Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

UNLABELLED: An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

Detection of Actinobacteria cultivated from environmental samples reveals bias in universal primers.

AIMS: The aims of this study were to develop media to cultivate actinomycetes, screen the resulting isolates with Actinobacteria-specific primers, and examine the efficacy of detection of the actinobacterial isolates with universal primers. METHODS AND RESULTS: Soil-extract medium was developed for a terrestrial bluff environment. Recovered isolates were subjected to polymerase chain reaction (PCR) with taxon-specific primers to identify Actinobacteria. Universal bacterial primers 24f and 1492r (modified and original versions) were used to amplify the 16S rRNA gene from the putative Actinobacteria. While both reverse primers failed to provide amplification products from 20% to 50% of the isolates, the 1492r primer detected Actinobacteria more effectively than 1492r-mod. The region of the gene containing the annealing site for the 1492r primers from 15 isolates that failed to amplify showed no differences in nucleotide sequence to the original 1492r primer. CONCLUSIONS: Universal 16S rRNA gene primers are not capable of amplifying this gene from all bacteria within an environmental sample. Some Actinobacteria may share 100% sequence similarity to universal primers but remain undetected. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are important for studies of particular taxa in environmental samples where reactions utilizing universal primers may not reveal the extent of their presence and diversity.