Journal of Microbiology

Journal Articles

Characterization of Newly Isolated Bacteriophages Targeting Carbapenem-Resistant Klebsiella pneumoniae: Bokyung Kim, Shukho Kim, Yoon-Jung Choi, Minsang Shin, Jungmin Kim; J. Microbiol. 2024;62(12):1133-1153. Published online December 10, 2024; DOI: https://doi.org/10.1007/s12275-024-00180-7

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Abstract: Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is increasingly resistant to carbapenems in clinical settings. This growing problem necessitates the development of alternative antibiotics, with phage therapy being one promising option. In this study, we investigated novel phages targeting carbapenem-resistant Klebsiella pneumoniae (CRKP) and evaluated their lytic capacity against clinical isolates of CRKP. First, 23 CRKP clinical isolates were characterized using Multi-Locus Sequence Typing (MLST), carbapenemase test, string test, and capsule typing. MLST classified the 23 K. pneumoniae isolates into 10 sequence types (STs), with the capsule types divided into nine known and one unknown type. From sewage samples collected from a tertiary hospital, 38 phages were isolated. Phenotypic and genotypic characterization of these phages was performed using Random Amplification of Polymorphic DNA-PCR (RAPD-PCR), transmission electron microscopy (TEM), and whole genome sequencing (WGS) analysis. Host spectrum analysis revealed that each phage selectively lysed strains sharing the same STs as their hosts, indicating ST-specific activity. These phages were subtyped based on their host spectrum and RAPD-PCR, identifying nine and five groups, respectively. Fourteen phages were selected for further analysis using TEM and WGS, revealing 13 Myoviruses and one Podovirus. Genomic analysis grouped the phages into three clusters: one closely related to Alcyoneusvirus, one to Autographiviridae, and others to Straboviridae. Our results showed that the host spectrum of K. pneumoniae-specific phages corresponds to the STs of the host strain. These 14 novel phages also hold promise as valuable resources for phage therapy against CRKP.

Enterococcus Phage vB_EfaS_HEf13 as an Anti-Biofilm Agent Against Enterococcus faecalis: Dongwook Lee, Jintaek Im, A Reum Kim, Woohyung Jun, Cheol-Heui Yun, Seung Hyun Han; J. Microbiol. 2024;62(8):683-693. Published online June 27, 2024; DOI: https://doi.org/10.1007/s12275-024-00150-z

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Abstract: Enterococcus faecalis is a Gram-positive bacterium that is frequently found in the periapical lesion of patients with apical periodontitis. Its biofilm formation in root canal is closely related to the development of refractory apical periodontitis by providing increased resistance to endodontic treatments. Phage therapy has recently been considered as an efficient therapeutic strategy in controlling various periodontal pathogens. We previously demonstrated the bactericidal capacities of Enterococcus phage vB_EfaS_HEf13 (phage HEf13) against clinically-isolated E. faecalis strains. Here, we investigated whether phage HEf13 affects biofilm formation and pre-formed biofilm of clinically-isolated E. faecalis, and its combinatory effect with endodontic treatments, including chlorhexidine (CHX) and penicillin. The phage HEf13 inhibited biofilm formation and disrupted pre-formed biofilms of E. faecalis in a dose- and time-dependent manner. Interestingly, phage HEf13 destroyed E. faecalis biofilm exopolysaccharide (EPS), which is known to be a major component of bacterial biofilm. Furthermore, combined treatment of phage HEf13 with CHX or penicillin more potently inhibited biofilm formation and disrupted pre-formed biofilm than either treatment alone. Confocal laser scanning microscopic examination demonstrated that these additive effects of the combination treatments on disruption of pre-formed biofilm are mediated by relatively enhanced reduction in thickness distribution and biomass of biofilm. Collectively, our results suggest that the effect of phage HEf13 on E. faecalis biofilm is mediated by its EPS-degrading property, and its combination with endodontic treatments more potently suppresses E. faecalis biofilm, implying that phage HEf13 has potential to be used as a combination therapy against E. faecalis infections.

[Protocol] Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering: Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho; J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024; DOI: https://doi.org/10.1007/s12275-024-00107-2

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Abstract

Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

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Characteristics of bioaerosols under high-ozone periods, haze episodes, dust storms, and normal days in Xi’an, China
Yiming Yang, Liu Yang, Xiaoyan Hu, Zhenxing Shen
Particuology.2024; 90: 140. CrossRef
Airborne desert dust and aeromicrobiology over the Turkish Mediterranean coastline
Dale W. Griffin, Nilgün Kubilay, Mustafa Koçak, Mike A. Gray, Timothy C. Borden, Eugene A. Shinn
Atmospheric Environment.2007; 41(19): 4050. CrossRef

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.): Hegde Veena , Sandesh K. Gowda , Rajeshwara N. Achur , Nayaka Boramuthi Thippeswamy; J. Microbiol. 2021;59(4):435-447. Published online February 25, 2021; DOI: https://doi.org/10.1007/s12275-021-0423-2

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Abstract

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1β, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1β, and nitric oxide.

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Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I
Dayeong Bae, Hana Hyeon, Eunkyoung Shin, Ji-Hyun Yeom, Kangseok Lee
Journal of Microbiology.2023; 61(2): 211. CrossRef
A systematic antidiarrhoeal evaluation of a vegetable root Begonia roxburghii and its marker flavonoids against nonpathogenic and pathogenic diarrhoea
Rupali S. Prasad, Nikhil Y. Yenorkar, Suhas R. Dhaswadikar, Saurabh K. Sinha, Nitish Rai, Pravesh Sharma, Onkar Kulkarni, Neeraj Kumar, Mahaveer Dhobi, Damiki Laloo, Shailendra S. Gurav, Prakash R. Itankar, Satyendra K. Prasad
Food Bioscience.2023; 53: 102672. CrossRef

Review

Dissection of plant microbiota and plant-microbiome interactions: Kihyuck Choi , Raees Khan , Seon-Woo Lee; J. Microbiol. 2021;59(3):281-291. Published online February 23, 2021; DOI: https://doi.org/10.1007/s12275-021-0619-5

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Abstract

Plants rooted in soil have intimate associations with a diverse array of soil microorganisms. While the microbial diversity of soil is enormous, the predominant bacterial phyla associated with plants include Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Plants supply nutrient niches for microbes, and microbes support plant functions such as plant growth, development, and stress tolerance. The interdependent interaction between the host plant and its microbes sculpts the plant microbiota. Plant and microbiome interactions are a good model system for understanding the traits in eukaryotic organisms from a holobiont perspective. The holobiont concept of plants, as a consequence of co-evolution of plant host and microbiota, treats plants as a discrete ecological unit assembled with their microbiota. Dissection of plant-microbiome interactions is highly complicated; however, some reductionist approaches are useful, such as the synthetic community method in a gnotobiotic system. Deciphering the interactions between plant and microbiome by this reductionist approach could lead to better elucidation of the functions of microbiota in plants. In addition, analysis of microbial communities’ interactions would further enhance our understanding of coordinated plant microbiota functions. Ultimately, better understanding of plantmicrobiome interactions could be translated to improvements in plant productivity.

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Journal Articles

Leucobacter coleopterorum sp. nov., Leucobacter insecticola sp. nov., and Leucobacter viscericola sp. nov., isolated from the intestine of the diving beetles, Cybister brevis and Cybister lewisianus, and emended description of the genus Leucobacter: Dong-Wook Hyun , Hojun Sung , Pil Soo Kim , Ji-Hyun Yun , Jin-Woo Bae; J. Microbiol. 2021;59(4):360-368. Published online January 26, 2021; DOI: https://doi.org/10.1007/s12275-021-0472-6

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Abstract

Three novel bacterial strains, HDW9AT, HDW9BT, and HDW9CT, isolated from the intestine of the diving beetles Cybister lewisianus and Cybister brevis, were characterized as three novel species using a polyphasic approach. The isolates were Gram-staining-positive, strictly aerobic, non-motile, and rod-shaped. They grew optimally at 30°C (pH 7) in the presence of 0.5% (wt/vol) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that they belong to the genus Leucobacter and are closely related to L. denitrificans M1T8B10T (98.4–98.7% sequence similarity). Average nucleotide identity (ANI) values among the isolates were 76.4–84.1%. ANI values for the isolates and the closest taxonomic species, L. denitrificans KACC 14055T, were 72.3–73.1%. The isolates showed ANI values of < 76.5% with all analyzable Leucobacter strains in the EzBioCloud database. The genomic DNA G + C content of the isolates was 60.3–62.5%. The polar lipid components were phosphatidylglycerol, diphosphatidylglycerol, and other unidentified glycolipids, phospholipids, and lipids. The major cellular fatty acids were anteiso- C15:0, iso-C16:0, and anteiso-C17:0. MK-10 was the major respiratory quinone, and MK-7 and MK-11 were the minor respiratory quinones. The whole-cell sugar components of the isolates were ribose, glucose, galactose, and mannose. The isolates harbored L-2,4-diaminobutyric acid, L-serine, L-lysine, L-aspartic acid, glycine, and D-glutamic acid within the cell wall peptidoglycan. Based on phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses, strains HDW9AT, HDW9BT, and HDW9CT represent three novel species within the genus Leucobacter. We propose the name Leucobacter coleopterorum sp. nov. for strain HDW9AT (= KACC 21331T = KCTC 49317T = JCM 33667T), the name Leucobacter insecticola sp. nov. for strain HDW9BT (= KACC 21332T = KCTC 49318T = JCM 33668T), and the name Leucobacter viscericola sp. nov. for strain HDW9CT (= KACC 21333T = KCTC 49319T = JCM 33669T).

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Biogenic Silver Nanoparticles Produced by Soil Rare Actinomycetes and Their Significant Effect on Aspergillus-derived mycotoxins
Mohamed N. Abd El-Ghany, Salwa A. Hamdi, Shereen M. Korany, Reham M. Elbaz, Ahmed N. Emam, Mohamed G. Farahat
Microorganisms.2023; 11(4): 1006. CrossRef
Leucobacter tenebrionis sp. nov., isolated from the gut of Tenebrio molitor
Yu Ying, Bo Yuan, Tingting Liu, Xiaoshuan Bai, Haifeng Zhao
International Journal of Systematic and Evolutionary Microbiology .2023;[Epub] CrossRef
Leucobacter allii sp. nov. and Leucobacter rhizosphaerae sp. nov., isolated from rhizospheres of onion and garlic, respectively
Seunghwan Kim, Tomomi Asano, Hanako Naito, Moriyuki Hamada, Hang-Yeon Weon, Soon-Wo Kwon, Jun Heo
International Journal of Systematic and Evolutionary Microbiology .2023;[Epub] CrossRef
Leucobacter chinensis sp. nov., with plant growth-promoting potential isolated from field soil after seven-years continuous maize cropping
Jie Zhu, Juan Che, Xin Jiang, Mingchao Ma, Dawei Guan, Li Li, Fengming Cao, Baisuo Zhao, Yaowei Kang, Ji Zhao, Delong Kong, Yiqing Zhou, Zhiyong Ruan, Jun Li
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Hongbo Zhang, Yanhui Yu, Jianhua Li, Pengtao Gong, Xiaocen Wang, Xin Li, Yidan Cheng, Xiuyan Yu, Nan Zhang, Xichen Zhang
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Lysobacter ciconiae sp. nov., and Lysobacter avium sp. nov., isolated from the faeces of an Oriental stork
So-Yeon Lee, Pil Soo Kim, Hojun Sung, Dong-Wook Hyun, Jin-Woo Bae
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Valid publication of new names and new combinations effectively published outside the IJSEM. Validation List no. 203
Aharon Oren, George M. Garrity
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Characterization of a Salmonella Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria: Shukho Kim , Sung-Hun Kim , Marzia Rahman , Jungmin Kim; J. Microbiol. 2018;56(12):917-925. Published online October 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8310-1

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Abstract

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.

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Review of phage display: A jack-of-all-trades and master of most biomolecule display
Brenda Pei Chui Song, Angela Chiew Wen Ch'ng, Theam Soon Lim
International Journal of Biological Macromolecules.2024; 256: 128455. CrossRef
Application of the lytic bacteriophage Rostam to control Salmonella enteritidis in eggs
Rahim Azari, Mohammad Hashem Yousefi, Zohreh Taghipour, Jeroen Wagemans, Rob Lavigne, Saeid Hosseinzadeh, Seyed Mohammad Mazloomi, Marta Vallino, Sepideh Khalatbari-Limaki, Enayat Berizi
International Journal of Food Microbiology.2023; 389: 110097. CrossRef
Isolation and genomic characterization of Vmp-1 using Vibrio mimicus as the host: A novel virulent bacteriophage capable of cross-species lysis against three Vibrio spp.
Bin Yang, Yang Wang, Lu Gao, Sheng-qi Rao, Wen-yuan Zhou, Zhen-quan Yang, Xin-an Jiao, Benjamin Kumah Mintah, Mokhtar Dabbour
Microbial Pathogenesis.2023; 174: 105948. CrossRef
A Review on the Antimicrobial Effect of Honey on Salmonella and Listeria monocytogenes: Recent Studies
Fatih Ramazan İSTANBULLUGİL, Nuri TAŞ, Ulaş ACARÖZ, Damla ARSLAN-ACAROZ, Ömer ÇAKMAK, Sezen EVRENKAYA, Zeki GÜRLER
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Characterization of a Diverse Collection of Salmonella Phages Isolated from Tennessee Wastewater
Daniel W. Bryan, Lauren K. Hudson, Jia Wang, Thomas G. Denes
PHAGE.2023; 4(2): 90. CrossRef
Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food
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Prevalence of Indigenous Antibiotic-Resistant Salmonella Isolates and Their Application to Explore a Lytic Phage vB_SalS_KFSSM with an Intra-Broad Specificity
Jaein Choe, Su-Hyeon Kim, Ji Min Han, Jong-Hoon Kim, Mi-Sun Kwak, Do-Won Jeong, Mi-Kyung Park
Journal of Microbiology.2023; 61(12): 1063. CrossRef
Statistical optimization of a podoviral anti-MRSA phage CCASU-L10 generated from an under sampled repository: Chicken rinse
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Characterization and Genome Study of a Newly Isolated Temperate Phage Belonging to a New Genus Targeting Alicyclobacillus acidoterrestris
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In Vitro and In Vivo Gastrointestinal Survival of Non-Encapsulated and Microencapsulated Salmonella Bacteriophages: Implications for Bacteriophage Therapy in Poultry
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Characterization of the narrow-spectrum bacteriophage LSE7621 towards Salmonella Enteritidis and its biocontrol potential on lettuce and tofu
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Research Support, Non-U.S. Gov'ts

Comparative Genomic Analysis of Bacteriophage EP23 Infecting Shigella sonnei and Escherichia coli: Ho-Won Chang , Kyoung-Ho Kim; J. Microbiol. 2011;49(6):927-934. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1577-0

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Abstract: Bacteriophage EP23 that infects Escherichia coli and Shigella sonnei was isolated and characterized. The bacteriophage morphology was similar to members of the family Siphoviridae. The 44,077 bp genome was fully sequenced using 454 pyrosequencing. Comparative genomic and phylogenetic analyses showed that EP23 was most closely related to phage SO-1, which infects Sodalis glossinidius and phage SSL-2009a, which infects engineered E. coli. Genomic comparison indicated that EP23 and SO-1 were very similar with each other in terms of gene order and amino acid similarity, even though their hosts were separated in the level of genus. EP23 and SSL-2009a displayed high amino acid similarity between their genes, but there was evidence of several recombination events in SSL-2009a. The results of the comparative genomic analyses further the understanding of the evolution and relationship between EP23 and its bacteriophage relatives.

Antibacterial Efficacy of Lytic Pseudomonas Bacteriophage in Normal and Neutropenic Mice Models: Birendra R. Tiwari , Shukho Kim , Marzia Rahman , Jungmin Kim; J. Microbiol. 2011;49(6):994-999. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1512-4

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61 Scopus

Abstract: Recently, lytic bacteriophages (phages) have been focused on treating bacterial infectious diseases. We investigated the protective efficacy of a novel Pseudomonas aeruginosa phage, PA1Ø, in normal and neutropenic mice. A lethal dose of P. aeruginosa PAO1 was administered via the intraperitoneal route and a single dose of PA1Ø with different multiplicities of infection (MOI) was treated into infected mice. Immunocompetent mice infected with P. aeruginosa PAO1 were successfully protected by PA1Ø of 1 MOI, 10 MOI or 100 MOI with 80% to 100% survival rate. No viable bacteria were found in organ samples after 48 h of the phage treatment. Phage clearing patterns were different in the presence or absence of host bacteria but PA1Ø disappeared from all organs after 72 h except spleen in the presence of host bacteria. On the contrary, PA1Ø treatment could not protect neutropenic mice infected with P. aeruginosa PAO1 even though could extend their lives for a short time. In in vitro phage-neutrophil bactericidal test, a stronger bactericidal effect was observed in phage-neutrophil co-treatment than in phage single treatment without neutrophils, suggesting phage-neutrophil co-work is essential for the efficient killing of bacteria in the mouse model. In conclusion, PA1Ø can be possibly utilized in future phage therapy endeavors since it exhibited strong protective effects against virulent P. aeruginosa infection.

Phenotypic Characterization and Genomic Analysis of the Shigella sonnei Bacteriophage SP18: Kyoung-Ho Kim , Ho-Won Chang , Young-Do Nam , Seong Woon Roh , Jin-Woo Bae; J. Microbiol. 2010;48(2):213-222. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-0055-4

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Abstract: A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum analysis, it is lytic to S. sonnei, but not to Shigella flexneri, Shigella boydii or members of the genera Escherichia and Salmonella. Pyrosequencing of the SP18 bacteriophage genome revealed a 170-kb length sequence. In total, 286 ORFs and 3 tRNA genes were identified, and 259 ORFs showed similarity (BLASTP e-value<0.001) to genes of other bacteriophages. The results from comparative genomic analysis indicated that the enterophage JS98, isolated from human stool, is the closest relative of SP18. Based on phylogenetic analysis of gp23 protein-coding sequences, dot plot comparison and BLASTP analysis of genomes, SP18 and JS98 appear to be closely related to T4-even phages. However, several insertions, deletions, and duplications indicate differences between SP18 and JS98. Comparison of duplicated gp24 genes and the soc gene showed that duplication events are responsible for the differentiation and evolution of T4-like bacteriophages.

An Examination of the Bacteriophages and Bacteria of the Namib Desert: Eric Prestel , Sylvie Salamitou , Michael S. DuBow; J. Microbiol. 2008;46(4):364-372. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0007-4

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Abstract: Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth''s terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55~65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94~98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and delta-Proteobacteria groups were also observed.

Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes: Boyun Jang , Yuna Jung , Dongbin Lim; J. Microbiol. 2007;45(6):593-596.; DOI: https://doi.org/2602 [pii]

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Abstract: In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

Isolation and Characterization of the Smallest Bacteriophage P4 Derivatives Packaged into P4-Size Head in Bacteriophage P2-P4 System: Kyoung-Jin Kim , Jaeho Song; J. Microbiol. 2006;44(5):530-536.; DOI: https://doi.org/2444 [pii]

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Abstract: Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasmids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size head, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.

Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography: Tau Chuan Ling , Chee Kin Loong , Wen Siang Tan , Beng Ti Tey , Wan Mohammad Wan Abdullah , Arbakariya Ariff; J. Microbiol. 2004;42(3):228-232.; DOI: https://doi.org/2084 [pii]

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Abstract: In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine^TM 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (H_o=15cm) of STREAMLINE DEAE (r=1.2 g/cm^3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.

An Analysis of the Arm-type Site Binding Domain of Bacteriophage γ Integrase: Cho , Eun Hee; J. Microbiol. 1995;33(2):165-170.

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Abstract: The 356 amino acid long lambda integrase protein of bacteriophage λ constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P_tac promoter and the lacI^q gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.

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