We performed metagenomic library transfer experiments in the specialized strain MPO555, which produces both the transcriptional activator NahR and the antitermination protein N. These experiments allowed us to identify six fosmids that encode carbenicillin resistance genes and confer CbR in the specialized strain. Four of the identified fosmids were also found among 100 CbR transconjugants obtained from direct selection using the bulk metagenomic library. The remaining two were isolated among the 100 CbR transconjugants derived from NalR derivatives of EPI300-T1 and MPO553, the latter a strain that produces both NahR and N but with a frameshift in gene N.
The metagenomic DNA was cloned into the pC1FOS vector, which carries the T7 promoter and gene 10 and a thnL transcription terminator, followed by the lambda phage N antitermination protein. Upon induction with salicylate, pC1FOS drives transcription of cloned metagenomic DNA by NahR and N antitermination. The thnL terminator prevents the N protein from terminating the transcription, which makes this vector suitable for obtaining high levels of transcription of metagenomic DNA.
During the metagenomic library transfer experiment, the pC1FOS vector and the metagenomic DNA were combined in a triparental mating of the EPI300-T1 host with the nalidixic acid-resistant NalR derivatives of EPI300-T1, MPO555 and MPO555 (see supplementary methods). The conjugation frequencies were comparable for each of these three strains. The resulting 100 CbR transconjugants were tested for carbenicillin resistance. Six of the recombinant fosmids, designated ETN1, possessed the carbenicillin resistance gene. ETN1 contains the blaC gene, which codes for a class-C b-lactamase, and a short Orf that is located downstream of blaC and is most similar to homologues in different Pseudomonas species.
Induction of the expression of gfp in the presence of arabinose in a culture of MPO555, which constitutively produces low levels of T7 RNA polymerase, resulted in very low basal levels of gfp expression. However, when pC1FOS was infected with MPO555 and the thnL terminator was replaced by a psal promoter that is induced in the presence of salicylate, expression of gfp increased dramatically, reaching nearly identical levels to those of the plasmid pMPO579, which contains the thnL terminator but not the psal promoter. Upon addition of salicylate, expression was induced by both the psal promoter and NahR, which is a marker for the presence of the required transcriptional activator in MPO555. The phenotype demonstrates that the thnL terminator in pC1FOS does not terminate transcription by T7 RNA polymerase, as has been observed for some thnL-containing plasmids. This observation is in agreement with the results from an earlier study on another thnL-containing pC1FOS derivative containing the thnL promoter and a trg inserted construct coding for the nahR transcriptional activator.
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