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

Abstract

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of 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 isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged 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 interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

FIG. 1

(A) E. coli biotin biosynthesis pathway; (B) genetic organization of the E. coli biotin biosynthesis operon.

FIG. 2

Scheme of the enrichment and cloning strategy used in this study. The samples were taken from five different microbial environments (an agriculture soil, a forest soil, horse excrement, volcanic soil, and sandy soil).

FIG. 3

Dot blot hybridization of DNA extracted from environmental samples and enrichment cultures. (A) Nylon membrane with DNA directly isolated from the HE sample and DNA from a corresponding HE enrichment culture, which was grown in the presence of avidin; 780 ng of DNA was spotted per dot, and hybridizations were done with the E. coli biotin biosynthesis genes as a DNA probe. (B) Nylon membrane loaded with DNA which was isolated from two different enrichment cultures inoculated with soil from an agricultural site (AS); one of the cultures was grown in the presence of added avidin, while no avidin was added to the other culture. DNA was extracted after 48 h of growth, and 930 ng of DNA was spotted per dot onto the nylon membranes. Hybridizations were performed overnight under high-stringency conditions at 68°C employing the bio genes on pCosAS1 as a DNA probe.

FIG. 4

Restriction maps of the central parts of the different biotin cosmids isolated in this work; arrows indicate locations and directions of transcription of the identified ORFs on the different cosmids. (A) pCosHE1 restriction map of the bio cosmid derived from horse excrement showing highest homologies to genes of E. herbicola; (B) pCosHE2 bio cosmid isolated from an enrichment culture derived from the same source as in panel A but highly similar to E. coli; (C) pCosAS1 restriction map of a bio cosmid isolated from an agricultural soil; (D) pCosFS1 restriction map of a bio cosmid isolated from a forest soil enrichment culture. The bio genes identified in panels C and D show highest similarities to E. herbicola and S. marcescens, respectively. Observed similarities for selected ORFS are listed in Table 4, together with the assigned GenBank accession numbers. For genes of pCosHE1 and of pCosFS1, only partial sequences were obtained during the shotgun sequencing approach. DNA restriction enzymes: N, NcoI, B, BamHI; E, EcoRI; Sm, SmaI; P, PstI; K, KpnI; H, HindIII; Sc, SacI.