Journal of Microbiology

Volume 43(1); February 2005

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

The Diversity of Culturable Organotrophic Bacteria from Local Solar Salterns: Sun-Hee Yeon , Won-Jin Jeong , Jin-Sook Park; J. Microbiol. 2005;43(1):1-10.; DOI: https://doi.org/2146 [pii]

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Abstract: We isolated and cultured bacteria inhabiting solar saltern ponds in Taean-Gun, Chungnam Province, Korea. All of the isolated 64 strains were found to be moderately halophilic bacteria, growing in a salt range of 2-20 %, with an optimal concentration of 5% salt. Bacterial diversity among the isolated halophiles was evaluated via RFLP analyses of PCR-amplified 16S rDNAs, followed by phylogenetic analysis of the partial 16S rDNA sequences. The combination of restriction enzyme digestions with HaeIII, CfoI, MspI and RsaI generated 54 distinct patterns. A neighbor-joining tree of the partial 16S rDNA sequences resulted in the division of the 64 strains into 2 major groups, 45 strains of [gammar]-Proteobacteria (70.3%) and 19 strains of Firmicutes (29.7%). The [alpha]-Proteobacteria and Cytophaga-Flavobacterium-Bacterioides groups, which were repeatedly found to exist in thalassohaline environments, were not represented in our isolates. The [gammar]-Proteobacteria group consisted of several subgroups of the Vibrionaceae (37.5%), Pseudoalteromonadaceae (10.9%), Halomonadaceae (7.8%), Alteromonadaceae (7.8%), and Idiomarinaceae (6.3%). Members of Salinivibrio costicola (29.7%) were the most predominant species among all of the isolates, followed by Halobacillus treperi (12.5%). Additionally, three new species candidates were found, based on similarities of the 16S rDNA sequences to those of previously published species.

Neovastat(Æ-941) inhibits the airway inflammation and hyperresponsiveness in a murine model of asthma: Sook-Young Lee , Soon-Young Paik , Su-Mi Chung ,; J. Microbiol. 2005;43(1):11-16.; DOI: https://doi.org/2145 [pii]

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Abstract: Matrix metalloproteinase (MMP)-9 plays an important role in the pathogenesis of bronchial asthma. Neovastat, having significant antitumor and antimetastatic properties, is classified as a naturally occurring multifunctional antiangiogenic agent. We evaluated the therapeutic effect of Neovastat on airway inflammation in a mouse model of asthma. BALB/c mice were immunized subcutaneously with ovalbumin (OVA) on days 0, 7, 14, and 21 and challenged with inhaled OVA on days 26, 29, and 31. Neovastat was administrated by gavage (5 mg/kg body weight) three times with 12 h intervals, beginning 30 min before OVA inhalation. On day 32, mice were challenged with inhaled methacholine, and enhanced pause (Penh) was measured as an index of airway hyperresponsiveness. The severity of airway inflammation was determined by differential cell count of bronchoalveolar lavage (BAL) fluid. The MMP-9 concentration in BAL fluid samples was measured by ELISA, and MMP-9 activity was measured by zymography. The untreated asthma group showed an increased inflammatory cell count in BAL fluid and Penh value compared with the normal control group. Mice treated with Neovastat had significantly reduced Penh values and inflammatory cell counts in BAL fluid compared with untreated asthmatic mice. Furthermore, mice treated with Neovastat showed significantly reduced MMP-9 concentrations and activity in BAL fluid. These results demonstrate that Neovastat might have new therapeutic potential for airway asthmatic inflammation.

Nematicidal Activity and Chemical Component of Poria cocos: Guo Hong Li , Yue Mao Shen , Ke Qin Zhang; J. Microbiol. 2005;43(1):17-20.; DOI: https://doi.org/2144 [pii]

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Abstract: Poria cocos, a famous traditional Chinese medicine, was found to have nematicidal activity in experiments searching for nematicidal fungi. The experiment showed it could kill 94.9% of the saprophytic nematode, Panagrellus redivivue, 92.6% of the root-knot nematode, Meloidogyne arenaria, and 93.5% of the pine nematode, Bursaphelenchus xylophilus, on PDA plate within 12 hours. According to the nematicidal activity, three new compounds, 2, 4, 6-triacetylenic octane diacid, 2, 4, 5, 6-tetrahydroxyhexanoic acid and 3, 4-dihydroxy-2-keto-n-butyl 2,4,5,6-tetrahydroxyhexanate, were isolated from submerged cultures of Poria cocos. Of these, 2, 4, 6-triacetylenic octane diacid could kill 83.9% Meloidogyne arenaria and 73.4% Panagrellus redivivus at 500 ppm within 12 hours. Here, it is reported for the first time that Poria cocos has nematicidal activity.

Purification and Characterization of NADPH-Dependent Cr(VI) Reductase from Escherichia coli ATCC 33456: Woo-Chul Bae , Han-Ki Lee , Young-Chool Choe , Deok-Jin Jahng , Sang-Hee Lee , Sang-Jin Kim , Jung-Hyun Lee , Byeong-Chul Jeong; J. Microbiol. 2005;43(1):21-27.; DOI: https://doi.org/2143 [pii]

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Abstract: A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37^oC. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61% higher activity than NADH. The K_m values for NADPH and NADH were determined to be 47.5 and 17.2 umol, and the V_max values 322.2 and 130.7 umol Cr(VI) min^-1mg^-1 protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag^2+, Cd^2+, Hg^2+, and Zn^2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.

Development of a Bottle-Free Multipurpose Incubator for Generating Various Bacterial Culture Conditions: Nam Woong Yang , Yong Lim; J. Microbiol. 2005;43(1):28-33.; DOI: https://doi.org/2142 [pii]

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Abstract: The purpose of this study was to develop a multipurpose incubator, without the gas cylinders (bottles) which are required for H_2 and CO_2 supplementation. In our bottle-free multipurpose incubator, the H_2 and CO_2 were generated by chemical reactions induced within the chamber. The reaction between sodium borohydride and acetic acid at a molar ratio of 1:1 was used to generate H_2, according to the following formula: 4NaBH_4 + 2CH_3COOH + 7H_2O → 2CH_3COONa + Na_2B_4O_7 + 16H_2, whereas the other reaction, citric acid and sodium bicarbonate at a 1:1 molar ratio, was used to generate CO_2, according to the following formula: C_6H_8O_7 + 3NaHCO_3 → Na_3(C_6H_5O_7) + 3H_2O + 3CO_2. Five species of obligate anaerobic bacteria, one strain of capnophilic bacterium, and one strain of microaerophilic bacterium were successfully cultured in the presence of their respective suitable conditions, all of which were successfully generated by our bottle-free multipurpose incubator. We conclude that, due to its greater safety, versatility, and significantly lower operating costs, this bottle-free multipurpose incubator can be used for the production of fastidious bacterial cultures, and constitutes a favorable step above existing anaerobic incubators.

Molecular Cloning and Expression of a Thermostable Xylose (Glucose) Isomerase Gene, xylA, from Streptomyces chibaensis J-59: Gil-Jae Joo , Jae-Ho Shin , Gun-Young Heo , Young-Mog Kim , In-Koo Rhee; J. Microbiol. 2005;43(1):34-37.; DOI: https://doi.org/2141 [pii]

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Abstract: In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85oC and the enzyme exhibited a high level of heat stability.

Journal Article

Optimization of Lactic Acid Production in SSF by Lactobacillus amylovorus NRRL B-4542 Using Taguchi Methodology: Pyde Acharya Nagarjun , Ravella Sreenivas Rao , Swargam Rajesham , Linga Venkateswar Rao; J. Microbiol. 2005;43(1):38-43.; DOI: https://doi.org/2140 [pii]

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Abstract: Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO_4 . 7H_20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO_4 . 7H_20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.

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

The Schizosaccharomyces pombe Gene Encoding [gamma]-Glutamyl Transpeptidase I Is Regulated by Non-fermentable Carbon Sources and Nitrogen Starvation: Hong-Gyum Kim , Hey-Jung Park , Hyun-Jung Kang , Hye-Won Lim , Kyunghoon Kim , Eun-Hee Park , Kisup Ahn , Chang-Jin Lim; J. Microbiol. 2005;43(1):44-48.; DOI: https://doi.org/2139 [pii]

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Abstract: In our previous study, the first structural gene (GGTI) encoding g-glutamyl transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of [beta]-galactosidase from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

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

Identification of Two-Component Regulatory Genes Involved in o-Xylene Degradation by Rhodococcus sp. Strain DK17: Dockyu Kim , Jong-Chan Chae , Gerben J. Zylstra , Ho-Yong Sohn , Gi-Seok Kwon , Eungbin Kim; J. Microbiol. 2005;43(1):49-53.; DOI: https://doi.org/2138 [pii]

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Abstract: Putative genes for a two-component signal transduction system (akbS and akbT) were detected near the alkylbenzene-degrading operon of Rhodococcus sp. DK17. Sequence analysis indicates that AkbS possesses potential ATP-binding and histidine autophosphorylation sites in the N- and C-terminal regions, respectively, and that AkbT has a typical response regulator domain. Mutant analysis combined with RT-PCR experiments further shows that AkbS is required to induce the expression of o-xylene dioxygenase in DK17.

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

Growth of Staphylococcus aureus with Defective Siderophore Production in Human Peritoneal Dialysate Solution: Ra-Young Park , Hui-Yu Sun , Mi-Hwa Choi , Young-Hoon Bae , Sung-Heui Shin; J. Microbiol. 2005;43(1):54-61.; DOI: https://doi.org/2137 [pii]

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Abstract: In this study, we attempted to determine the effects of iron-availability and the activity of the bacterial iron-uptake system (IUS) on the growth of Staphylococcus aureus in human peritoneal dialysate (HPD) solution. A streptonigrin-resistant S. aureus (SRSA) strain, isolated from S. aureus ATCC 6538, exhibited defective siderophore production, thereby resulting in ineffective uptake of iron from low iron-saturated transferrin. The growth of both strains was stimulated in HPD solution supplemented with FeCl_3 and holotransferrin, but growth was inhibited in HPD solution which had been supplemented with apotransferrin and dipyridyl. The SRSA strain grew less robustly than did its parental strain in both iron-supplemented HPD solution and regular HPD solution. These results indicate that iron-availability and siderophore-mediated IUS activity in particular, the ability to produce siderophores and thus capture iron from low iron-saturated transferrin play critical roles in the growth of S. aureus in HPD solution. Our results also indicated that the possibility of using iron chelators as therapeutic or preventive agents warrants further evaluation.

Laboratory Diagnosis of Invasive Candidiasis: Arjuna N.B. Ellepola , Christine J. Morrison; J. Microbiol. 2005;43(1):65-84.

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Abstract: Invasive candidiasis is associated with high morbidity and mortality. Clinical diagnosis is complicated by a lack of specific clinical signs and symptoms of disease. Laboratory diagnosis is also complex because circulating antibodies to Candida species may occur in normal individuals as the result of commensal colonization of mucosal surfaces thereby reducing the usefulness of antibody detection for the diagnosis of this disease. In addition, Candida species antigens are often rapidly cleared from the circulation so that antigen detection tests often lack the desired level of sensitivity. Microbiological confirmation is difficult because blood cultures can be negative in up to 50% of autopsy-proven cases of deep-seated candidiasis or may only become positive late in the infection. Positive cultures from urine or mucosal surfaces do not necessarily indicate invasive disease although can occur during systemic infection. Furthermore, differences in the virulence and in the susceptibility of the various Candida species to antifungal drugs make identification to the species level important for clinical management. Newer molecular biological tests have generated interest but are not yet standardized or readily available in most clinical laboratory settings nor have they been validated in large clinical trials. Laboratory surveillance of at-risk patients could result in earlier initiation of antifungal therapy if sensitive and specific diagnostic tests, which are also cost effective, become available. This review will compare diagnostic tests currently in use as well as those under development by describing their assets and limitations for the diagnosis of invasive candidiasis.

Genetic and Environmental Control of Salmonella Invasion: Craig Altier; J. Microbiol. 2005;43(1):85-92.

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Abstract: An early step in the pathogenesis of non-typhoidal Salmonella species is the ability to penetrate the intestinal epithelial monolayer. This process of cell invasion requires the production and transport of secreted effector proteins by a type III secretion apparatus encoded in Salmonella pathogenicity island I (SPI-1). The control of invasion involves a number of genetic regulators and environmental stimuli in complex relationships. SPI-1 itself encodes several transcriptional regulators (HilA, HilD, HilC, and InvF) with overlapping sets of target genes. These regulators are, in turn, controlled by both positive and regulators outside SPI-1, including the two-component regulators BarA/SirA and PhoP/Q, and the csr post-transcriptional control system. Additionally, several environmental conditions are known to regulate invasion, including pH, osmolarity, oxygen tension, bile, Mg^2+ concentration, and short chain fatty acids. This review will discuss the current understanding of invasion control, with emphasis on the interaction of environmental factors with genetic regulators that leads to productive infection.

The Viable but Nonculturable State in Bacteria: James D. Oliver; J. Microbiol. 2005;43(1):93-100.

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Abstract: It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

Quorum Sensing and Quorum-Quenching Enzymes: Yi-Hu Dong , Lian-Hui Zhang; J. Microbiol. 2005;43(1):101-109.

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Abstract: To gain maximal benefit in a competitive environment, single-celled bacteria have adopted a community genetic regulatory mechanism, known as quorum sensing (QS). Many bacteria use QS signaling systems to synchronize target gene expression and coordinate biological activities among a local population. N-acylhomoserine lactones (AHLs) are one family of the well-characterized QS signals in Gram-negative bacteria, which regulate a range of important biological functions, including virulence and biofilm formation. Several groups of AHL-degradation enzymes have recently been identified in a range of living organisms, including bacteria and eukaryotes. Expression of these enzymes in AHL-dependent pathogens and transgenic plants efficiently quenches the microbial QS signaling and blocks pathogenic infections. Discovery of these novel quorum quenching enzymes has not only provided a promising means to control bacterial infections, but also presents new challenges to investigate their roles in host organisms and their potential impacts on ecosystems.

Salmonella Invasion Gene Regulation: A Story of Environmental Awareness: Bradley D. Jones; J. Microbiol. 2005;43(1):110-117.

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Abstract: Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. A critical virulence determinant of Salmonella is the ability to invade mammalian cells. The expression of genes required for invasion is tightly regulated by environmental conditions and a variety of regulatory genes. The hilA regulator encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. Work from several laboratories has highlighted that regulation of hilA expression is a key point for controlling expression of the invasive phenotype. A number of positive regulators of hilA expression have been identified including csrAB, sirA/barA, pstS, hilC/sirC/sprA, fis, and hilD. HilD, an AraC/XylS type transcriptional regulator, is of particular importance as a mutation in hilD results in a 14-fold decrease in chromosomal hilA::Tn5lacZY-080 expression and a 53-fold decrease in invasion of HEp-2 cells. It is believed that HilD directly regulates hilA expression as it has been shown to bind to hilA promoter sequences. In addition, our research group, and others, have identified genes (hilE, hha, pag, and lon) that negatively affect hilA transcription. HilE appears to be an important Salmonella-specific regulator that plays a critical role in inactivating hilA expression. Recent work in our lab has been directed at understanding how environmental signals that affect hilA expression may be processed through a hilE pathway to modulate expression of hilA and the invasive phenotype. The current understanding of this complex regulatory system is reviewed.

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