Molecular characterisation of ABC-type multidrug efflux systems in Bifidobacterium longum

Administration of probiotic bacteria such as Bifidobacterium spp. can prevent antibiotic associated diarrhoea since they can survive the often harsh conditions of the gut. In Bifidobacterium longum subsp. longum(T) NCIMB 702259, two gene clusters, with homology to the ATP-binding cassette (ABC) family of efflux transporters, were identified and studied to assess their functional contribution to antibiotic resistance. Both gene clusters contained two genes encoding putative efflux transporters and a regulator gene, upstream of the structural genes. Reverse transcriptase analysis indicated that the genes in each cluster were transcribed as operons, one where all three genes, including a putative MarR-type regulator were transcribed together (BLLJ_1496/1495/1494), and the other where the two ABC-type transporter genes (BLLJ_1837/1836) were co-transcribed, but excluded the putative regulator (BLLJ_1838). Heterologous expression of the cloned BLLJ_1837/1836 transporter genes in Lactococcus lactis conferred resistance to erythromycin and tetracycline by increasing the minimum inhibitory concentration between 1.5 and 3 fold. The presence of these genes also allowed a 16% increase in the efflux of Hoechst 33342 from L. lactis cells containing the two transporter genes, BLLJ_1837-6. In B. longum, an increase in the levels of transcription of 3.3 fold was observed for BLLJ_1837 in the presence of erythromycin, as measured by multiplex quantitative PCR. In contrast to this, the expression of the genes of the BLLJ_1495/1494 operon in L. lactis did not show significant drug resistance functionality. Gel shift experiments showed that in the BLLJ_1495/1494 operon, the putative MarR-type regulator protein (BLLJ_1496) bound with high affinity to the DNA sequence upstream of the operon in which it was located but this was not erythromycin dependent. This study demonstrated the occurrence of a drug inducible, ABC-type transporter system (BLLJ_1837/1836) in B. longum as well as a putative MarR-type DNA binding protein (BLLJ_1496).