Journal of Microbiology

Volume 45(1); February 2007

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

Comparison of Bacterial Composition between Human Saliva and Dental Unit Water System: Eun-Hyoung Jeon , Ji-Hye Han , Tae-Young Ahn; J. Microbiol. 2007;45(1):1-5.; DOI: https://doi.org/2500 [pii]

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Abstract: The bacterial compositions between the dental unit water system and human saliva were characterized and compared by direct sequence analysis of 16S rDNA clone libraries. Based on the species richness estimation, bacterial diversity in the dental unit water system (DUW) was more diverse than that of the human saliva (HS). The Chao1 estimates of species richness in HS and DUW samples were 12.0 and 72.4, respectively. The total numbers of OTUs observed in the combined libraries accounted for 83% (HS) and 59% (DUW) of the Chao1 diversity estimate as defined at the 80% similarity threshold. Based on the sequence analysis, the phylum Proteobacteria was the major group in both clone libraries at phylum level. DUW clone library contained 80.0% Proteobacteria, 8.0% Bacteroides, 4.0% Nitrospira, 4.0% Firmicutes, 2.0% Planctomycetes and 2.0% Acidobacteria. On the other hand, human saliva (HS) clone library contained 55.5% Proteobacteria, 36.1% Firmicutes and 8.4% Bacteroides. The majority of bacteria identified belonged to phylum Proteobacteria in both samples. In dental unit water system (DUW), Alphaproteobacteria was detected as the major group. There was no evidence of the bacterial contamination due to a dental treatment. Most sequences were related to microorganisms derived from biofilm in oligotrophic environments.

Production and Biological Activity of Laidlomycin, Anti-MRSA/VRE Antibiotic from Streptomyces sp. CS684: Jin Cheol Yoo , Jun Ho Kim , Jung Wan Ha , Nae Soo Park , Jae Kyung Sohng , June Woo Lee , Seong Chan Park , Mi Sun Kim , Chi Nam Seong; J. Microbiol. 2007;45(1):6-10.; DOI: https://doi.org/2499 [pii]

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Abstract: Culture broth of a streptomycete isolate, Streptomyces sp. CS684 showed antibacterial activity on methicilin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Among purified substances from the organism, CSU-1, which is active against MRSA and VRE, is a C37H62O12Na (M+, 721.3875), and identified as laidlomycin. The anti-MRSA and anti-VRE activity of CSU-1 was stronger than oxacillin and vancomycin. Phylogenetic analysis showed that strain CS684 is very similar to Streptomyces ardus NRRL 2817T, whereas the ability of Streptomyces sp. CS684 to produce laidlomycin was shown to be unique.

Evaluation of Endophytic Colonization of Citrus sinensis and Catharanthus roseus Seedlings by Endophytic Bacteria: Paulo Teixeira Lacava , Welington Luiz Araujo , Joao Lucio Azevedo; J. Microbiol. 2007;45(1):11-14.; DOI: https://doi.org/2498 [pii]

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Abstract: Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.

Differential Response of Etiolated Pea Seedlings to Inoculation with Rhizobacteria Capable of Utilizing 1-Aminocyclopropane-1-Carboxylate or L-Methionine: Baby Shaharoona , Muhammad Arshad , Azeem Khalid; J. Microbiol. 2007;45(1):15-20.; DOI: https://doi.org/2497 [pii]

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Abstract: The majority of soil microorganisms can derive ethylene from L-methionine (L-MET), while some rhizobacteria can hydrolyze 1-aminocyclopropane-1-carboxylate (ACC) due to their ACC-deaminase activity. In this study, three strains having either ACC-deaminase activity (Pseudomonas putida biotype A, A7), or the ability to produce ethylene from L-MET (Acinetobacter calcoaceticus, M9) or both (Pseudomonas fluorescens, AM3) were used for inoculation. The highly ethylene specific bioassay of a classical “triple” response in pea seedlings was used to investigate the effect of the inoculation with the rhizobacteria in the presence of 10 mM ACC or L-MET. The exogenous application of ACC had a concentration-dependent effect on the etiolated pea seedlings in creating the classical “triple” response. The inoculation with P. putida diluted the effect of ACC, which was most likely due to its ACC-deaminase activity. Similarly, the application of Co2+ reduced the ACC-imposed effect on etiolated pea seedlings. In contrast, the inoculation of A. calcoaceticus or P. fluorescens in the presence of L-MET caused a stronger classical “triple” response in etiolated pea seedlings; most likely by producing ethylene from L-MET. This is the first study, to our knowledge, reporting on the comparative effect of rhizobacteria capable of utilizing ACC vs L-MET on etiolated pea seedlings.

Comparative Genomics Profiling of Clinical Isolates of Helicobacter pylori in Chinese Populations Using DNA Microarray: Yue-Hua Han , Wen-Zhong Liu , Yao-Zhou Shi , Li-Qiong Lu , Shudong Xiao , Qing-Hua Zhang , Guo-Ping Zhao; J. Microbiol. 2007;45(1):21-28.; DOI: https://doi.org/2496 [pii]

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Abstract: In order to search for specific genotypes related to this unique phenotype, we used whole genomic DNA microarray to characterize the genomic diversity of Helicobacter pylori (H. pylori) strains isolated from clinical patients in China. The open reading frame (ORF) fragments on our microarray were generated by PCR using gene-specific primers. Genomic DNA of H. pylori 26695 and J99 were used as templates. <br>Thirty-four H. pylori isolates were obtained from patients in Shanghai. Results were judged based on ln(x) transformed and normalized Cy3/Cy5 ratios. Our microarray included 1882 DNA fragments corresponding to 1636 ORFs of both sequenced H. pylori strains. Cluster analysis, revealed two diverse regions in the H. pylori genome that were not present in other isolates. Among the 1636 genes, 1091 (66.7%) were common to all H. pylori strains, representing the functional core of the genome. Most of the genes found in the H. pylori functional core were responsible for metabolism, cellular processes, transcription and biosynthesis of amino acids, functions that are essential to H. pylori’s growth and colonization in its host. In contrast, 522 (31.9%) genes were strain-specific genes that were missing from at least one strain of H. pylori. Strainspecific genes primarily included restriction modification system components, transposase genes, hypothetical proteins and outer membrane proteins. These strain-specific genes may aid the bacteria under specific circumstances during their long-term infection in genetically diverse hosts. Our results suggest 34 H. pylori clinical strains have extensive genomic diversity. Core genes and strain-specific genes both play essential roles in H. pylori propagation and pathogenesis. Our microarray experiment may help select relatively significant genes for further research on the pathogenicity of H. pylori and development of a <br>vaccine for H. pylori.

Identification of Genes Differentially Expressed in RAW264.7 Cells Infected by Salmonella typhimurium Using PCR Method: Kyung Ho Kang , Jung A Song , Dong-Jun Shin , Hyon E Choy , Yeongjin Hong; J. Microbiol. 2007;45(1):29-33.; DOI: https://doi.org/2495 [pii]

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Abstract: Salmonella typhimurium, causing mouse typhoid, infects hosts such as macrophage cells, and proliferates in intracellular vacuoles causing infected cells to trigger numerous genes to respond against the infection. In this study, we tried to identify such genes in RAW264.7 cells by using the PCR screening method with degenerate primers. Fourteen genes were found to be differentially expressed after a 4 h infection in which the expression of 8 genes increased while expression of the others decreased. Most of the genes were involved in proinflammatory responses such as cytokines production and cell death. The mutation in msbB gene encoding the myristoyl transferase in lipid A of lipopolysaccharide (LPS) resulted in much lower toxicity to the inoculated animals. We compared the expression of the identified genes in wild-type and msbB-mutated S. typhimurium infections and found that Lyzs encoding lysozyme type M was differentially expressed. This gene is quite likely to be related to bacterial survival in the host cells.

Requirement of Bni5 Phosphorylation for Bud Morphogenesis in Saccharomyces cerevisiae: Sung Chang Nam , Hyeran Sung , Yeon Bok Chung , Chong-Kil Lee , Dong Hun Lee , Sukgil Song; J. Microbiol. 2007;45(1):34-40.; DOI: https://doi.org/2494 [pii]

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Abstract: In budding yeast, G2/M transition is tightly correlated with bud morphogenesis regulated by Swe1 and septin that plays as a scaffold to recruits protein components. BNI5 isolated as a suppressor for septin defect is implicated in septin organization and cytokinesis. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we show that Bni5 phosphorylation is required for mitotic entry regulated by Swe1 pathway. Bni5 modification was evident from late mitosis to G1 phase, and CIP treatment in vitro of affinity-purified Bni5 removed the modification, indicative of phosphorylation on Bni5. The phosphorylation-deficient mutant of BNI5 (bni5-4A) was defective in both growth at semi-restrictive temperature and suppression of septin defect. Loss of Bni5 phosphorylation resulted in abnormal bud morphology and cell cycle delay at G2 phase, as evidenced by the formation of elongated cells with multinuclei. However, deletion of Swe1 completely eliminated the elongated-bud phenotypes of both bni5 deletion and bni5-4A mutants. These results suggest that the bud morphogenesis and mitotic entry are positively regulated by phosphorylation-dependent function of Bni5 which is under the control of Swe1 morphogenesis pathway.

Defining the N-Linked Glycosylation Site of Hantaan Virus Envelope Glycoproteins Essential for Cell Fusion: Feng Zheng , Lixian Ma , Lihua Shao , Gang Wang , Fengzhe Chen , Ying Zhang , Song Yang; J. Microbiol. 2007;45(1):41-47.; DOI: https://doi.org/2493 [pii]

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Abstract: The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.

Development of a Virus Concentration Method and its Application for the Detection of Noroviruses in Drinking Water in China: Junyi Liu , Qingping Wu , Xiaoxia Kou; J. Microbiol. 2007;45(1):48-52.; DOI: https://doi.org/2492 [pii]

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Abstract: A new procedure for the concentration of nonoviruses from water samples has been developed. This procedure (calcium flocculation-citrate dissolution method) uses the following steps: virus flocculation formed by treatment with 1 M CaCl2 and 1 M Na2HPO4, virus release by sodium citrate dissolution (0.3 M Na citrate, pH 3.5), and virus re-concentration by ultrafiltration. When reverse transcription (RT)-PCR was performed after the procedure, the overall detection sensitivity for seeded noroviruses in a one liter drinking water sample was as low as 1 RT-PCR unit, which is equal to a 10-6 dilution of the virus sample. This approach showed at least a 5-fold-higher sensitivity than the current method with its three steps of adsorption-elution-concentration. The newly developed procedure was used to test different brands of bottled drinking water from China for putative contamination with noroviruses. A total of 144 samples were analyzed; all of the samples were negative for norovirus specific nucleic acids.

The Antimicrobial Activity of Essential Oil from Dracocephalum foetidum against Pathogenic Microorganisms: Saet Byoul Lee , Kwang Hyun Cha , Su Nam Kim , Shataryn Altantsetseg , Sanduin Shatar , Oidovsambuu Sarangerel , Chu Won Nho; J. Microbiol. 2007;45(1):53-57.; DOI: https://doi.org/2491 [pii]

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Abstract: A number of essential oils from Mongolian aromatic plants are claimed to have antimicrobial activities. The essential oil of Dracocephalum foetidum, a popular essential oil used in Mongolian traditional medicine, was examined for its antimicrobial activity. Eight human pathogenic microorganisms including B. subtilis, S. aureus, M. lutens, E. hirae, S. mutans, E. coli, C. albicans, and S. cerevisiae were examined. The essential oil of Dracocephalum foetidum exhibited strong antimicrobial activity against most of the pathogenic bacteria and yeast strains that were tested; by both the agar diffusion method and the minimum inhibitory concentration (MIC) assay (MIC range was 26-2592 μg/ml). Interestingly, Dracocephalum foetidum even showed antimicrobial activity against methicilin-resistant Staphylococcus aureus (MRSA) strains. We also analyzed the chemical composition of the oil by GC-MS and identified several major components, including n-Mentha-1,8-dien-10-al, limonene, geranial, and neral.

Studies on Synonymous Codon and Amino Acid Usage Biases in the Broad-Host Range Bacteriophage KVP40: Keya Sau , Sanjib Kumar Gupta , Subrata Sau , Subhas Chandra Mandal , Tapash Chandra Ghosh; J. Microbiol. 2007;45(1):58-63.; DOI: https://doi.org/2490 [pii]

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Abstract: In this study, the relative synonymous codon and amino acid usage biases of the broad-host range phage, KVP40, were investigated in an attempt to understand the structure and function of its proteins/proteincoding genes, as well as the role of its tRNAs. Synonymous codons in KVP40 were determined to be ATrich at the third codon positions, and their variations are dictated principally by both mutational bias and translational selection. Further analysis revealed that the RSCU of KVP40 is distinct from that of its Vibrio hosts, V. cholerae and V. parahaemolyticus. Interestingly, the expression of the putative highly expressed genes of KVP40 appear to be preferentially influenced by the abundant host tRNA species, whereas the tRNAs expressed by KVP40 may be required for the efficient synthesis of all its proteins in a diverse array of hosts. The data generated in this study also revealed that KVP40 proteins are rich in low molecular weight amino acid residues, and that these variations are influenced primarily by hydropathy, mean molecular weight, aromaticity, and cysteine content.

The Diversity of Multi-drug Resistance Profiles in Tetracycline-Resistant Vibrio Species Isolated from Coastal Sediments and Seawater: Farzana Ashrafi Neela , Lisa Nonaka , Satoru Suzuki; J. Microbiol. 2007;45(1):64-68.; DOI: https://doi.org/2489 [pii]

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Abstract: In this study we examined the multi-drug resistance profiles of the tetracycline (TC) resistant genus Vibrio to determine its susceptibility to two β-lactams, ampicillin (ABPC), and mecillinam (MPC), as well as to macrolide, erythromycin (EM). The results showed various patterns of resistance among strains that were isolated from very close geographical areas during the same year, suggesting diverse patterns of drug resistance in environmental bacteria from this area. In addition, the cross-resistance patterns suggested that the resistance determinants among Vibrio spp. are acquired differently within the sediment and seawater environments.

Genetic Characterization of the Escherichia coli O66 Antigen and Functional Identification of its wzy Gene: Jiansong Cheng , Bin Liu , David A. Bastin , Weiqing Han , Lei Wang , Lu Feng; J. Microbiol. 2007;45(1):69-74.; DOI: https://doi.org/2488 [pii]

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Abstract: Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.

Analysis of Substitution Events in HIV-1 vif Gene of the Korean Clade: Chan Seung Park , Mi Sook Kim , Hyun Ah Yi , Dong Hun Lee , Keon Myung Lee , Chan Hee Lee; J. Microbiol. 2007;45(1):75-78.; DOI: https://doi.org/2487 [pii]

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Abstract: Nucleotide and amino acid substitution pattern in vif gene of the Korean clade of HIV-1 isolated from Koreans were analyzed using consensus sequences. At nucleotide level, transition/transversion substitution ratio was 1.88, and nonsynonymous/synonymous substitution ratio was 2.67, suggesting a divergent <br>evolution in the Korean clade. At amino acid level, there were 17 substitutions and G→E substitution at position 37 may be responsible for change in predicted secondary structure.

Nucleotide Sequence and Secondary Structure of 5S rRNA from Sphingobium chungbukense DJ77: Hae-Ryong Kwon , Young-Chang Kim; J. Microbiol. 2007;45(1):79-82.; DOI: https://doi.org/2486 [pii]

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Abstract: The 5S rRNA gene from Sphingobium chungbukense DJ77 was identified. The secondary structure of the 199-base-long RNA was proposed. The two-base-long D loop was the shortest among all of the known 5S rRNAs. The U19-U64 non-canonical pair in the helix II region was uniquely found in strain DJ77 among all of the sphingomonads.

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