Electron microscopic examination of the dormant spore and the sporulation of Paenibacillus motobuensis strain MC10.

We previously reported a new species Paenibacillus motobuensis. The type strain MC10 was stained gram-negative, but had a gram-positive cell wall structure and its spore had a characteristic star shape. The spore and sporulation process of P. motobuensis strain MC10 were examined by electron microscopy using the technique of freeze-substitution in thin sectioning. The structure of the dormant spore was basically the same as that of the other Bacillus spp. The core of the spore was enveloped with two main spore components, the cortex and the spore coat. In thin section, the spore showed a star-shaped image, which was derived from the structure of the spore coat, which is composed of three layers, namely the inner, middle and outer spore coat. The middle coat was an electron-dense thick layer and had a characteristic ridge. By scanning electron microscopic observation, the ridges were seen running parallel to the long axis of the oval-shaped spore. The process of sporulation was essentially the same as that of the other Bacillus spp. The forespore was engulfed by the mother cell membrane, then the spore coat and the cortex were accumulated in the space between the mother cell membrane and forespore membrane. The mother cell membrane seemed to participate in the synthesis of the spore coat. MC10 strain showed almost identical heat resistance to that of B. subtilis.

Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures.

AIMS: To characterize the alcohol-surviving bacterial isolate ARBG1 from in vitro grapes (Vitis vinifera). METHODS AND RESULTS: Two bacterial strains that survived in covert form in grape cultures were isolated from the spent alcohol used for disinfecting the tools of which one (ARBG2) was characterized earlier. The present study describes characterization of the second isolate, ARBG1. Nutrient agar (NA)-derived colonies of ARBG1 displayed consistently Gram-negative staining rods (2-4x0.5-0.6 micro) substantiated by KOH mucoid thread test. Older cultures (3-7 days) showed emergence of Gram-negative staining, oblong, phase-refractile cells with ellipsoidal spores. The growth and sporulation were modified by growth medium and incubation temperature with the optimum around 37 degrees C. Identification attempts involving microscopic, biochemical, Biolog or fatty acid profiling approaches brought in mixed and inconclusive results. PCR amplification of 16S rDNA was not successful with the standard primers 27F and 1492R but with 27F and a modified primer ARBG1-RP1. The identity of the isolate was established as Brevibacillus sp. based on partial 16S rDNA sequence data from eight single colonies with Gram-positive Brevibacillus choshinensis as the closest match (99.5%). Spotting tests on NA employing spore suspension in aqueous ethanol (0%, 25%, 50%, 60%, 70%, 80% or 90%, v/v) indicated unhindered bacterial-survival in alcohol for 1 month, and that at 2 or 4 months revealed 90% ethanol as more sporicidal than lower levels, corroborated by plating results. Grape microcuttings inoculated with ARBG1 showed substantial general colonization of shoots, roots and medium but low endophytic colonization. CONCLUSIONS: The rare type of spore-producing consistently Gram-negative bacterial isolate ARBG1 was identified as Brevibacillus sp. based on 16S rDNA sequence similarity. The alcohol-defying organism was nonpathogenic and survived in covert form in grape cultures. Aqueous 90% ethanol appeared more sporicidal than lower levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of an unusual endospore-forming Gram-negative bacterium, observation that some bacteria may fall outside the purview of standard 16S rDNA primers, elucidation of the threats of covert bacteria in plant tissue cultures and alcohol-mediated lateral transmission of spore formers, and the revelation that 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria.

Role of membrane-bound thiol-disulfide oxidoreductases in endospore-forming bacteria.

Thiol-disulfide oxidoreductases catalyze formation, disruption, or isomerization of disulfide bonds between cysteine residues in proteins. Much is known about the functional roles and properties of this class of redox enzymes in vegetative bacterial cells but their involvement in sporulation has remained unknown until recently. Two membrane-embedded thiol-disulfide oxidoreductases, CcdA and StoA/SpoIVH, conditionally required for efficient production of Bacillus subtilis heat-resistant endospores, have now been identified. Properties of mutant cells lacking the two enzymes indicate new aspects in the molecular details of endospore envelope development. This mini-review presents an overview of membrane-bound thiol-disulfide oxidoreductases in the Gram-positive bacterium B. subtilis and endospore synthesis. Accumulated experimental findings on CcdA and StoA/SpoIVH are reviewed. A model for the role of these proteins in endospore cortex biogenesis in presented.

Ultrastructural organization of Alicyclobacillus tolerans strain K1T cells.

Electron microscopy examinations of thin sections and freeze-fracture replicas revealed the specific ultrastructural features of Alicyclobacillus tolerans strain K1(T). In particular, the cell wall displayed an ultrastructure typical of gram-positive bacteria and consisted of a thin murein layer (50-60 A in thickness); cells exhibited a surface S-layer constituted by large hexagonally packed (p6-symmetry) rod-shaped subunits of 150-160 A in diameter and 200 A in height. In the cytoplasmic membrane, there were intramembrane vesicular structures that sometimes appeared as large leaflets in the central part. The cytoplasm contained numerous vesicular inclusions covered with a monolayered wall, dissimilar to bilamellar lipid membranes. Endospore coats displayed an intricate structure and consisted of three thick layers; the outer layer had an unusual fine structure; the exosporium was also found.

Heliophilum fasciatum gen. nov. sp. nov. and Heliobacterium gestii sp. nov.: endospore-forming heliobacteria from rice field soils.

Two new taxa of phototrophic heliobacteria are described: Heliobacterium gestii sp. nov. and Heliophilum fasciatum gen. nov. sp. nov. Both organisms were isolated from dry paddy soils. Cells of H. gestii were motile spirilla; cells of H. fasciatum formed cell bundles that were motile as units. Both organisms produced endospores; H. gestii endospores contained dipicolinic acid and elevated levels of calcium. As with other heliobacteria, bacteriochlorophyll g was produced in both organisms and no intracytoplasmic photosynthetic membranes were observed. Growth of H. gestii and H. fasciatum occurred under both photoheterotrophic and chemotrophic conditions; nitrogen fixation also occurred in both organisms. H. gestii and H. fasciatium showed a phylogenetic relationship to the "low GC" line of gram-positive Bacteria, but H. fasciatum was distinct from H. gestii and all other heliobacteria. The ability of H. gestii and H. fasciatum to form endospores might be a significant ecological advantage for survival in their rice soil habitat.