Genetic characterization of an oligopeptide transport system from Lactobacillus delbrueckii subsp. bulgaricus

Abstract.

The operon of the putative lactobacillar oligopeptide transport system (Opp) from Lactobacillus delbrueckii subsp. bulgaricus B14 was cloned and characterized. The opp operon was found to consist of five genes, oppD, oppF, oppB, oppC and oppA ₁ . In addition, an oppA ₁ homolog, oppA ₂ , was found downstream of the operon. Sequence comparisons of the L. delbrueckii subsp. bulgaricus Opp system with other bacterial transport systems revealed the highest similarity to the oligopeptide transport system of Lactococcus lactis. Northern analyses of opp mRNAs revealed 6.1-kb and 2.1-kb transcripts, confirming that, in addition to the operon structure oppDFBCA ₁ , the oppA ₁ gene was also expressed as a monocistronic transcript. The oppA ₂ gene was expressed as a separate 2.1-kb monocistronic transcript with a low expression level. Primer-extension mapping of the 5′end of oppDFBCA ₁ mRNA revealed two adjacent transcriptional start sites, and primer extension analyses of oppA ₁ and oppA ₂ mRNAs confirmed the location of the predicted promoters of these genes. For complementation analysis, oppA ₁ alone and the operon constructs oppDFBCA ₁ and oppDFBCA ₂ were fused with the nisA promoter and expressed in Lactococcus lactis NZ9000ΔoppA strain. Only the L. delbrueckii subsp. bulgaricus oppDFBCA ₁ genes were able to complement the L. lactis oppA mutation.