Direct cloning from enrichment cultures, a reliable strategy for isolationof complete operons and genes from microbial consortia

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular lev el and to be explored as potential sources for biotechnology processes, We have used a combined approach of enrichment culture and direct cloning to c onstruct cosmid libraries with large (>30-kb) inserts from microbial consor tia. Enrichment cultures were inoculated with samples from five environment s, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichmen t cultures and used to construct cosmid libraries; each library consisted o f between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments iso lated from the consortia organisms. These three libraries were used to comp lement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta (bio -uvrB). Initial screens resulted in the isolation of seven different comple menting cosmid clones, carrying biotin biosynthesis operons, Biotin biosynt hesis capabilities and growth under defined conditions of four of these clo nes were studied. Biotin measured in the different culture supernatants ran ged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram- negative bacteria. In addition, random sequencing identified other interest ing open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdop terin cofactors in bacteria (moaABCDE).