The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos. Large amounts of several types of contaminants are released into the environment due to industrial activities and accidental spills. Many of these contaminants, such as aromatic hydrocarbons, have complex and stable chemical structures, which make them prevail in the environment for a long time, thus resulting in the contamination of ecosystems. Biological treatment of industrial wastewater and bioremediation of contaminated soils and water are therefore critical to prevent or combat this contamination.Standardized culturing techniques have been successfully applied for many years to isolate many different types of bacteria that are capable of utilizing a variety of aromatic hydrocarbons and to characterize their biodegradation pathways and the coding genes responsible for these capabilities, which have shown that bacteria can adapt to utilize a plethora of organic contaminants, including xenobiotics. The aerobic biodegradation of aromatics has been well documented and has been shown to follow two pathways, involving either intradiol or extradiol cleavage of the aromatic rings of di-or trihydroxylated intermediates. The intradiol dioxygenases that have been described to date appear to belong to the same superfamily, but extradiol dioxygenases (Edos) of three different phylogenetic origins have been reported (1, 2). Type I extradiol dioxygenases are more numerous and belong to the vicinal oxygen chelate superfa...