Journal of Microbiology

Volume 47(5); October 2009

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

Antarcticimonas flava gen. nov., sp. nov., Isolated from Antarctic Coastal Seawater: Seung-Jo Yang , Hyun-Myung Oh , Sangyun Chung , Jang-Cheon Cho; J. Microbiol. 2009;47(5):517-523. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0225-4

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Abstract: A marine bacterium, designated IMCC3175T, was isolated from a seawater sample collected off the Antarctic coast. The strain was Gram-negative, obligately aerobic, carotenoid pigment-containing, and rod-shaped bacterium that divided by binary fission. As determined by 16S rRNA gene sequence comparisons, the most closely related genera were Formosa (92.9~93.3%), Bizionia (91.6~93.2%), Gaetbulibacter (91.5~92.8%), Sediminibacter (92.7%), Yeosuana (92.6%), Subsaximicrobium (92.1~92.2%), and Gillisia (89.5~92.2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a monophyletic clade together with the genera Sediminibacter and Subsaximicrobium but represented an independent phyletic line in this clade of the family Flavobacteriaceae. The DNA G+C content of the strain was 37.3 mol%. The major respiratory quinone was MK-6 and the predominant cellular fatty acids were C16:1 ω7c and/or iso-C15:0 2-OH (12.8%), anteiso-C15:0 (9.4%), and iso-C16:1 (9.4%). Low 16S rRNA gene sequence similarity, formation of a distinct phylogenetic branch, and several phenotypic characteristics, including a narrow range of temperature and salinity for growth, differentiated strain IMCC3175T from other related genera in the family Flavobacteriaceae. Therefore the name Antarcticimonas flava gen. nov., sp. nov. is proposed, with strain IMCC3175T (=KCCM 42713T =NBRC 103398T) as the type strain.

Paenibacillus filicis sp. nov., Isolated from the Rhizosphere of the Fern: Byung-Chun Kim , Mi Na Kim , Kang Hyun Lee , Sun Beom Kwon , Kyung Sook Bae , Kee-Sun Shin; J. Microbiol. 2009;47(5):524-529. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0266-8

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Abstract: A Gram-positive and endospore-forming bacterial strain, designated S4T, was isolated from the rhizosphere of ferns in Daejeon, Republic of Korea. This isolate is strictly aerobic, motile, and rod-like in shape, and it is positive for catalase, oxidase, esterase lipase, and β-galactosidase activities. In addition, this strain grows when cultured at temperatures between 15 and 37°C and at pH values ranging from 5.5 to 9.0. The DNA G+C content was determined to be 53.2 mol%. Strain S4T has meso-diaminopimelic acid in the cell-wall peptidoglycan; it also contains menaquinone 7 (MK-7) as the predominant isoprenoid quinone and anteiso-C15:0 (57.5%), iso-C16:0 (11.3%), and C16:0 (9.4%) as the major cellular fatty acids. Phylogenetic analysis based on alignments of the 16S rRNA gene sequence showed that S4T is affiliated with a cluster of strains within the genus Paenibacillaceae and is most closely related to Paenibacillus chinjuensis WN9T, with 96.8% similarity. Based on the phylogenetic and phenotypic characteristics of strain S4T, we believe that this isolate should be distinguished from all type species of the genus Paenibacillus and should thus represent a novel taxon within the genus Paenibacillus. We propose naming this type species Paenibacillus filicis sp. nov. for the rhizosphere isolate; the type strain will be known as S4T (=KCTC 13693T =KACC 14197T =JCM 16417T).

Paenibacillus pinihumi sp. nov., a Cellulolytic Bacterium Isolated from the Rhizosphere of Pinus densiflora: Byung-Chun Kim , Kang Hyun Lee , Mi Na Kim , Eun-Mi Kim , Moon-Soo Rhee , O-Yu Kwon , Kee-Sun Shin; J. Microbiol. 2009;47(5):530-535. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0270-z

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Abstract: A novel cellulolytic bacterium, strain S23T, was isolated from the rhizosphere of the pine trees in Daejeon, Republic of Korea. This isolate was Gram-positive, strictly aerobic, rod-shaped, catalase-negative, oxidase- positive, motile by means of peritrichous flagella, and tested positive for alkaline phosphatase, esterase lipase, leucine arylamidase, α-galactosidase, and β-galactosidase activities. The DNA G+C content was 49.5mol%. The main cellular fatty acids were anteiso-C15:0 (51.9%), iso-C16:0 (14.7%), and iso-C15:0 (13.2%). The major isoprenoid quinone was menaquinone 7 (MK-7). Diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. Comparative 16S rRNA gene sequence analysis showed that this strain clustered with Paenibacillus species. The 16S rRNA gene sequence similarity values between S23T and other Paenibacillus species were between 89.9% and 95.9%, and S23T was most closely related to Paenibacillus tarimensis SA-7-6T. On the basis of phylogenetic and phenotypic properties of strain S23T, the isolate is considered as a novel species belonging to the genus Paenibacillus. Therefore, the name, Paenibacillus pinihumi sp. nov., is proposed for the rhizosphere isolate; the type strain is S23T (=KCTC 13695T =KACC 14199T =JCM 16419T)

Nitroreductase II Involved in 2,4,6-Trinitrotoluene Degradation: Purification and Characterization from Klebsiella sp. C1: Jung-Hye Shin , Hong-Gyu Song; J. Microbiol. 2009;47(5):536-541. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-008-0171-6

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Abstract: Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. C1 have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen- insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-aminodinitrotoluene).

Characterization of a Novel β-Glucosidase-Like Activity from a Soil Metagenome: Chengjian Jiang , Gefei Ma , Shuangxi Li , Tingting Hu , Zhiqun Che , Peihong Shen , Bing Yan , Bo Wu; J. Microbiol. 2009;47(5):542-548. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0024-y

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Abstract: We report the cloning of a novel β-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgl1C and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known β-glucosidases. The putative β-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacІ; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant Bgl1C protein hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. The maximum activity for Bgl1C protein occurred at pH 8.0 and 42°C using p-nitrophenyl-β-D-glucoside as the substrate. A CaCl2 concentration of 1 mM was required for optimal activity. The putative β-glucosidase had an apparent Km value of 0.19 mM, a Vmax value of 4.75 U/mg and a kcat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of Bgl1C has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.

Journal Article

Analysis and Identification of ADP-Ribosylated Proteins of Streptomyces coelicolor M145: András Penyige , Judit Keser&# , Ferenc Fazakas , Iván Schmelczer , Krisztina Szirák , György Barabás , Sándor Biró; J. Microbiol. 2009;47(5):549-556. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0032-y

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Abstract: Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD+ to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD+/NADH or NADP+/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SCO5477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SCO1968 are secreted hydrolases. Dehydrogenases are encoded by SCO4824 and SCO4771. The other targets are GlnA (glutamine synthetaseI., SCO2198) and SpaA (starvation-sensing protein encoded by SCO7629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.

Research Support, Non-U.S. Gov't

Overexpression of Outer Membrane Protein OprT and Increase of Membrane Permeability in phoU Mutant of Toluene-Tolerant Bacterium Pseudomonas putida GM730: Kyunghee Lee , Juna Jung , Kwang Kim , Dongwon Bae , Dongbin Lim; J. Microbiol. 2009;47(5):557-562. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0105-y

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Abstract: Eight toluene-sensitive mutants were previously isolated from the toluene-tolerant bacterium Pseudomonas putida GM730. One of these mutants was TOS6, in which Tn5 had been inserted into phoU. Susceptibility to multiple antibiotics, as well as toluene sensitivity, was increased in the phoU mutant of P. putida GM730. We compared the outer membrane proteins from the phoU mutant and wild-type via two-dimensional gel electrophoresis. A 45 kDa protein was dramatically overexpressed as the result of phoU inactivation, and this protein was identified by peptide mass fingerprinting and microsequencing as a conserved hypothetical protein consisting of 414 amino acids. The protein, designated as OprT, harbors a signal sequence and extended β-sheets, both of which are features common to the bacterial porins. The rate of ethidium bromide accumulation in TOS6 was higher than in GM730, which indicates that the TOS6 membranes may be more permeable to ethidium bromide than are the membranes of GM730. We propose that the toluene sensitivity and increased antibiotic susceptibility observed in the phoU mutant may be attributable to increased membrane permeability.

Journal Article

Response Surface Analysis for the Production of an Enantioselective Lipase from Aspergillus niger by Solid-State Fermentation: Fabiano Jares Contesini , Vania Castriani Fernades da Silva , Rafael Ferreira Maciel , Rosemary Joana de Lima , Francisco Fábio Cavalcante Barros , Patrícia de Oliveira Carvalho; J. Microbiol. 2009;47(5):563-571. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-008-0279-8

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Abstract: The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: water content, glucose, yeast extract, peptone, olive oil, temperature, NaH2PO4, KH2PO4, MgSO4․7H2O, CaCl2, NaCl, and MnSO4. The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH2PO4, and KH2PO4, which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2PO4 (4.0%). The predicted lipase activity was 33.03 U/ml and the experimental data confirmed the validity of the model. The enzymatic activity was expressed as µmoles of oleic acid released per minute of reaction (µmol/min).

Research Support, Non-U.S. Gov't

Ligand-Receptor Recognition for Activation of Quorum Sensing in Staphylococcus aureus: Li-Chun Chen , Li-Tse Tsou , Feng-Jui Chen; J. Microbiol. 2009;47(5):572-581. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0004-2

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Abstract: The accessory gene regulator (agr) locus controls many of the virulence toxins involved in Staphylococcus aureus pathogenesis, and can be divided into four specificity groups. AgrC is the only group-specific receptor to mediate both intra-group activation and inter-group inhibition. We studied the ligand-receptor recognition of the agr system in depth by using a luciferase reporter system to identify the key residues responsible for AgrC activation in two closely related agr groups, AgrC-I, and AgrC-IV. Fusion PCR and site-directed mutagenesis were used to screen for functional residues of AgrC. Our data suggest that for AgrC-IV activation, residue 101 is critical for activating the receptor. In contrast, the key residues for the activation of AgrC-I are located at residues 49~59, 107, and 116. However, three residue changes, T101A, V107S, I116S, are sufficient to convert the AIP recognizing specificity from AgrC-IV to AgrC-I.

Validation Study

Development and Validation of a Recombinant Nucleocapsid Protein-Based ELISA for Detection of the Antibody to Porcine Reproductive and Respiratory Syndrome Virus: Jia-Qi Chu , Xu-Min Hu , Myung-Cheol Kim , Chang-Sik Park , Moo-Hyung Jun; J. Microbiol. 2009;47(5):582-588. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0033-x

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Abstract: Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.

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

Identification of a Novel Linear B-Cell Epitope in the M Protein of Avian Infectious Bronchitis Coronaviruses: Junji Xing , Shengwang Liu , Zongxi Han , Yuhao Shao , Huixin Li , Xiangang Kong; J. Microbiol. 2009;47(5):589-599. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0104-z

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Abstract: This report describes the identification of a novel linear B-cell epitope at the C-terminus of the membrane (M) protein of avian infectious bronchitis virus (IBV). A monoclonal antibody (MAb) (designated as 15E2) against the IBV M protein was prepared and a series of 14 partially-overlapping fragments of the IBV M gene were expressed with a GST tag. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using MAb 15E2 to identify the epitope. A linear motif, 199FATFVYAK206, which was located at the C-terminus of the M protein, was identified by MAb 15E2. ELISA and western blotting also showed that this epitope could be recognized by IBV-positive serum from chicken. Given that 15E2 showed reactivity with the 199FATFVYAK206 motif, expressed as a GST fusion protein, in both western blotting and in an ELISA, we proposed that this motif represented a linear B-cell epitope of the M protein. The 199FATFVYAK206 motif was the minimal requirement for reactivity as demonstrated by analysis of the reactivity of 15E2 with several truncated peptides that were derived from the motif. Alignment and comparison of the 15E2-defined epitope sequence with the sequences of other coronaviruses indicated that the epitope is well conserved among chicken and turkey coronaviruses. The identified epitope should be useful in clinical applications and as a tool for the further study of the structure and function of the M protein of IBV.

Production of and Applications for a Polyclonal IgY Diagnostic Reagent Specific for Mycobacterium avium subsp. paratuberculosis: Sung Jae Shin , Seung-Sub Lee , Elizabeth J. B. Manning , Michael T. Collins; J. Microbiol. 2009;47(5):600-609. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0052-7

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Abstract: Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-labeled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immuno- magnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.

Retracted Publication

Identification and Characterization of a Novel Antibacterial Peptide, Avian β-Defensin 2 from Ducks: Deying Ma , Ruiqin Wang , Wenyan Liao , Zongxi Han , Shengwang Liu; J. Microbiol. 2009;47(5):610-618. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0068-z

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33 Citations

Abstract: In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, -20°C to 100°C) and pH values (range, 3 to 12).

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

The Photodynamic Effect of Methylene Blue and Toluidine Blue on Candida albicans Is Dependent on Medium Conditions: Gabriela Guimarães Carvalho , Monalisa Poliana Felipe , Maricilia Silva Costa; J. Microbiol. 2009;47(5):619-623. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0059-0

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48 Citations

Abstract: Due to the increased number of immunocompromised patients, the infections associated with the pathogen of the genus Candida and other fungi have increased dramatically. Photodynamic antimicrobial chemotherapy (PACT) has been presented as a potential antimicrobial therapy, in a process that combines light and a photosensitizing drug, which promotes a phototoxic response by the treated cells. In this work, we studied the effects of the different medium conditions during PACT, using either methylene blue (MB) or toluidine blue (TB) on Candida albicans. The inhibition of the growth produced by PACT was decreased for different pH values (6.0, 7.0, and 8.0) in a buffered medium. The phototoxic effects were observed only in the presence of saline (not buffered medium). PACT was modulated by calcium in a different manner using either MB or TB. Also when using MB both verapamil or sodium azide were able to decrease the phototoxic effects on the C. albicans. These results show that PACT is presented as a new and promising antifungal therapy, however, new studies are necessary to understand the mechanism by which this event occurs.

Identification of the Vibrio vulnificus ahpC1 Gene and Its Influence on Survival under Oxidative Stress and Virulence: Woon Ki Baek , Hyun Sung Lee , Man Hwan Oh , Myung Jin Koh , Kun-Soo Kim , Sang Ho Choi; J. Microbiol. 2009;47(5):624-632. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0130-x

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Abstract: Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpC1, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H2O2. In order to analyze the role of AhpC1 in the pathogenesis of V. vulnificus, a mutant, in which the ahpC1 gene was disrupted, was constructed by allelic exchanges. The ahpC1 mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H2O2 and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpC1 reduced H2O2 in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpC1 is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpC1 mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpC1 mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpC1 and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.

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