• Microbiology and Biotechnology Letters
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• v.49 no.4
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• pp.510-518
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• 2021

Bacteriophages are considered excellent sensing elements for platforms detecting bacteria. However, their lytic cycle has restricted their efficacy. Here, we used the minor coat protein pIII domain (N1N2) of phage CTXφ to construct a novel surface plasmon resonance (SPR) biosensor that could detect Vibrio cholerae. N1N2 harboring the domains required for phage adsorption and entry was obtained from Escherichia coli using recombinant protein expression and purification. SDS-PAGE revealed an approximate size of 30 kDa for N1N2. Dot blot and transmission electron microscopy analyses revealed that the protein bound to the host V. cholerae but not to non-host E. coli K-12 cells. Next, we used amine-coupling to develop a novel recombinant N1N2 (rN1N2)-functionalized SPR biosensor by immobilizing rN1N2 proteins on gold substrates and using SPR to monitor the binding kinetics of the proteins with target bacteria. We observed rapid detection of V. cholerae in the range of approximately 10³ to 10⁹ CFU/ml but not of E. coli at any tested concentration, thereby confirming that the biosensor exhibited differential recognition and binding. The results indicate that the novel biosensor can rapidly monitor a target pathogenic microorganism in the environment and is very useful for monitoring food safety and facilitating early disease prevention.

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2075-2088
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• 2017

Salmonella is one of the principal causes of foodborne outbreaks. As traditional control methods have shown less efficacy against emerging Salmonella serotypes or antimicrobial-resistant Salmonella, new approaches have been attempted. The use of lytic phages for the biocontrol of Salmonella in the food industry has become an attractive method owing to the many advantages offered by the use of phages as biocontrol agents. Phages are natural alternatives to traditional antimicrobial agents; they have proven effective in the control of bacterial pathogens in the food industry, which has led to the development of different phage products. The treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases, and ultimately promotes safe environments for animal and plant food production, processing, and handling. After an extensive investigation of the current literature, this review focuses predominantly on the efficacy of phages for the successful control of Salmonella spp. in foods. This review also addresses the current knowledge on the pathogenic characteristics of Salmonella, the prevalence of emerging Salmonella outbreaks, the isolation and characterization of Salmonella-specific phages, the effectiveness of Salmonella-specific phages as biocontrol agents, and the prospective use of Salmonella-specific phages in the food industry.

• Journal of Life Science
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• v.28 no.1
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• pp.78-82
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• 2018

Detection of pathogenic microorganisms takes several days by conventional methods. It is necessary to assess microorganisms in a timely manner to reduce the risk of spreading infection. For this purpose, bacteriophages are chosen for use as a biosensing tool due to their host specificity, wide abundance, and safety. However, their lytic cycle limits their efficacy as biosensors. Phage proteins involved in binding to bacteria could be a robust alternative in resolving this drawback. Here, a fragment of tail protein J (residues 784 to 1,132) of phage lambda fused with 6X His-tag (6HN-J) at its N-terminus was cloned, overexpressed, purified, and characterized for its binding with microorganisms. The purified protein demonstrated a size of about 38 kDa in sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and bound with anti-His monoclonal antibodies. It bound specifically to Escherichia coli K-12, and not Salmonella typhimurium, Bacillus subtilis, or Pseudomonas aeruginosa in dot blotting. Binding of the protein to E. coli K-12 inhibited about 50% of the in vivo adsorption of the phage lambda to host cells at a concentration of $1{\mu}g/ml$ 6HN-J protein and almost 100% at $25{\mu}g/ml$ 6HN-J. The results suggest that a fusion viral protein could be utilized as a biosensing element (e.g., protein chips) for detecting microorganisms in real time.

• BMB Reports
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• v.36 no.6
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• pp.586-592
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• 2003

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the $\beta$-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli $\sigma^{70}$-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early $P_{left}$ promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the $P_{left}$ equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli $\sigma^{70}$ promoters.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.385-393
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• 2016

Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50℃, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits.

• Korean Journal of Microbiology
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• v.54 no.2
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• pp.149-151
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• 2018

Clostridium perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming pathogenic bacterium, which belongs to the Clostridiaceae family. C. perfringens causes diseases including food poisoning in vertebrates and intestinal tract of humans. Bacteriophages that can kill target bacteria specifically have been considered as one of control methods for bacterial pathogens. Here, we report a draft genome sequence of the bacteriophage CP3 effective to C. perfringens. The phage genome comprises 52,068 bp with a G + C content of 34.0%. The draft genome has 74 protein-coding genes, 29 of which have predicted functions from BLASTp analysis. Others are conserved proteins with unknown functions. No RNAs were found in the genome.

• Korean Journal of Microbiology
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• v.54 no.2
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• pp.152-154
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• 2018

Klebsiella pneumoniae is a Gram-negative, rod-shape bacterium causing disease in human and animal lungs. K. pneumoniae has been often found to gain antimicrobial resistance, thus it has been difficult to treat K. pneumoniae infection with antibiotics. For such infection, bacteriophage can provide an alternative approach for pathogenic bacterial infection with antimicrobial resistance, because of its sensitivity and specificity to the host bacteria. Bacteriophage KP1 was isolated in sewage and showed specific infectivity to K. pneumoniae. Here, we report the draft genome sequence of Klebsiella pneumoniae phage KP1. The draft genome of KP1 is 167,989 bp long, and the G + C content is 39.6%. The genome has 295 predicted ORFs and 14 tRNA genes. In addition, it encodes various enzymes which involve in lysis of the host cell such as lysozyme and holin.

• Journal of Microbiology and Biotechnology
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• v.32 no.6
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• pp.816-823
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• 2022

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

• Korean Journal of Microbiology
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• v.34 no.4
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• pp.248-253
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• 1998

Bacteriophage N4, a lytic phage specific for Esherichia coli K12 strain encodes single-stranded DNA-binding protein, N4SSB (bacteriophage N4-coded single-stranded DNA-binding protein). N4SSB protein is originally identified as a protein required for N4 DNA replication. N4SSB protein is also required for N4 late transcription, which is catalyzed by E. coli ${\sigma}^{70}$ RNA polymerase. N4 late transcription does not occur until N4SSB protein is synthesized. Recently it is reported that N4SSB protein is essential for N4 DNA recombination. Therefore N 4SSB protein is a multifunctional protein required for N4 DNA replication, late transcription, and N4 DNA recombination. In this study, a variety of mutant N4SSB proteins containing internal deletions or substitutions were constructed to define and characterize domains important for N4 DNA replication, late transcription, and N4 DNA recombination. Test for the ill vivo activity of these mutant N4SSBs for N4 DNA replication, late transcription, and N4 DNA recombination was examined. The results suggest that C-terminal 7 amino acid residues are important for the activity of N4SSB. Three lysine residues, which are contained in this region play important roles on N4SSB activity.