Role of the operon orf12 13 14 in the immunity to aureocin A53, a bacteriocin with a potencial use against bovine mastitis pathogens

Janaína dos Santos Nascimento¹, Maria do Carmo de Freire Bastos¹, Morten Skaugen²  and Ingolf F. Nes².

Departamento de Microbiologia Geral, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil¹ and Laboratory of Microbial Gene Technology, Agricultural University of Norway, Ås, Norway².

Aureocin A53 is a bacteriocin produced by Staphylococcus aureus A53, encoded on a 10.4 kb plasmid called pRJ9. This bacteriocin is active against Streptococcus, coagulase-negative Staphylococcus and S. aureus strains involved in bovine mastitis, and it can also inhibit Listeria monocytogenes, an important food-borne pathogen that has been also described as a bovine mastitis causative agent. Previous studies showed that the plasmid pRJ9 carries 14 orfs which are preceded by a putative ribosome binding site at an adequate distance for initiation of translation. One segment of this plasmid (nucleotides 1 to 4256, orf1 to orf6), encodes functions related to the plasmid mobilisation and replication. The second part of the plamid (nucleotides 4257 to 10406, orf7  to orf14) appears to carry the genes involved in the production of aureocin A53. The orfs12, 13 and14 seems to be organized in an operon structure encoding to a three-component ABC transporter system that in some cases appear to play a role in immunity of some bacteriocin producing bacteria.

In this work, we have investigated the role of the putative operon orf12 13 14 in the immunity to aureocin A53. A 5.8 kb EcoRI-BamH1 DNA fragment from pRJ9 containing the sequence of orf9-13 (orf9 encodes the aureocin) was cloned into the plasmid pELS100, an Escherichia coli-Lactobacillus shuttle/expression vector. This construction (called plasmid pJN2) was electroporated into Lactobacillus sake DSM20017, which is sensitive to aureocin A53. The expression of immunity was tested by exposing the recombinant strain Lb. sake DSM20017(pJN2) to varying amount of aureocin and the results were compared with the aureocin sensitivity of the mother strain. The test was carried out in a Bioscreen C Plate Reader equipment. The strain DSM20017 (pJN2) became immune to aureocin A53, since it was not inhibited by any dilution of the bacteriocin, in contrast to the original strain that was inhibited by. The production of aureocin A53 by Lactobacillus sake DSM20017 harbouring the plasmid pJN2 was also evaluated. This strain was able to inhibit Micrococcus luteus ATCC4698, the main indicator strain to aureocin A53. This result suggests that the operon orf12 13 14 may also encode the transport function associated with aureocin A53 production.