In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E.coli

ABSTRACT: BACKGROUND: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E.coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. RESULTS: In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E.coli BL21/pET28a-lipAB and two dual expression plasmid systems E.coli BL21/pETDuet-lipA-lipB and E.coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E.coli BL21/pET28a-lipAB and E.coli BL21/pETDuet-lipA-lipB were 1300U/L and 3200U/L, respectively, while the activity of the lipase expressed by E.coli BL21/pACYCDuet-lipA-lipB was up to 8500U/L. The lipase LipA had an optimal temperature of 30[degree sign]C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. CONCLUSIONS: The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.     

Stereoselectivity of lipases: esterification reactions of octadecylglycerol.

Stereoselectivity of several triacylglycerol lipases (EC 3.1.1.3) has been investigated in the enzymatic esterification of rac-1-O-octadecylglycerol with oleic acid in the presence of organic solvents, such as hexane. X-1(3)-O-Octadecylmonooleoylglycerols were the only products formed with most lipases; considerable proportions of X-1(3)-O-octadecyldioleoylglycerols were also formed with the lipase from Candida cylindracea. The mixtures of unesterified enantiomeric substrates, i.e., X-1(3)-O-octadecylglycerols were converted to their 3,5-dinitrophenylurethane derivatives and subsequently resolved into sn-1 and sn-3 enantiomers by HPLC on a chiral stationary phase (Sumichiral OA 2100). The data on enantiomeric excess (ee) and enantiomeric ratio (E) in the unesterified substrate revealed for the lipases from porcine pancreas, Rhizopus sp., Pseudomonas sp., Candida cylindracea, Chromobacterium viscosum and Penicillium cyclopium a distinct preference for 1-O-octadecyl-sn-glycerol over its enantiomer indicating stereoselectivity for the sn-3 position. For the lipase from Rhizomucor miehei a slight stereoselectivity for the sn-1 position was observed. Solvents, such as diethyl ether and dichloromethane, strongly inhibited the esterification reaction, but the enzymatic activity could be restored upon removal of such solvents by washing with hexane indicating reversible inhibition.     

假单胞菌M18中藤黄绿菌素合成限速酶基因的鉴定及表达优化

【目的】假单胞菌株M18中负责抗真菌剂藤黄绿菌素(Plt)合成的结构基因包括pltLABCDEFG、pltM。为了鉴定Plt合成限速酶的基因,分别将9个结构基因过表达。【方法】以M18菌株染色体DNA为模板,PCR扩增这9个Plt合成基因的编码区,分别克隆到穿梭载体pME6032的tac启动子下游,构建9个结构基因的过表达载体,并转入假单胞菌株M18中。在KMB培养基中进行Plt发酵分析。【结果】分别携带pltC、pltD、pltF过表达载体的菌株与携带空质粒的菌株相比,Plt产量分别提高了96%、78%、75%。对重组菌株进行IPTG诱导浓度和诱导时间的优化,确定IPTG最佳诱导浓度为1.0 mmol/L,最佳诱导时间为培养6 h。【结论】pltC、pltD、pltF分别编码的Ⅰ型聚酮合成酶、卤化酶、乙酰CoA合成酶可能为Plt生物合成的限速酶。按照优化条件发酵,携带pltD、pltF过表达质粒的菌株产量分别上升77.5%、159.1%。     

Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp. Strain HR199.

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding beta-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsOmegaGm and Pseudomonas sp. strain HRechOmegaKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatOmegaKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a beta-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.     

Bacteriocin activity of Pseudomonas sp. on enteropathogenic bacteria in an artificial aquatic system

The bacteriocin of Pseudomonas sp. strain R10 was active in vitro against several enteropathogenic bacteria: enterotoxigenic Escherichia coli, Salmonella typhimurium, Salmonella typhi, Shigella flexneri and Shigella sonnei. Pseudomonas sp. R10 was studied for 5 d in an aquatic system in the presence of strains of the enteropathogenic species mentioned. All the target strains were inhibited in the presence of bacteriocinogenic Pseudomonas sp. R10 and the highest antibacterial action was observed on the second day. Similar results were obtained with the partially purified bacteriocin, although the greatest bactericidal action, in all the studied target strains, was observed on the first day and the bacterial recounts were slightly higher overall.     

Prokaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase in the 2-nitrobenzoate degradation pathway of Pseudomonas fluorescens strain KU-7

The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes. The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe(2+) but is inhibited by other metal ions, such as Zn(2+), Cu(2+), and Cd(2+). The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.     

Phytase activity in some groups of bacteria. Search for and cloning of genes for bacterial phytases

A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.     

Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production,characterization and application

Abstract

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58?U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91?U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53?U/g) and Aspergillus niger (INCQS 40015)/frit (34.76?U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).     

Isolation rate of gram negative microflora and its sensitivity to antibiotics in hemoblastosis patients

A total of 67 patients with blood system diseases and infectious complications were examined. During the period of the examination 139 microorganisms were isolated. Of these gram negative microorganisms constituted 51%, gram positive microorganisms--34.8% and fungal flora--14.2%. Most frequently the following gram negative microorganisms were isolated from the patients: Pseudomonas sp. (including P. aeruginosa), Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae. All isolated microorganisms retained sensitivity to imipenem, with the exception of individual strains of Pseudomonas sp.; the latter exhibited sensitivity to amicacin and ceftazidim. Cefotaxime was active with respect to 75% of K. pneumoniae strains and all E. coli strains, ciprofloxacin was active with respect to 43% of E. coli strains, 80% of K. pneumoniae strains and 83.4% of Pseudomonas sp. strains, cefepim was active with respect to 85.7% of Pseudomonas sp. strains and all E. coli strains, ceftazidim was active with respect to all Pseudomonas sp. and E. coli strains. 75% of K. pneumoniae strains, 77.8% of Pseudomonas sp. strains and 86% of E. coli strains retained sensitivity to amicacin. 25% of K. pneumoniae strains required testing for ESBL production.     

Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

Biosynthesis of synthons in two-liquid-phase media

The Pseudomonas oleovorans alkane hydroxylase and xylene oxygenase from Pseudomonas putida are versatile mono-oxygenases for stereo- and regioselective oxidation of aliphatic and aromatic hydrocarbons. Pseudomonas oleovorans and alkanol dehydrogenase deficient mutants of Pseudomonas have previously been used to produce alkanols from various alkanes and optically active epoxides from alkenes. Similarly, P. putida strains have been used to produce aromatic alcohols, aromatic acids, and optically active styrene oxides. A limitation in the use of Pseudomonas strains for bioconversions is that these strains can degrade some of the products formed. To counter this problem, we have constructed Escherichia coli recombinants, which contain the alk genes from the OCT plasmid of P. oleovorans [E. coli HB101 (pGEc47)] and the xylMA genes from the TOL plasmid of P. putida mt-2 [E. coli HB101 (pGB63)], encoding alkane hydroxylase and xylene oxygenase, respectively. Escherichia coli HB101 (pGEc47) was used to produce octanoic acid from n-octane and E. coli HB101 (pBG63) was put to use for the oxidation of styrene to styrene oxide in two-liquid phase biocatalysis at high cell densities. The alk(+) recombinant strain E. coli HB101 (pGEc47) was grown to 40 g/L cell dry mass in the presence of n-octane, which was converted to octanoic acid by the alkane oxidation system, the product accumulating in the aqueous phase. The xyl(+) recombinant E. coli HB101 (pBG63) was grown to a cell density of 26 g/L cell dry mass in the presence of around 7% (v/v) n-dodecane, which contained 2% (v/v) styrene. The recombinant E. coli (xyl(+)) converted styrene to (S)-(+)-styrene oxide at high enantiomeric excess (94% ee) and this compound partitioned almost exclusively into the organic phase. Using these high-cell-density two-liquid-phase cultures, the products accumulated rapidly, yielding high concentrations of products (50 mM octanoic acid and 90 mM styrene oxide) in the respective phases. (c) 1996 John Wiley & Sons, Inc.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Coastal Seawater Bacteria Harbor a Large Reservoir of Plasmid-Mediated Quinolone Resistance Determinants in Jiaozhou Bay, China

Diversity and prevalence of plasmid-mediated quinolone resistance determinants were investigated in environmental bacteria isolated from surface seawater of Jiaozhou Bay, China. Five qnr gene alleles were identified in 34 isolates by PCR amplification, including qnrA3 gene in a Shewanella algae isolate, qnrB9 gene in a Citrobacter freundii isolate, qnrD gene in 22 Proteus vulgaris isolates, qnrS1 gene in 1 Enterobacter sp. and 4 Klebsiella spp. isolates, and qnrS2 gene in 1 Pseudomonas sp. and 4 Pseudoalteromonas sp. isolates. The qnrC, aac(6')-Ib-cr, and qepA genes could not be detected in this study. The 22 qnrD-positive Proteus vulgaris isolates could be differentiated into four genotypes based on ERIC-PCR assay. The qnrS1 and qnrD genes could be transferred to Escherichia coli J53 Azi(R) or E. coli TOP10 recipient strains using conjugation or transformation methods. Among the 34 qnr-positive isolates, 30 had a single point mutation in the QRDRs of GyrA protein (Ala67Ser, Ser83Ile, or Ser83Thr), indicating that cooperation of chromosome- and plasmid-mediated resistance contributed to the spread and evolution of quinolone resistance in this coastal bay. Eighty-five percent of the isolates were also found to be resistant to ampicillin, and bla(CMY), bla(OXY), bla(SHV), and bla(TEM) genes were detected in five isolates that also harbored the qnrB9 or qnrS1 gene. Our current study is the first identification of qnrS2 gene in Pseudoalteromonas and Pseudomonas strains, and qnrD gene in Proteus vulgaris strains. High prevalence of diverse qnr genes in Jiaozhou Bay indicates that coastal seawater may serve as an important reservoir, natural source, and dissemination vehicle of quinolone resistance determinants.