Bacillus pumilus is a Gram-positive bacterium with a wide range of attributed applications, namely as a plant growth promoting rhizobacteria (PGPR), animal, and human probiotic. However, a rare putative role in human diseases has been reported, namely in food poisoning or as anthrax-like cutaneous infectious agent. This species is difficult to distinguish from its closely related species on the basis of phenotypic or biochemical characteristics and 16S rRNA gene sequences. In this study, the phylogenetic analysis of gyrB and rpoB gene sequences of a collection of isolates previously identified as B. pumilus, assigned most of them (93%, 38 of 41 isolates) to B. safensis or to the new recently described B. invictae. Moreover, we extended the previously reported recognized habitats of these species and unveiled a human health or biotechnological relevance (e.g. as implicated in food poisoning or PGPR) for them. Additionally, we demonstrated that both B. safensis and B. invictae species encompass a clonally diverse population, which can justify their great adaptation ability to different niches, with evidence of clonal-host specificity.

A Gram-positive, rod-shaped, endospore-forming Bacillus isolate, Bi.(FFUP1) (T), recovered in Portugal from a health product was subjected to a polyphasic study and compared with the type strains of Bacillus pumilus, Bacillus safensis, Bacillus altitudinis and Bacillus xiamenensis, the phenotypically and genotypically most closely related species. Acid production from cellobiose, D-glucose and D-mannose and absence of acid production from D-arabinose, erythritol, inositol, maltose, mannitol, raffinose, rhamnose, sorbitol, starch and L-tryptophan discriminated this new isolate from the type strains of the most closely related species. Additionally, a significant different protein and carbohydrate signature was evidenced by spectroscopic techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Fourier transform IR spectroscopy with attenuated total reflectance. Using a chemometric approach, the score plot generated by principal component analysis clearly delineated the isolate as a separate cluster. The quinone system for strain Bi.(FFUP1) (T) comprised predominantly menaquinone MK-7 and major polar lipids were diphosphatidylglycerol, an unidentified phospholipid and an unidentified glycolipid. Strain Bi.(FFUP1) (T) showed ? 99% 16S rRNA gene sequence similarity to B. safensis FO-036b(T), B. pumilus (7061(T) and SAFR-032), B. altitudinis 41KF2b(T) and B. xiamenensis HYC-10(T). Differences in strain Bi.FFUP1 (T) gyrB and rpoB sequences in comparison with the most closely related species and DNA-DNA hybridization experiments with Bi.FFUP1 (T) and B. pumilus ATCC 7061(T), B. safensis FO-036b(T), B. altitudinis 41KF2b(T) and B. xiamenensis HYC-10(T) gave relatedness values of 39.6% (reciprocal 38.0%), 49.9% (reciprocal 42.9%), 61.9% (reciprocal 52.2%) and 61.7% (reciprocal 49.2%), respectively, supported the delineation of strain Bi.(FFUP1) (T) as a representative of a novel species of the genus Bacillus, for which the name Bacillus invictae sp. nov. is proposed, with strain Bi.(FFUP1) (T) (=DSM 26896(T) =CCUG 64113(T)) as the type strain.

Bacteria closely related to Bacillus pumilus cannot be distinguished from such other species as B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus simply by 16S rRNA gene sequence. In this report, 76 marine strains were subjected to phylogenetic analysis based on 7 housekeeping genes to understand the phylogeny and biogeography in comparison with other origins. A phylogenetic tree based on the 7 housekeeping genes concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB was constructed and compared with trees based on the single genes. All these trees exhibited a similar topology structure with small variations. Our 79 strains were divided into 6 groups from A to F; Group A was the largest and contained 49 strains close to B. altitudinis. Additional two large groups were presented by B. safensis and B. pumilus respectively. Among the housekeeping genes, gyrB and pyrE showed comparatively better resolution power and may serve as molecular markers to distinguish these closely related strains. Furthermore, a recombinant phylogenetic tree based on the gyrB gene and containing 73 terrestrial and our isolates was constructed to detect the relationship between marine and other sources. The tree clearly showed that the bacteria of marine origin were clustered together in all the large groups. In contrast, the cluster belonging to B. safensis was mainly composed of bacteria of terrestrial origin. Interestingly, nearly all the marine isolates were at the top of the tree, indicating the possibility of the recent divergence of this bacterial group in marine environments. We conclude that B. altitudinis bacteria are the most widely spread of the B. pumilus group in marine environments. In summary, this report provides the first evidence regarding the systematic evolution of this bacterial group, and knowledge of their phylogenetic diversity will help in the understanding of their ecological role and distribution in marine environments.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

Yanyanku and Ikpiru made by the fermentation of Malcavene bean (Hibiscus sabdariffa) are used as functional additives for Parkia biglobosa seed fermentations in Benin. A total of 355 aerobic endospore-forming bacteria (AEFB) isolated from Yanyanku and Ikpiru produced in northern and southern Benin were identified using phenotypic and genotypic methods, including GTG5-PCR, M13-PCR, 16S rRNA, gyrA and gyrB gene sequencing. Generally, the same 5-6 species of the genus Bacillus predominated: Bacillus subtilis (17-41% of isolates), Bacillus cereus (8-39%), Bacillus amyloliquefaciens (9-22%), Bacillus licheniformis (3-26%), Bacillus safensis (8-19%) and Bacillus altitudinis (0-19%). Bacillus aryabhattai, Bacillus flexus, and Bacillus circulans (0-2%), and species of the genera Lysinibacillus (0-14%), Paenibacillus (0-13%), Brevibacillus (0-4%), and Aneurinibacillus (0-3%) occurred sporadically. The diarrheal toxin encoding genes cytK-1, cytK-2, hblA, hblC, and hblD were present in 0%, 91% 15%, 34% and 35% of B. cereus isolates, respectively. 9% of them harbored the emetic toxin genetic determinant, cesB. This study is the first to identify the AEFB of Yanyanku and Ikpiru to species level and perform a safety evaluation based on toxin gene detections. We further suggest, that the gyrA gene can be used for differentiating the closely related species Bacillus pumilus and B. safensis.

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable �]-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified �]-1,3-1,4-glucanase was observed to be stable at 60 �XC and retain more than 90% activity when incubated for 2 h at 60 �XC and remain about 75% and 44% activity after incubating at 70 �XC and 80 �XC for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 �XC. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of �]-1,3-1,4-glucanase showed a homology of 98% with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported �]-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on �]-1,3-1,4-glucanase produced by B. altitudinis.