A New Protein Antimicrobial Isolated from the Genome of an Uncharacterized Soil Streptomycete

March 2016
Topics: Genetics, Disease Transmission, Public Health, Diseases
Dr. Heath Farris, The MITRE Corporation
Haley Skaer, The MITRE Corporation
Alfred Steinberg, The MITRE Corporation
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The genomes of uncultivated bacteria represent a fertile reservoir of molecular diversity. Streptomyces, a medically and industrially important genus of soil bacteria, produce many useful antibiotics and enzymes, contain large prokaryotic genomes, and produce secondary metabolites with exceptional functionality. Among these, protein antimicrobials have received attention as natural food preservatives, agricultural biocontrol agents, and pharmaceutical alternatives to antibiotics.

In this study, the previously uncharacterized Streptomyces sp. isolate 212 bacterium was cultivated from rich organic soil samples collected from a woodland bluff environment in Alabama. Production of protein antimicrobials from the isolate was evaluated using a standard line inoculum assay and zymogram analyses of concentrated growth supernatant. Whole genome extracts from the isolate were analyzed by de novo GS/FLX 454 pyrosequencing, and putative genes within the draft quality genome were annotated. The recombinant gene coding for the Mitrecin A protein was identified, synthesized, and heterologously over-expressed in E. coli. Mitrecin A was purified by affinity and size exclusion chromatography. Well-diffusion and dye-release assays were used to determine the tolerances of purified Mitrecin A activity against ranges of temperature, pH, and salinity.

Streptomyces sp. isolate 212, a novel soil bacterium, produces multiple species of bacteriolytic enzymes as determined by zymogram analyses of growth supernatant. The genome of the streptomycete, estimated by pyrosequencing, is approximately 10 Mbp with a GC content of 68%. Annotation of the draft quality genome identified a suite of putative bacteriolytic genes, including the gene for Mitrecin A. Synthesis and over-expression of the gene followed by multi-step purification of the gene product resulted in a 14.3 kDa cationic protein with bacteriolytic activity against the medically important genera of Salmonella, Vibrio, and Yersinia.

Note: This paper originally appeared in 2012, in a slightly different format.

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