Genome Sequence of Ralstonia pseudosolanacearum SL1931, a Causal Phytopathogen of Bacterial Wilt Disease in Capsicum annuum and Nicotiana benthamiana.

Here, we report the genome sequence of Ralstonia pseudosolanacearum (R. solanacearum phylotype I) strain SL1931 (KACC10711), isolated from pepper (Capsicum annuum L.) stems; R. solanacearum is the causal pathogen of bacterial wilt. Strain SL1931 had a different type III effector profile than that of the reference genome strain GMI1000.

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper.

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

Population changes and growth modeling of Salmonella enterica during alfalfa seed germination and early sprout development.

This study examined the effects of alfalfa seed germination on growth of Salmonella enterica. We investigated the population changes of S. enterica during early sprout development. We found that the population density of S. enterica, which was inoculated on alfalfa seeds was increased during sprout development under all experimental temperatures, whereas a significant reduction was observed when S. enterica was inoculated on fully germinated sprouts. To establish a model for predicting S. enterica growth during alfalfa sprout development, the kinetic growth data under isothermal conditions were collected and evaluated based on Baranyi model as a primary model for growth data. To elucidate the influence of temperature on S. enterica growth rates, three secondary models were compared and found that the Arrhenius-type model was more suitable than others. We believe that our model can be utilized to predict S. enterica behavior in alfalfa sprout and to conduct microbial risk assessments.

Analysis of Genetic and Pathogenic Diversity of Ralstonia solanacearum Causing Potato Bacterial Wilt in Korea.

The Ralstonia solanacearum species complex (RSSC) can be divided into four phylotypes, and includes phenotypically diverse bacterial strains that cause bacterial wilt on various host plants. This study used 93 RSSC isolates responsible for potato bacterial wilt in Korea, and investigated their phylogenetic relatedness based on the analysis of phylotype, biovar, and host range. Of the 93 isolates, twenty-two were identified as biovar 2, eight as biovar 3, and sixty-three as biovar 4. Applied to the phylotype scheme, biovar 3 and 4 isolates belonged to phylotype I, and biovar 2 isolates belonged to phylotype IV. This classification was consistent with phylogenetic trees based on 16S rRNA and egl gene sequences, in which biovar 3 and 4 isolates clustered to phylotype I, and biovar 2 isolates clustered to phylotype IV. Korean biovar 2 isolates were distinct from biovar 3 and 4 isolates pathologically as well as genetically - all biovar 2 isolates were nonpathogenic to peppers. Additionally, in host-determining assays, we found uncommon strains among biovar 2 of phylotype IV, which were the tomato-nonpathogenic strains. Since tomatoes are known to be highly susceptible to RSSC, to the best of our knowledge this is the first report of tomato-nonpathogenic potato strains. These results imply the potential prevalence of greater RSSC diversity in terms of host range than would be predicted based on phylogenetic analysis.

Development of a Screening Method and Device for the Detection of Escherichia coli from Agri-Food Production Environments and Fresh Produce.

This study was conducted to develop a screening method using Colilert-18 and a device for the detection of E. coli from agri-food production environments and fresh vegetables. The specificity and sensitivity of Colilert-18 by temperature (37°C and 44°C) were evaluated with 38 E. coli and 78 non-E. coli strains. The false-positive rate was 3.8% (3/78) and 0% (0/78) at 37°C and 44°C, respectively. The detection limit of E. coli at 37°C at <1.0 log CFU/250 ml was lower than that at 44°C. The efficiency of the developed device, which comprised an incubator equipped with a UV lamp to detect E. coli in the field, was evaluated by measuring the temperature and UV lamp brightness. The difference between the set temperature and actual temperature of the developed device was about 1.0°C. When applying the developed method and device to various samples, including utensils, gloves, irrigation water, seeds, and vegetables, there were no differences in detection rates of E. coli compared with the Korean Food Code method. For sanitary disposal of culture samples after experiments, the sterilization effect of sodium dichloroisocyanurate (NaDCC) tablets was assessed for use as a substitute for an autoclave. The addition of one tablet of NaDCC per 50 ml was sufficient to kill E. coli cultured in Colilert-18. These results show that the developed protocol and device can efficiently detect E. coli from agri-food production environments and vegetables.

Clavibacter michiganensis subsp. capsici subsp. nov., causing bacterial canker disease in pepper.

Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies. One of these subspecies is C. michiganensis subsp. michiganensis, which causes bacterial canker disease in tomato. Bacterial strains showing very similar canker disease symptoms to those of a strain originally classified as C. michiganensis have been isolated from pepper. In this paper, we reclassified strains isolated from pepper. On the basis of phylogenetic analysis with 16S rRNA gene sequences, the strains isolated from pepper were grouped in a separate clade from other subspecies of C. michiganensis. Biochemical, physiological and genetic characteristics of strain PF008T, which is the representative strain of the isolates from pepper, were examined in this study. Based on multi-locus sequence typing and other biochemical and physiological features including colony color, utilization of carbon sources and enzyme activities, strain PF008T was categorically differentiated from eight subspecies of C. michiganensis. Moreover, genome analysis showed that the DNA G+C content of strain PF008T is 73.2 %. These results indicate that PF008T is distinct from other known subspecies of C. michiganensis. Therefore, we propose a novel subspecies, C. michiganensis subsp. capsici, causing bacterial canker disease in pepper, with a type strain of PF008T (=KACC 18448T=LMG 29047T).

An HrpB-dependent but type III-independent extracellular aspartic protease is a virulence factor of Ralstonia solanacearum.

The host specificity of Ralstonia solanacearum, the causal organism of bacterial wilt on many solanaceous crops, is poorly understood. To identify a gene conferring host specificity of the bacterium, SL341 (virulent to hot pepper but avirulent to potato) and SL2029 (virulent to potato but avirulent to hot pepper) were chosen as representative strains. We identified a gene, rsa1, from SL2029 that confers avirulence to SL341 in hot pepper. The rsa1 gene encoding an 11.8-kDa protein possessed the perfect consensus hrp(II) box motif upstream of the gene. Although the expression of rsa1 was activated by HrpB, a transcriptional activator for hrp gene expression, Rsa1 protein was secreted in an Hrp type III secretion-independent manner. Rsa1 exhibited weak homology with an aspartic protease, cathepsin D, and possessed protease activity. Two specific aspartic protease inhibitors, pepstatin A and diazoacetyl-d,l-norleucine methyl ester, inhibited the protease activity of Rsa1. Substitution of two aspartic acid residues with alanine at positions 54 and 59 abolished protease activity. The SL2029 rsa1 mutant was much less virulent than the wild-type strain, but did not induce disease symptoms in hot pepper. These data indicate that Rsa1 is an extracellular aspartic protease and plays an important role for the virulence of SL2029 in potato.

Characterization of a new bacteriocin, Carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21.

Two different bacteriocins, carotovoricin and carocin S1, had been found in Pectobacterium carotovorum subsp. carotovorum, which causes soft-rot disease in diverse plants. Previously, we reported that the particular strain Pcc21, producing only one high-molecular-weight bacteriocin, carried a new antibacterial activity against the indicator strain Pcc3. Here, we report that this new antibacterial activity is due to a new bacteriocin produced by strain Pcc21 and named carocin D. Carocin D is encoded by the caroDK gene located in the genomic DNA together with the caroDI gene, which seems to encode an immunity protein. N-terminal amino acid sequences of purified carocin D were determined by Edman degradation. In comparison with the primary translation product of caroDK, it was found that 8 amino acids are missing at the N terminus. This finding proved that carocin D is synthesized as a precursor peptide and that 8 amino acids are removed from its N terminus during maturation. Carocin D has two putative translocation domains; the N-terminal and C-terminal domains are homologous to those of Escherichia coli colicin E3 and Pseudomonas aeruginosa S-type pyocin, respectively. When caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3, the bacteria became resistant to this bacteriocin. Carocin D has one putative DNase domain at the extreme C terminus and showed DNase activity in vitro. This bacteriocin had slight tolerance to heat but not to proteases. The caroDK gene was present in only 5 of 54 strains of P. carotovorum subsp. carotovorum. These results indicate that carocin D is a third bacteriocin found in P. carotovorum subsp. carotovorum, and this bacteriocin can be readily expressed in carocin D-sensitive nonpathogenic bacteria, which may have high potential as a biological control agent in the field.

Cloning of circular DNAs from microorganisms using a novel plasmid capture system.

Plasmid capture system (PCS) facilitates cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in microorganisms including invertebrate pathogens.

Isolation and characterization of bacteriophages specific for Campylobacter jejuni.

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni-specific bacteriophages (CPS1-6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni, but not other Gram-negative bacteria, including C. coli, Escherichia coli, Salmonella spp., and Gram-positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni.

Diverse Antibacterial activity of Pectobacterium carotovorum subsp.carotovorum isolated in Korea.

Fifty-four Pectobacterium carotovorum subsp. carotovorum strains isolated in Korea were characterized by a spectrum of antibacterial activities against 7 indicator strains chosen to represent various regions and host plants. All P. carotovorum subsp. carotovorum isolates tested could be grouped into 4 classes depending on the pattern of antibacterial substance production. All tested strains had DNA fragment(s) homologous to the genes encoding carotovoricin and 21 of them had genes homologous to DNA invertase. Sixteen strains had genes homologous to the genes encoding carocin S1. Several isolates produced antibacterial substances active against strains in Brenneria, Pantoea, and Pectobacterium genera that belonged formerly to the genus Erwinia. Strains in Pseudomonas or Xanthomonas sp. were not sensitive to the antibacterial substances produced by P. carotovorum subsp. carotovorum, except for X. albilineans that was sensitive to antibacterial substances produced by most strains in P. carotovorum subsp. carotovorum and P. betavasculorum KACC10056. These results demonstrated the diverse patterns of antibacterial substance production and the possibility of the existence of new antibacterial substance(s) produced by P. carotovorum subsp. carotovorum isolated in Korea.

Avirulence gene diversity of Xanthomonas axonopodis pv. glycines isolated in Korea.

The hybridization patterns with the avrBs3 gene that is known to determine the recognition of host specificity were used to study the diversity of Xanthomonas axonopodis pv. glycines causing bacterial leaf pustule in soybean. A total of 155 strains were isolated from diverse tissues of soybean cultivars collected in Korea and were classified into six different type strains of OcsF, SL1017, SL1018, SL1045, SL1157, and SL2098 according to the patterns of avrBs3-homologous bands. When these type strains were inoculated on various cultivars, most of the Korean strains mildly induced disease symptoms on the resistant CNS1 cultivars. Unlike other type strains, strain SL2098, which appeared not to contain any avrBs3 homolog, induced only a few pustules on even highly susceptible cultivars. When a plasmid carrying the 3.7-kb avrBs3-homologous gene from strain SL1045 was introduced into SL2098, the transformant could not recover the pathogenicity in susceptible host plants. However, when avrBs3-homologous genes of strain SL1018 were mutated by transposon mutagenesis, one of the mutants in which a 5.2-kb chromosomal band homologous to avrBs3 was disrupted could not induce the hypersensitive response on resistant cultivars such as William82 or CNS2. Our results suggest that the avrBs3 homologs may play important roles in the pathogenicity of Xanthomonas axonopodis pv. glycines and the recognition of soybean cultivars.

Genetic Diversity and Distribution of Korean Isolates of Ralstonia solanacearum.

Genetic diversity among 478 isolates of Ralstonia solanacearum collected from various plants in Korea between 1997 and 2005 was determined based on biovar, pathogenicity, amplified fragment length polymorphism (AFLP), 16S rRNA, endoglucanase, hrpB, and mutS gene sequence analyses. Of the isolates, 440 belonged to biovars 1, 3, or 4, and 38 belonged to biovar 2. Biovar N2 isolates were not found. The biovar 1 and 2 isolates were found mainly in southern Korea, whereas the biovar 3 and 4 isolates were widely distributed throughout all nine provinces. AFLP analysis divided the 109 representative Korean isolates into six clusters that were distinct from most of the foreign isolates. Grouping of 8 representative isolates based on their 16S rRNA gene sequences indicated that biovars 1, 3, and 4 belonged to division 1, while biovar 2 belonged to subdivision 2b. Sequence analysis of the endoglucanase, hrpB, and mutS genes from the same isolates indicated that the biovar 1, 3, and 4 isolates belonged to phylotype I, while the biovar 2 isolate belonged to phylotype IV. This study is the first comprehensive analysis of genetic diversity among Korean isolates of R. solanacearum.

One-pot enzymatic synthesis of UDP-D-glucose from UMP and glucose-1-phosphate using an ATP regeneration system.

Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.