[Show abstract][Hide abstract] ABSTRACT: Due to abundant contamination in various foods, the pathogenesis of Bacillus cereus has been widely studied in physiological and molecular level. B. cereus FORC_005 was isolated from a Korean side dish, soy sauce braised fish-cake with quail-egg in South Korea. While 21 complete genome sequences of B. cereus has been announced to date, this strain was completely sequenced, analyzed, and compared with other complete genome sequences of B. cereus to elucidate the distinct pathogenic features of a strain isolated in South Korea. The genomic DNA containing a circular chromosome consists of 5,349,617-bp with a GC content of 35.29 %. It was predicted to have 5170 open reading frames, 106 tRNA genes, and 42 rRNA genes. Among the predicted ORFs, 3892 ORFs were annotated to encode functional proteins (75.28 %) and 1278 ORFs were predicted to encode hypothetical proteins (748 conserved and 530 non-conserved hypothetical proteins). This genome information of B. cereus FORC_005 would extend our understanding of its pathogenesis in genomic level for efficient control of its contamination in foods and further food poisoning.
Preview · Article · Nov 2015 · Standards in Genomic Sciences
[Show abstract][Hide abstract] ABSTRACT: Cronobacter sakazakii is an important pathogen with high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach could be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989-bp DNA, including 231 predicted open reading frames (ORFs), with a GC content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence, toxin, or lysogen formation, but more than 80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE gel electrophoresis and MALDI-TOF/MS revealed that seven phage structural proteins are indeed present, supporting ORF prediction. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with C. sakazakii clinical isolate or food isolate, revealing the complete growth inhibition of the isolates by the addition of the phage CR5 when multiplicity of infection (MOI) was 10^5.
No preview · Article · Oct 2015 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus.
[Show abstract][Hide abstract] ABSTRACT: The strain FOL01T was isolated from traditionally-fermented Korean jogaejeotgal (clam-fermented seafood). Phylogenetic sequence analysis of 16S rDNA from FOL01T revealed that it is closely related to Weissella thailandensis FS61-1T and W. paramesenteroides ATCC 33313T with 99.39% and 98.50% sequence identities, respectively. API and VITEK analyses showed that strain FOL01T can be separated from its nearest phylogenetic relatives with respect to carbohydrate fermentation and antibiotic resistance. Subsequent Amplified Ribosomal DNA Restriction Analysis (ARDRA) of 16S rDNAs and HaeIII-restriction enzyme (RE) profiling of genomic DNAs revealed different band patterns. In addition, DNA-DNA hybridization (DDH) of genomic DNAs showed 63.9% relatedness. Analysis of the composition of cellular fatty acids confirmed that strain FOL01T differs from close relatives and supports the proposal to assign this organism to a novel species of Weissella. Based on these results, strain FOL01T could be classified as a novel species of the genus Weissella, for which the name Weissella jogaejeotgali sp. nov is proposed. The type strain of Weissella jogaejeotgali is FOL01T (= KCCM 43128T = JCM 30589T).
No preview · Article · Sep 2015 · International Journal of Systematic and Evolutionary Microbiology
[Show abstract][Hide abstract] ABSTRACT: Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of its stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionicbacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain, B. subtilis ZA400, with the highest fibrinolytic activity was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, Western blot and MALDI-TOF analyses, showing 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40°C), metal ions (Mn(2+), Ca(2+), K(2+), and Mg(2+)), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may be even useful as a new bacterial starter for manufacturing functional fermented foods.
No preview · Article · Sep 2015 · Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Korean fermented foods are known to be beneficial for human health. Bacterial community studies have been conducted to figure out what roles the bacteria used to ferment these foods play in food fermentation. The metagenomic approach identifies both culturable and unculturable bacterial compositions, but this technique is limited in its ability to accurately determine the bacterial species from short 16S rRNA PCR products. In this study, we revisited the culture-dependent method using a relatively large number of bacterial isolates in an attempt to overcome the problem of bacterial identification, accepting that the unculturable bacterial population in each fermented food would be undetectable. Eight Korean fermented foods including kimchi, jeotgal, and meju were collected, and 1589 fermenting bacterial strains were randomly isolated. Bacteria were grouped by banding pattern using arbitrary-primed (AP) PCR prior to bacterial identification and sorted into 219 groups; 351 strains were not grouped because there was no identical AP-PCR band pattern. 16S rRNA sequence analysis identified the bacterial compositions of the fermented foods. As dominant genera, Lactobacillus and Leuconostoc strains were detected in four kimchi samples, Staphylococcus in three jeotgal samples, and Enterococcus and Bacillus in the meju sample. Interestingly, S. Equorum was most dominant in saeu-jeotgal, indicating that it is halophilic and may enhance the fermentation flavor. Further comparative analysis of this study with previous metagenomic results revealed that bacterial communities in fermented foods are highly similar at the genus level but often differ at the species level. This bacterial community study is useful for understanding the roles and functional properties of fermenting bacteria in the fermentation process of Korean fermented foods.
No preview · Article · Jun 2015 · Journal of the Korean Society for Applied Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori), a potential ribosomal binding site (RBS), and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, four different PCR products (MR1, ori+RBS+repA; MR2, RBS+repA; MR2', repA; MR3, fragment of repA) were obtained and cloned into pUC19 (pKUCm1, pKUCm2, pKUCm2', and pKUCm3, respectively) containing the chloramphenicol acetyltransferase (CAT) gene. These constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5 × 10(5) CFU/μg, 1.3 × 10(1) CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria.
Preview · Article · Feb 2015 · Frontiers in Microbiology
[Show abstract][Hide abstract] ABSTRACT: Amylosucrase (AS), a glucosyltransferase of the glycoside hydrolase 13 family, does not require a nucleotide-activated sugar as a glucosyl-donor. A gene (drpas) of Deinococcus radiopugnans encoding a putative AS was identified and cloned for characterization. The amino acid sequence revealed that drpas exhibited 76 and 74% identities with AS genes from D. geothermalis and D. radiodurans, respectively. Recombinant DRpAS exhibited AS activities. The ratios of hydrolysis, polymerization, and isomerization reactions were 5.8:82.7:11.5 at 40°C. The DRpAS was highly thermostable and produced more polymerization products than AS from D. radiodurans although the optimum growth temperature of both strains is 30°C.
No preview · Article · Dec 2014 · Food science and biotechnology
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus aureus is an important food-borne pathogen that causes a diverse diseases ranging from minor infections to life threatening conditions in humans and animals. To further understand its pathogenesis, the genome of the strain S. aureus FORC_001 was isolated from a contaminated food. Its genome consists of 2,886,017bp double-stranded DNA with a GC content of 32.8%. It is predicted to contain 2,728 open reading frames (ORFs), 57 tRNAs, and 6 rRNA operons, including 1 additional 5S rRNA gene. Comparative phylogenetic tree analysis of 40 complete S. aureus genome sequences using average nucleotide identity (ANI) revealed that the strain FORC_001 belonged to Group I. The closest phylogenetic match was S. aureus MRSA252 according to a whole genome ANI (99.87%), suggesting that they might share a common ancestor. Comparative genome analysis of FORC_001 and MRSA252 revealed two non-homologous regions: Regions I and II. The presence of various antibiotic resistance genes, including the SCCmec cluster in Region I of MRSA252, suggests that this strain might have acquired the SCCmec cluster to adapt to specific environments containing methicillin. Region II of both genomes contains prophage regions but their DNA sequence identity is very low, suggesting that the prophages might differ. This is the first report of the complete genome sequence of S. aureus isolated from a real food-borne outbreak in South Korea. This report would be helpful to extend our understanding about genome, general characteristics, and virulence factors of S. aureus for further studies of pathogenesis, rapid detection, and epidemiological investigation in food-borne outbreak.
Full-text · Article · Oct 2014 · Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas axonopodis pv. glycines 8ra is a causal agent of bacterial pustule disease in soybean. This bacterium possesses transcription activator-like (TAL) effectors which are useful for genetic/protein engineering applications in higher organisms including plants and humans. Here, we report that the draft genome sequence consists of 5,337,885-bp double-stranded DNA encoding 4,674 open reading frames (ORFs) in 13 different contigs. This genome sequence would be useful in applications of TAL effectors in genetic engineering and in elucidating virulence factors against plants.
Full-text · Article · Jun 2014 · Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Metagenomic analysis of human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new long-range 16S rRNA universal PCR primer set targeting V6 to V9 regions with 580-bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.
No preview · Article · Apr 2014 · Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: PM1, a novel virulent bacteriophage that infects Pectobacterium carotovorum subsp. carotovorum, was isolated. Its morphological features were examined by electron microscopy, which indicated that this phage belongs to the family Myoviridae. It has a 55,098-bp genome, including a 2,665-bp terminal repeat. A total of 63 open reading frames (ORFs) were predicted, but only 20 ORFs possessed homology with functional proteins. There is one tRNA coding region, and the GC-content of the genome is 44.9 %. Most ORFs in bacteriophage PM1 showed high homology to enterobacteria phage ΦEcoM-GJ1 and Erwinia phage νB EamM-Y2. Like these bacteriophages, PM1 encodes an RNA polymerase, which is a hallmark of T7-like phages. There is no integrase or repressor, suggesting that PM1 is a virulent bacteriophage.
Full-text · Article · Mar 2014 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Due to the emergence of antibiotic-resistant strains, bacteriophages are considered to be an alternative approach for the control of pathogens. In this study, the bacteriophages BPS10C and BPS13 were isolated and characterized to investigate their ability to control food-borne pathogenic Bacillus cereus. Phage BPS13 exhibited slightly higher host lysis activity compared with phage BPS10C. In addition, phage BPS13 exhibited greater stability under various pH and temperature conditions. To extend our knowledge of the lysis of B. cereus by these phages, their genomes were completely sequenced and analyzed, revealing that these phage genomes encode endolysin and two tail lysins, which are likely involved in host lysis and invasion mechanisms, respectively. These lysis-related proteins may increase the bactericidal activities of these phages, suggesting that they may be good candidates for the potential control of B. cereus.
Full-text · Article · Mar 2014 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Thermococcus sp. strain ES1 is an anaerobic, hyperthermophilic archaeon from a hydrothermal vent that catabolizes sugars and peptides and produces H2S from S°, H2, acetate and CO2 as its primary metabolites. We present the complete genome sequence of this strain (1,957,742bp) with a focus on its substrate utilization and metabolite production capabilities. The sequence will contribute to the development of heterotrophic archaea for bioenergy production and biogeochemical modeling in hydrothermal environments.
No preview · Article · Jan 2014 · Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: The Bacillus cereus group phages infecting B. cereus, B. anthracis, and B. thuringiensis (Bt) have been studied at the molecular level and, recently, at the genomic level to control the pathogens B. cereus and B. anthracis and to prevent phage contamination of the natural insect pesticide Bt. A comparative phylogenetic analysis has revealed three different major phage groups with different morphologies (Myoviridae for group I, Siphoviridae for group II, and Tectiviridae for group III), genome size (group I > group II > group III), and lifestyle (virulent for group I and temperate for group II and III). A subsequent phage genome comparison using a dot plot analysis showed that phages in each group are highly homologous, substantiating the grouping of B. cereus phages. Endolysin is a host lysis protein that contains two conserved domains: a cell-wall-binding domain (CBD) and an enzymatic activity domain (EAD). In B. cereus sensu lato phage group I, four different endolysin groups have been detected, according to combinations of two types of CBD and four types of EAD. Group I phages have two copies of tail lysins and one copy of endolysin, but the functions of the tail lysins are still unknown. In the B. cereus sensu lato phage group II, the B. anthracis phages have been studied and applied for typing and rapid detection of pathogenic host strains. In the B. cereus sensu lato phage group III, the B. thuringiensis phages Bam35 and GIL01 have been studied to understand phage entry and lytic switch regulation mechanisms. In this review, we suggest that further study of the B. cereus group phages would be useful for various phage applications, such as biocontrol, typing, and rapid detection of the pathogens B. cereus and B. anthracis and for the prevention of phage contamination of the natural insect pesticide Bt.
Full-text · Article · Nov 2013 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: A novel Vibrio vulnificus-infecting bacteriophage, SSP002, belonging to the Siphoviridae family, was isolated from the coastal area of the Yellow Sea of South Korea. Host range analysis revealed that the growth
inhibition of phage SSP002 is relatively specific to V. vulnificus strains from both clinical and environmental samples. In addition, a one-step growth curve analysis and a bacteriophage stability
test revealed a latent period of 65 min, a burst size of 23 ± 2 PFU, as well as broad temperature (20°C to 60°C) and pH stability
(pH 3 to 12) ranges. A Tn5 random transposon mutation of V. vulnificus and partial DNA sequencing of the inserted Tn5 regions revealed that the flhA, flhB, fliF, and fleQ mutants are resistant to SSP002 phage infection, suggesting that the flagellum may be the host receptor for infection. The
subsequent construction of specific gene-inactivated mutants (flhA, flhB, fliF, and fleQ) and complementation experiments substantiated this. Previously, the genome of phage SSP002 was completely sequenced and
analyzed. Comparative genomic analysis of phage SSP002 and Vibrio parahaemolyticus phage vB_VpaS_MAR10 showed differences among their tail-related genes, supporting different host ranges at the species level,
even though their genome sequences are highly similar. An additional mouse survival test showed that the administration of
phage SSP002 at a multiplicity of infection of 1,000 significantly protects mice from infection by V. vulnificus for up to 2 months, suggesting that this phage may be a good candidate for the development of biocontrol agents against V. vulnificus infection.
Full-text · Article · Nov 2013 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: To understand phage infection and host cell lysis mechanisms in pathogenic Salmonella, a novel Salmonella enterica serovar Typhimurium-targeting bacteriophage, SPN9CC, belonging to the Podoviridae family was isolated and characterized. The phage infects S. Typhimurium via the O antigen of lipopolysaccharide (LPS) and forms clear plaques with cloudy centers due to lysogen formation.
Phylogenetic analysis of phage major capsid proteins revealed that this phage is a member of the lysogen-forming P22-like
phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions
and host cell lysis gene clusters show very low levels of identity, suggesting that lysogen formation and host cell lysis
mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding
holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in S. Typhimurium and Escherichia coli hosts revealed that collaboration of these lysis proteins is important for the lysis of both hosts and that holin is a key
protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a ΔcI mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting
that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge
assays revealed that SPN9CCM has shorter eclipse/latency periods and a larger burst size, as well as higher host cell lysis
activity, than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol
agents similar to virulent phages.
Full-text · Article · Nov 2013 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: For molecular study of marine bacteria Pseudoalteromonas phenolica using bacteriophage, a novel bacteriophage, TW1, belonging to the family Siphoviridae, was isolated, and its genome was completely sequenced and analyzed. The phage TW1 genome consists of 39,940-bp-length double-stranded DNA with a GC content of 40.19 %, and it was predicted to have 62 open reading frames (ORFs), which were classified into functional groups, including phage structure, packaging, DNA metabolism, regulation, and additional function. The phage life style prediction using PHACTS showed that it may be a temperate phage. However, genes related to lysogeny and host lysis were not detected in the phage TW1 genome, indicating that annotation information about P. phenolica phages in the genome databases may not be sufficient for the functional prediction of their encoded proteins. This is the first report of a P. phenolica-infecting phage, and this phage genome study will provide useful information for further molecular research on P. phenolica and its phage, as well as their interactions.
Full-text · Article · Jul 2013 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus aureus is a well-known pathogen that causes several serious diseases in humans and animals. As part of the efforts to control this pathogen, a newly isolated bacteriophage, SA12, which specifically targets S. aureus, was characterized, and its genome was completely sequenced. Host range and bacteriophage challenge tests demonstrated its specific and efficient host lysis of S. aureus. The genome of phage SA12 consists of 42,902 bp length double-stranded DNA with 58 predicted ORFs-encoding phage structure, DNA manipulation, packaging, host lysis, and regulation proteins. The characterization and genome study of phage SA12 in this report is useful for understanding S. aureus-targeting bacteriophages and provides basic information for the further development of phage-based biocontrol agents against S. aureus.