Influence of core divisome proteins on cell division in Streptomyces venezuelae ATCC 10712.

The sporulating, filamentous soil bacterium Streptomyces venezuelae ATCC 10712 differentiates under submerged and surface growth conditions. In order to lay a solid foundation for the study of development-associated division for this organism, a congenic set of mutants was isolated, individually deleted for a gene encoding either a cytoplasmic (i.e. ftsZ) or core inner membrane (i.e. divIC, ftsL, ftsI, ftsQ, ftsW) component of the divisome. While ftsZ mutants are completely blocked for division, single mutants in the other core divisome genes resulted in partial, yet similar, blocks in sporulation septum formation. Double and triple mutants for core divisome membrane components displayed phenotypes that were similar to those of the single mutants, demonstrating that the phenotypes were not synergistic. Division in this organism is still partially functional without multiple core divisome proteins, suggesting that perhaps other unknown lineage-specific proteins perform redundant functions. In addition, by isolating an ftsZ2p mutant with an altered -10 region, the conserved developmentally controlled promoter was also shown to be required for sporulation-associated division. Finally, microscopic observation of FtsZ-YFP dynamics in the different mutant backgrounds led to the conclusion that the initial assembly of regular Z rings does not per se require the tested divisome membrane proteins, but the stability of Z rings is dependent on the divisome membrane components tested. The observation is consistent with the interpretation that Z ring instability likely results from and further contributes to the observed defects in sporulation septation in mutants lacking core divisome proteins.

Ultrastructural analysis of spores from diverse Bacillales species isolated from Brazilian soil.

Many species in the order Bacillales form a specialized cell type called a spore that is resistant to a range of environmental stresses. Transmission electron microscopy (TEM) reveals that the spore is comprised of a series of concentric shells, surrounding an interior compartment harbouring the spore DNA. The outermost of these shells varies considerably in morphology among species, likely reflecting adaptations to the highly diverse niches in which spores are found. To better characterize the variation in spore ultrastructure among diverse species, we used TEM to analyse spores from a collection of 23 aerobic spore-forming bacteria from the Solo do Distrito Federal (SDF strains), spanning the genera Bacillus, Lysinibacillus, Paenibacillus and Brevibacillus, isolated from soil from central Brazil. We found that the structures of these spores varied widely, as expected. Interestingly, even though these isolates are novel strains of each species, they were structurally very similar to the known examples of each species in the literature. Because in most cases, the species we analysed are poorly characterized, our data provide important evidence regarding which structural features are likely to be constant within a taxon and which are likely to vary.

Description of a new anaerobic thermophilic bacterium, Thermoanaerobacterium butyriciformans sp. nov.

Strain USBA-019T, an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4×3.0-7.0µm. Optimal growth occurs at 50-55°C (35-65°C). Optimum pH is 5.0-5.5 (4.0-8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019T, Th. thermostercoris DSM22141T and Th. aotearoense DMS10170T found DNA-DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019T (=CMPUJ U-019T=DSM 101588T).

Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils.

The diversity of thermophilic bacteria was investigated in four hot springs, three salt marshes and 12 desert sites in Morocco. Two hundred and forty (240) thermophilic bacteria were recovered, identified and characterized. All isolates were Gram positive, rod-shaped, spore forming and halotolerant. Based on BOXA1R-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by the genus Bacillus (97.5%) represented by B. licheniformis (119), B. aerius (44), B. sonorensis (33), B. subtilis (subsp. spizizenii (2) and subsp. inaquosurum (6)), B. amyloliquefaciens (subsp. amyloliquefaciens (4) and subsp. plantarum (4)), B. tequilensis (3), B. pumilus (3) and Bacillus sp. (19). Only six isolates (2.5%) belonged to the genus Aeribacillus represented by A. pallidus (4) and Aeribacillus sp. (2). In this study, B. aerius and B. tequilensis are described for the first time as thermophilic bacteria. Moreover, 71.25%, 50.41% and 5.41% of total strains exhibited high amylolytic, proteolytic or cellulolytic activity respectively.

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production.

Bacillus subtilis spores have important biotechnological applications; however, achieving both, high spore cell densities and sporulation efficiencies in fermentation, is poorly reported. In this study, medium components and culture conditions were optimized with different statistical methods to increase spore production of the plant growth promoting rhizobacteria B. subtilis EA-CB0575. Key medium components were determined with Plackett-Burman (PB) design, and the optimum concentration levels of two components (glucose, MgSO4·7H2O) were optimized with a full factorial and central composite design, achieving 1.37 × 10(9) CFU/mL of spore cell density and 93.5 % of sporulation efficiency in shake flask. The optimized medium was used to determine the effect of culture conditions on spore production at bioreactor level, finding that maintaining pH control did not affect significantly spore production, while the interaction of agitation and aeration rates had a significant effect on spore cell density. The overall optimization generated a 17.2-fold increase in spore cell density (8.78 × 10(9) CFU/mL) and 1.9-fold increase in sporulation efficiency (94.2 %) compared to that of PB design. These results indicate the potential of B. subtilis EA-CB0575 to produce both, high spore cell densities and sporulation efficiencies, with very low nutrient requirements and short incubation period which can represent savings of process production.

Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils

The diversity of thermophilic bacteria was investigated in four hot springs, three salt marshes and 12 desert sites in Morocco. Two hundred and forty (240) thermophilic bacteria were recovered, identified and characterized. All isolates were Gram positive, rod-shaped, spore forming and halotolerant. Based on BOXA1R-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by the genus Bacillus (97.5%) represented by B. licheniformis (119), B. aerius (44), B. sonorensis (33), B. subtilis (subsp. spizizenii (2) and subsp. inaquosurum (6)), B. amyloliquefaciens (subsp. amyloliquefaciens (4) and subsp. plantarum (4)), B. tequilensis (3), B. pumilus (3) and Bacillus sp. (19). Only six isolates (2.5%) belonged to the genus Aeribacillus represented by A. pallidus (4) and Aeribacillus sp. (2). In this study, B. aerius and B. tequilensis are described for the first time as thermophilic bacteria. Moreover, 71.25%, 50.41% and 5.41% of total strains exhibited high amylolytic, proteolytic or cellulolytic activity respectively.

Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils

The diversity of thermophilic bacteria was investigated in four hot springs, three salt marshes and 12 desert sites in Morocco. Two hundred and forty (240) thermophilic bacteria were recovered, identified and characterized. All isolates were Gram positive, rod-shaped, spore forming and halotolerant. Based on BOXA1R-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by the genus Bacillus (97.5%) represented by B. licheniformis (119), B. aerius (44), B. sonorensis (33), B. subtilis (subsp. spizizenii (2) and subsp. inaquosurum (6)), B. amyloliquefaciens (subsp. amyloliquefaciens (4) and subsp. plantarum (4)), B. tequilensis (3), B. pumilus (3) and Bacillus sp. (19). Only six isolates (2.5%) belonged to the genus Aeribacillus represented by A. pallidus (4) and Aeribacillus sp. (2). In this study, B. aerius and B. tequilensis are described for the first time as thermophilic bacteria. Moreover, 71.25%, 50.41% and 5.41% of total strains exhibited high amylolytic, proteolytic or cellulolytic activity respectively.(AU)

The Clostridium difficile exosporium cysteine (CdeC)-rich protein is required for exosporium morphogenesis and coat assembly.

Clostridium difficile is an important nosocomial pathogen that has become a major cause of antibiotic-associated diarrhea. There is a general consensus that C. difficile spores play an important role in C. difficile pathogenesis, contributing to infection, persistence, and transmission. Evidence has demonstrated that C. difficile spores have an outermost layer, termed the exosporium, that plays some role in adherence to intestinal epithelial cells. Recently, the protein encoded by CD1067 was shown to be present in trypsin-exosporium extracts of C. difficile 630 spores. In this study, we renamed the CD1067 protein Clostridium difficile exosporium cysteine-rich protein (CdeC) and characterized its role in the structure and properties of C. difficile spores. CdeC is expressed under sporulation conditions and localizes to the C. difficile spore. Through the construction of an ΔcdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ΔcdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) ΔcdeC spores were more sensitive to lysozyme, ethanol, and heat treatment than wild-type spores; and (iv) despite the almost complete absence of the exosporium layer, ΔcdeC spores adhered at higher levels than wild-type spores to intestinal epithelium cell lines (i.e., HT-29 and Caco-2 cells). Collectively, these results indicate that CdeC is essential for exosporium morphogenesis and the correct assembly of the spore coat of C. difficile.

Bacillus subtilis endospores at high purity and recovery yields: optimization of growth conditions and purification method.

Bacillus subtilis endospores have applications in different fields including their use as probiotics and antigen delivery vectors. Such specialized applications frequently require highly purified spore preparations. Nonetheless, quantitative data regarding both yields and purity of B. subtilis endospores after application of different growth conditions and purification methods are scarce or poorly reported. In the present study, we conducted several quantitative and qualitative analyses of growth conditions and purification procedures aiming generation of purified B. subtilis spores. Based on two growth media and different incubations conditions, sporulation frequencies up to 74.2 % and spore concentrations up to 7 × 10(9) spores/ml were achieved. Application of a simplified spore isolation method, in which samples were incubated with lysozyme and a detergent, resulted in preparations with highly purified spores at the highest yields. The present study represents, therefore, an important contribution for those working with B. subtilis endospores for different biotechnological purposes.

Caloramator boliviensis sp. nov., a thermophilic, ethanol-producing bacterium isolated from a hot spring.

A novel moderately thermophilic, anaerobic, ethanol-producing bacterial strain, 45B(T), was isolated from a mixed sediment water sample collected from a hot spring at Potosi, Bolivia. The cells were straight to slightly curved rods approximately 2.5 µm long and 0.5 µm wide. The strain was Gram-stain-variable, spore-forming and monotrichously flagellated. Growth of the strain was observed at 45-65 °C and pH 5.5-8.0, with optima of 60 °C and pH 6.5. The substrates utilized by strain 45B(T) were xylose, cellobiose, glucose, arabinose, sucrose, lactose, maltose, fructose, galactose, mannose, glycerol, xylan, carboxymethylcellulose and yeast extract. The main fermentation product from xylose and cellobiose was ethanol (0.70 and 0.45 g ethanol per gram of consumed sugar, respectively). Acetate, lactate, propionate, carbon dioxide and hydrogen were also produced in minor quantities. 1,3-Propanediol was produced when glycerol-containing medium was supplemented with yeast extract. The major cellular fatty acids were anteiso-C(15:0), C(16:0), iso-C(16:0), C(15:1), iso-C(14:0), C(13:0) and C(14:0). The polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminoglycolipid and 15 other unidentified lipids were predominant. The DNA G+C content of strain 45B(T) was 32.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain 45B(T) is located within the Gram-type positive Bacillus-Clostridium branch of the phylogenetic tree. On the basis of morphological and physiological properties and phylogenetic analysis, strain 45B(T) represents a novel species, for which the name Caloramator boliviensis sp. nov. is proposed; the type strain is 45B(T) (=DSM 22065(T)=CCUG 57396(T)).

New approach for petroleum hydrocarbon degradation using bacterial spores entrapped in chitosan beads.

Spores of Bacillus subtilis LAMI008 were entrapped in 3-mm chitosan beads and cross-linked with 0.3% glutaraldehyde for n-hexadecane biodegradation and biosurfactant recovery. When exposed to nutrients, the spores generated vegetative cells without morphological alterations as revealed by atomic force microscopy. The entrapped cells degraded almost 100% of 1% of n-hexadecane in medium supplemented with 1% glucose and produce biosurfactant within 48 h, as well as free cells. The number of viable cells inside the beads was maintained throughout the n-hexadecane degradation process and the released biosurfactant was not used as a carbon source. Entrapment of bacterial spores in chitosan beads overcomes problems with stability, storage, and long term cell viability encountered with vegetative cells. This approach can potentially be utilized for biodegradation of complex compounds by entrapping spores of different species of bacteria.

Proteocatella sphenisci gen. nov., sp. nov., a psychrotolerant, spore-forming anaerobe isolated from penguin guano.

A novel, obligately anaerobic, psychrotolerant bacterium, designated strain PPP2T, was isolated from guano of the Magellanic penguin (Spheniscus magellanicus) in Chilean Patagonia. Cells were Gram-stain-positive, spore-forming, straight rods (0.7-0.8x3.0-5.0 microm) that were motile by means of peritrichous flagella. Growth was observed at pH 6.7-9.7 (optimum pH 8.3) and 2-37 degrees C (optimum 29 degrees C). Growth was observed between 0 and 4% (w/v) NaCl with optimum growth at 0.5% (w/v). Strain PPP2T was a catalase-negative chemo-organoheterotroph that was capable of fermentative metabolism. Peptone, bacto-tryptone, Casamino acids, oxalate, starch, chitin and yeast extract were utilized as substrates. The major metabolic products were acetate, butyrate and ethanol. Strain PPP2T was resistant to ampicillin, but sensitive to tetracycline, chloramphenicol, rifampicin, kanamycin, vancomycin and gentamicin. The DNA G+C content of strain PPP2T was 39.5 mol%. Phylogenetic analysis revealed that strain PPP2T was related most closely to Clostridium sticklandii SR (approximately 90% 16S rRNA gene sequence similarity). On the basis of phylogenetic analysis and phenotypic characteristics, strain PPP2T is considered to represent a novel species of a new genus, for which the name Proteocatella sphenisci gen. nov., sp. nov. is proposed. The type strain of Proteocatella sphenisci is PPP2T (=ATCC BAA-755T=JCM 12175T=CIP 108034T).

Pseudoxanthomonas mexicana sp. nov. and Pseudoxanthomonas japonensis sp. nov., isolated from diverse environments, and emended descriptions of the genus Pseudoxanthomonas Finkmann et al. 2000 and of its type species.

Three mesophilic bacteria (strains AMX 26B(T), UR374_02 and 12-3(T)) isolated respectively from an anaerobic digester, human urine and urban riverside soil were characterized. Cells were Gram-negative, motile, non-sporulating, straight to curved rods with one polar flagellum and had a strictly respiratory metabolism with O(2) as the preferential terminal electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all strains clustered within the Xanthomonadaceae branch of the Proteobacteria. Isolates AMX 26B(T) and UR374_02 exhibited 100 % 16S rRNA gene sequence similarity and both were related to strain 12-3(T) (99.6 % similarity). The closest relative of all the isolates was Pseudoxanthomonas broegbernensis DSM 12573(T) (similarity 97.1-97.5 %), and they were equidistantly related to Xanthomonas species (95.4-96.6 %), Stenotrophomonas species (95.3-96.1 %) and Pseudoxanthomonas taiwanensis ATCC BAA-4040(T) (95.3-95.4 %). Chemotaxonomic and biochemical data (branched-chain cellular fatty acid pattern without C(13 : 0) iso 3-OH, ubiquinone with eight isoprenoid units, limited range of substrates used, ability to reduce nitrite but not nitrate with the production of N(2)O) supported their affiliation to the genus Pseudoxanthomonas. The results of DNA-DNA hybridization and/or phenotypic analysis allowed them to be differentiated from the two Pseudoxanthomonas species with validly published names and showed that strain 12-3(T) was genomically and phenotypically distinct from the other two isolates. On the basis of these results, two novel species of the genus Pseudoxanthomonas are proposed: Pseudoxanthomonas mexicana sp. nov., consisting of strains AMX 26B(T) (=ATCC 700993(T)=CIP 106674(T)=JCM 11524(T)) (type strain) and UR374_02 (=DSM 15133), and Pseudoxanthomonas japonensis sp. nov., consisting of strain 12-3(T) (=CCUG 48231(T)=CIP 107388(T)=JCM 11525(T)). The report of these two novel species leads to the emendation of the description of the genus Pseudoxanthomonas and the re-evaluation of the phenotype of P. broegbernensis DSM 12573(T) necessitates the emendation of its description.

Actinomadura mexicana sp. nov. and Actinomadura meyerii sp. nov., two novel soil sporoactinomycetes.

The taxonomic position of two soil isolates, strains A288(T) and A290(T) [provisionally assigned to the genus Actinomadura] was clarified in a polyphasic study. The organisms showed a combination of chemotaxonomic and morphological properties typical of actinomadurae. They also formed distinct phyletic lines in the 16S rRNA Actinomadura gene tree; strain A288(T) was associated with A. nitritigenes whereas strain A290(T) was closely related to a group that consisted of A. citrea, A. coerulea, A. glauciflava, A. luteofluorescens and A. verrucosospora. Strains A288(T) and A290(T) showed key phenotypic features which readily distinguish them from one another and from representatives of related validly described species of Actinomadura. It is proposed that the two organisms be classified as new species of the genus Actinomadura. The names proposed for the new taxa are Actinomadura mexicana (A290(T) = DSM 44485(T) = NRRL B-24203(T)), and Actinomadura meyerii (A288(T) = DSM 44485(T) = NRRL B-24203(T)).

A Bacillus thuringiensis isolate possessing a spore-associated filament.

We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure. Nucleotide sequence from the isolate's 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group. Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation. The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body. Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.