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Archives of Microbiology (v.188, #2)

Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai–Tibet plateau and in other zones of China by Feng Ling Kan; Zhong Yi Chen; En Tao Wang; Chang Fu Tian; Xin Hua Sui; Wen Xin Chen (pp. 103-115).

Qinghai–Tibet plateau is the highest place in the world and the environment in that plateau is hard for animals and plants, with low temperature, low concentration of oxygen and high solar radiation. In this study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai–Tibet plateau and in loess plateau were comparatively characterized. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA–DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium sp., Phyllobacterium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from Qinghai–Tibet plateau and from the loess plateau were mixed in these species or groups. Oxytropis spp. and Medicago archiducis-nicolai grown in Qinghai–Tibet plateau were recorded as new hosts for R. leguminosarum, as well as Oxytropis glabra and Medicago lupulina for S. fredii. In addition, strains resistant to high alkaline (pH 11) and high concentration of NaCl (3–5%, w/v) were found in each of the rhizobial species. This was the first systematic study of rhizobia isolated from Qinghai–Tibet plateau

Keywords: Qinghai–Tibet plateau; Alfalfa; Vicia ; Rhizobia ; Endophyte

Styrene lower catabolic pathway in Pseudomonas fluorescens ST: identification and characterization of genes for phenylacetic acid degradation by Patrizia Di Gennaro; Silvia Ferrara; Ilaria Ronco; Enrica Galli; Guido Sello; Maddalena Papacchini; Giuseppina Bestetti (pp. 117-125).

Pseudomonas fluorescens ST is a styrene degrading microorganism that, by the sequential oxidation of the vinyl side chain, converts styrene to phenylacetic acid. The cluster of styrene upper pathway catabolic genes (sty genes) has been previously localized on a chromosomal region. This report describes the isolation, sequencing and analysis of a new chromosomal fragment deriving from the ST strain genomic bank that contains the styrene lower degradative pathway genes (paa genes), involved in the metabolism of phenylacetic acid. Analysis of the paa gene cluster led to the description of 14 putative genes: a gene encoding a phenylacetyl-CoA ligase (paaF), the enzyme required for the activation of phenylacetic acid; five ORFs encoding the subunits of a ring hydroxylation multienzymatic system (paaGHIJK); the gene paaW encoding a membrane protein of unknown function; five genes for a β-oxidation-like system (paaABCDE), involved in the steps following the aromatic ring cleavage; a gene encoding a putative permease (paaL) and a gene (paaN) probably involved in the aromatic ring cleavage. The function of some of the isolated genes has been proved by means of biotransformation experiments.

Keywords: Styrene lower catabolic pathway; Phenylacetate degradation; Pseudomonas

Xanthan is not essential for pathogenicity in citrus canker but contributes to Xanthomonas epiphytic survival by Germán Dunger; Verónica M. Relling; María Laura Tondo; Máximo Barreras; Luis Ielpi; Elena G. Orellano; Jorgelina Ottado (pp. 127-135).

Xanthan-deficient mutants of Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, were generated by deletion and marker exchange of the region encoding the carboxy-terminal end of the first glycosyltransferase, GumD. Mutants of gumD did not produce xanthan and remained pathogenic in citrus plants to the same extent as wild-type bacteria. The kinetics of appearance of initial symptoms, areas of plant material affected, and growth of bacteria inside plant tissue throughout the disease process were similar for both wild-type and mutant inoculations. Moreover, exopolysaccharide deficiency did not impair the ability of the bacteria to induce hypersensitive response on non-host plants. Apart from variations in phenotypic aspects, no differences in growth or survival under different stress conditions were observed between the xanthan-deficient mutant and wild-type bacteria. However, gumD mutants displayed impaired survival under oxidative stress during stationary phase as well as impaired epiphytic survival on citrus leaves. Our results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv. citri and non-host plants, but facilitates the maintenance of bacteria on the host plant, possibly improving the efficiency of colonization of distant tissue.

Keywords: Xanthomonas ; Citrus canker; Xanthan

Analysis of diurnal and vertical microbial diversity of a hypersaline microbial mat by Laura Villanueva; Antoni Navarrete; Jordi Urmeneta; David C. White; Ricardo Guerrero (pp. 137-146).

Microbial mats are prokaryotic communities that provide model systems to analyze microbial diversity and ecophysiological interactions. Community diversity of microbial mat samples was assessed at 8:00 a.m. and 3:00 p.m. in a combined analysis consisting of 16S rRNA-denaturing gradient gel electrophoresis (DGGE) and phospholipid fatty acid (PLFA) profiles. The divergence index determined from PLFA and DGGE data showed that depth-related differences have a greater influence on diversity than temporal variations. Shannon and Simpson indices yielded similar values in all samples, which suggested the stable maintenance of a structurally diverse microbial community. The increased diversity observed at 3:00 p.m. between 2.5 and 4 mm can be explained mainly by diversification of anaerobic microorganisms, especially sulfate-reducing bacteria. In the afternoon sampling, the diversity index reflected a higher diversity between 4 and 5.5 mm depth, which suggested an increase in the diversity of strict anaerobes and fermenters. The results are consistent with the conclusion that hypersaline microbial mats are characterized by high degree of diversity that shifts in response to the photobiological adaptations and metabolic status of the microbial community.

Keywords: Microbial mat; PLFA; DGGE; Lipid analysis; Diversity indices

Dual effect of organic acids as a function of external pH in Oenococcus oeni by Yoann Augagneur; Jean-François Ritt; Daniel M. Linares; Fabienne Remize; Raphaëlle Tourdot-Maréchal; Dominique Garmyn; Jean Guzzo (pp. 147-157).

In this study we analyzed under various pH conditions including low pH, the effects of l-malic acid and citric acid, combined or not, on the growth, the proton motive force components and the transcription level of selected genes of the heterolactic bacterium Oenococcus oeni. It is shown here that l-malate enhanced the growth yield at pH equal or below 4.5 while the presence of citrate in media led to a complete and unexpected inhibition of the growth at pH 3.2. Nevertheless, whatever the growth conditions, both l-malate and citrate participated in the enhancement of the transmembrane pH gradient, whereas the membrane potential decreased with the pH. These results suggested that it was not citrate that was directly responsible for the inhibition observed in cultures done at low pH, but probably its end products. This was confirmed since, in media containing l-malate, the addition of acetate substantially impaired the growth rate of the bacterium and slightly the membrane potential and pH gradient. Finally, study of the expression of genes involved in the metabolism of organic acids showed that at pH 4.5 and 3.2 the presence of l-malate led to an increased amount of mRNA of mleP encoding a malate transporter.

Keywords: Oenococcus oeni ; l-malate; Citrate; Intracellular pH; Membrane potential; Real-time PCR

Mycelial forms of Pseudallescheria boydii present ectophosphatase activities by Tina Kiffer-Moreira; Ana Acacia S. Pinheiro; Márcia R. Pinto; Fabiano F. Esteves; Thais Souto-Padrón; Eliana Barreto-Bergter; José R. Meyer-Fernandes (pp. 159-166).

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl₂, MnCl₂ and ZnCl₂ were able to increase the (p-NPP) hydrolysis while CdCl₂ and CuCl₂ inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.

Keywords: Pseudallescheria boydii ; Phosphatase activities; Ecto-enzymes

Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys conoides by Jinkui Yang; Juan Li; Lianming Liang; Baoyu Tian; Ying Zhang; Chunmei Cheng; Ke-Qin Zhang (pp. 167-174).

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2°C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65°C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.

Keywords: Arthrobotrys conoides ; Protease purification; Nematicidal activity; Gene cloning; Sequence analysis

Thermosensitivity of the Saccharomyces cerevisiae gpp1gpp2 double deletion strain can be reduced by overexpression of genes involved in cell wall maintenance by Iwona Wojda; Jan-Paul Bebelman; Teresa Jakubowicz; Marco Siderius (pp. 175-184).

A Saccharomyces cerevisiae strain in which the GPP1 and GPP2 genes, both encoding glycerol-3-phosphate phosphatase isoforms, are deleted, displays both osmo- and thermosensitive (ts) phenotypes. We isolated genes involved in cell wall maintenance as multicopy suppressors of the gpp1gpp2 ts phenotype. We found that the gpp1gpp2 strain is hypersensitive to cell wall stress such as treatment with β-1,3-glucanase containing cocktail Zymolyase and chitin-binding dye Calcofluor-white (CFW). Sensitivity to Zymolyase was rescued by overexpression of SSD1, while CFW sensitivity was rescued by SSD1, FLO8 and WSC3—genes isolated as multicopy suppressors of the gpp1gpp2 ts phenotype. Some of the isolated suppressor genes (SSD1, FLO8) also rescued the lytic phenotype of slt2 deletion strain. Additionally, the sensitivity to CFW was reduced when the cells were supplied with glycerol. Both growth on glycerol-based medium and overexpression of SSD1, FLO8 or WSC3 had additive suppressing effect on CFW sensitivity of the gpp1gpp2 mutant strain. We also confirmed that the internal glycerol level changed in cells exposed to cell wall perturbation.

Keywords: Saccharomyces cerevisiae ; Heat shock; PKC pathway; Cell wall stress; Glycerol

Synthetic esters recognized by glucuronoyl esterase from Schizophyllum commune by Silvia Špániková; Monika Poláková; Dušan Joniak; Ján Hirsch; Peter Biely (pp. 185-189).

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.

Keywords: Glucuronoyl esterase; 4-O-methyl-d-glucuronic acid; Lignin-carbohydrate complexes

Characterization of a novel T4-type Stenotrophomonas maltophilia virulent phage Smp14 by Chiy-Rong Chen; Ching-Hsuan Lin; Juey-Wen Lin; Chi-I Chang; Yi-Hsiung Tseng; Shu-Fen Weng (pp. 191-197).

Stenotrophomonas maltophilia (Sm), with most of the isolates being resistant to multidrugs, is an opportunistic bacterium causing nosocomial infections. In this study, a novel virulent Sm phage, Smp14, was characterized. Electron microscopy showed that Smp14 resembled members of Myoviridae and adsorbed to poles of the host cells during infection. It lysed 37 of 87 clinical Sm isolates in spot test, displayed a latent period of ca. 20 min, and had a burst size of ca. 150. Its genome (estimated to be 160 kb by PFGE), containing m4C and two unknown modified bases other than m5C and m6A as identified by HPLC, resisted to digestion with many restriction endonucleases except MseI. These properties indicate that it is a novel Sm phage distinct from the previously reported phiSMA5 which has a genome of 250 kb digestible with various restriction enzymes. Sequencing of a 16 kb region revealed 12 ORFs encoding structural proteins sharing 15–45% identities with the homologues from T4-type phages. SDS-PAGE displayed 20 virion proteins, with the most abundant one being the 39 kDa major capsid protein (gp23), which had the N-terminal 52 amino acids removed. Phylogenetic analysis based on gp23 classified Smp14 into a novel single-membered T4-type subgroup.

Keywords: S. maltophilia ; Virulent phage; T4-type; Unknown modified bases; Major capsid protein; Phylogenetic analysis

Thiosulfate oxidation by a moderately thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus by Daisuke Miyake; Shin-ichi Ichiki; Miyako Tanabe; Takahiro Oda; Hisao Kuroda; Hirofumi Nishihara; Yoshihiro Sambongi (pp. 199-204).

The moderately thermophilic Betaproteobacterium, Hydrogenophilus thermoluteolus, not only oxidizes hydrogen, the principal electron donor for growth, but also sulfur compounds including thiosulfate, a process enabled by sox genes. A periplasmic extract of H. thermoluteolus showed significant thiosulfate oxidation activity. Ten genes apparently involved in thiosulfate oxidation (soxEFCDYZAXBH) were found on a 9.7-kb DNA fragment of the H. thermoluteolus chromosome. The proteins SoxAX, which represent c-type cytochromes, were co-purified from the cells of H. thermoluteolus; they enhanced the thiosulfate oxidation activity of the periplasmic extract when added to the latter.

Keywords: Hydrogenophilus thermoluteolus ; Thiosulfate oxidation; Sox genes

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Archives of Microbiology (v.188, #2)