Journal of Microbiology

Volume 53(8); August 2015

Review

MINIREVIEW] Regulation of Escherichia coli RNase III activity: Boram Lim , Minji Sim , Howoon Lee , Seogang Hyun , Younghoon Lee , Yoonsoo Hahn , Eunkyoung Shin , Kangseok Lee; J. Microbiol. 2015;53(8):487-494. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5323-x

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12 Citations

Abstract: Bacterial cells respond to changes in the environment by adjusting their physiological reactions. In cascades of cellular responses to stresses of various origins, rapid modulation of RNA function is known to be an effective biochemical adaptation. Among many factors affecting RNA function, RNase III, a member of the phylogenetically highly conserved endoribonuclease III family, plays a key role in posttranscriptional regulatory pathways in Escherichia coli. In this review, we provide an overview of the factors affecting RNase III activity in E. coli.

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

Aliisedimentitalea scapharcae gen. nov., sp. nov., isolated from ark shell Scapharca broughtonii: Young-Ok Kim , Sooyeon Park , Bo-Hye Nam , Dong-Gyun Kim , Sung-Min Won , Ji-Min Park , Jung-Hoon Yoon; J. Microbiol. 2015;53(8):495-502. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5075-7

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Abstract: A Gram-negative, aerobic, non-spore-forming, motile and ovoid or rod-shaped bacterial strain, designated MA2-16T, was isolated from ark shell (Scapharca broughtonii) collected from the South Sea, South Korea. Strain MA2-16T was found to grow optimally at 30캜, at pH 7.0?.0 and in the presence of 2.0% (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain MA2-16T clustered with the type strain of Sedimentitalea nanhaiensis. The novel strain exhibited a 16S rRNA gene sequence similarity value of 97.1% to the type strain of S. nanhaiensis. In the neighbour- joining phylogenetic tree based on gyrB sequences, strain MA2-16T formed an evolutionary lineage independent of those of other taxa. Strain MA2-16T contained Q-10 as the predominant ubiquinone and C18:1 ?c and 11-methyl C18:1 ?c as the major fatty acids. The major polar lipids of strain MA2-16T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA2- 16T was 57.7 mol% and its DNA-DNA relatedness values with the type strains of S. nanhaiensis and some phylogenetically related species of the genera Leisingera and Phaeobacter were 13?4%. On the basis of the data presented, strain MA2-16T is considered to represent a novel genus and novel species within the family Rhodobacteraceae, for which the name Aliisedimentitalea scapharcae gen. nov., sp. nov. is proposed. The type strain is MA2-16T (=KCTC 42119T =CECT 8598T).

Journal Article

Description of a novel pectin-degrading bacterial species Prevotella pectinovora sp. nov., based on its phenotypic and genomic traits: Brigita Nograsek , Tomaz Accetto , Lijana Fanedl , Gorazd Avgustin; J. Microbiol. 2015;53(8):503-510. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5142-0

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Abstract: Five strictly anaerobic Gram-negative bacterial strains, P4-65, P4-76T, P5-60, P5-119, and P5-125, presumably belonging to the genus Prevotella were isolated from pig fecal samples. Strains were tested for various phenotypic traits and nearcomplete genome sequences were obtained and analyzed. Phylogenetic analysis based on 16S rRNA gene sequences and multilocus sequence analysis based on five conserved genes confirmed that the strains belong to the genus Prevotella, revealing that they represent a novel and discrete lineage distinct from other known species of this genus. The size of the genome of the isolated strains is 3?.3 Mbp, and the DNA G+C content is 47.5?8.1 mol%. The isolates are strictly anaerobic, rod-shaped with rounded ends, non-motile and non-spore-forming. The main fermentation products are succinate and acetate, with minor concentrations of isovalerate, propionate and isobutyrate. Hydrogen is also produced. Major cellular fatty acids consist of anteiso-C15:0 and iso-C15:0, and a number of additional acids are present in lower concentrations. A substantial portion of genes involved in carbohydrate utilization is devoted to pectin degradation and utilization, while those supporting growth on xylan in ruminal Prevotella could not have been revealed. On the basis of the presented results, a novel species, Prevotella pectinovora sp. nov. is proposed. The type strain is P4-76T (=DSM 29996T =ZIM B1020T).

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

Isolation of an algicide from a marine bacterium and its effects against the toxic dinoflagellate Alexandrium catenella and other harmful algal bloom species: Yun Sook Kim , Hong-Joo Son , Seong-Yun Jeong; J. Microbiol. 2015;53(8):511-517. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5303-1

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27 Citations

Abstract: The aim of this study was to isolate and identify bacteria demonstrating an algicidal effect against Alexandrium catenella and to determine the activity and range of any algicide discovered. The morphological and biochemical attributes of an algicidal bacterium, isolate YS-3, and analysis of its 16S rRNA gene sequence revealed it to be a member of the genus Brachybacterium. This organism, designated Brachybacterium sp. YS-3, showed the greatest effect against A. catenella cells of all bacteria isolated, and is assumed to produce secondary metabolites. When 10% solutions of culture filtrates from this strain were applied to A. catenella cultures, over 90% of cells were killed within 9 h. Bioassay-guided isolation of the algicide involved led to the purification and identification of an active compound. Based on physicochemical and spectroscopic data, including nuclear magnetic resonance and mass analyses, this compound was identified as 1-acetyl-β-carboline. This algicide showed significant activity against A. catenella and a wide range of harmful algal bloom (HAB)-forming species. Taken together, our results suggest that Brachybacterium sp. YS-3 and its algicide represent promising candidates for use in HAB control.

Journal Article

Characterization of MocR, a GntR-like transcriptional regulator, in Bradyrhizobium japonicum: its impact on motility, biofilm formation, and soybean nodulation: May Nyan Taw , Hae-In Lee , Sang-Ho Lee , Woo-Suk Chang; J. Microbiol. 2015;53(8):518-525. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5313-z

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Abstract: Bradyrhizobium japonicum is a Gram-negative soil bacterium that can fix nitrogen into ammonia by developing a symbiotic relationship with the soybean plant. MocR proteins make up a subfamily of GntR superfamily, one of the most widely distributed and prolific groups of the helix-turn-helix transcription factors. In this study, we constructed a mutant strain for mocR (blr6977) to investigate its role in cellular processes and symbiosis in B. japonicum. Although growth rate and morphology of the mutant were indistinguishable from those of the wild type, the mutant showed significant differences in motility and attachment (i.e., biofilm formation) from the wild type. The mutant displayed a decrease in biofilm formation, but was more motile than the wild type. The inactivation of mocR did not affect the number of nodules on soybean roots, but caused delayed nodulation. Delayed nodulation intrigued us to study competitiveness of the mutant infecting soybeans. The mutant was less competitive than the wild type, indicating that delayed nodulation might be due to competitiveness. Gene expressions of other MocR subfamily members were also compared between the wild type and mutant strains. None of the mocR-like genes examined in this study were differentially expressed between both strains.

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

Role of the extracytoplasmic function sigma factor CarQ in oxidative response of Bradyrhizobium japonicum: Anchana Thaweethawakorn , Dylan Parks , Jae-Seong So , Woo-Suk Chang; J. Microbiol. 2015;53(8):526-534. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5308-9

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Abstract: As a nitrogen-fixing bacterium, Bradyrhizobium japonicum can establish a symbiotic relationship with the soybean plant (Glycine max). To be a successful symbiont, B. japonicum must deal with plant defense responses, such as an oxidative burst. Our previous functional genomics study showed that carQ (bll1028) encoding extracytoplasmic function (ECF) sigma factor was highly expressed (107.8-fold induction) under oxidative stress. Little is known about the underlying mechanisms of how CarQ responds to oxidative stress. In this study, a carQ knock-out mutant was constructed using site-specific mutagenesis to identify the role of carQ in the oxidative response of B. japonicum. The carQ mutant showed a longer generation time than the wild type and exhibited significantly decreased survival at 10 mM H2O2 for 10 min of exposure. Surprisingly, there was no significant difference in expression of oxidative stress-responsive genes such as katG and sod between the wild type and carQ mutant. The mutant also showed a significant increase in susceptibility to H2O2 compared to the wild type in the zone inhibition assay. Nodulation phenotypes of the carQ mutant were distinguishable compared to those of the wild type, including lower numbers of nodules, decreased nodule dry weight, decreased plant dry weight, and a lower nitrogen fixation capability. Moreover, desiccation of mutant cells also resulted in significantly lower percent of survival in both early (after 4 h) and late (after 24 h) desiccation periods. Taken together, this information will provide an insight into the role of the ECF sigma factor in B. japonicum to deal with a plant-derived oxidative burst.

Functional properties of the major outer membrane protein in Stenotrophomonas maltophilia: Yih-Yuan Chen , Han-Chiang Wu , Juey-Wen Lin , Shu-Fen Weng; J. Microbiol. 2015;53(8):535-543. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5202-5

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Abstract: Stenotrophomonas maltophilia is an opportunistic pathogen that is closely associated with high morbidity and mortality in debilitated and immunocompromised individuals. Therefore, to investigate the pathogenesis mechanism is urgently required. However, there are very few studies to evaluate the functional properties of outer membrane protein, which may contribute to the pathogenesis in S. maltophilia. In this study, three abundant proteins in the outer membrane fraction of S. maltophilia were identified by liquid chromatography- tandem mass spectrometry as OmpW1, MopB, and a hypothetical protein. MopB, a member of the OmpA family, was firstly chosen for functional investigation in this study because many OmpA-family proteins are known to be involved in pathogenesis and offer potential as vaccines. Membrane fractionation analyses demonstrated that MopB was indeed the most abundant outer membrane protein (OMP) in S. maltophilia. For functional studies, the mopB mutant of S. maltophilia (SmMopB) was constructed by insertional mutation. MopB deficiency resulted in a change in the protein composition of OMPs and altered the architecture of the outer membrane. The SmMopB strain exhibited reduced cytotoxicity toward L929 fibroblasts and was more sensitive to numerous stresses, including human serum, sodium dodecyl sulfate, and hydrogen peroxide compared with wildtype S. maltophilia. These results suggest that MopB may be a good candidate for the design of vaccines or anti-MopB drugs for controlling serious nosocomial infections of multidrug- resistant S. maltophilia, especially in immunosuppressed patients.

Journal Articles

The role of programmed death ligand 1 pathway in persistent biomaterial-associated infections: Agnieszka Magrys , Jolanta Paluch-Oles , Agnieszka Bogut , Michal Kielbus , Dorota Plewik , Maria Koziol-Montewka; J. Microbiol. 2015;53(8):544-552. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5022-7

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Abstract: Staphylococcus epidermidis is commonly involved in biomaterial- associated infections. Bacterial small colony variants (SCV) seem to be well adapted to persist intracellularly in professional phagocytes evading the host immune response. We studied the expression of PD-L1/L2 on macrophages infected with clinical isolates of S. epidermidis SCV and their parent wild type (WT) strains. The cytokine pattern which is triggered by the examined strains was also analysed. In the study, we infected macrophages with S. epidermidis WT and SCV strains. Persistence and release from macrophages were monitored via lysostaphin protection assays. Moreover, the effect of IFN-γ pre-treatment on bacterial internalisation was investigated. Expression of PD-L1/L2 molecules was analysed with the use of FACS. Inflammatory reaction was measured by IL-10, TNF-α ELISAs, and transcriptional induction of TNF-α. Our study revealed that clinical SCV isolates were able to persist and survive in macrophages for at least 3 days with a low cytotoxic effect and a reduced proinflammatory response as compared to WT strains. Bacteria upregulated PD-L1/L2 expression on macrophages as compared to non-stimulated cells. The results demonstrated that the ability of S. epidermidis SCVs to induce elevated levels of anti-inflammatory cytokine, IL-10, and reduced transcriptional induction of TNF-α, together with expression of PD-L1 on macrophages and the ability to persist intracellularly without damaging the host cell could be the key factor contributing to chronicity of SCV infections.

Antagonistic effect of peptidoglycan of Streptococcus sanguinis on lipopolysaccharide of major periodontal pathogens: Sung-Hoon Lee; J. Microbiol. 2015;53(8):553-560. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5319-6

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16 Citations

Abstract: Streptococcus sanguinis is often found in subgingival biofilm including periodontopathogens, and is correlated with a delay in colonization by periodontopathogens. However, the effect of S. sanguinis on inflammation induced by periodontopathogens is poorly understood. Thus, this study investigated the effect of S. sanguinis peptidoglycan (PGN) on induction of TNF-α, IL-6, and IL-8 expression by lipopolysaccharide (LPS) of periodontal pathogens. LPS was extracted from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia, and PGN was isolated from S. sanguinis. THP-1 cells, a monocytic cell-line, were cotreated with LPS of the periodontal pathogens and S. sanguinis PGN, and then the expression of inflammatory cytokines was analyzed by real-time RT-PCR. To analyze the underlying mechanism, the binding assay of the LPS to CD14 or LPS-binding protein (LBP) was performed in the presence or absence of the PGN after coating recombinant human CD14 and LBP on EIA plate. The PGN inhibited the binding of LPS to CD14 and LBP in a dose-dependent manner. Also, THP-1 cells were co-treated with the LPS in the presence of N-acetylmuramic acid and N-acetylglucosamine, as components of PGN, and the competition binding assay to CD14 and LBP was performed. N-acetylmuramic acid inhibited the induction of inflammatory cytokine expression by LPS and the binding of LPS to CD14 or LBP whereas Nacetylglucosamine did not show such effect. Collectively, the
results
suggest that S. sanguinis PGN inhibited the cytokine expression induced by the LPS of periodontopathogens due to the inhibition of LPS binding to LBP and CD14. N-acetylmuramic acid of PGN may play a role in inhibition of the LPS binding of periodontopathogens to CD14 and LBP.

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

Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication: Young-Eui Kim , Mi Young Park , Kyeong Jin Kang , Tae Hee Han , Chan Hee Lee , Jin-Hyun Ahn; J. Microbiol. 2015;53(8):561-569. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5301-3

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Abstract: The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112- 113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112- 113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112- 113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112- 113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our
results
demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

Molecular characterization of mammalian-adapted Korean-type avian H9N2 virus and evaluation of its virulence in mice: Kuk Jin Park , Min-Suk Song , Eun-Ha Kim , Hyeok-il Kwon , Yun Hee Baek , Eun-hye Choi , Su-Jin Park , Se Mi Kim , Young-il Kim , Won-Suk Choi , Dae-Won Yoo , Chul-Joong Kim , Young Ki Choi; J. Microbiol. 2015;53(8):570-577. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5329-4

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Abstract: Avian influenza A virus (AIV) is commonly isolated from domestic poultry and wild migratory birds, and the H9N2 subtype is the most prevalent and the major cause of severe disease in poultry in Korea. In addition to the veterinary concerns regarding the H9N2 subtype, it is also considered to be the next potential human pandemic strain due to its rapid evolution and interspecies transmission. In this study, we utilize serial lung-to-lung passage of a low pathogenic avian influenza virus (LPAI) H9N2 (A/Ck/Korea/163/04, WT163) (Y439-lineage) in mice to increase pathogenicity and investigate the potential virulence marker. Mouse-adapted H9N2 virus obtained high virulence (100% mortality) in mice after 98 serial passages. Sequence results show that the mouse adaptation (ma163) possesses several mutations within seven gene segments (PB2, PA, HA, NP, NA, M, and NS) relative to the wild-type strain. The HA gene showed the most mutations (at least 11) with one resulting in the loss of an N-glycosylation site (at amino acid 166). Moreover, reverse genetic studies established that an E627K substitution in PB2 and the loss of the N-glycosylation site in the HA protein (aa166) are critical virulence markers in the mouse-adapted H9N2 virus. Thus, these results add to the increasing body of mutational analysis data defining the function of the viral polymerase and HA genes and their roles in mammalian host adaptation. To our knowledge, this is first report of the generation of a mammalian-adapted Korea H9N2 virus (Y493-lineages). Therefore, this study offers valuable insights into the molecular evolution of the LPAI Korean H9N2 in a new host and adds to the current knowledge of the molecular markers associated with increased virulence.

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