Journal of Microbiology

Journal Article

Deletion of IRC19 Causes Defects in DNA Double-Strand Break Repair Pathways in Saccharomyces cerevisiae.: Ju-Hee Choi, Oyungoo Bayarmagnai, Sung-Ho Bae; J. Microbiol. 2024;62(9):749-758. Published online July 12, 2024; DOI: https://doi.org/10.1007/s12275-024-00152-x

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Abstract: DNA double-strand break (DSB) repair is a fundamental cellular process crucial for maintaining genome stability, with homologous recombination and non-homologous end joining as the primary mechanisms, and various alternative pathways such as single-strand annealing (SSA) and microhomology-mediated end joining also playing significant roles under specific conditions. IRC genes were previously identified as part of a group of genes associated with increased levels of Rad52 foci in Saccharomyces cerevisiae. In this study, we investigated the effects of IRC gene mutations on DSB repair, focusing on uncharacterized IRC10, 19, 21, 22, 23, and 24. Gene conversion (GC) assay revealed that irc10Δ, 22Δ, 23Δ, and 24Δ mutants displayed modest increases in GC frequencies, while irc19Δ and irc21Δ mutants exhibited significant reductions. Further investigation revealed that deletion mutations in URA3 were not generated in irc19Δ mutant cells following HO-induced DSBs. Additionally, irc19Δ significantly reduced frequency of SSA, and a synergistic interaction between irc19Δ and rad52Δ was observed in DSB repair via SSA. Assays to determine the choice of DSB repair pathways indicated that Irc19 is necessary for generating both GC and deletion products. Overall, these results suggest a potential role of Irc19 in DSB repair pathways, particularly in end resection process.

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

Novel Mutations in CYP51B from Penicillium digitatum Involved in Prochloraz Resistance: Jinlong Wang , Jinhui Yu , Jing Liu , Yongze Yuan , Na Li , Muqing He , Ting Qi , Geng Hui , Li Xiong , Deli Liu; J. Microbiol. 2014;52(9):762-770. Published online August 2, 2014; DOI: https://doi.org/10.1007/s12275-014-4112-2

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46 Citations

Abstract: Green mold caused by Penicillium digitatum is one of the most serious postharvest diseases of citrus fruit, and it is ubiquitous in all citrus growing regions in the world. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in the biological kingdom and a prime target of antifungal drugs. Mutations in CYP51s have been found to be correlated with resistance to azole fungicides in many fungal species. To investigate the mechanism of resistance to prochloraz (PRC) in P. digitatum, the PRC sensitivity was determined in vitro in this study to assess the sensitivity of 78 P. digitatum isolates collected in Hubei province. The results showed that 25 isolates were prochloraz-resistant (PRC-R), including six high-resistant (HR) strains, twelve medium-resistant (MR) and seven low-resistant (LR) strains. A sequence analysis showed no consistent point mutations of PdCYP51A in the PRC-R strains, but four substitutions of CYP51B were found, Q309H in LR strains, Y136H and Q309H in HR strains, and G459S and F506I in MR strains, which corresponded to the four sensitivity levels. Based on the sequence alignment analysis and homology modeling followed by the molecular docking of the PdCYP51B protein, the potential correlation between the mutations and PRC resistance is proposed.

Identification and Characterization of an Autolysin Gene, atlA, from Streptococcus criceti: Haruki Tamura , Arisa Yamada , Hirohisa Kato; J. Microbiol. 2012;50(5):777-784. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2187-1

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Abstract: AtlA of Streptococcus mutans is a major autolysin and belongs to glycoside hydrolase family 25 with cellosyl of Streptomyces coelicolor. The autolysin gene (atlA) encoding AtlA was identified from S. criceti. AtlA of S. criceti comprises the signal sequence in the N-terminus, the putative cell-wallbinding domain in the middle, and the catalytic domain in the C-terminus. Homology modeling analysis of the catalytic domain of AtlA showed the resemblance of the spatial arrangement of five amino acids around the predicted catalytic cavity to that of cellosyl. Recombinant AtlA and its four point mutants, D655A, D747A, W831A, and D849A, were evaluated on zymogram of S. criceti cells. Lytic activity was destroyed in the mutants D655A and D747A and diminished in the mutants W831A and D849A. These results suggest that Asp655 and Asp747 residues are critical for lytic activity and Trp831 and Asp849 residues are also associated with enzymatic activity.

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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6 Citations

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.

Characterization of Osh3, an Oxysterol-binding Protein, in Filamentous Growth of Saccharomyces cerevisiae and Candida albicans: Hyang-sook Hur , Ji-Ho Ryu , Kwang-Hoon Kim , Jinmi Kim; J. Microbiol. 2006;44(5):523-529.; DOI: https://doi.org/2445 [pii]

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Abstract: OSH3 is one of the seven yeast homologues of the oxysterol binding proteins (OSBPs) which have the major binding affinity to the oxysterols and function as regulator of cholesterol biosynthesis in mammals. Mutational analysis of OSH3 showed that OSH3 plays a regulatory role in the yeast-to-hyphal transition through its oxysterol-binding domain in Saccharomyces cerevisiae. The OSH3 gene was also identified in the pathogenic yeast Candida albicans. Deletion of OSH3 caused a defect in the filamentous growth, which is the major cause of the C. albicans pathogencity. The filamentation defect of the mutation in the MAPK-associated transcription factor, namely cph1Δ was suppressed by overexpression of OSH3. These findings suggest the regulatory roles of OSH3 in the yeast filamentous growth and the functional conservations of OSH3 in S. cerevisiae and C. albicans.

Sequence Analysis of NS4 Region of HCV Isolated from Korean Patient: Paik, Sang Hoon , Lee, Young Ik , Kim, Won Bae , Yang, Jai Myung; J. Microbiol. 1995;33(3):260-266.

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Abstract: Hepatitis C virus (HCV) has been considered as a major causative agent of post-transfusion related non-A, non-B hepatitis. In this study, the cDNA sequence of NS4 region of HCV (HCV-S) obtained from a Korean patient's plasma was determined. Comparative nucleotide sequence analysis between to type II. 67.2% homology to type III, and 66.4% homology to type IV. The putative amino acid sequence homologies to types I, II, III, and IV were 82.8-84.7%, 92.5-95.1%. 72.5% and 71.1% respectively. This data strongly suggests that HCV-S should be classified as type II. Significant similarities of hydrophobicity profiles and putative transmembranous domains were found in HCV-S and four major prototypes, indicating that the protein structure is similar in spite of the heterogeneities of intertype homologies at the level of the primary nucleotide and amino acid sequences.

Cloning and Sequence Analysis of the hpaD Gene Responsible for Homoprotocatechuate 2,3-Dioxygenase from Pseudomonas sp. DJ-12: Sang-Mahn Lee , Jong-Chan Chae , Youngsoo Kim , Chi-Kyung Kim; J. Microbiol. 2001;39(4):334-337.

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Abstract: The degradative pathway of homoprotocatechuate (HPC) is the bacterial route whereby 3,4-dihydroxyphenylacetic acid is catabolized to pyruvate and succinate by a series of sequential reactions. The HPC is catalized by homoprotocatechuate 2,3-dioxygenase (HPC-2,3O) to form 5-carboxymethyl-2-hydroxy-muco semialdehyde. In this study, the hpaD gene encoding HPC-2,3O was cloned from the chromosomal DNA of Pseudomonas sp. DJ-12 and its nucleotide sequence was analyzed. The open reding frame of hpaD gene was found to be composed of 864 nucleotide pairs and to encode a polypeptide with 287 amino acid residues. The deduced amino acid sequence of the HPC-2,3O from Pseudomonas sp. DJ-12 exhibited 60~64% homology with those of the corresponding enzymes from E. coli, Salmonella enterica, and Klebsiella pneumoniae.

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