the same quorum sensing type (pherotype). We sampled isolates of Bacillus subtilis from soil on a microgeographical scale and identified three ecologically distinct phylogenetic groups (ecotypes) and three pherotypes. Each pherotype was strongly associated with a different ecotype, suggesting that it is usually not adaptive for one ecotype to 'listen' to the signalling of another. Each ecotype, however, contained one or more minority pherotypes shared with the other B. subtilis ecotypes and with more distantly related species taxa. The pherotype diversity within ecotypes is consistent with two models: first, a pherotype cycling model, whereby minority pherotypes enter a population through horizontal genetic transfer and increase in frequency through cheating the social interaction; and second, an occasional advantage model, such that when two ecotypes are each below their quorum densities, they may benefit from listening to one another. This is the first survey of pherotype diversity in relation to ecotypes and it will be interesting to further test the hypotheses raised and supported here, and to explore other bacterial systems for the role of ecological divergence in fostering pherotype diversity.