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International Biodeterioration & Biodegradation 08/2013; 82:24-32. · 2.07 Impact Factor
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ABSTRACT: Multiple acidophilic xylanolytic enzymes were produced by Penicillium oxalicum GZ-2 during growth on wheat straw, rice straw, corn stover, and wheat bran. The expression of xylanase isoforms was dependent on substrate type and nitrogen source. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that wheat straw was the best inducer, resulting in the highest xylanase (115.2U/mL) and β-xylosidase (89mU/mL) activities during submerged fermentation. The optimum temperature and pH for xylanase activity were 50°C and 4.0, respectively; however, the crude xylanase enzymes exhibited remarkable stability over a broad pH range and showed more than 90% activity at 50°C for 30min at pH 4.0-8.0. The results revealed that P. oxalicum GZ-2 is a promising acidophilic xylanase-producing microorganism that has great potential to be used in biofuels, animal feed, and food industry applications.
Bioresource technology 07/2012; 123:117-24. · 4.25 Impact Factor
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ABSTRACT: Mai Po Nature Reserve is the largest mangrove ecosystem and the most polluted coastal water body in Hong Kong. Plasmids screening of 100 Vibrio isolates randomly showed 45 % of them contained 1-3 plasmids. These plasmid(s)-bearing isolates could be divided into 12 groups based on their plasmid profiles. Phylogenetic analysis of the partial 16S rRNA gene sequences confirmed that all plasmid(s)-bearing isolates belonged to Vibrio cholerae. Full DNA sequences of the plasmids in Groups I (pVCG1.1 and pVCG1.2), II (pVCG2.1), III (pVCG3.2) and IV (pVCG4.1) have been determined and the results showed that pVCG1.1, pVCG2.1 and pVCG3.2 were almost identical. Plasmids pVCG1.1, pVCG1.2 and pVCG4.1 are comprised of 4,439, 2,357 and 2,163 bp with the overall G+C content of 45.57, 53.54 and 43.09 %, respectively. pVCG1.1 is a novel plasmid, and plasmids pVCG1.2 and pVCG4.1 showed homology of replication initiation proteins to that of the theta type replicons. Attempts to cure the plasmids from their hosts were unsuccessful. These data suggest that plasmids of Vibrio spp. are a significant gene reservoir in the marine ecosystem.
Ecotoxicology 06/2012; 21(6):1661-8. · 2.36 Impact Factor
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ABSTRACT: Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.
In this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of bgl3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.
An native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.
Microbial Cell Factories 02/2012; 11:25. · 3.55 Impact Factor
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ABSTRACT: The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.
PLoS ONE 01/2012; 7(2):e31693. · 4.09 Impact Factor
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ABSTRACT: Various parameters were measured during the period of composting of dairy manure and rice chaff in different ratios (dairy manure/rice chaff=V/V, pile 1: 75/25; pile 2: 80/20; pile 3: 85/15) to evaluate their suitability as indicators for the composting process. The temperature in pile 1 increased rapidly and remained above 60 °C for 30 days, while the temperature in pile 3 increased slowly relative to the other two piles. Furthermore, the degradation of organic substrates, as indicated by the reduction of C/N ratio, was rapid in pile 1 (below 20% 28 days after beginning of the composting). The major fluctuations of various water-soluble fractions in all piles were observed during the first 3 weeks, and the results in general showed that the highest microbial populations and enzymatic activities also appeared in this phase. Various parameters indicated that the rapid composting method was a feasible one for treating agricultural wastes.
Bioresource technology 07/2011; 102(19):9040-9. · 4.25 Impact Factor
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ABSTRACT: Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.
Protein Expression and Purification 06/2011; 79(2):176-86. · 1.59 Impact Factor
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ABSTRACT: A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His(6)-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652 IU/L after 2 days of cultivation in the induction medium. The His(6)-tagged rNC-CDH1 was purified through a one-step Ni-NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 745 1IU/L (0.89 mg protein/mL), with a specific CDH activity of 8.37 IU/mg. The purity of the enzyme was examined by SDS-PAGE, and a single band corresponding to a molecular weight of about 120 kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25-70°C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K(m) value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k(cat) remains relatively constant when the chain length changes.
Protein Expression and Purification 01/2011; 75(1):63-9. · 1.59 Impact Factor
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ABSTRACT: Bacterial type IV secretion systems (T4SS) perform two fundamental functions related to pathogenesis: the delivery of effector molecules to eukaryotic target cells, and genetic exchange. Two T4SSs have been identified in Burkholderia cenocepacia K56-2, a representative of the ET12 lineage of the B. cepacia complex (Bcc). The plant tissue watersoaking (Ptw) T4SS encoded on a resident 92 kb plasmid is a chimera composed of VirB/D4 and F-specific subunits, and is responsible for the translocation of effector(s) that have been linked to the Ptw phenotype. The bc-VirB/D4 system located on chromosome II displays homology to the VirB/D4 T4SS of Agrobacterium tumefaciens. In contrast to the Ptw T4SS, the bc-VirB/D4 T4SS was found to be dispensable for Ptw effector(s) secretion, but was found to be involved in plasmid mobilization. The fertility inhibitor Osa did not affect the secretion of Ptw effector(s) via the Ptw system, but did disrupt the mobilization of a RSF1010 derivative plasmid.
Microbiology 10/2009; 155(Pt 12):4005-13. · 3.06 Impact Factor
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ABSTRACT: A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine-cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.
Marine Biotechnology 12/2008; 11(4):456-62. · 3.43 Impact Factor
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ABSTRACT: A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30 degrees C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10(5)-10(7) CFU ml(-1)), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30 degrees C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg(-1) MP and 25 mg kg(-1) carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.
Biodegradation 09/2007; 18(4):403-12. · 2.02 Impact Factor
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ABSTRACT: A marine bacterium was isolated from Mai Po Nature Reserve of Hong Kong and identified as Vibrio cholerae MP-1. It contains a small plasmid designated as pVC of 3.8 kb. Four open reading frames (ORFs) are identified on the plasmid, but none of them shows homology to any known protein. Database search indicated that a 440 bp fragment is 96% identical to a fragment found in a small plasmid of another V. cholerae. Further experiments demonstrated that a 2.3 kb EcoRI fragment containing the complete ORF1, partial ORF4 and their intergenic region could self-replicate. Additional analyses revealed that sequence upstream of ORF1 showed the features characteristic of theta type replicons. Protein encoded by ORF1 has two characteristic motifs existed in most replication initiator proteins (Rep): the leucine zipper (LZ) motif located at the N-terminal region and the alpha helix-turn-alpha helix motif (HTH) located at the C-terminal end. The results suggest that pVC replicates via the theta type mechanism and is likely a novel type of theta replicon.
The Journal of Microbiology 07/2007; 45(3):193-8. · 1.10 Impact Factor
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ABSTRACT: Seven organophosphorus pesticide-degrading bacteria harboring the methyl parathion degrading (mpd) gene were isolated from a methyl parathion contaminated site. In this study, the 4.7 kb mpd gene cluster, conserved in all seven bacteria capable of degrading methyl parathion, was cloned and further analysis revealed that this cluster contained five ORFs and the mpd gene was associated with a mobile element, IS6100. In addition to mpd gene ORF and tnpA ORF, three other ORFs showed high homology to the permease component of ABC-type transport system, the general secretion pathway protein B, and the RNA polymerase sigma 70 factor, respectively. The mpd genes of these 7 strains were subcloned and expressed in E. coli, SDS-PAGE and zymogram analysis showed that two expression products of mpd genes in E. coli were found, but the one without signal peptide showed the hydrolytic activities. Our evidences collectively suggest that mpd gene cluster may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains, and comparisons of GC content of the cluster and respective host's chromosome.
Biodegradation 11/2006; 17(5):465-72. · 2.02 Impact Factor
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ABSTRACT: The long-term effects of methylparathion contamination on the diversity of soil microbial community was investigated by a culture-independent approach using small subunit ribosomal RNA (SSU rRNA) gene-based cloning. Microbial DNA extracted from both the control soil sample and methylparathion contaminated soil sample was subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences. From the PCR amplification product, clone libraries were constructed for both samples. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and HhaI. A total of 603 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, the phylotype richness, frequency distribution (evenness) of the two clone libraries were compared by using a variety of diversity indices. Phylogenetic analysis of the sequences of the dominant phylotypes revealed that the bacterial communities changed noticeably. In the control soil, the dominant bacterial groups included a member of a novel bacterial division, the bacillus genus, and a member of alpha-proteobacteria, while in methylparathion contaminated soil, the dominant phylotypes were replaced by a member of the flexibactera-cytophaga-bacteroides division and two members of the gamma-proteobacteria subdivision. This is the first report of the long-term effects of methylparathion (one of the major pesticides widely used in developing countries) on soil microbial community diversity and structure by a culture-independent method, and provides the evidences to assess the long-term environmental toxicological effects of methylparathion from the microbial community viewpoint.
Ecotoxicology 09/2006; 15(6):523-30. · 2.36 Impact Factor
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ABSTRACT: Seven methyl parathion-degrading bacteria were isolated from a long-term methyl parathion contaminated soil and were found to belong to the genera Pseudaminobacter, Achromobacter, Brucella, and Ochrobactrum. Southern blot analysis using an mpd gene probe revealed that their hydrolase genes were similar to the mpd gene from Plesiomonas sp. strain M6 and were all located on the chromosome. Gene libraries were constructed from genomic DNA of each of the 7 organophosphorus pesticide-degrading bacteria, and their mpd genes were cloned and sequenced. Sequence analysis revealed that their hydrolase genes were conserved, and that the G+C content of the mpd genes were distinctly different from that of the chromosome-located 16S rRNA gene, suggesting that the mpd gene could be transferred and expressed among a variety of bacterial hosts.
Canadian Journal of Microbiology 05/2005; 51(4):337-43. · 1.36 Impact Factor
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ABSTRACT: 3.00 kg(a. i) x hm(-2) phoxin and 2.63 kg(a. i) x hm(-2) methyl parathion were respectively applied to control the Taeniothrips alliorum on Chinese chive. Compared to no pesticide treatment, the decline rate of the insect density was 98.28% and 98.39% at the 3rd day after spraying pesticides, and 89.94% and 94.04% at the 20th day after spraying pesticides, respectively. At the 3rd day after spraying 15.00, 18.00 and 21.00 kg(a. i) x hm(-2) phoxin, the insect density of Bradysia odoriphaga decreased 80.77%, 93.10% and 96.98%, and at the 35th day after spraying, it decreased 92.44%, 95.05% and 96.81%, respectively. The application of pesticide-degrading bacterium had not any effect on controlling insect pests, but could markedly degrade pesticide. At the 3rd day after spraying 45.00 L x hm(-2) pesticide-degrading bacterium to control Taeniothrips alliorum, the degradion rate of phoxin and methyl parathion was 99.52% and 98.83%, and at the 3rd after spraying 75.00 L x hm(-2) pesticide-degrading bacterium to control Bradysia odoriphaga, the degradation rate of three concentrations of phoxin was 100%, 100% and 99.69%, respectively.
Ying yong sheng tai xue bao = The journal of applied ecology / Zhongguo sheng tai xue xue hui, Zhongguo ke xue yuan Shenyang ying yong sheng tai yan jiu suo zhu ban 09/2004; 15(8):1459-62.
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ABSTRACT: Degradation of methyl parathion in soil and Chinese chive by strain DLL-1 was studied. Usage of methyl parathion at 7.5, 15, and 22.5 kg(a.i.).hm-2 resulted in the average amount of residue of 0.663, 1.270, and 1.901 mg.kg-1 in Chinese chive respectively. The natural degradation rate was 98.94%, 96.44%, and 96.04% corresponding to the 3 levels of usage. The amount of pesticide residue could be significantly decreased through the application of high effective degrading microbial agents. The amount of pesticide residue in Chinese chive and soil was 0.269 and 0.099 mg.kg-1 with the usage of 75 kg.hm-2 of degradation bacterium, which was decreased by 78.82% and 98.68% compared with the control. Increasing the bacterium usage led to the increase of degradation rate. Usage of degradation bacterium more than 75 kg.hm-2 did not increase the degradation rate further. The best time of the application of the degrading microbe was 3 days after the application of the pesticide.
Ying yong sheng tai xue bao = The journal of applied ecology / Zhongguo sheng tai xue xue hui, Zhongguo ke xue yuan Shenyang ying yong sheng tai yan jiu suo zhu ban 03/2004; 15(2):295-8.
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ABSTRACT: A Pseudomonas strain P3 was isolated in this work. P3 can grow with p-nitrophenol as the sole carbon and nitrogen source. Growth in media with nitrogen, P3 can degrade p-nitrophenol to accumulate nitrite in the culture media. P3 can utilize a series of aromatic compounds as sole carbon sources. Different heavy metal ions have different effects on the degradation of p-Nitrophenol by P3. Glucose had no effect on the degradation of p-nitrophenol, while trace yeast extract greatly increased the degradation rate. Methyl parathion hydrolase gene mpd was clone into P3 by conjugation and genetically engineered bacterium PM was obtained. Methyl parathion hydrolase was expressed by PM. PM could grew on methyl parathion as sole carbon source. PM could degrade methyl parathion with relatively high activity and stability.
ACTA MICROBIOLOGICA SINICA 03/2002; 42(1):19-26.
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ABSTRACT: Fifty environmental isolates of Vibrio species were isolated from water samples of Mai Po Nature Reserve and the Cape d'Aguilar Marine Reserve in Hong Kong and screened for the presence of plasmid. Mai Po is a wastewater-impacted area while the Cape d'Aguilar Marine Reserve is pristine natural marine water. Plasmid was found in Vibrio isolates from both sites at similar frequencies and each site showed distinctive plasmid profiles. These plasmid-bearing Vibrio isolates were identified as different species of the Vibrio genus by both biochemical test and subsequently full-length 16S rRNA sequences. Antibiotic resistance test showed that all these plasmid-bearing Vibrio isolates showed multiple resistance to 21 antibiotics tested. In addition, selective isolates also showed tolerance to 10 microM Hg 2+ in culture medium and they generally harbored large plasmid(s) (>30 kb). Our results show that the high frequency of plasmid in Vibrio species of both polluted and pristine environments may be ecologically important to the survival of these bacteria in the environment. The specific functioning of the cryptic plasmids remains the focus of current investigations.
Antonie van Leeuwenhoek 89(3-4):307-15. · 2.09 Impact Factor
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ABSTRACT: Water samples were collected from several previously determined sampling sites at Mai Po Nature Reserve (22°29′N to 22°31′N and 113°59′E to 114°03′E) and Cape d'Aguilar Marine Reserve (22°12′N and 114°15′E) of Hong Kong. Fifty environmental isolates of Vibrio species were isolated from water samples of Mai Po Nature Reserve and the Cape d'Aguilar Marine Reserve in Hong Kong and they were screened for the presence of plasmids. Mai Po is a wastewater-impacted area while the Cape d'Aguilar Marine Reserve is a pristine marine water. Plasmids were found in Vibrio isolates from both sites at similar frequencies and each site had distinctive plasmid profiles. These plasmid-bearing Vibrio isolates were identified to be different species of the Vibrio species by both biochemical test and subsequent full-length 16S rRNA sequences. Antibiotic resistance test showed that all these plasmid-bearing Vibrio isolates showed multiple resistance to the 21 antibiotics tested. In addition, selective isolates also showed tolerance to 10 µM Hg 2+ in culture medium and these isolates generally harbored large plasmid(s) (>30 kb). Among the plasmids detected, a novel cryptic plasmid, pVC, from an environmental isolate of Vibrio cholerae MP-1 from Mai Po Nature Reserve in Hong Kong has been isolated. The complete nucleotide sequence analysis (3,806 bp) revealed three major putative open reading frames (ORFs). Our results showed that high frequency of plasmid in Vibrio species of both polluted and pristine environments may be ecologically important to the survival of these bacteria in the environment. At the same time, public awareness of the associated problem with these bacteria should also be acknowledged. The specific functioning of the cryptic plasmids remains the focus of our current investigations.
