Journal of Microbiology

Volume 42(2); June 2004

Journal Articles

Changes in Membrane Fatty Acid Composition during Entry of Vibrio vulnificus into the Viable But Nonculturable State: Ashley P. Day , James D. Oliver; J. Microbiol. 2004;42(2):69-73.; DOI: https://doi.org/2043 [pii]

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Abstract: Vibrio vulnificus, a Gram-negative bacterium found in estuarine waters, is responsible for over 95% of all seafood-related deaths in the United States. As a result of a temperature downshift to 5^oC, this organism enters the viable but nonculturable (VBNC) state. Changes in the membrane fatty acid (FA) composition of V. vulnificus may be a contributing factor to the ability of this organism to enter into and survive in the VBNC state. This hypothesis was tested by incubating the organism at 5^oC in artificial sea water and analyzing the cells’ FAs during the initial hours of temperature and nutrient downshift. Prior to downshift, the predominant FAs were 16:0, 16:1 and 18:0. During the first four hours of downshift, statistically significant changes occurred in 15:0, 16:1, 16:0, 17:0, and 18:0. These results indicate that changes in FA composition occur prior to entry of V. vulnificus into the VBNC state, suggesting that the ability to maintain membrane fluidity may be a factor in this physiological response. Cells in which fatty acid synthesis was inhibited did not survive, indicating that active fatty acid metabolism is essential for entry of cells into the VBNC state.

The Viable But Nonculturable State of Kanagawa Positive and Negative Strains of Vibrio parahaemolyticus: Tonya C. Bates , James D. Oliver; J. Microbiol. 2004;42(2):74-79.; DOI: https://doi.org/2042 [pii]

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Abstract: Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP^+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP^-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media. We speculated that KP^+ and KP^- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP^+ strains isolated from estuarine waters. The response of eleven KP^+ and KP^- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP^+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.

Molecular Investigation of Two Consecutive Nosocomial Clusters of Candida tropicalis Candiduria Using Pulsed-Field Gel Electrophoresis: Joon Rho , Jong Hee Shin , Jeong Won Song , Mi-Ra Park , Seung Jung Kee , Sook Jin Jang , Young Kyu Park , Soon Pal Suh , Dong Wook Ryang; J. Microbiol. 2004;42(2):80-86.; DOI: https://doi.org/2041 [pii]

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Abstract: Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.

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

Genetic and Phenotypic Diversity of (R/S)-Mecoprop [2-(2-Methyl-4-Chlorophenoxy)Propionic Acid]-Degrading Bacteria Isolated fromSoils: Jong-Sung Lim , Mee-Kum Jung , Mi-Soon Kim , Jae-Hyung Ahn , Jong-Ok Ka; J. Microbiol. 2004;42(2):87-93.; DOI: https://doi.org/2040 [pii]

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Abstract: Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer.

Degradation of Phenanthrene by Trametes versicolor and Its Laccase: Mun-Jung Han , Hyoung-Tae Choi , Hong-Gyu Song; J. Microbiol. 2004;42(2):94-98.; DOI: https://doi.org/2039 [pii]

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Abstract: Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30^oC. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/ l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T. versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'- azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.

Biochemical Quantitation of PM2 Phage DNA as a Substrate for Endonuclease Assay: Yoo Jin Joo , Hee-Ju Kim , Jae Yung Lee , Joon Kim; J. Microbiol. 2004;42(2):99-102.; DOI: https://doi.org/2038 [pii]

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Abstract: Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4^oC; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD_600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.

Factors Influencing Preferential Utilization of RNA Polymerase Containing Sigma-38 in Stationary-Phase Gene Expression in Escherichia coli: Eun Young Kim , Min-Sang Shin , Joon Haeng Rhee , Hyon E. Choy; J. Microbiol. 2004;42(2):103-110.; DOI: https://doi.org/2037 [pii]

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Abstract: In order to understand the molecular basis of selective expression of stationary-phase genes by RNA polymerase containing [sigma]^38 (E[sigma]^38) in Escherichia coli, we examined transcription from the stationaryphase promoters, katEP, bolAP, hdeABP, csgBAP, and mcbP, in vivo and in vitro. Although these promoters are preferentially recognized in vivo by E[sigma]^38, they are transcribed in vitro by both E[sigma]^38 and E[sigma]^70 containing the major exponential [sigma], [sigma]^70. In the presence of high concentrations of glutamate salts, however, only E[sigma]^38 was able to efficiently transcribe from these promoters, which supports the concept that the promoter selectivity of [sigma]^38 -containing RNA polymerase is observed only under specific reaction conditions. The examination of 6S RNA, which is encoded by the ssr1 gene in vivo, showed that it reduced E[sigma]^70 activity during the stationary phase, but this reduction of activity did not result in the elevation of E[sigma]^38 activity. Thus, the preferential expression of stationary-phase genes by E[sigma]^38 is unlikely the consequence of selective inhibition of E[sigma]^70 by 6S RNA.

Growth Inhibition of Escherichia coli during Heterologous Expression of Bacillus subtilis Glutamyl-tRNA Synthetase that Catalyzes the Formation of Mischarged Glutamyl-tRNA_1^Gln: Ji-Won Baick , Jang-Ho Yoon , Suk Namgoong , Dieter S?l , Sung-Il Kim; J. Microbiol. 2004;42(2):111-116.; DOI: https://doi.org/2036 [pii]

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Abstract: It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA_1^Gln with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-tRNA_1^ Gln formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA_1^Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA_1^ Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding GlutRNA^Gln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli GlutRNA_1^Gln, and converts it to the cognate Gln-tRNA_1^Gln species. B. subtilis GluRS-dependent Glu-tRNA_1^Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

Antibody Response to Crude Cell Lysate of Propionibacterium acnes and Induction of Pro-Inflammatory Cytokines in Patients with Acne and Normal Healthy Subjects: Basal , E. , A. , Kaushal , G.P.; J. Microbiol. 2004;42(2):117-125.; DOI: https://doi.org/2035 [pii]

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Abstract: Propionibacterium acnes (P. acnes) plays an important role in the disease pathogenesis of acne vulgaris, a disorder of pilosebaceous follicles, seen primarily in the adolescent age group. In the present study, the presence of antibodies against P. acnes (MTCC1951) were detected in acne patient (n=50) and disease free controls (n=25) using dot-ELISA and Western blot assay. The ability of P. acnes to induce proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), obtained from acne patients and healthy subjects, were also analysed. The patients (n=26) who were culture positive for skin swab culture, were found to have a more advanced disease and higher antibody titres (1:4000 to >1:16000) compared to the P. acnes negative patients (n=24) and normal controls (n=25). An analysis of patients' sera by western blot assay recognized a number of antigenic components of P. acnes, ranging from 29 to 205 kDa. The major reactive component was an approximately 96 kDa polypeptide, which was recognised in 92% (24 of 26) of the patients sera. Further, the P. acnes culture supernatant, crude cell lysate and heat killed P. acnes whole cells, obtained from 72-h incubation culture, were observed to be able to induce significant amounts of IL-8 and tumor necrosis factor alpha (TNF-[alpha]) by the PBMCs in both the healthy subjects and patients, as analysed by cytokine-ELISA. The levels of cytokines were significantly higher in the patients than the healthy subjects. A major 96 kDa polypeptide reactant was eluted from the gel and was found to cause dose dependent stimulation of the productions of IL-8 and TNF-[alpha]. Thus, the above results suggest that both humoral and pro-inflammatory responses play major roles in the pathogenesis of acne.

Expression and Purification of a Recombinant scFv towards the Exotoxin of the Pathogen, Burkholderia pseudomallei: Kue-Peng Lim , HongBin Li , Sheila Nathan; J. Microbiol. 2004;42(2):126-132.; DOI: https://doi.org/2034 [pii]

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Abstract: A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30^oC until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30^oC for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

Free Fatty Acid Accumulation by Mesophilic Lactic Acid Bacteria in Cold-Stored Milk: Hayri Co kun , Eda Ondul; J. Microbiol. 2004;42(2):133-138.; DOI: https://doi.org/2033 [pii]

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Abstract: This study was aimed to determine the accumulation of free fatty acid by mesophilic lactic acid bacteria (Lactococcus lactis subsp. lactis 1471, Lactococcus lactis subsp. cremoris 1000 and Lactobacillus casei 111) in cold-stored milk. According to the results, all cold-stored milks had higher acid degree values than those of fresh milk. This phenomenon showed that a slight increase occurred in the accumulation of free fatty acids as a result of spontaneous lipolysis during cold storage. All lactic acid bacteria showed good performance in production of titratable acidity, which increased during fermentation of the milk (fresh and stored milks). Moreover, as the storage time was prolonged, more free fatty acid accumulation was obtained from the fermentation of the cold-stored milk by the investigated lactic acid bacteria. The control milk, which was without lactic acid bacteria, showed no change in the accumulation of free fatty acid during fermentation. From this result, it can be suggested that longer cold-storage time can induce higher free fatty acid accumulation in milk by lactic acid bacteria.

Isolation of Citrobacter sp. Mutants Defective in Decolorization of Brilliant Green by Transposon Mutagenesis: Moon-Sun Jang , Young-Mi Lee , Yong-Lark Choi , Young-Su Cho , Young-Choon Lee; J. Microbiol. 2004;42(2):139-142.; DOI: https://doi.org/2032 [pii]

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Abstract: To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5- inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

DD1.5k, the Gene Preferentially Expressed in Bloodstream Isolates of Vancomycin-Resistant Enterococcus faecium: Seung-Han Kim , Dong-Gun Lee , Jin-Hong Yoo , Su-Mi Choi , Jung-Hyun Choi , Wan-Shik Shin , Kyungwon Lee , Dongeun Yong , Wee Gyo Lee , Byung-S. Youn , Moon-Won Kang; J. Microbiol. 2004;42(2):143-146.

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Abstract: Vancomycin-resistant Enterococcus faecium (VREFM) is becoming a threatening pathogen. We identified a gene called DD1.5K by differential display-PCR, which was preferentially expressed in the bloodstream isolates of VREFM. Due to its amino acid similarity to transfer complex protein, trsE, and tissue-specific expression, this gene may be involved in virulence of VREFM.

Identification of [sigma]^B-Dependent Promoters Using Consensus-Directed Search of Streptomyces coelicolor Genome: Eun-Jin Lee , You-Hee Cho , Hyo-Sub Kim , Jung-Hye Roe; J. Microbiol. 2004;42(2):147-151.; DOI: https://doi.org/2030 [pii]

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Abstract: [sigma]^B plays an important role in both osmoprotection and proper differentiation in Streptomyces coelicolor A3(2). We searched for candidate members of the [sigma]^B regulon from the genome database, using the consensus promoter sequence (GNNTN_14-16GGGTAC/T). The list consists of 115 genes, and includes all the known [sigma]^B target genes and many other genes whose functions are related to stress protection and differentiation.

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.

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