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Tetracycline resistance-encoding plasmid from Bacillus sp. strain #24, isolated from the marine sponge Haliclona simulans.

ABSTRACT: Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans.

SUBMITTER: Phelan RW

PROVIDER: S-EPMC3019722 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Tetracycline resistance-encoding plasmid from Bacillus sp. strain #24, isolated from the marine sponge Haliclona simulans.

Phelan Robert W RW Clarke Charles C Morrissey John P JP Dobson Alan D W AD O'Gara Fergal F Barbosa Teresa M TM

Applied and environmental microbiology 20101105 1

Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans. ...[more]

PMID: 21057017

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Project description:BACKGROUND:Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome. RESULTS:A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C ?-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3-12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 ?mol/min, respectively. CONCLUSION:We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.

Project description:Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.

Dataset Information

Tetracycline resistance-encoding plasmid from Bacillus sp. strain #24, isolated from the marine sponge Haliclona simulans.

Publications

Tetracycline resistance-encoding plasmid from Bacillus sp. strain #24, isolated from the marine sponge Haliclona simulans.

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