Journal of Microbiology

Journal Articles

Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11: Wonsik Lee; J. Microbiol. 2022;60(12):1123-1129. Published online November 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2476-2

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Abstract: Transposon mutant libraries are an important resource to study bacterial metabolism and pathogenesis. The fitness analysis of mutants in the libraries under various growth conditions provides important clues to study the physiology and biogenesis of structural components of a bacterial cell. A transposon library in conjunction with next-generation sequencing techniques, collectively named transposon sequencing (Tnseq), enables high-throughput genome profiling and synthetic lethality analysis. Tn-seq has also been used to identify essential genes and to study the mode of action of antibacterials. To construct a high-density transposon mutant library, an efficient delivery system for transposition in a model bacterium is essential. Here, I describe a detailed protocol for generating a high-density phage-based transposon mutant library in a Staphylococcus aureus strain, and this protocol is readily applicable to other S. aureus strains including USA300 and MW2.

Full-repertoire comparison of the microscopic objects composing the human gut microbiome with sequenced and cultured communities: Edmond Kuete Yimagou , Jean-Pierre Baudoin , Rita Abou Abdallah , Fabrizio Di Pinto , Jacques Yaacoub Bou Khalil , Didier Raoult; J. Microbiol. 2020;58(5):377-386. Published online April 11, 2020; DOI: https://doi.org/10.1007/s12275-020-9365-3

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Abstract: The study of the human gut microbiome is essential in microbiology and infectious diseases as specific alterations in the gut microbiome might be associated with various pathologies, such as chronic inflammatory disease, intestinal infection and colorectal cancer. To identify such dysregulations, several strategies are being used to create a repertoire of the microorganisms composing the human gut microbiome. In this study, we used the “microscomics” approach, which consists of creating an ultrastructural repertoire of all the cell-like objects composing stool samples from healthy donors using transmission electron microscopy (TEM). We used TEM to screen ultrathin sections of 8 resin-embedded stool samples. After exploring hundreds of micrographs, we managed to elaborate ultrastructural categories based on morphological criteria or features. This approach explained many inconsistencies observed with other techniques, such as metagenomics and culturomics. We highlighted the value of our cultureindependent approach by comparing our microscopic images to those of cultured bacteria and those reported in the literature. This study helped to detect “minimicrobes” Candidate Phyla Radiation (CPR) for the first time in human stool samples. This “microscomics” approach is non-exhaustive but complements already existing approaches and adds important data to the puzzle of the microbiota.

Taxonomic description and draft genome of Pseudomonas sediminis sp. nov., isolated from the rhizospheric sediment of Phragmites karka: Pratiksha Behera , Madhusmita Mahapatra , Arman Seuylemezian , Parag Vaishampayan , V. Venkata Ramana , Neetha Joseph , Amaraja Joshi , Yogesh Shouche , Mrutyunjay Suar , Ajit K. Pattnaik , Gurdeep Rastogi; J. Microbiol. 2018;56(7):458-466. Published online June 14, 2018; DOI: https://doi.org/10.1007/s12275-018-7549-x

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Abstract: The taxonomic position of a Gram-stain-negative, rod-shaped bacterial strain, designated PI11T , isolated from the rhizospheric sediment of Phragmites karka was characterized using a polyphasic approach. Strain PI11T could grow optimally at 1.0% NaCl concentration with pH 7.0 at 30°C and was positive for oxidase and catalase but negative for hydrolysis of starch, casein, and esculin ferric citrate. Phylogenetic analysis of 16S rRNA gene sequences indicated that the strain PI11T belonged to the genus Pseudomonas sharing the highest sequence similarities with Pseudomonas indoloxydans JCM 14246T (99.72%), followed by, Pseudomonas oleovorans subsp. oleovorans DSM 1045T (99.29%), Pseudomonas toyotomiensis JCM 15604T (99.15%), Pseudomonas chengduensis DSM 26382T (99.08%), Pseudomonas oleovorans subsp. lubricantis DSM 21016T (99.08%), and Pseudomonas alcaliphila JCM 10630T (99.01%). Experimental DNA-DNA relatedness between strain PI11T and P. indoloxydans JCM 14246T was 49.4%. The draft genome of strain PI11T consisted of 4,884,839 bp. Average nucleotide identity between the genome of strain PI11T and other closely related type strains ranged between 77.25–90.74%. The polar lipid pattern comprised of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine. The major (> 10%) cellular fatty acids were C18:1 ω6c/ω7c, C16:1 ω6c/ω7c, and C16:0. The DNA G + C content of strain PI11T was 62.4 mol%. Based on the results of polyphasic analysis, strain PI11T was delineated from other closely related type strains. It is proposed that strain PI11T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas sediminis sp. nov. is proposed. The type strain is PI11T (= KCTC 42576T = DSMZ 100245T).

Characterization of siderophore produced by Pseudomonas syringae BAF.1 and its inhibitory effects on spore germination and mycelium morphology of Fusarium oxysporum: Sumei Yu , Chunying Teng , Jinsong Liang , Tao Song , Liying Dong , Xin Bai , Yu Jin , Juanjuan Qu; J. Microbiol. 2017;55(11):877-884. Published online October 27, 2017; DOI: https://doi.org/10.1007/s12275-017-7191-z

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26 Citations

Abstract: In this study, an antagonistic bacterium against Fusarium oxysporum was identified and designated as Pseudomonas syringae strain BAF.1 on the basis of 16S rDNA sequence analysis and physiological-biochemical characteristics. It produced catechol-species siderophore at a molecular weight of 488.59 Da and a maximum amount of 55.27 μg/ml with glucose as a carbon source and asparagine as a nitrogen source at a C/N ratio of 10:1, 30°C and pH 7. The siderophore exhibited prominent antagonistic activity against Fusarium oxysporum with a maximum inhibition rate of 95.24% and had also suppressive effects on other kinds of 11 phytopathogenic fungi in the absence of FeCl3·6H2O. Spore germination was completely inhibited by 50 μl of the siderophorecontaining solution, and the ultrastructures of mycelia and spores were also considerably suppressed by siderophore treatment as established by electron microscopy observation. These results indicate that the siderophore produced by Pseudomonas syringae BAF.1 could be potentially used for biocontrol of pathogenic Fusarium oxysporum.

Research Support, Non-U.S. Gov'ts

The hrp pathogenicity island of Pseudomonas syringae pv. tomato DC3000 is induced by plant phenolic acids: Jun Seung Lee , Hye Ryun Ryu , Ji Young Cha , Hyung Suk Baik; J. Microbiol. 2015;53(10):725-731. Published online October 2, 2015; DOI: https://doi.org/10.1007/s12275-015-5256-4

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Abstract: Plants produce a wide array of antimicrobial compounds, such as phenolic compounds, to combat microbial pathogens. The hrp PAI is one of the major virulence factors in the plant pathogen, Pseudomonas syringae. A major role of hrp PAI is to disable the plant defense system during bacterial invasion. We examined the influence of phenolic compounds on hrp PAI gene expression at low and high concentrations. There was approximately 2.5 times more hrpA and hrpZ mRNA in PtoDC3000 that was grown in minimal media (MM) supplemented with 10 μM of ortho-coumaric acid than in PtoDC3000 grown in MM alone. On the other hand, a significantly lower amount of hrpA mRNA was observed in bacteria grown in MM supplemented with a high concentration of phenolic compounds. To determine the regulation pathway for hrp PAI gene expression, we performed qRTPCR using gacS, gacA, and hrpS deletion mutants.

The Role of Wheat Germ Agglutinin in the Attachment of Pseudomonas sp. WS32 to Wheat Root: Jian Zhang , Liyuan Meng , Yuanyuan Cao , Huiping Chang , Zhongyou Ma , Leni Sun , Ming Zhang , Xinyun Tang; J. Microbiol. 2014;52(12):1020-1024. Published online November 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4089-x

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Abstract: Wheat germ agglutinin (WGA), which is secreted on the surface of wheat root, has been defined as a protein that reversibly and non-enzymatically binds to specific carbohydrates. However, little attention has been paid to the function of WGA in the attachment of bacteria to their host plants. The aim of this study was to investigate the role of WGA in the attachment of Pseudomonas sp. WS32 to wheat roots. Wheat roots were initially treated with double-distilled water, WGA-H (WGA solution that was heated at 100°C for 15 min) and WGA, independently. Subsequently, the roots were coincubated with cell solutions (109 cells/ml). A dilution plate
method
using a solid nutrient medium was employed to determine the adsorption of WS32 to wheat roots. WGA was labeled with fluorescein isothiocyanate and detected using the fluorescent in situ hybridization (FISH) technique. The number of adsorptive WS32 cells on wheat roots was significantly increased when the wheat roots were pretreated with WGA, compared with the control treatment (p = 0.01). However, WGA-H failed to increase the amount of bacterial cells that attached to the wheat roots because of the loss of its physiological activity. The FISH assay also revealed that more cells adhered to WGA-treated wheat roots than to control or WGA-H-treated roots. The results indicated that WGA can mediate Pseudomonas strain WS32’s adherence to wheat seedling roots. The findings of this study provide a better understanding of the processes involved in plant-microbe interactions.

Journal Article

Characterization of Nitrogen-Fixing Bacteria Isolated from Field-Grown Barley, Oat, and Wheat: Anastasia Venieraki , Maria Dimou , Eleni Vezyri , Io Kefalogianni , Nikolaos Argyris , Georgia Liara , Panagiotis Pergalis , Iordanis Chatzipavlidis , Panagiotis Katinakis; J. Microbiol. 2011;49(4):525-534. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0457-y

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30 Citations

Abstract: Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.

Research Support, Non-U.S. Gov'ts

Isolation and Characterization of Biogenic Amine-Producing Bacteria in Fermented Soybean Pastes: Jin Seok Moon , Seung Kee Cho , Hwa Young Choi , Ji Eun Kim , So-Young Kim , Kyung-Ju Cho , Nam Soo Han; J. Microbiol. 2010;48(2):257-261. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-0040-y

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20 Citations

Abstract: Biogenic amines (BAs) are produced primarily by microorganisms found in fermented foods and are often implicated in food poisoning. BA-producing bacteria found in fermented soybean pastes were isolated and characterized using a decarboxylating medium and multiplex PCR analysis. Two BA-producing bacteria were isolated from traditional soybean pastes: one was a histamine-producing Clostridium strain, and the other was a tyramine-producing Pseudomonas strain. The Clostridium strain was determined to be a potent histamine producer among the cultures tested. Synthesis of tyramine by Pseudomonas sp. T1 was observed for the first time in this study.

Note] Comparative Analysis of 2,4,6-Trinitrotoluene (TNT)-Induced Cellular Responses and Proteomes in Pseudomonas sp. HK-6 in Two Types of Media: Yun-Seok Cho , Bheong-Uk Lee , Hyung-Yeel Kahng , Kye-Heon Oh; J. Microbiol. 2009;47(2):220-224. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0108-0

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Abstract: TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.

Impact of cry1AC-Carrying Brassica rapa subsp. pekinensis on Leaf Bacterial Community: Young Tae Kim , Kang Seon Lee , Moon Jung Kim , Seung Bum Kim; J. Microbiol. 2009;47(1):33-39. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0254-4

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Abstract: The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of transgene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.

Journal Articles

S5 Lipase : An Organic Solvent Tolerant Enzyme: Raja Noor Zaliha Raja Abdul Rahman , Syarul Nataqain Baharum , Abu Bakar Salleh , Mahiran Basri; J. Microbiol. 2006;44(6):583-590.; DOI: https://doi.org/2470 [pii]

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Abstract: In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.

Stabilities of Artificially Transconjugated Plasmids for the Bioremediation of Cocontaminated Sites: Kyung Pyo Yoon; J. Microbiol. 2005;43(2):196-203.; DOI: https://doi.org/2161 [pii]

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Abstract: Here, we attempted to evaluate the activity of artificially transconjugated multiple plasmids in “designer biocatalysts” for the bioremediation of cocontaminated sites under nonselective conditions. We observed profound losses in the percent survivals of artificially transconjugated plasmid activity (66 - 78% loss immediately after freeze-drying, 99.45 - 99.88% loss by the end of 6 months storage) in reconstituted Pseudomonas sp. KM12TC. Such unpredictable high losses of this particular plasmid appeared to clearly be a deleterious effect. However, even after 6 months of storage, the cells remained able to degrade 95% of phenol within 9 days, and the full efflux of ^73As, as compared to that of the non-freeze-dried cells, was successfully achieved 4 to 9 days later. These results indicate that “stable designer biocatalysts” can remain viable, even after freeze-drying and 6 months of storage.

Research Support, Non-U.S. Gov't

Occurrence of the strA-strB Streptomycin Resistance Genes in Pseudomonas Species Isolated from Kiwifruit Plants: Hyo Shim Han , Young Jin Koh , Jae-Seoun Hur , Jae Sung Jung; J. Microbiol. 2004;42(4):365-368.; DOI: https://doi.org/2096 [pii]

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Abstract: The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA→CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gln to Arg.

Journal Article

Characterization of Protocatechuate 4,5-Dioxygenase Induced from p-Hydroxybenzoate-Cultured Pseudomonas sp. K82: Sung-Ho Yun , Chi-Young Yun , Seung Il Kim; J. Microbiol. 2004;42(2):152-155.; DOI: https://doi.org/2029 [pii]

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Abstract: Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, phydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 ([alpha]subunit and [beta] subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer ([alpha]_1[beta]_1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15^oC. PCR amplification of these two subunits of PCD4,5 revealed that the [alpha] subunit and [beta] subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.

cloning of Gene Encoding for Siderophore biosynthesis in Fluorescent Pseudomonas sp.: Koh, Han Cheol , Ha, Sung Cheol , Na, Jung A , Kim, Ho Sang , Yeo, Myeong Gu , Lee, Jung Sup , Kim, Sung Jun , Park, yeal; J. Microbiol. 1995;33(1):28-33.

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Abstract: Pseudomonas sp. strain PY002, isolated from soil, was mutagenized with a transposon Tn5(21). To screening of siderophore biosunthesis defective mutant, 138 kanamycin resistant mutants were tested of growth on MKB medium supplemented with iron chelator(bipydidyl and EDDHA) and in vitro antibiosis. Among 138 mutants, 32 mutants do not excreted a siderophore and lose their antibiotic activity. So, these mutants were designated Flu^-Sid^-. A gene bank of DNA from Pseudomonas sp. strain PY002 was constructed using the broad-host range conjugative cosmid pLAFR3. The recombinant cosmids contained insert DNA averaging 21 kb in length and the frequence of transduction into E. coli HB101 per 1㎍ of insert DNA was 9 × 10³. Nonfluorescent mutants were complemented by mating the gene bank en masse and identifying the 108 fluorescent transconjugants. Restriction enzyme analysis of these complemented transconjugants revealed three different types and they were named pCOM61, pCOM91 and pCOM97. Sizes of their insert DNA were 30kb, 26kb and 28kb, respectively.

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