Tymoszewska A., Aleksandrzak-Piekarczyk T., Diep D., Bardowski J.
Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

Bacteriocins are currently the subject of intensive study worldwide due to their great potential as a second-generation antibiotics or natural food preservatives. Bacteriocin-producing bacteria (such as lactic acid bacteria) can also be use as probiotics with health-giving effects for human and animals. As bacteriocins are highly diverse in terms of their primary and secondary structures, physico-chemical properties, inhibitory spectra, etc, it is therefore expected that they likely recognize different receptors on target membrane. So far, 5 receptors are known. Among them, the mannose phosphotransferase system (Man-PTS) serves as a receptor for all class IIa bacteriocins as well as class IId bacteriocin - lactococcin A (lcnA). In this study, we searched for genes encoding proteins engaged in conferring the sensitivity to the class IId bacteriocin - garvieacin Q (garQ) produced by Lactococcus garvieae BCC 43578. Among them, we expected to specify gene(s) coding for potential garQ receptor(s).

The activity spectrum of garQ was determined by the spot-on lawn inhibition spectrum assay. Spontaneous mutants resistant to garQ were obtained through the induction of sensitive L. garvieae strain by the presence of the bacteriocin in the solid medium. The degree of resistance in the garQ resistant mutants was estimated by the microtiter plate assay. Whole genome sequencing of mutants was carried out by the method of Illumina sequencing. The deletion of identified genes was performed through a double cross-over between the chromosome (target genes) and the targeting plasmid. The deleted genes were complemented using nisin-controlled expression system involving pNZ9530 and pNZ8037 plasmids.

The results showed wide activity spectrum of garQ, which was active against several bacterial species from the Carnobacterium, Enterococcus, Lactobacillus, Lactococcus and Leuconostoc genus. The obtained 20 L. garvieae garQ resistant mutants were over 1024-fold more resistant than the wild-type strain. Analyses of the 9 sequenced mutants genomes revealed mutations in genes encoding subunits IIC (4 mutants) or IID (5 mutants) of Man-PTS and included nonsense or nonsynonymous mutations. Deletion of the Man-PTS operon resulted in the full resistance to garQ. Complementation of the membrane located IIC and IID components was sufficient to restore the sensitivity to garQ.

The results of this study enable us to propose IIC and IID components of Man-PTS as receptors for garQ. This is second to date documented case, that the Man-PTS is a receptor for class IId bacteriocin. Importantly, no similarity in primary structures and in the activity spectrum between the garQ and lcnA bacteriocins suggest that they both may interact with the Man-PTS by different mechanisms, however confirmation of this hypothesis requires further research.

Keywords: Bacteriocin biology, Resistance to bacteriocins, Receptors for bacteriocins, Man-PTS, Garvieacin Q, Probiotic

Tymoszewska A., et al. (2016). Garvieacin Q – the wide-spectrum class IID bacteriocin that targets Man-PTS. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 76.). IPC2016

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