Characterization of LysB4, an endolysin from the Bacillus cereus-infecting bacteriophage B4

<p>Abstract</p> <p>Background</p> <p><it>Bacillus cereus </it>is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. The incidence of <it>B. cereus </it>food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against <it>B. cereus </it>need to be developed. Endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents.</p> <p>Results</p> <p>The endolysin from <it>B. cereus </it>phage B4, designated LysB4, was identified and characterized. <it>In silico </it>analysis revealed that this endolysin had the VanY domain at the N terminus as the catalytic domain, and the SH3_5 domain at the C terminus that appears to be the cell wall binding domain. Biochemical characterization of LysB4 enzymatic activity showed that it had optimal peptidoglycan hydrolase activity at pH 8.0-10.0 and 50°C. The lytic activity was dependent on divalent metal ions, especially Zn²⁺. The antimicrobial spectrum was relatively broad because LysB4 lysed Gram-positive bacteria such as <it>B. cereus, Bacillus subtilis </it>and <it>Listeria monocytogenes </it>and some Gram-negative bacteria when treated with EDTA. LC-MS analysis of the cell wall cleavage products showed that LysB4 was an <smcaps>L</smcaps>-alanoyl-<smcaps>D</smcaps>-glutamate endopeptidase, making LysB4 the first characterized endopeptidase of this type to target <it>B. cereus</it>.</p> <p>Conclusions</p> <p>LysB4 is believed to be the first reported <smcaps>L</smcaps>-alanoyl-<smcaps>D</smcaps>-glutamate endopeptidase from <it>B. cereus</it>-infecting bacteriophages. The properties of LysB4 showed that this endolysin has strong lytic activity against a broad range of pathogenic bacteria, which makes LysB4 a good candidate as a biocontrol agent against <it>B. cereus </it>and other pathogenic bacteria.</p

Colanic Acid Is a Novel Phage Receptor of Pectobacterium carotovorum subsp. carotovorum Phage POP72

The emergence and widespread nature of pathogen resistance to antibiotics and chemicals has led to the re-consideration of bacteriophages as an alternative biocontrol agent in several fields, including agriculture. In this study, we isolated and characterized a novel bacteriophage, POP72, that specifically infects Pectobacterium carotovorum subsp. carotovorum (Pcc), which frequently macerates agricultural crops. POP72 contains a 44,760 bp double-stranded DNA genome and belongs to the family Podoviridae. To determine the phage receptor for POP72, a random mutant library of Pcc was constructed using a Tn5 transposon and screened for resistance against POP72 infection. Most of the resistant clones had a Tn5 insertion in various genes associated with colanic acid (CA) biosynthesis. The phage adsorption rate and CA production decreased dramatically in the resistant clones. Complementation of the clones with the pUHE21-2 lacIq vector harboring genes associated with CA biosynthesis restored their sensitivity to POP72, as well as their ability to produce CA. These results suggest that CA functions as a novel phage receptor for POP72. The application of POP72 protected Chinese cabbage from Pcc infection, suggesting that phage POP72 would be an effective alternative antimicrobial agent to protect agricultural products from Pcc

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

Isolation and Genomic Characterization of the T4-Like Bacteriophage PM2 Infecting Pectobacterium carotovorum subsp. carotovorum

In order to control Pectobacterium carotovorum subsp. carotovorum, a novel virulent bacteriophage PM2 was isolated. Bacteriophage PM2 can infect 48% of P. carotovorum subsp. carotovorum and 78% of P. carotovorum subsp. brasilliensis but none of atrosepticum, betavasculorum, odoriferum and wasabiae isolates had been infected with PM2. PM2 phage belongs to the family Myoviridae, and contains a large head and contractile tail. It has a 170,286 base pair genome that encodes 291 open reading frames (ORFs) and 12 tRNAs. Most ORFs in bacteriophage PM2 share a high level of homology with T4-like phages including IME08, RB69, and JS98. Phylogenetic analysis based on the amino acid sequence of terminase large subunits confirmed that PM2 is classified as a T4-like phage. It contains no integrase- or no repressor-coding genes related to the lysogenic cycle, and lifestyle prediction using PHACT software suggested that PM2 is a virulent bacteriophage

The Interaction of Human Enteric Pathogens with Plants

There are an increasing number of outbreaks of human pathogens related to fresh produce. Thus, the growth of human pathogens on plants should be explored. Human pathogens can survive under the harsh environments in plants, and can adhere and actively invade plants. Plant-associated microbiota or insects contribute to the survival and transmission of enteric pathogens in plants. Human enteric pathogens also trigger plant innate immunity, but some pathogens–such as Salmonella–can overcome this defense mechanism

Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster.

The ability of Pseudomonas syringae pv. syringae 61 to elicit the hypersensitive response in nonhost plant species has been linked to a cluster of hrp/hrm genes whose expression appears to be environmentally regulated. To understand the genetic organization of this hrp/hrm gene cluster and its expression during the interaction with nonhost plant species better, we constructed a set of chromosomal hrp-uidA fusions in P. syringae pv. syringae 61 by Tn5-gusA1 mutagenesis of the cloned hrp/hrm gene cluster and transferred them into the genome by marker exchange mutagenesis. Complementation analysis employing plasmid-borne Tn5-gusA1 insertions and previously characterized chromosomal TnphoA mutations defined at least eight apparent transcriptional units within the hrp/hrm cluster, several of which were multicistronic. The expression of hrp-uidA fusions in seven of these apparent hrp transcriptional units increased following inoculation into tobacco leaves. Enhanced expression from a representative fusion was detected 1 h after inoculation of tobacco leaves. The induction observed in planta was similar to the levels detected following culture of the bacteria in minimal-salts medium: irrespective of the carbon source. Complex amino acid sources, such as peptone, repressed the expression of P. syringae pv. syringae 61 hrp genes at levels exceeding 0.028%. The results indicate that enhanced expression of hrp genes occurs early in the interaction with nonhost plant species in an apparent response to altered nutritional conditions

Simple Detection of Cochliobolus Fungal Pathogens in Maize

Northern corn leaf spot and southern corn leaf blight caused by Cochliobolus carbonum (anamorph, Bipolaris zeicola) and Cochliobolus heterostrophus (anamorph, Bipolaris maydis), respectively, are common maize diseases in Korea. Accurate detection of plant pathogens is necessary for effective disease management. Based on the polyketide synthase gene (PKS) of Cochliobolus carbonum and the nonribosomal peptide synthetase gene (NRPS) of Cochliobolus heterostrophus, primer pairs were designed for PCR to simultaneously detect the two fungal pathogens and were specific and sensitive enough to be used for duplex PCR analysis. This duplex PCR-based method was found to be effective for diagnosing simultaneous infections from the two Cochliobolus species that display similar morphological and mycological characteristics. With this method, it is possible to prevent infections in maize by detecting infected seeds or maize and discarding them. Besides saving time and effort, early diagnosis can help to prevent infections, establish comprehensive management systems, and secure healthy seeds

Genetic Diversity of Pectobacterium carotovorum subsp. brasiliensis Isolated in Korea

The plant pathogenic bacterial genus Pectobacteirum consists of heterogeneous strains. The P. carotovorum species is a complex strain showing divergent characteristics, and a new subspecies named P. carotovorum subsp. brasiliensis has been identified recently. In this paper, we re-identified the P. carotovorum subsp. brasiliensis isolates from those classified under the subspecies carotovorum and newly isolated P. carotovorum subsp. brasiliensis strains. All isolates were able to produce plant cell-wall degrading enzymes such as pectate lyase, polygalacturonase, cellulase and protease. We used genetic and biochemical methods to examine the diversity of P. carotovorum subsp. brasiliensis isolates, and found genetic diversity within the brasiliensis subsp. isolates in Korea. The restriction fragment length polymorphism analysis based on the recA gene revealed a unique pattern for the brasiliensis subspecies. The Korean brasiliensis subsp. isolates were divided into four clades based on pulsed-field gel electrophoresis. However, correlations between clades and isolated hosts or year could not be found, suggesting that diverse brasiliensis subsp. isolates existed

Potato Yield Gaps in North Korea and Strategies to Close the Gaps

Potato has become one of the staple crops to improve food security in North Korea since the late 1990s. However, the potato yield has been stagnated around 11&ndash;12 t ha&minus;1 for several decades, and a food shortage is still a primary issue in North Korea. Yield gap analyses were carried out using the SUBSTOR-potato model to quantify the potato yield gaps and explore the potential ways to close the yield gaps in two different cropping seasons in North Korea (early- and main-season potatoes). Yield gaps were estimated to be around 80% for both early- and main-season potatoes. Early-season potato yield was substantially determined by water or nitrogen supplies, depending on the year&rsquo;s weather condition (i.e., with or without spring drought). Irrigation during the vegetative stage could effectively reduce the year-to-year variation in yield as well as the yield gap (+7.0 t ha&minus;1, +66.1%). Meanwhile, additional nitrogen fertilizer in the early-season potatoes was less effective compared to that in the main-season potatoes. For the main-season potatoes, where precipitation was sufficient, the primary limiting factor of yield was nitrogen supply. Since heavy rainfall aggravated nitrogen leaching, additional nitrogen fertilizer is recommended as a top dressing rather than a basal dressing. Additional top dressing at 50 days after planting with the current amount of nitrogen fertilizer was expected to increase the main-season potato yield by 42.0 t ha&minus;1 (+191.4%). This study highlights that the primary limiting factor of potato yield may differ between the cropping seasons. Therefore, our findings suggest that different agronomic strategies should be applied for different cropping seasons to improve potato production in North Korea, where agronomic resources are limited