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Archives of Microbiology (v.190, #6)

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells by Augusto Etchegaray; Carolina de Castro Bueno; Itamar Soares de Melo; Siu Mui Tsai; Marli de Fátima Fiore; Maria Estela Silva-Stenico; Luiz Alberto Beraldo de Moraes; Omar Teschke (pp. 611-622).

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL⁻¹) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1–0.2 μm²) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.

Keywords: Antimicrobial peptides; Non-ribosomal peptides; Lipopeptides; Iturin; Surfactin; Atomic force microscope; Bacillus subtilis ; Xanthomonas

Inactivation of Burkholderia pseudomallei bsaQ results in decreased invasion efficiency and delayed escape of bacteria from endocytic vesicles by Veerachat Muangsombut; Supaporn Suparak; Pornpan Pumirat; Suwat Damnin; Paiboon Vattanaviboon; Visith Thongboonkerd; Sunee Korbsrisate (pp. 623-631).

Burkholderia pseudomallei, an infectious Gram-negative bacterium, is the causative pathogen of melioidosis. In the present study, a B. pseudomallei strain with mutation in the bsaQ gene, encoding a structural component of the type III secretion system (T3SS), was constructed. This bsaQ mutation caused a marked decrease in secretion of BopE effector and BipD translocator proteins into culture supernatant. The B. pseudomallei bsaQ mutant also exhibited decreased efficiencies of plaque formation, invasion into non-phagocytic cells and multinucleated giant cell (MNGC) development in a J774A.1 macrophage cell line. Co-localization of the bacteria and lysosome-associated membrane glycoprotein-1 (LAMP-1) containing vesicles suggested that defects in MNGC formation may result from the delayed ability of this B. pseudomallei mutant to escape from the vacuoles of macrophages.

Keywords: Burhkolderia pseudomallei ; Type III secretion system; BsaQ; Invasion; Escaping endocytic vesicles; Multinucleated giant cell

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis by N. de la Fuente-Salcido; Ma. Guadalupe Alanís-Guzmán; D. K. Bideshi; R. Salcedo-Hernández; M. Bautista-Justo; J. E. Barboza-Corona (pp. 633-640).

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.

Keywords: Bacillus thuringiensis ; Bacteriocins; Antibacterial activity; Enhancement

Isolation and characterization of porins from Desulfovibrio piger and Bilophila wadsworthia: structure and gene sequencing by Ofir Avidan; Elena Kaltageser; Izabella Pechatnikov; Hannah M. Wexler; Alla Shainskaya; Yeshayahu Nitzan (pp. 641-650).

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95°C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small nonspecific channels with an exclusion limit of 260–300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic β-strands. This research presents porins which were isolated and characterized for the first time from bacteria belonging to the Desulfovibrionaceae family.

Keywords: Porins; Desulfovibrio piger ; Bilophila wadsworthia

Identification of three proteins up-regulated by raw starch in Cytophaga sp. by Rong-Jen Shiau; Yu-Der Wen; Chii-Ling Jeang (pp. 651-655).

Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars. We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin (cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide an insight into how Cytophaga sp. cells respond to raw starch stimulation.

Keywords: Cytophaga sp.; Raw starch; Two dimensional-gel electrophoresis

Diverse bacteria isolated from root nodules of wild Vicia species grown in temperate region of China by Xia Lei; En Tao Wang; Wen Feng Chen; Xin Hua Sui; Wen Xin Chen (pp. 657-671).

In the present study, a total of 154 bacterial strains isolated from nodules of eighteen Vicia species mainly grown in the temperate Chinese provinces were characterized by ARDRA, ITS PCR–RFLP, BOX-PCR, sequencing of 16S rDNA, nodC, nifH, atpD and glnII, and nodulation tests. The results demonstrated that most of the R. leguminosarum strains were effective microsymbionts of the wild Vicia species, while genomic species related to Rhizobium gallicum, Mesorhizobium huakuii, Ensifer meliloti and Bradyrhizobium spp. were symbiotic bacteria occasionally nodulating with Vicia species. In addition, fourteen strains related to Agrobacterium, Phyllobacterium, Ensifer, Shinella and R. tropici, as well as 22 strains of R. leguminosarum might be nodule endophytes without symbiotic genes. Diverse symbiotic gene lineages were found among the test strains and a strong association was found among the symbiotic gene types and genomic species, indicating the absence of lateral gene transfer. These results greatly enlarged the rhizobial spectrum of Vicia species.

Keywords: Diversity; Phylogeny; Rhizobia; Vicia ; Symbiotic gene; Housekeeping gene

Characterization of 4-nonylphenol-degrading bacterial consortium obtained from a textile wastewater pretreatment plant by Diana Di Gioia; Laura Salvadori; Giulio Zanaroli; Ester Coppini; Fabio Fava; Claudia Barberio (pp. 673-683).

4-Nonylphenol (4-NP) isomers are toxic and recalcitrant compounds often resulting, together with short-chain ethoxylated nonylphenol (NPnEO, where n is the number of ethylene oxide units), from NPnEO biodegradation in conventional activated sludge plants. In this work, a microbial consortium, defined as Consortium A, capable of removing 100 mg/L of 4-NP with no accumulation of metabolites with aromatic moiety was isolated from textile wastewaters after enrichment with 4-NP. The consortium showed remarkable degradation activities toward several short-chain NPnEO congeners. Culture-dependent techniques were used to isolate from the consortium twenty-six strains assigned to seven different amplified ribosomal DNA restriction analysis groups. Two- and three-member cocultures were prepared with the strains showing highest 4-NP-degrading capabilities, but neither the single strains nor the cocultures were as efficient in 4-NP degradation as Consortium A. FISH was used to characterize the microbial composition of Consortium A: it evidenced a strong occurrence of Proteobacteria and, in particular, of Gammaproteobacteria along with a relevant stability of the culture. Therefore, the isolated consortium has the potential of being used in the development of a biotechnological process for the tertiary treatment of effluents of activated sludge plants fed with NPnEO-contaminated wastewaters.

Keywords: 4-Nonylphenol; Nonylphenol ethoxylates; FISH; Wastewaters

Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein by Iris Lüke; Gareth Butland; Kevin Moore; Grant Buchanan; Verity Lyall; Shirley A. Fairhurst; Jack F. Greenblatt; Andrew Emili; Tracy Palmer; Frank Sargent (pp. 685-696).

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO₂ and H₂. The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation.

Keywords: Bacterial respiration; Formate dehydrogenase; Enzyme biosynthesis; Molecular chaperone

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Archives of Microbiology (v.190, #6)