Journal of Microbiology

Volume 46(1); February 2008

Review

The Use of the Rare UUA Codon to Define "Expression Space" for Genes Involved in Secondary Metabolism, Development and Environmental Adaptation in Streptomyces: Keith F. Chater , Govind Chandra; J. Microbiol. 2008;46(1):1-11.; DOI: https://doi.org/10.1007/s12275-007-0233-1

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118 Citations

Abstract: In Streptomyces coelicolor, bldA encodes the only tRNA for a rare leucine codon, UUA. This tRNA is unnecessary for growth, but is required for some aspects of secondary metabolism and morphological development, as revealed by the phenotypes of bldA mutants in diverse streptomycetes. This article is a comprehensive review of out understanding of this unusual situation. Based on information from four sequenced genomes it now appears that, typically, about 2~3% of genes in any one streptomycete contain a TTA codon, most having been acquired through species-specific horizontal gene transfer. Among the few widely conserved TTA-containing genes, mutations in just one, the pleiotropic regulatory gene adpA, give an obvious phenotype: such mutants are defective in aerial growth and sporulation, but vary in the extent of their impairment in secondary metabolism in different streptomycetes. The TTA codon in adpA is largely responsible for the morphological phenotype of a bldA mutant of S. coelicolor. AdpA-dependent targets include several genes involved in the integrated action of extracellular proteases that, at least in some species, are involved in the conversion of primary biomass into spores. The effects of bldA mutations on secondary metabolism are mostly attributable to the presence of TTA codons in pathway-specific genes, particularly in transcriptional activator genes. This is not confined to S. coelicolor-it is true for about half of all known antibiotic biosynthetic gene sets from streptomycetes. Combined microarray and proteomic analysis of liquid (and therefore non-sporulating) S. coelicolor bldA mutant cultures revealed effects of the mutation during rapid growth, during transition phase, and in stationary phase. Some of these effects may be secondary consequences of changes in the pattern of ppGpp accumulation. It is argued that the preferential accumulation of the bldA tRNA under conditions in which growth is significantly constrained has evolved to favour the expression of genes that confer adaptive benefits in intermittently encountered sub-optimal environments. The evolution of this system may have been a secondary consequence of the selective pressure exerted by bacteriophage attack. Some biotechnological implications of bldA phenomenology are considered.

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

Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Community Profiles in the Rhizosphere of cry1AC-carrying Brassica rapa subsp. pekinensis: Sera Jung , Semi Park , Daeha Kim , Seung Bum Kim; J. Microbiol. 2008;46(1):12-15.; DOI: https://doi.org/10.1007/s12275-007-0190-8

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Abstract: The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.

Diversity and Metal Tolerance of Nematode-Trapping Fungi in Pb-Polluted Soils: Ming-He Mo , Wei-Min Chen , Hao-Ran Yang , Ke-Qin Zhang; J. Microbiol. 2008;46(1):16-22.; DOI: https://doi.org/10.1007/s12275-007-0174-8

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16 Citations

Abstract: The diversity of nematode-trapping fungi (NTF) in two lead (Pb) mines in Yunnan Province, China was investigated in 2004. In total, 20 species belonging to five genera were identified from 500 samples collected at the Lanping and the Huize mines. Pb concentrations ranged from 216~7,150 mg/kg for the former and 132~13,380 mg/kg for the latter, respectively. The fungi were divided into five groups based on different trapping mechanisms. The trapping-net producer group contained the largest number of species, with nine. Two predators, Dactylellina ellipsosporum and Arthrobotrys oligospora, were found at frequencies of 32.85% and 15.41%, respectively. The diversity indexes of NTF were positively correlated with Pb pollution levels in both the Lanping Mine (r=0.66) and the Huize Mine (r=0.72), suggesting that the distribution of NTF was not negatively affected by Pb contamination. For most strains of a given species, there was no significant difference (P>0.01) in the Pb tolerance between the strains isolated from habitats with low or high Pb concentrations. However, Pb toxicity exerted adverse effects on trap formation and predacious capability of fungi. We discuss the possible metal tolerance mechanisms and their relationships to the survival strategy of NTF in Pb-polluted environments.

Removal of Heavy Metals by an Enriched Consortium: Eun Young Lee , Joung Soo Lim , Kyung Hwan Oh , Jae Yeon Lee , Seog Ku Kim , Yoo Kyung Lee , Keun Kim; J. Microbiol. 2008;46(1):23-28.; DOI: https://doi.org/10.1007/s12275-007-0131-6

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Abstract: An enriched consortium obtained from lake-sediment was developed for the removal of heavy metals such as Cu, Pb, Cr, Ni, and Zn from heavy metal-contaminated water. The removal efficiency of heavy metals in a shaking condition was generally higher than that in the static state. After the fifteenth enrichment with assorted heavy metals, the removal efficiencies in the shaking and static condition at an average concentration of 100 mg/L of each heavy metal were approximately 99~100% and 95~100%, respectively, depending on the type of heavy metal. An aerobically grown, pure culture isolated from an enriched culture was analyzed by 16S rRNA sequencing and identified as Ralstonia sp. HM-1. This strain was found to remove various heavy metals with an efficiency of approximately 97~100% at an average concentration of 200 mg/L of each heavy metal.

Genetic Variation and Geographic Distribution of Megalocytiviruses: Jun-Young Song , Shin-Ichi Kitamura , Sung-Ju Jung , Toshiaki Miyadai , Shinji Tanaka , Yutaka Fukuda , Seok-Ryel Kim , Myung-Joo Oh; J. Microbiol. 2008;46(1):29-33.; DOI: https://doi.org/10.1007/s12275-007-0184-6

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63 Citations

Abstract: Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.

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

A Novel Archaeal Group in the Phylum Crenarchaeota Found Unexpectedly in an Eukaryotic Survey in the Cariaco Basin: Sun-Ok Jeon , Tae-Seok Ahn , Sun-Hee Hong; J. Microbiol. 2008;46(1):34-39.; DOI: https://doi.org/10.1007/s12275-007-0247-8

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Abstract: Archaea have been found in many more diverse habitats than previously believed due in part to modern molecular approaches to discovering microbial diversity. We report here an unexpected expansion of the habitat diversity of the Archaea in the Cariaco Basin we found using a primer set designed for 18S eukaryotic rDNA sequence analysis. The results presented here expand the originally identified 9 archaeal clones reported in this environment using bacterial/archaeal primers to 152 archaeal clones: 67 (18 OTU) of these clones were found at a depth of 900 m of station A while 71 (9 OTU) of them were at a depth of between 300~335 m of station B&C depending upon which location the samples were taken. We used three phylogenetic analysis methods and detected 20 phylotypes belonging to a single previously unreported group distantly related to the Crenarchaeota. Also, we determined that the original nine sequences did not fall into any of the known phyla of the Archaea suggesting that they may represent a novel group within the Kingdom Archaea. Thus, from these two studies, we suggest that Archaea in the Cariaco Basin could be unique; however, further studies using archaeal-specific primers and the design of new primers as well as the systematic use of several different primer combinations may improve the chances of understanding the archeal diversity in the Cariaco Basin.

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

Arthrobacter soli sp. nov., a Novel Bacterium Isolated from Wastewater Reservoir Sediment: Seong Woon Roh , Youlboong Sung , Young-Do Nam , Ho-Won Chang , Kyoung-Ho Kim , Jung-Hoon Yoon , Che Ok Jeon , Hee-Mock Oh , Jin-Woo Bae; J. Microbiol. 2008;46(1):40-44.; DOI: https://doi.org/10.1007/s12275-007-0239-8

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73 Citations

Abstract: A novel Gram-positive bacterium, designated SYB2T, was isolated from wastewater reservoir sediment, and a polyphasic taxonomic study was conducted based on its morphological, physiological, and biochemical features, as well as the analysis of its 16S rRNA gene sequence. During the phylogenetic analysis of the strain SYB2T, results of a 16S rRNA gene sequence analysis placed this bacterium in the genus Arthrobacter within the family Micrococcaceae. SYB2T and Arthrobacter protophormiae ATCC 19271T, the most closely related species, both exhibited a 16S rRNA gene sequence similarity of 98.99%. The genomic DNA G+C content of the novel strain was found to be 62.0 mol%. The predominant fatty acid composition was anteiso-C15:0, anteiso-C17:0, iso-C16:0, and iso-C15:0. Analysis of 16S rRNA gene sequences and DNA-DNA relatedness, as well as physiological and biochemical tests, showed genotypic and phenotypic differences between strain SYB2T and other Arthrobacter species. The type strain of the novel species was identified as SYB2T (= KCTC 19291T= DSM 19449T).

Isolation and Taxonomic Characterization of a Novel Type I Methanotrophic Bacterium: Hee Gon Kim , Gui Hwan Han , Chi-Yong Eom , Si Wouk Kim; J. Microbiol. 2008;46(1):45-50.; DOI: https://doi.org/10.1007/s12275-008-0017-2

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Abstract: A methane-oxidizing bacterium was isolated from the effluent of manure and its molecular and biochemical properties were characterized. The isolate was aerobic, Gram-negative, and non-motile. The organism had a type I intracytoplasmic membrane structure and granular inclusion bodies. The outer cell wall surface (S-layers) was tightly packed with cup-shaped structures. Colonies were light yellow on nitrate mineral salt agar medium. In addition, the organism was catalase and oxidase positive. The isolate used the ribulose monophosphate (RuMP) pathway for carbon assimilation, and was able to utilize methane and methanol as a sole carbon and energy source, however, it could not utilize any other organic compounds that were tested. The cells grew well in a mixture of methane and air (methane:air=1:1, v/v) in a compulsory circulation diffusion system, and when grown under those conditions, the optimum pH was approximately 7.0 and the optimal temperature was 30°C. In addition, the specific growth rate and generation time were 0.13 per h and 5.43 h, respectively, when grown under the optimum conditions. The major ubiquinone was Q-8, and the G+C mol% of the DNA was 55.3. Phylogenetic analyses based on the 16S rRNA gene sequence comparisons showed that this bacterium belongs to a group of type I methanotrophs, and that it is most closely related to Methylomicrobium, with a sequence similarity of 99%. Therefore, the isolate was named Methylomicrobium sp. HG-1.

Purification and Characterization of Thermostable β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris Grown on Microcrystalline Cellulose: Jeong-Jun Yoon , Ki-Yeon Kim , Chang-Jun Cha; J. Microbiol. 2008;46(1):51-55.; DOI: https://doi.org/10.1007/s12275-007-0230-4

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51 Citations

Abstract: An extracellular β-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC- MS/MS suggested that the protein has high homology with fungal β-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-β-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified β-glucosidase was observed at pH 4.5 and 70°C. The F. palustris protein exhibited half-lives of 97 h at 55°C and 15 h at 65°C, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-β-lactoside, p-nitrophenyl-β-xyloside, p-nitrophenyl-α-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the β-glucosidase from F. palustris can be classified as an aryl-β-glucosidase with cellobiase activity.

5’ Untranslated Region of the Pseudomonas putida WCS358 Stationary Phase Sigma Factor rpoS mRNA is Involved in RpoS Translational Regulation: Branko Jovcic , Iris Bertani , Vittorio Venturi , Ljubisa Topisirovic , Milan Kojic; J. Microbiol. 2008;46(1):56-61.; DOI: https://doi.org/10.1007/s12275-007-0127-2

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Abstract: The σS subunit of RNA polymerase is a central regulator which governs the expression of a host of stationary phase-induced and osmotically regulated genes in Gram-negative bacteria. The Pseudomonas putida rpoS gene is transcribed as a monocistronic rpoS mRNA with a 368 nucleotide-long 5’ untranslated region (5’ UTR). In this study, we investigate the posttranscriptional control of RpoS synthesis using rpoS-lacZ transcriptional and translational fusions consisting of the native promoter and deletions of 5’ UTR or insertion into UTR. The differing activity of constructed translational fusions strongly indicated that the 5’ UTR is involved in the translational regulation of RpoS expression in the stationary phase. The results obtained herein demonstrated that the structure of UTR performs an important function in the translational regulation of the rpoS gene.

Journal Article

Molecular Characteristics of Two Laccase from the Basidiomycete Fungus Polyporus brumalis: Sun-Hwa Ryu , A-Young Lee , Myungkil Kim; J. Microbiol. 2008;46(1):62-69.; DOI: https://doi.org/10.1007/s12275-007-0110-y

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Abstract: Two laccase cDNAs, pblac1 and pblac2, were cloned from a white-rot fungus strain, Polyporus brumalis (KFRI 20912). The cloned cDNAs consisted of 1,829 bp and 1,804 bp, and their open reading frames encoded proteins of 520 and 524 amino acids, with calculated molecular masses of approximately 55.9 kDa and 56 kDa, respectively. The deduced amino acid sequences of each protein showed 70% similarity. The copper binding regions were conserved in both proteins, as in other fungal laccases. RT-PCR analysis revealed that the transcript levels of the two laccases increased progressively in shallow stationary culture liquid medium. The transcript level of each laccase was induced when the fungus was exposed to di-butyl phthalate (DBP), suggesting that the two laccases are involved in DBP degradation. The overexpression of the pblac1 gene was derived by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase, using a homologous system. The activity of laccase in the transformants was significantly higher than that of the wild type. The identification of these laccase cDNAs was a first step to characterize the molecular events related to the lignin degradation ability of this basidiomycetous fungus, as well as the degradation of many recalcitrant xenobiotics.

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

Nitrogen Depletion Causes Up-Regulation of Glutathione Content and γ-Glutamyltranspeptidase in Schizosaccharomyces pombe: Seung-Hyun Song , Chang-Jin Lim; J. Microbiol. 2008;46(1):70-74.; DOI: https://doi.org/10.1007/s12275-007-0244-y

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Abstract: This work aims to elucidate the relationship between nitrogen depletion and Glutathione (GSH) level in Schizosaccharomyces pombe. The total GSH level was much higher in the Pap1-positive KP1 cells than in the Pap1-negative TP108-3C cells, suggesting that synthesis of GSH is dependent on Pap1. When the Pap1-positive KP1 cells were transferred to the nitrogen-depleted medium, total GSH level significantly increased up to 6 h and then slightly declined after 9 h. Elevation of the total GSH level was observed to be much less with the Pap1-negative cells. However, glucose deprivation was not able to enhance the GSH level in the KP1 cells. Activity of γ-glutamyltranspeptidase (γ-GT), an enzyme in the first step of GSH catabolism, also increased during nitrogen depletion. The total GSH level was more significantly enhanced in the KP1 cells overexpressing γ-GT2 than γ-GT1 during nitrogen starvation. Reactive oxygen species (ROS) levels were not changed during nitrogen starvation in both Pap1-positive and Pap1-negative cells. Collectively, nitrogen depletion causes up-regulation of GSH synthesis and γ-GT in a Pap1-dependent manner.

The Mutation of a Novel Saccharomyces cerevisiae SRL4 Gene Rescues the Lethality of rad53 and lcd1 Mutations by Modulating dNTP Levels: Do-Hee Choi , Young-Mi Oh , Sung-Hun Kwon , Sung-Ho Bae; J. Microbiol. 2008;46(1):75-80.; DOI: https://doi.org/10.1007/s12275-008-0013-6

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Abstract: The SRL4 (YPL033C) gene was initially identified by the screening of Saccharomyces cerevisiae genes that play a role in DNA metabolism and/or genome stability using the SOS system of Escherichia coli. In this study, we found that the srl4Δ; mutant cells were resistant to the chemicals that inhibit nucleotide metabolism and evidenced higher dNTP levels than were observed in the wild-type cells in the presence of hydroxyurea. The mutant cells also showed a significantly faster growth rate and higher dNTP levels at low temperature (16 oC) than were observed in the wild-type cells, whereas we detected no differences in the growth rate at 30oC. Furthermore, srl4Δ was shown to suppress the lethality of mutations of the essential S phase checkpoint genes, RAD53 and LCD1. These results indicate that SRL4 may be involved in the regulation of dNTP production by its function as a negative regulator of ribonucleotide reductase.

Hepatitis C Virus (HCV) Genotyping by Annealing Reverse Transcription-PCR Products with Genotype-Specific Capture Probes: Jungmin Rho , Jong Soon Ryu , Wonhee Hur , Chang Wook Kim , Jeong Won Jang , Si Hyun Bae , Jong Young Choi , Sung Key Jang , Seung Kew Yoon; J. Microbiol. 2008;46(1):81-87.; DOI: https://doi.org/10.1007/s12275-007-0121-8

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18 Citations

Abstract: The genotype of the hepatitis C virus (HCV) strain infecting a given patient is an important predictive factor for the clinical outcome of chronic liver disease and its response to anti-viral therapeutic agents. We herein sought to develop a new easy, sensitive and accurate HCV genotyping method using annealing genotype- specific capture probes (AGSCP) in an automation-friendly 96-well plate format. The validation of our new AGSCP was performed using the Standard HCV Genotype Panel. We then used both our AGSCP and the commercially available INNO-LiPA assay to analyze the HCV genotypes from 111 Korean patients. Discordant results were analyzed by direct sequencing. AGSCP successfully genotyped the standard panel. The genotypes of 111 patient samples were also obtained successfully by AGSCP and INNO-LiPA. We observed a high concordance rate (93 matched samples, 83.8%) between the two assays. Sequencing analysis of the 18 discordant results revealed that the AGSCP had correctly identified 12 samples, whereas the INNO- LiPA had correctly identified only 6. These results collectively indicate that AGSCP assay is a convenient and sensitive method for large-scale genotyping, and it may be a promising tool for the determination of HCV and other genotypes in clinical settings.

Characterization and Signature Pattern Analysis of Korean Clade HIV-1 Using nef Gene Sequences: Chan Seung Park , Dong Hun Lee , Keon Myung Lee , Chan-Hee Lee; J. Microbiol. 2008;46(1):88-94.; DOI: https://doi.org/10.1007/s12275-007-0156-x

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Abstract: Phylogenetic studies of the HIV-1 gene sequences isolated from Korean patients have suggested that most of Korean isolates belong to the subtype B strain. This study aims to characterize the Korean clade by molecular phylogenetic analysis using all of the Korean nef gene sequences registered in the NCBI GenBank (N=422), in addition to 41 reference strains and 94 foreign isolates. Through phylogenetic analyses, we verified that most of the Korean isolates belonged to the subtype B, where 78.8% are clustered exclusively of foreign isolates. This cluster has been named the Korean clade subtype B (KCB) in order to distinguish it from other subtype B clusters. Genetic distance analysis suggested that the KCB cluster was more homogeneous and clearly distinctive from the non-Korean clade subtype B (NKCB). Comparison of consensus amino acid sequences from KCB and NKCB revealed that characteristic KCB signature amino acid patterns composed of 11 amino acid residues, whose frequencies in the KCB were significantly higher than in the NKCB. The KCB signature amino acid residues were critical in identifying KCB from NKCB, since substitution of the NKCB sequences with KCB signature amino acids relocated them to the Koran clade, and vice versa.

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