Journal of Microbiology

Volume 48(3); June 2010

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

Antimicrobial Susceptibilities of Coagulase-Negative Staphylococci (CNS) and Streptococci from Bovine Subclinical Mastitis Cases: Emel Banu Buyukunal Bal , Suleyman Bayar , Mehmet Ali Bal; J. Microbiol. 2010;48(3):267-274. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9373-9

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Abstract: The prevalence and antimicrobial susceptibilities of Staphylococci and Streptococci were assessed from subclinical mastitis cases. One hundred Coagulase-Negative Staphylococci (CNS) and 34 Streptoccocci were identified. The most frequently isolated species were Staphylococcus haemolyticus (27%) and Staphylococcus simulans (24%). Susceptible CNS species revealed the highest resistance to penicillin G (58%), ampicillin (48%), neomycin (20%), and oleandomycin (14%). CNS methicillin resistance rates within 82 isolates were 21.95% and 1.22% by disk diffusion and PCR methods, respectively. These results suggested the disk diffusion
method
was more prone to yield false positives. Partial sequencing of the 16S rRNA region from the mecA carrying isolate (S. haemolyticus) was homologous with S. haemolyticus sequences/accessions obtained from GenBank. However, the mecA gene sequence from this isolate was more closely allied with the S. aureus mecA gene of human origins. Identical sequence data was acquired from the National Center for Biotechnology Information (NCBI) database, suggesting horizontal gene transfer between the two species. CNS β-lactamase activity within 81 isolates was 29.63%. The most frequently isolated Streptococcus species were S. uberis (52%) and S. agalactiae (15%). Oleandomycin was the least effective antimicrobial agent on these isolates with 59% susceptibility. Results indicated that CNS and Streptococci exhibited various antimicrobial resistance responses. Consequently, isolation and identification of udder pathogens in herds suffering from subclinical agents is essential to select the most effective antimicrobial agent. Moreover, multiple resistance features of methicillin resistant (MR) isolates should be considered during antimicrobial susceptibility tests.

Isolation, Characterization, and Abundance of Filamentous Members of Caldilineae in Activated Sludge: Dae-No Yoon , Soo-Je Park , So-Jeong Kim , Che Ok Jeon , Jong-Chan Chae , Sung-Keun Rhee; J. Microbiol. 2010;48(3):275-283. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9366-8

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49 Citations

Abstract: Chloroflexi are currently believed to serve as backbone forming agents in the activated sludge of wastewater treatment plants (WWTPs). In this study, we isolated and characterized filamentous bacteria in the class Caldilineae of the phylum Chloroflexi in municipal WWTPs. Diversity analysis using Chloroflexi-specific 16S rRNA gene clone libraries showed that 97% of the clones belonged to the subdivision Anaerolineae comprising the two classes Anaerolineae (95%) and Caldilineae (2%). Clones of Caldilineae were related to a thermophilic filament Caldilinea aerophila with 93% 16S rRNA gene sequence similarity. We obtained filamentous isolates classified into the class Caldilineae showing the best match to C. aerophila with 89% 16S rRNA gene sequence similarity. Isolates showed no ability to assimilate glucose or N-acetylglucosamine or to degrade biopolymers which were observed in filamentous Chloroflexi of WWTPs. The assessment of relative abundance based on quantitative PCR of the 16S rRNA gene indicated that members of the class Caldilineae comprised 12-19% of the Chloroflexi in the activated sludge. Additionally, fluorescence in situ hybridization experiments showed that diverse filamentous Caldilineae inhabit the activated sludge of municipal WWTPs. These findings yield insight into the role of filamentous mesophilic Caldilinea in stabilizing flocs of activated sludge in a wide range of WWTPs.

Assessment of Soil Fungal Communities Using Pyrosequencing: Young Woon Lim , Byung Kwon Kim , Changmu Kim , Hack Sung Jung , Bong-Soo Kim , Jae-Hak Lee , Jongsik Chun; J. Microbiol. 2010;48(3):284-289. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9369-5

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107 Citations

Abstract: Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporic Ascomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.

Kinetic Evaluation of Products Inhibition to Succinic Acid Producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130ZT: Qiang Li , Dan Wang , Yong Wu , Maohua Yang , Wangliang Li , Jianmin Xing , Zhiguo Su; J. Microbiol. 2010;48(3):290-296. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9262-2

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Abstract: Succinic acid is one of the platform compounds and its production via natural feedstocks has drawn worldwide concerns. To evaluate the inhibitory effects of fermentation products on the growth of Actinobacillus succinogenes 130ZT and Escherichia coli NZN111, AFP111, BL21, fermentations with addition of individual products in medium were carried out. The cell growth was inhibited when the concentrations of formate, acetate, lactate, and succinate were at range of 8.8-17.6 g/L, 10-40 g/L, 9-18 g/L, and 10-80 g/L, respectively. For these two species of bacteria, E. coli was more resistant to acid products than A. succinogenes, while both endured succinate rather than by-products. As a result of end product inhibition, succinate production yield by A. succinogenes decreased from 1.11 to 0.49 g/g glucose. Logistic and Monod mathematical models were presented to simulate the inhibition kinetics. The Logistic model was found more suitable for describing the overall synergistic inhibitory effects.

Journal Article

Characterization of Cultivated Fungi Isolated from Grape Marc Wastes Through the Use of Amplified rDNA Restriction Analysis and Sequencing: Spyridon Ntougias , Nektarios Kavroulakis , Kalliope K. Papadopoulou , Constantinos Ehaliotis , Georgios I. Zervakis; J. Microbiol. 2010;48(3):297-306. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9193-y

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Abstract: Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7×106 per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.

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

Gaetbulibacter jejuensis sp. nov., Isolated from Seawater: You-Sung Oh , Sang-Bin Lim , Hyung-Yeel Kahng , Byoung-Jun Yoon , Duck-Chul Oh , Jae-Ho Joa , Dong-Heon Lee; J. Microbiol. 2010;48(3):307-311. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9232-8

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Abstract: A novel marine bacterium, designated strain CNURIC014T was isolated from coastal seawater of Jeju Island in Korea. Strain CNURIC014T formed yellow colonies on marine agar 2216 and the cells were Gram-negative, non-motile, strictly aerobic, rod-shaped. The temperature, pH and NaCl ranges for growth were 15-37°C, pH 6.0-9.0 and 1.0-7.0% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNURIC014T was most closely related to Gaetbulibacter marinus and Gaetbulibacter saemankumensis, with a sequence similarity of 95.1% and 94.6%, respectively. The DNA G+C content of the strain was 33.1 mol% and the major respiratory quinone was menaquinone-6. The major cellular fatty acids were iso-C15:1 (22.8%), iso-C15:0 (18.8%), summed feature 3 (iso-C15:0 2-OH/C16:1 ω7c, 12.9%) and iso-C17:0 3-OH (11.5%). On the basis of phenotypic, phylogenetic, and genotypic data, strain CNURIC014T represents a novel species within the genus Geatbulibacter, for which the name Gaetbulibacter jejuensis sp. nov. is proposed. The type strain is CNURIC014T (=KCTC 22615T =JCM 15976T).

Oceanobacillus manasiensis sp. nov., a Moderately Halophilic Bacterium Isolated from the Salt Lakes of Xinjiang, China: Lei Wang , Wen-Yan Liu , Zhi-Jing Gu , San-Feng Chen , Su-Sheng Yang; J. Microbiol. 2010;48(3):312-317. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-0135-5

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10 Citations

Abstract: Three Gram reaction positive, rod-shaped, moderately motile halophilic bacterial strains, designated YD3-56T, YD16, and YH29, were isolated from the sediments of Manasi and Aiding salt lakes in the Xinjiang region of China, respectively. The strains grew optimally at 30-37°C, pH 8-11, in the presence of 5-10% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to members of the genus Oceanobacillus, exhibiting 99.1-99.2% similarity to O. kapialis KCTC 13177T, 99.2-99.3% to O. picturae KCTC 3821T, and 94.2-96% sequence similarity to other described Oceanobacillus species. SDS-PAGE of whole cell proteins preparations demonstrated that the strains exhibited high similarity to each other, but distinguished from O. kapialis KCTC 13177T and O. picturae KCTC 3821T (75%). DNA-DNA hybridization revealed that the similarity between the representative strain YD3-56T and O. kapialis KCTC 13177T was 35.3%, and the similarity between YD3-56T and O. picturae KCTC 3821T was 22.3%. Chemotaxonomic analysis of the strains showed menaquinone-7 was the predominant respiratory quinine. Major cellular fatty acids were anteiso-C15:0 and anteiso-C17:0. The polar lipid pattern for strain YD3-56T predominantly contained phosphatidylcholine, and trace to moderate amounts of phosphatidyl ethanolamine and hydroxy-phosphatidyl ethanolamine. The diamino acid in murein was meso-diaminopimelic acid. The DNA G+C content of the strains was 39.7-40.1 mol%. On the basis of these results, the three strains should be classified as a novel species of the genus Oceanobacillus, for which the name Oceanobacillus manasiensis sp. nov. has been proposed, with the type strain as YD3-56T (=CGMCC 1.9105T =NBRC 105903T).

Cel8H, a Novel Endoglucanase from the Halophilic Bacterium Halomonas sp. S66-4: Molecular Cloning, Heterogonous Expression, and Biochemical Characterization: Xiaoluo Huang , Zongze Shao , Yuzhi Hong , Ling Lin , Chanjuan Li , Fei Huang , Hui Wang , Ziduo Liu; J. Microbiol. 2010;48(3):318-324. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-009-0188-5

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28 Citations

Abstract: A recombinant Escherichia coli clone expressing an endoglucanase was identified from a genomic library of the halophilic bacterium Halomonas sp. S66-4, and the enzyme was designated Cel8H. The cel8H gene consisted of 1,053 bp and encoded 350 amino acids sharing the highest identity of 48% to other known endoglucanases. The protein was expressed in E. coli BL21 (DE3) and purified to homogeneity. The purified recombinant enzyme had an optimal activity of 4.9 U/mg at pH 5 and 45°C toward the substrate carboxymethylcellulose. It exhibited extraordinary properties which differed from endoglucanases reported previously at the point of high salt tolerance above 5 M, simultaneously with high pH stability at pH 4-12 and high temperature stability at 40-60°C. Various substrate tests indicated that the enzyme hydrolyzes β-1,4-glucosidic bonds specifically.

Identification and Characterization of a Novel Bacterial ATP-Sensitive K+ Channel: Seung Bum Choi , Jong-Uk Kim , Hyun Joo , Churl K. Min; J. Microbiol. 2010;48(3):325-330. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9231-9

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Abstract: Five bacterial species that are most likely to have putative prokaryotic inward rectifier K+ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K+ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K+ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed inXenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K+ current with characteristics similar to those of the ATP-sensitive K+ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.

Production, Partial Characterization, and Immobilization in Alginate Beads of an Alkaline Protease from a New Thermophilic Fungus Myceliophthora sp.: Letícia Maria Zanphorlin , Fernanda Dell Antonio Facchini , Filipe Vasconcelos , Rafaella Costa Bonugli-Santos , André Rodrigues , Lara Durães Sette , Eleni Gomes , Gustavo Orlando Bonilla-Rodriguez; J. Microbiol. 2010;48(3):331-336. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9269-8

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Abstract: Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9 (SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

Altered Protein Expression Patterns of Mycobacterium tuberculosis Induced by ATB107: Hongbo Shen , Enzhuo Yang , Feifei Wang , Ruiliang Jin , Shengfeng Xu , Qiang Huang , Honghai Wang; J. Microbiol. 2010;48(3):337-346. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9315-6

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Abstract: ATB107 is a potent inhibitor of indole-3-glycerol phosphate synthase (IGPS). It can effectively inhibit the growth of clinical isolates of drug-resistant Mycobacterium tuberculosis strains as well as M. tuberculosis H37Rv. To investigate the mechanism of ATB107 action in M. tuberculosis, two-dimensional gel electrophoresis coupled with MALDI-TOF-MS analysis (2-DE-MS) was performed to illustrate alterations in the protein expression profile in response to ATB107. Results show that ATB107 affected tryptophan biosynthesis by decreasing the expression of protein encoded by Rv3246c, the transcriptional regulatory protein of MtrA belonging to the MtrA-MtrB two-component regulatory system, in both drug-sensitive and drug-resistant virulent strains. ATB107 might present a stress condition similar to isoniazid (INH) or ethionamide for M. tuberculosis since the altered expression in response to ATB107 of some genes, such as Rv3140, Rv2243, and Rv2428, is consistent with INH or ethionamide treatment. After incubation with ATB107, the expression of 2 proteins encoded by Rv0685 and Rv2624c was down-regulated while that of protein encoded by Rv3140 was up-regulated in all M. tuberculosis strains used in this study. This may be the common response to tryptophan absence; however, relations to ATB107 are unknown and further evaluation is warranted.

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

Phenotypic Diversity of Escherichia coli O157:H7 Strains Associated with the Plasmid O157: Ji Youn Lim , Joon Bae Hong , Haiqing Sheng , Smriti Shringi , Rajinder Kaul , Thomas E. Besser , Carolyn J. Hovde; J. Microbiol. 2010;48(3):347-357. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9228-4

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Abstract: Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

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

Genetic Diversity of Chromosomal Metallo-β-Lactamase Genes in Clinical Isolates of Elizabethkingia meningoseptica from Korea: Jong Hwa Yum , Eun Young Lee , Sung-Ho Hur , Seok Hoon Jeong , Hyukmin Lee , Dongeun Yong , Yunsop Chong , Eun-Woo Lee , Patrice Nordmann , Kyungwon Lee; J. Microbiol. 2010;48(3):358-364. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9308-5

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23 Citations

Abstract: This study was performed to characterize the chromosomal metallo-β-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5α. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the blaGOB genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of blaGOB gene, including 10 novel variants (blaGOB-8 to blaGOB-17). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5α. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.

Proteomic Analysis of Hyphae-Specific Proteins That Are Expressed Differentially in cakem1/cakem1 Mutant Strains of Candida albicans: Kang-Hoon Lee , Seung-Yeop Kim , Jong-Hwan Jung , Jinmi Kim; J. Microbiol. 2010;48(3):365-371. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9155-4

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Abstract: The yeast-to-hyphal transition is a major virulence factor in the fungal pathogen Candida albicans. Mutations in the CaKEM1 gene, which encodes a 5′-3′ exoribonuclease responsible for mRNA degradation, show a defect in hyphal growth. We applied two-dimensional gel electrophoresis to identify hyphae-specific proteins that have altered expressions in the presence of the cakem1 mutation. Eight proteins, Eno1, Eps1, Fba1, Imh3, Lpd1, Met6, Pdc11, and Tsa1 were upregulated during hyphal transition in wild-type but not in cakem1/cakem1 mutant cells. A second group of proteins, Idh1, Idh2, and Ssb1, showed increased levels of expression in cakem1/cakem1 mutant cells when compared to wild-type cells. Overexpression of Lpd1, a component of the pyruvate dehydrogenase complex, caused slight hyperfilamentation in a wild-type strain and suppressed the filamentation defect of the cakem1 mutation. The Ssb1 protein, which is a potential heat shock protein, and the Imh3 protein, which is a putative enzyme in GMP biosynthesis also showed the filamentation-associated phenotypes.

Helicobacter pylori γ-Glutamyltranspeptidase Induces Cell Cycle Arrest at the G1-S Phase Transition: Kyung-Mi Kim , Seung-Gyu Lee , Jung-Min Kim , Do-Su Kim , Jea-Young Song , Hyung-Lyun Kang , Woo-Kon Lee , Myung-Je Cho , Kwang-Ho Rhee , Hee-Shang Youn , Seung-Chul Baik; J. Microbiol. 2010;48(3):372-377. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9293-8

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Abstract: In our previous study, we showed that Helicobacter pylori γ-glutamyltranspeptidase (GGT) is associated with H. pylori-induced apoptosis through a mitochondrial pathway. To better understand the role of GGT in apoptosis, we examined the effect of GGT on cell cycle regulation in AGS cells. To determine the effect of recombinant GGT (rGGT) on cell cycle distribution and apoptosis, rGGT-treated and untreated AGS cells were analyzed in parallel by flow cytometry using propidium iodide (PI). We found that rGGT inhibited the growth of AGS cells in a time-dependent manner, and that the pre-exposure of cells to a caspase-3 inhibitor (z-DEVD-fmk) effectively blocked GGT-induced apoptosis. Cell cycle analysis showed G1 phase arrest and apoptosis in AGS cells following rGGT treatment. The rGGT-mediated G1 phase arrest was found to be associated with down-regulation of cyclin E, cyclin A, Cdk 4, and Cdk 6, and the up-regulation of the cyclindependent kinase (Cdk) inhibitors p27 and p21. Our results suggest that H. pylori GGT induces cell cycle arrest at the G1-S phase transition.

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