- 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
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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
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J. Microbiol. 2006;44(2):192-199.
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DOI: https://doi.org/2365 [pii]
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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.
- Genetic Organization of the dhlA Gene Encoding 1,2-Dichloroethane Dechlorinase from Xanthobacter flavus UE15
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Ji-Sook Song , Dong-Hun Lee , Kyoung Lee , Chi-Kyung Kim
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J. Microbiol. 2004;42(3):188-193.
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DOI: https://doi.org/2091 [pii]
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Xanthobacter flavus strain UE15 was isolated in wastewater obtained from the Ulsan industrial complex, Korea. This strain functions as a 1,2-dichloroethane (1,2-DCA) degrader, via a mechanism of hydrolytic dechlorination, under aerobic conditions. The UE15 strain was also capable of dechlorinating other chloroaliphatics, such as 2-chloroacetic acid and 2-chloropropionic acid. The dhlA gene encoding 1,2-DCA dechlorinase was cloned from the genomic DNA of the UE15 strain, and its nucleotide sequence was determined to consist of 933 base pairs. The deduced amino acid sequence of the DhlA dechlorinase exhibited 100% homology with the corresponding enzyme from X. autotrophicus GJ10, but only 27 to 29% homology with the corresponding enzymes from Rhodococcus rhodochrous, Pseudomonas pavonaceae, and Mycobacterium sp. strain GP1, which all dechlorinate haloalkane compounds. The UE15 strain has an ORF1 (1,356 bp) downstream from the dhlA gene. The OFR1 shows 99% amino acid sequence homology with the transposase reported from X. autotrophicus GJ10. The transposase gene was not found in the vicinity of the dhlA in the GJ10 strain, but rather beside the dhlB gene coding for haloacid dechlorinase. The dhlA and dhlB genes were confirmed to be located at separate chromosomal loci in the Xanthobacter flavus UE15 strain as well as in X. autotrophicus GJ10. The dhlA and transposase genes of the UE15 strain were found to be parenthesized by a pair of insertion sequences, IS1247, which were also found on both sides of the transposase gene in the GJ10 strain. This unique structure of the dhlA gene organization in X. flavus strain UE15 suggested that the dechlorinase gene, dhlA, is transferred with the help of the transposase gene.
- Comparative Analysis of Cyanobacterial Communities from Polluted Reservoirs in Korea
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Jin-Book Kim , Mi-Sook Moon , Dong-Hun Lee , Sung-Taik Lee , Marco Bazzicalupo , Chi-Kyung Kim
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J. Microbiol. 2004;42(3):181-187.
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DOI: https://doi.org/2092 [pii]
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Cyanobacteria are the dominant phototrophic bacteria in water environments. Here, the diversity of cyanobacteria in seven Korean reservoir waters where different levels of algal blooms were observed during the summer of 2002, was examined by T-RFLP analysis. The number of T-RF bands in the HaeIII T-RFLP profiles analyzed from those water samples ranged from 20 to 44. Of these, cyanobacteria accounted for 6.1 to 27.2% of the total bacteria. The water samples could be clustered into 2 groups according to the Dice coefficient of the T-RF profiles. The eutrophic Dunpo and oligotrophic Chungju reservoirs were selected, and several representative clones from both reservoir waters analyzed for the nucleotide sequences of their 16S rDNA. The major clones were found to belong to the Microcystis and Anabaena species in the waters from the Dunpo and Chungju reservoirs, respectively, which was in agreement with the T-RFLP result. That is, the Microcystis and Anabaena species were dominant in the eutrophic and polluted Dunpo and oligotrophic Chungju reservoir waters, respectively. These results indicated that there is a correlation between prevalence of cyanobacterial species and levels of pollution in reservoir waters.
- Characteristics of Several Bacterial Isolates Capable of Degrading Chloroaliphatic Compounds via Hydrolytic Dechlorination
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Ji-Sook Song , Dong-Hun Lee , Kyoung Lee , Chi-Kyung Kim
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J. Microbiol. 2003;41(4):277-283.
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Haloaliphatic hydrocarbons have been widely used as solvents and ingredients of pesticides and herbicides. However, when these compounds contaminate the environment, they can be very hazardous to animals and humans because of their potential toxicity and carcinogenicity. Therefore, lots of studies have been made for microbial degradation of those pollutant chemicals. In this study, 11 bacterial strains capable of degrading 1,2-dichloroethane (1,2-DCA), 2-chloropropionic acid (2-CPA), 2,3-dichloropropionic acid (2,3-DCPA), and 2-monochloroacetic acid (2-MCA) by hydrolytic dechlorination under aerobic conditions were isolated from wastewaters and rice paddy soil samples. Their morphological and biochemical characteristics and their degradation capabilities of haloaliphatic hydrocarbons were examined. On the basis of the 16S rDNA sequences, 8 different kinds of microbial species, including Pseudomonas plecoglossicida, Xanthobacter flavus, Ralstonia eutropha, were identified. All of the isolated strains can degrade MCA. In particular, strains UE-2 and UE-15 degraded 1,2-DCA, and strain CA-11 degraded 2,3-DCPA, which are hardly degraded by other strains.
- Structural Analysis of the fcbABC Gene Cluster Responsible for Hydrolytic Dechlorination of 4-Chlorobenzoate from pJS1 Plasmid of Comamonas sp. P08
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Jeong-Soon Lee , Kyoung Lee , Jong-Ok Ka , Jong-Chan Chae , Chi-Kyung Kim
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J. Microbiol. 2003;41(2):89-94.
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Bacterial strain No. P08 isolated from wastewater at the Cheongju industrial complex was found to be capable of degrading 4-chlorobenzoate under aerobic condition. P08 was identified as Comamonas sp. from its cellular fatty acid composition and 16S rDNA sequence. The fcb genes, responsible for the hydrolytic dechlorination of 4-chlorobenzoate, were cloned from the plasmid pJS1 of Comamonas sp. P08. The fcb gene cluster of comamonas sp. P08 was organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC. This organization of the fcb genes was very similar to that of the fcb genes carried on the chromosomal DNA of Pseudomonas sp. DJ-12. However, it differed from the fcbA-fcbB-fcbC ordering of Arthrobacter sp. SU. The nucleotide sequences of the fcbABC genes of strain P08 showed 98% and 53% identities to those of Pseudomonas sp. DJ-12 and Arthrobacter sp. SU, respectively. This suggests that the fcb genes might have been derived from Pseudomonas sp. DJ-12 to form plasmid pJS1 in Comamonas sp. P08, or that the fcb genes in strain DJ-12 were transposed from Comamonas sp. P08 plasmid.
- Cloning and Sequence Analysis of the hpaD Gene Responsible for Homoprotocatechuate 2,3-Dioxygenase from Pseudomonas sp. DJ-12
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Sang-Mahn Lee , Jong-Chan Chae , Youngsoo Kim , Chi-Kyung Kim
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J. Microbiol. 2001;39(4):334-337.
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The degradative pathway of homoprotocatechuate (HPC) is the bacterial route whereby 3,4-dihydroxyphenylacetic acid is catabolized to pyruvate and succinate by a series of sequential reactions. The HPC is catalized by homoprotocatechuate 2,3-dioxygenase (HPC-2,3O) to form 5-carboxymethyl-2-hydroxy-muco semialdehyde. In this study, the hpaD gene encoding HPC-2,3O was cloned from the chromosomal DNA of Pseudomonas sp. DJ-12 and its nucleotide sequence was analyzed. The open reding frame of hpaD gene was found to be composed of 864 nucleotide pairs and to encode a polypeptide with 287 amino acid residues. The deduced amino acid sequence of the HPC-2,3O from Pseudomonas sp. DJ-12 exhibited 60~64% homology with those of the corresponding enzymes from E. coli, Salmonella enterica, and Klebsiella pneumoniae.
- Toxic Effects of Catechol and 4-Chlorobenzoate Stresses on Bacterial Cells
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Sang-Ho Park , Yeon-Ja Ko , Chi-Kyung Kim
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J. Microbiol. 2001;39(3):206-212.
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Catechol and 4-chlorobenzoate (4CBA) which are produced from the biodegradation of a variety of aromatic and chloroaromatics have been recognized as toxic to living organisms. In this study, the toxic effects of catechol and 4-chlorobenzoate on gram-positive and -negative bacteria were examined in terms of survival, morphology, change in fatty acids and membrane protein composition. The survival rate of the organisms during treatment for 6 h was decreased, as the concentration of each aromatic was increased. Escherichia coli and Pseudomonas cells treated with catechol and 4CBA at concentrations causing a significant decrease in their viability, showed destructive openings in their cell envelopes. Bacillus subtilis treated with the aromatics were reduced in cell size and Staphylococcus aureus cells displayed irregular rod shapes with wrinkled surfaces. The bacterial cells treated with 20 mM catechol showed increases in unsaturated fatty acids, but several saturated fatty acids were decreased. In the E. coli cells treated with 20 mM catechol, inner membrane proteins of 150 kDa and 105 kDa were decreased. But several kinds of the inner and outer membrane proteins were increased. In B. subtilis treated with 20 mM catechol, several kinds of proteins were increased or decreased in membrane proteins.
- Cloning and Sequence Analysis of the xylL Gene Responsible for 4CBA-Dihydrodiol Dehydrogenase from Pseudomonas sp. S-47
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Dong-Woo Park , Youngsoo Kim , Sang-Mahn Lee , Jong-Ok Ka , Chi-Kyung Kim
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J. Microbiol. 2000;38(4):275-280.
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Pseudomonas sp. S-47 is capable of catabolizing 4-chlorobenzoate (4CBA) as carbon and energy sources under aerobic conditions via the meta-cleavage pathway. 4CBA-dioxygenase and 4CBA-dihydrodiol dehydrogenase (4CBA-DD) catalyzed the degradation of 4CBA to produce 4-chlorocatechol in the pathway. In this study, the xylL gene encoding 4CBA-DD was cloned from the chromosomal DNA of Pseudomonas sp. S-47 and its nucleotide sequence was analyzed. The xylL gene was found to be composed of 777 nucleotide pairs and to encode a polypeptide of 28 kDa with 258 amino acid residues. The deduced amino acid sequence of the dehydrogenase (XylL) from strain S-47 exhibited 98% and 60% homologies with those of the corresponding enzymes, Pseudomonas putida mt-2 (XylL) and Acinetobacter calcoaceticus (BenD), respectively. However, the amino acid sequences show 30% or less homology with those of Pseudomonas putida (BnzE), Pseudomonas putida F1 (TodD), Pseudomonas pseudoalcaligenes KF707 (BphB), and Pseudomonas sp. C18 (NahB). Therefore, the 4CBA-dihydrodiol dehydrogenase of strain S-47 belongs to the group I dehydrogenase involved in the degradation of mono-aryls with a carboxyl group
- Construction of a Bioluminescent Reporter Using the luc Gene and meta-Cleavage Dioxygenase Promoter for Detection of Catecholic Compounds
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Sang-Ho Park , Dong-Hun Lee , Kye-Heon Oh , Chi-Kyung Kim
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J. Microbiol. 2000;38(3):183-186.
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Several types of bioluminescent reporter strains have been developed for the detection and monitoring of pollutant aromatics contaminating the environment. In this study, a bioluminescent reporter strain, E. coli SHP3, was constructed by fusing the luc gene of firefly luciferase with the promoter of pcbC responsible for the meta-cleavage of aromatic hydrocarbons. The bioluminescence expressed by the luc gene in the reporter was well triggered by the promoter when it was exposed to 2,3-dihydroxybiphenyl (2,3-DHBP) at 0.5 to 1 mM concentrations. The bioluminescent response was more extensive when the reporter strain was exposed to 5 mM catechol and 2 mM 4-chlorocatechol. These different types of bioluminescent responses by E. coli SHP3 appeared to be characterized by the nature of the aromatics to stress. Since E. coli SHP3 responded to 2,3-DHBP quite sensitively, this reporter strain could be applied for detecting some catecholic pollutants.
- Microbial Degradation of Monohydroxybenzoic Acids
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Timmanagouda B. Karegoudar , Chi-Kyung Kim
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J. Microbiol. 2000;38(2):53-61.
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Hydroxybenzoic acids are the most important intermediates in the degradative pathways of various aromatic compounds. Microorganisms catabolize aromatic compounds by converting them to hydroxylated intermediates and then cleave the benzene nucleus with ring dioxygenases. Hydroxylation of the benzene nucleus of an aromatic compound is an essential step for the initiation and subsequent disintegration of the benzene ring. The incorporation of two hydroxyl groups is essential for the labilization of the benzene nucleus. Monohydroxybenzoic acids such as 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid through hydroxylation yield terminal aromatic intermediates like catechol, protocatechuic acid, gentisic acid, or pyrocatechuic acid that are susceptible for subsequent oxygenative cleavage of the benzene ring. These terminal aromatic intermediates are further degraded to cellular components through ortho-and /or meta-cleavage pathways and finally lead to the formation of constituents of the TCA cycle. Many groups of microorganisms have been isolated as degraders of hydroxybenzoic acids with diverse degradative routes and specific enzymes involved in their metabolic pathways. Various microorganisms carry out unusual non-oxidative decarboxylation of aromatic acids and convert them to respective phenols which have been documented. Further, Pseudomonas and Bacillus spp. are the most ubiquitous microorganisms, being the principal components of microflora of most soil and water environments.
- Development of Molecular Biological Methods to Analyze Bacterial Species Diversity in Freshwater and Soil Ecosystems
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Dong-Hun Lee , Sung-Ae Noh , Chi-Kyung Kim
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J. Microbiol. 2000;38(1):11-17.
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A new method was developed for the rapid analysis of diverse bacterial species in the natural envi-ronment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indig-enous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats.
- Assessing Survival and Detection of Catechol-bidegrading Strains in Waste Water microcosms
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Soo-Jin Choi , Min-Sup Song , Soo-youn Lee , Seong-Karp Hong , Kyung-Hee Min , Jong-Ok Ka , Chi-Kyung Kim , Young-Keun Park
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J. Microbiol. 1998;36(4):239-248.
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Catechol-degrading bacteria, Escherichia coli JM101 carrying pIB1343 (strain pIB1343) and Flavimonas orizihabitans KHi (strain KH1), were chosen as model bacteria to estimate the sur vival and catechol-degradability in waste water microcosms. Before their application to ecosystem, survival and catechol degradation of these bacteria were investigated in waste water microcosms. It was found that strain pIB1343 adapted much faster to waste water environments and degraded catechol in the case of Namdong samples, whereas the strain KH1 degraded catechol much faster in Sihwa samples. When catechol was added to the microcosms, indigenous microorganisms in Sihwa samples used catechol as a carbon and energy source much better than those in Namdong samples. A modified filter extraction technique was used to obtain the high-yield purified DNA from 50 ml of waste water samples and the extracted DNA (polymorphic DNA [20~23 kb]) was of sufficient quantity and quality for the amplification. PCR was performed with catA-specicic promers, C120U and C120L, which specifically detected the catA gene encoding catechol 1,2-dioxygenase in waste water microcosms, and then the 320 bp PCR products were amplified. PCR products were quantified by densitomenter. Using the standard curve of detection limit by catA-specific PCR products, the number of catA genes for their corresponding intensities of PCR products was obtained. the number of total catA genes PCR in waste water microcosms was correalated with catechol degradation.
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