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- Isolation and Characterization of Novel Lipase Gene LipHim1 from the DNA Isolated from Soil Samples
- Pavan Kumar Pindi , Raja Srinath Rebba , Theetha L. Pavankumar
- J. Microbiol. 2014;52(5):384-388. Published online April 11, 2014
- DOI: https://doi.org/10.1007/s12275-014-3302-2
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- 12 Citations
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Abstract
- Metagenomics is a magnificent tool to isolate genes from unknown/uncharacterized species and also from organisms that cannot be cultured. In this study, we constructed a meta-genomic library from isolated DNA of soil samples collected from Palamuru University campus premises, in Mahabub-nagar district of Andhra Pradesh, India. We isolated a novel lipase gene LipHim1, which has an open reading frame of 591 base pairs and encodes ~23 kDa protein consisting of 196 amino acids. The Lipase LipHim1 showed maximum 32% homology at the protein level with the extracellular Aeromonas hydrophila lipase (Class II, GDSL family) and was significantly different from all other known lipases. The isolated lipase catalyzed the hydrolysis of fatty acid esters of polyoxyethylene sorbitan such as Tween 60. Our results in-dicate that the isolated lipase gene is novel.
- Genome-Wide Enrichment Screening Reveals Multiple Targets and Resistance Genes for Triclosan in Escherichia coli
- Byung Jo Yu , Jung Ae Kim , Hyun Mok Ju , Soo-Kyung Choi , Seung Jin Hwang , Sungyoo Park , EuiJoong Kim , Jae-Gu Pan
- J. Microbiol. 2012;50(5):785-791. Published online November 4, 2012
- DOI: https://doi.org/10.1007/s12275-012-2439-0
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- 19 Citations
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Abstract
- Triclosan is a widely used biocide effective against different microorganisms. At bactericidal concentrations, triclosan appears to affect multiple targets, while at bacteriostatic concentrations, triclosan targets FabI. The site-specific antibioticlike mode-of-action and a widespread use of triclosan in household products claimed to possibly induce cross-resistance to other antibiotics. Thus, we set out to define more systematically the genes conferring resistance to triclosan; A genomic library of Escherichia coli strain W3110 was constructed and enriched in a selective medium containing a lethal concentration of triclosan. The genes enabling growth in the presence of triclosan were identified by using a DNA microarray and confirmed consequently by ASKA clones overexpressing the selected 62 candidate genes. Among these, forty-seven genes were further confirmed to enhance the resistance to triclosan; these genes, including the FabI target, were involved in inner or outer membrane synthesis, cellsurface material synthesis, transcriptional activation, sugar phosphotransferase (PTS) systems, various transporter systems, cell division, and ATPase and reductase/dehydrogenase reactions. In particular, overexpression of pgsA, rcsA, or gapC conferred to E. coli cells a similar level of triclosan resistance induced by fabI overexpression. These results indicate that triclosan may have multiple targets other than well-known FabI and that there are several undefined novel mechanisms for the resistance development to triclosan, thus probably inducing cross antibiotic resistance.
- Screening and Characterization of a Cellulase Gene from the Gut Microflora of Abalone Using Metagenomic Library
- Duwoon Kim , Se-Na Kim , Keun Sik Baik , Seong Chan Park , Chae Hong Lim , Jong-Oh Kim , Tai-Sun Shin , Myung-Joo Oh , Chi Nam Seong
- J. Microbiol. 2011;49(1):141-145. Published online March 3, 2011
- DOI: https://doi.org/10.1007/s12275-011-0205-3
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- 15 Citations
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Abstract
- A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAM11E10). A shotgun clone library was constructed using the positive clone (pAM11E10) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAM11L9). The pAM11L9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAM11) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAM11) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAM11 were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.
- Molecular Characterization of TEM-type [beta]-Lactamases Identified in Cold-Seep Sediments of Edison Seamount (South of Lihir Island, Papua New Guinea)
- Jae Seok Song , Jeong Ho Jeon , Jung Hun Lee , Seok Hoon Jeong , Byeong Chul Jeong , Sang-Jin Kim , Jung-Hyun Lee , Sang Hee Lee
- J. Microbiol. 2005;43(2):172-178.
- DOI: https://doi.org/2165 [pii]
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Abstract
- To determine the prevalence and genotypes of [beta]-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced [beta]-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum [beta]-lactamase. 837- and 259-bp fragments specific to bla_TEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent [beta]-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type [beta]-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type [beta]-lactamases of the pre-antibiotic era indicates that TEM-type [beta]-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.
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