Journal of Microbiology

Volume 47(2); April 2009

Research Support, Non-U.S. Gov'ts

Analysis of nifH Gene Diversity in Red Soil Amended with Manure in Jiangxi, South China: Qihui Teng , Bo Sun , Xinrui Fu , Shunpeng Li , Zhongli Cui , Hui Cao; J. Microbiol. 2009;47(2):135-141. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0184-1

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34 Citations

Abstract: In order to understand the community structure of diazotrophs in red soil and effects of organic manure application on the structure, four nifH gene libraries were constructed: the control (CK), low manure (LM), high manure (HM), and high manure adding lime (ML). Totally 150 nifH gene clones were screened and grouped into 21 clusters by RFLP analysis. Existence of dominant patterns was observed in all libraries, which counted for over 96% of clones in library HM and about 56~72% in other three libraries. The nifH sequences of the dominant patterns in all libraries were most similar to sequences of the cyanobacteria. nifH genes showed high diversity in red soil, dispersing throughout the nifH clades (alpha-, beta-, and gamma-Proteobacteria, Firmicutes, cyanobacteria, Verrucomicrobia, and posited group). Bradyrhizobium and Burkholderia were also important diazotrophs in low fertility soil samples. Low manure treatment increased the diversity of nifH genes compared with CK and high manure treatments. Manure and lime treatment led to obvious community succession. Total N to available P ratio, total carbon, and K concentrations were the main factors affecting the diversity of diazotrophs in red soil.

Adaptation and Cross-Adaptation of Listeria monocytogenes and Salmonella enterica to Poultry Decontaminants: Alicia Alonso-Hernando , Rosa Capita , Miguel Prieto , Carlos Alonso-Calleja; J. Microbiol. 2009;47(2):142-146. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0237-5

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Abstract: Information on the potential for acquired reduced susceptibility of bacteria to poultry decontaminants occurring is lacking. Minimal Inhibitory Concentrations (MICs) were established for assessing the initial susceptibility and the adaptative and cross-adaptative responses of four bacterial strains (Listeria monocytogenes serovar 1/2a, L. monocytogenes serovar 4b, Salmonella enterica serotype Typhimurium, and S. enterica serotype Enteritidis) to four poultry decontaminants (trisodium phosphate, acidified sodium chlorite -ASC-, citric acid, and peroxyacetic acid). The initial susceptibility was observed to differ among species (all decontaminants) and between Salmonella strains (ASC). These inter- and intra-specific variations highlight (1) the need for strict monitoring of decontaminant concentrations to inactivate all target pathogens of concern, and (2) the importance of selecting adequate test strains in decontamination studies. MICs of ASC (0.17±0.02 to 0.21±0.02 mg/ml) were higher than the U.S. authorized concentration when applied as a pre-chiller or chiller solution (0.05 to 0.15 mg/ml). Progressively increasing decontaminant concentrations resulted in reduced susceptibility of strains. The highest increase in MIC was 1.88 to 2.71-fold (ASC). All decontaminants were shown to cause cross-adaptation of strains between both related and unrelated compounds, the highest increase in MIC being 1.82-fold (ASC). Our results suggest that the in-use concentrations of ASC could, in certain conditions, be ineffective against Listeria and Salmonella strains. The adaptative and cross-adaptative responses of strains tested to poultry decontaminants are of minor concern. However, the observations being presented here are based on in vitro studies, and further research into practical applications are needed in order to confirm these findings.

Characterization of Plant-Growth Promoting Diazotrophic Bacteria Isolated from Field Grown Chinese Cabbage under Different Fertilization Conditions: Woo-Jong Yim , Selvaraj Poonguzhali , Munusamy Madhaiyan , Pitchai Palaniappan , M. A. Siddikee , Tongmin Sa; J. Microbiol. 2009;47(2):147-155. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0201-4

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25 Citations

Abstract: Diazotrophic bacteria isolated from the rhizosphere of Chinese cabbage were assessed for other plant growth promoting characteristics viz., production of IAA, ethylene, ACC deaminase, phosphate solubilization, and gnotobiotic root elongation. Their effect on inoculation to Chinese cabbage was also observed under growth chamber conditions. A total of 19 strains that showed higher nitrogenase activity identified by 16S rRNA gene sequence analysis were found to be the members of the genera Pseudomonas and Agrobacterium belonging to α- and γ-Proteobacteria groups. These strains were also efficient in producing IAA and ACC deaminase though they produced low levels of ethylene and no phosphate solubilization. In addition, inoculation of selected diazotrophic bacterial strains significantly increased seedling length, dry weight, and total nitrogen when compared to uninoculated control. The colonization of crop plants by diazotrophic bacteria can be affected by many biotic and abiotic factors, and further studies are oriented towards investigating the factors that could influence the establishment of a selected bacterial community.

Henriciella marina gen. nov., sp. nov., a Novel Member of the Family Hyphomonadaceae Isolated from the East Sea: Zhe-Xue Quan , Dan-Ning Zeng , Yi-Ping Xiao , Seong Woon Roh , Young-Do Nam , Ho-Won Chang , Jung-Hoon Yoon , Hee-Mock Oh , Jin-Woo Bae; J. Microbiol. 2009;47(2):156-161. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0290-0

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16 Citations

Abstract: A bacterial strain, designated Iso4T, was isolated from the East Sea of Korea and was subjected to a polyphasic taxonomy study including phenotypic and chemotaxonomic characteristics as well as 16S rRNA gene sequence analysis. Cells of the strain were Gram-negative, motile, non-budding, non-stalked, and strictly aerobic. Strain Iso4T grew optimally at 20°C in the presence of 1~2% (w/v) NaCl and at pH 6.9~7.6. The major respiratory quinone was Q-10 and the major cellular fatty acids were C18:1 ω7c (53.5%), C17:1 ω5c (11.7%), C17:1 ω6c (8.1%), C16:0 (7.8%), C17:0 (4.8%), C15:0 (2.9%), and C16:1 ω5c (2.2%). The DNA G+C content of strain Iso4T was 56.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Iso4T formed a monophyletic clade in the family Hyphomonadaceae, supported by high bootstrap value and was most closely related to the genus Hyphomonas (92~94%), a member of marine bacteria in the family. The phenotypic, genotypic, and chemotaxonomic evidences also suggest strain Iso4T represents a novel genus and species in the family Hyphomonadaceae, for which the name Henriciella gen. nov., sp. nov. is proposed. The type strain is Iso4T (=KCTC 12513T =DSM 19595T =JCM 15116T).

Halorubrum cibi sp. nov., an Extremely Halophilic Archaeon from Salt-Fermented Seafood: Seong Woon Roh , Jin-Woo Bae; J. Microbiol. 2009;47(2):162-166. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-009-0016-y

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26 Citations

Abstract: Strain B31T is a Gram-staining-negative, motile, and extremely halophilic archaeon that was isolated from salt-fermented seafood. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were determined. Phylogenetic analysis of its 16S rRNA gene sequence and composition of its major polar lipids placed this archaeon in the genus Halorubrum of the family Halobacteriaceae. Strain B31T showed 97.3, 97.2, and 96.9% 16S rRNA similarity to the type strains of Halorubrum alkaliphilum, Hrr. tibetense, and Hrr. vacuolatum, respectively. Its major polar lipids were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Genomic DNA from strain B31T has a 61.7 mol% G+C content. Analysis of 16S rRNA gene sequences, as well as physiological and biochemical tests, identified genotypic and phenotypic differences between strain B31T and other Halorubrum species. The type strain of the novel species is B31T (=JCM 15757T =DSM 19504T).

Burkholderia sp. KCTC 11096BP as a Newly Isolated Gibberellin Producing Bacterium: Gil-Jae Joo , Sang-Mo Kang , Muhammad Hamayun , Sang-Kuk Kim , Chae-In Na , Dong-Hyun Shin , In-Jung Lee; J. Microbiol. 2009;47(2):167-171. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0273-1

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41 Citations

Abstract: We isolated 864 bacteria from 553 soil samples and bioassayed them on cucumber and crown daisy for plant growth promotion. A new bacterial strain, Burkholderia sp. KCTC 11096BP gave maximum growth promotion and was selected for further investigations. The culture filtrate of this bacterium was thus analyzed for the presence of gibberellins and we found physiologically active gibberellins were found (GA1, 0.23 ng/100 ml; GA3, 5.11 ng/100 ml and GA4, 2.65 ng/100 ml) along with physiologically inactive GA9, GA12, GA15, GA20, and GA24. The bacterial isolate also solubilised tricalcium phosphate and lowered the pH of the medium during the process. The isolate was identified as a new strain of Burkholderia through phylogenetic analysis of 16S rDNA sequence. Gibberellin production capacity of genus Burkholderia is reported for the first time in current study.

Iso-Superoxide Dismutase in Deinococcus grandis, a UV Resistant Bacterium: Na-Rae Yun , Young Nam Lee; J. Microbiol. 2009;47(2):172-177. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0221-0

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Abstract: Deinococcus grandis possesses two types of superoxide dismutase (SOD, E. C. 1.15.1.1.) that show distinct electrophoretic behavior, one that migrates slowly and the other that migrates rapidly (SOD-1 and SOD-2, respectively). In this study, SOD-1 was uniformly and abundantly detected, regardless of growth phase, whereas SOD-2 was not detected during early growth, but was detectable from the exponential growth phase. In addition, a substantial increase in SOD-2 was observed in cells that were treated with potassium superoxide or UV, which suggests that SOD-2 is an inducible protein produced in response to stressful environments. Insensitivity of SOD-1 to both H2O2 and cyanide treatment suggests that SOD-1 is MnSOD. However, SOD-2 would be FeSOD, since it lost activity in response to H2O2 treatment, but not to cyanide. Localization studies of D. grandis iso-SODs in sucrose-shocked cells suggest that SOD-1 is a membrane-associated enzyme, whereas SOD-2 is a cytosolic enzyme. In conclusion, SOD-1 seems to be an essential constitutive enzyme for viability and SOD-2 appears to be an inducible enzyme that is probably critical for survival upon UV irradiation and oxidative stress.

Intracellular Substrates of a Heme-Containing Ascorbate Oxidase in Pleurotus ostreatus: Seung-Rock Lee , Woo-Jeong Joo , Yong-Un Baek , Youn-Kyong Lee , Seong-Woon Yu , Yeon-Ran Kim , Kee-Oh Chay , Seung-Hyun Cho , Sa-Ouk Kang; J. Microbiol. 2009;47(2):178-186. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0307-8

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Abstract: A novel heme-containing ascorbate oxidase isolated from oyster mushroom, Pleurotus ostreatus, catalyzes oxidation of ascorbic acid (Kim et al., 1996). In this report, we describe the identification of intracellular substrates of the enzyme in the mushroom. Six compounds, which can serve as substrate of the heme-containing ascorbate oxidase, were identified as L-ascorbic acid, D-erythroascorbic acid, 5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid, 5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid, 5-methyl-5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid, and 5-methyl-5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid. All of the compounds were oxidized at a significant rate by the heme-containing ascorbate oxidase. Oxidation of the compounds produced equimolar amounts of hydrogen peroxide per mole of substrate.

Translocation of Green Fluorescent Protein to Cyanobacterial Periplasm Using Ice Nucleation Protein: Wipa Chungjatupornchai , Sirirat Fa-aroonsawat; J. Microbiol. 2009;47(2):187-192. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0188-x

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Abstract: The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.

Identification of a Methyltransferase Encoded by Gene ste16 and Its Function in Ebosin Biosynthesis of Streptomyces sp. 139: Hong-Guan Xie , Yong-Gang Bao , Li-ping Bai , Jun-Jie Shan , Rong Jiang , Yang Zhang , Lian-Hong Guo , Ren Zhang , Yuan Li; J. Microbiol. 2009;47(2):193-200. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0195-y

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Abstract: Streptomyces sp. 139 generates a novel exopolysaccharide (EPS) designated as Ebosin, which exerts an antagonistic effect on IL-1R in vitro and anti-rheumatic arthritis activity in vivo. A ste gene cluster for Ebosin biosynthesis consisting of 27 ORFs was previously identified in our laboratory. In this paper, ste16 was expressed in Escherichia coli BL21 and the recombinant protein was purified, which has the ability to catalyze the transfer of the methyl group from S-adenosylmethionine (AdoMet) to dTDP-4-keto-6-deoxy-D-glucos, which was thus identified as a methyltransferase. In order to determine the function of ste16 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition of EPS-m generated by the mutant strain Streptomyces sp. 139 (ste16-) was found to differ from that of Ebosin. The IL-1R antagonist activity of EPS-m was markedly lower than that of Ebosin. These experimental results have shown that the ste16 gene codes for a methyltransferase which is involved in Ebosin biosynthesis.

Phylogeny of a Novel “Helicobacter heilmannii” Organism from a Japanese Patient with Chronic Gastritis Based on DNA Sequence Analysis of 16S rRNA and Urease Genes: Takehisa Matsumoto , Masatomo Kawakubo , Mayumi Shiohara , Toshiko Kumagai , Eiko Hidaka , Kazuyoshi Yamauchi , Kozue Oana , Kenji Matsuzawa , Hiroyoshi Ota , Yoshiyuki Kawakami; J. Microbiol. 2009;47(2):201-207. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0313-x

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Abstract: “Helicobacter heilmannii” is an uncultivable spiral-shaped bacterium inhabiting the human gastric mucosa. It is larger and more tightly-coiled than H. pylori. We encountered a patient with chronic gastritis infected a “H. heilmannii”-like organism (HHLO), designated as SH6. Gastric mucosa derived from the patient was orally ingested by specific pathogen free mice. Colonization of the mice by SH6 was confirmed by electron microscopy of gastric tissue specimens. In an attempt to characterize SH6, 16S rRNA and urease genes were sequenced. The 16S rRNA gene sequence was most similar (99.4%; 1,437/1,445 bp) to HHLO C4E from a cheetah. However, the urease gene sequence displayed low similarity (81.7%; 1,240/1,516 bp) with HHLO C4E. Taxonomic analysis disclosed that SH6 represents a novel strain and should constitute a novel taxon in the phylogenetic trees, being discriminated from any other taxon, with the ability of infecting human gastric mucosa.

Characterization of a Baculovirus Newly Isolated from the Tea Slug Moth, Iragoidae fasciata: Li-Rong Yang , Xiao Qiang , Bao-Qin Zhang , Mei-Jun Tang , Chuan-Xi Zhang; J. Microbiol. 2009;47(2):208-213. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0253-5

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Abstract: The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5B1-4. Bioassays on lethal concentration (LC50) and lethal time (LT50) were conducted to test the susceptibility of I. fasciata larvae to the virus.

Expression of c-Myc Is Related to Host Cell Death Following Salmonella typhimurium Infection in Macrophage: Jihyoun Seong , Hong Hua Piao , Phil Yeoul Ryu , Youn Uck Kim , Hyon E Choy , Yeongjin Hong; J. Microbiol. 2009;47(2):214-219. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0308-7

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Abstract: It has been known that ornithine decarboxylase (ODC) induced by the binding of c-Myc to odc gene is closely linked to cell death. Here, we investigated the relationship between their expressions and cell death in macrophage cells following treatment with Salmonella typhimurium or lipopolysaccharide (LPS). ODC expression was increased by bacteria or LPS and repressed by inhibitors against mitogen-activated protein kinases (MAPKs) in Toll-like receptor 4 (TLR4) signaling pathway. In contrast, c-Myc protein level was increased after treatment with bacteria, but not by treatment with LPS or heat-killed bacteria although both bacteria and LPS increased the levels of c-myc mRNA to a similar extent. c-Myc protein level is dependent upon bacterial invasion because treatment with cytochalasin D (CCD), inhibitors of endocytosis, decreased c-Myc protein level. The cell death induced by bacteria was significantly decreased after treatment of CCD or c-Myc inhibitor, indicating that cell death by S. typhimurium infection is related to c-Myc, but not ODC. Consistent with this conclusion, treatment with bacteria mutated to host invasion did not increase c-Myc protein level and cell death rate. Taken together, it is suggested that induction of c-Myc by live bacterial infection is directly related to host cell death.

Note] Comparative Analysis of 2,4,6-Trinitrotoluene (TNT)-Induced Cellular Responses and Proteomes in Pseudomonas sp. HK-6 in Two Types of Media: Yun-Seok Cho , Bheong-Uk Lee , Hyung-Yeel Kahng , Kye-Heon Oh; J. Microbiol. 2009;47(2):220-224. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0108-0

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Abstract: TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 <br>cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.

Note] Enhanced Expression of Chitinase during the Autolysis of Mushroom in Coprinellus congregatus: Hyangsoon Lim , Hyoung T. Choi; J. Microbiol. 2009;47(2):225-228. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0247-3

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Abstract: Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregatus, produced mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregatus that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.

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