Journal of Microbiology

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Comparative genomic analysis of Geosporobacter ferrireducens and its versatility of anaerobic energy metabolism: Man-Young Jung , So-Jeong Kim , Jong-Geol Kim , Heeji Hong , Joo-Han Gwak , Soo-Je Park , Yang-Hoon Kim , Sung-Keun Rhee; J. Microbiol. 2018;56(5):365-371. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7451-6

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Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H+ and Na+ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na+-dependent F-type ATPase and H+- dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.

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Overexpression of Outer Membrane Protein OprT and Increase of Membrane Permeability in phoU Mutant of Toluene-Tolerant Bacterium Pseudomonas putida GM730: Kyunghee Lee , Juna Jung , Kwang Kim , Dongwon Bae , Dongbin Lim; J. Microbiol. 2009;47(5):557-562. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0105-y

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Abstract: Eight toluene-sensitive mutants were previously isolated from the toluene-tolerant bacterium Pseudomonas putida GM730. One of these mutants was TOS6, in which Tn5 had been inserted into phoU. Susceptibility to multiple antibiotics, as well as toluene sensitivity, was increased in the phoU mutant of P. putida GM730. We compared the outer membrane proteins from the phoU mutant and wild-type via two-dimensional gel electrophoresis. A 45 kDa protein was dramatically overexpressed as the result of phoU inactivation, and this protein was identified by peptide mass fingerprinting and microsequencing as a conserved hypothetical protein consisting of 414 amino acids. The protein, designated as OprT, harbors a signal sequence and extended β-sheets, both of which are features common to the bacterial porins. The rate of ethidium bromide accumulation in TOS6 was higher than in GM730, which indicates that the TOS6 membranes may be more permeable to ethidium bromide than are the membranes of GM730. We propose that the toluene sensitivity and increased antibiotic susceptibility observed in the phoU mutant may be attributable to increased membrane permeability.

Nitroreductase II Involved in 2,4,6-Trinitrotoluene Degradation: Purification and Characterization from Klebsiella sp. C1: Jung-Hye Shin , Hong-Gyu Song; J. Microbiol. 2009;47(5):536-541. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-008-0171-6

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Abstract: Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. C1 have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen- insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-aminodinitrotoluene).

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 <br>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.

Identification and Expression of the cym, cmt, and tod Catabolic Genes from Pseudomonas putida KL47: Expression of the Regulatory todST Genes as a Factor for Catabolic Adaptation: Kyoung Lee , Eun Kyeong Ryu , Kyung Soon Choi , Min Chul Cho , Jae Jun Jeong , Eun Na Choi , Soo O Lee , Do-Young Yoon , Ingyu Hwang , Chi-Kyung Kim; J. Microbiol. 2006;44(2):192-199.; DOI: https://doi.org/2365 [pii]

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Abstract: Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene (C1-C4), biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99%) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

Selection of Suitable Packing Material for Biofiltration of Toluene, m- and p-Xylene Vapors: Young-Sook Oh , Sung-Chan Choi; J. Microbiol. 2000;38(1):31-35.

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Abstract: A suitable packing material for biofiltration of monoaromatic solvent vapors was selected among various types of packing materials such as peat, bark chips, vermiculite, and Hydroballs. A previously isolated strain, Pseudomonas pseudoalcaligenes BTXO2, which could utilize toluene, m- and p-xylene as carbon and energy sources was used as a biofilter inoculum. Four glass biofilters (6 cm dia. X 60 cm) were individually packed with each of the packing materials and solvent vapors were passed through the columns. During three weeks of peat biofilter operation, average removal efficiencies of toluene, m-and p-xylene were 90.4%, 95.3%, and 82.1%, respectively. With the other packings, the efficiencies were in the range of 10.1 to 58.6% which were significantly lower than those of the peat biofilter. The peat biofilter was continually operated for approximately nine months and the biofilter sustained its degradation activity during the operation period with minimal maintenance. At steady state, average removal rates of toluene, m- and p-xylene vapors were estimated as 14.2, 5.5, and 8.1 g m^-3 packing h^-1, respectively.

Simultaneous Utilization of Two Different Pathways in Degradation of 2,4,6-Trinitrotoluene by White Rot Fungus Irpex lacteus: Hyoun-Young Kim , Hong-Gyu Song; J. Microbiol. 2000;38(4):250-254.

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Abstract: This study confirmed that white rot fungus Irpex lacteus was able to metabolize 2,4,6-trinitrotoluene (TNT) with two different initial transformations. In one metabolic pathway of TNT a nitro group was removed from the aromatic ring of TNT. Hydride-Meisenheimer complexes of TNT (H^- -TNT), colored dark red, were confirmed as the intermediate in this transformation by comparison with the synthetic compounds. 2,4-Dinitrotoluene as a following metabolic product was detected, and nitrite produced by denitration of H^- -TNT supported this transformation. In the other TNT pathway, nitro groups in TNT were successively reduced to amine groups via hydroxylamines. Hydroxylamino-dinitrotoluenes and amino-dinitrotoluenes were identified as the intermediates. The activity of a membrane-associated aromatic nitroreductase was detected in the cell-free extract of I. lacteus. This enzyme catalyzed the nitro group reduction of TNT with NADPH as a cofactor. Enzyme activity was not observed in the presence of molecular oxygen.

Transformations of 2,4,6-Trinitrotoluene in Various Conditions by Klebsiella sp. Strain C1 Isolated from Activated Sludge: Chong-Suk Chang , Hyoun-Young Kim , Yang-Mi Kang , Kyung Sook Bae , Hong-Gyu Song; J. Microbiol. 2002;40(3):193-198.

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Abstract: Several 2,4,6-Trinitrotoluene (TNT) degrading bacteria were isolated from an activated sludge by an enrichment culture technique, and their TNT removal activities were examined. Among the isolates, strain C1 showed the highest degrading capability, and completely removed 100 or 200 mg l^-1 of TNT within 6 hours of incubation. This bacterium was identified as Klebsiella sp. The effects of different carbon sources on the removal of the parent TNT by Klebsiella sp. C1 were negligible, but the transformation rates of TNT metabolites such as amino-dinitrotoluenes and diamino-nitrotoluenes were higher with fructose addition compared to glucose addition. When nitrate was used as the nitrogen source, the degradation rates of TNT and hydroxylamino-dinitrotoluenes were higher than those with the ammonium addition. Although the TNT removal rate of Klebsiella sp. C1 was slightly higher in anaerobic conditions, the further transformations of TNT metabolites were more favorable in aerobic conditions.

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