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Alcaligenes sp. HPC 1271 demonstrated antibacterial activity against multidrug resistant bacteria, Enterobacter sp., resistant to sulfamethoxazole, ampicillin, azithromycin, and tetracycline, as well as against Serratia sp. GMX1, resistant to the same antibiotics with the addition of netilmicin. The cell-free culture supernatant was analyzed for possible antibacterials by HPLC, and the active fraction was further identified by LC-MS. Results suggest the production of tunicamycin, a nucleoside antibiotic. The draft genome of this bacterial isolate was analyzed, and the 4.2 Mb sequence data revealed six secondary metabolite-producing clusters, identified using antiSMASH platform as ectoine, butyrolactone, phosphonate, terpene, polyketides, and nonribosomal peptide synthase (NRPS). Additionally, the draft genome demonstrated homology to the tunicamycin-producing gene cluster and also defined 30 ORFs linked to protein secretion that could also play a role in the antibacterial activity observed. Gene expression analysis demonstrated that both NRPS and dTDP-glucose 4,6-dehydratase gene clusters are functional and could be involved in antibacterial biosynthesis.

We report a draft genome sequence of Alcaligenes sp. strain HPC1271, which demonstrates antimicrobial activity against multidrug-resistant bacteria. Antibiotic production by Alcaligenes has not been frequently reported, and hence, the availability of the genome sequence should enable us to explore new antibiotic-producing gene clusters.

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.