Torres Zapata, Irimar

Profile Picture

Filters

20121
Reset filters

Settings

Citations

Publication Search Results

Now showing 1 - 1 of 1
  • Thumbnail Image
    Publication
    Generation of large-insert metagenomic libraries from subtropical hypersaline microbial mats and their screening for antibiotic resistance
    (2012) Torres Zapata, Irimar; Ríos Velázquez, Carlos; College of Arts and Sciences - Sciences; Santos Flores, Carlos J.; Martínez Cruzado, Juan C.; Department of Biology; Griggs, Gayle W.
    Most of the microbial diversity in an environment cannot be studied through laboratory standard media culture methods. For this, an emerging science, coined “Metagenomics”, has been developed as an effort to access the majority of microorganisms. It involves the isolation and further characterization of microbial genomes and their study from different perspectives, using sequence-based and function-based analyses. Metagenomic studies of several environments (i.e. soil, water, animals, among others) have unraveled novel activities with industrial, biotechnological, and biomedical potential. Recently (during the last decade), unique environments such as microbial mats have also been subjected to metagenomic approaches. Microbial mats are organo-sedimentary structures found in extreme environments harboring highly metabolically diverse microorganisms. While macro and microscopic analyses as well as geomicrobiological studies have been performed with the microbial mats of Cabo Rojo Salterns, non-metagenomic studies were done to these ecosystems at that point. Our study presented the generation of large-insert metagenomic libraries from two subtropical hypersaline microbial mats (benthic and ephemeral) during the dry and rainy seasons. Also, due to the increase in antimicrobial resistant clinical isolates threatening human health, the metagenomic libraries were monitored by a functional-based analysis for the search of antibiotic resistance. Using an indirect extraction method, total DNA from microbial mats samples was isolated. DNA fragments of more than 20 Kbp were cloned into fosmids, packed in vitro and transduced into a host strain. Four metagenomic libraries were generated with a total of 64,600 clones with inserts ranging from 20-100 Kbp. The libraries were screened for resistance to ampicillin, tetracycline, spectinomycin, gentamicin and kanamycin. Only gentamicin and kanamycin resistant clones were isolated with inserts of approximately 30 Kbp and 40 Kbp, respectively. The restriction analysis and the retransformation of the fosmid into an isogenic strain confirmed the presence of an insert responsible for the resistance. The gentamicin resistant clone was mutagenized and further characterized by primer walking. The in silico analysis suggested the presence of five open reading frames from which two were related to antibiotic resistance genes. These included a 16S rRNA methyltransferase and an N-acetyltransferase most related (less than 40 % of identity with NCBI protein database) to Chloroflexus and uncultured prokaryote enzymes, respectively. Our data confirmed metagenomics as an emerging technology to unravel novel microbial strategies for biomedical application such as antibiotic resistance in environments as unique as microbial mats.