Abstract
Mannose phosphotransferase system (Man-PTS) serves as a receptor for several bacteriocins in sensitive bacterial cells, namely subclass IIa bacteriocins (pediocin-like; pediocins) and subclass IId ones - lactococcin A (LcnA), lactococcin B (LcnB) and garvicin Q (GarQ). Here, to identify the receptor for three other narrow-spectrum subclass IId bacteriocins - garvicins A, B and C (GarA-C) Lactococcus garvieae mutants resistant to bacteriocins were generated and sequenced to look for mutations responsible for resistance. Spontaneous mutants had their whole genome sequenced while in mutants obtained by integration of pGhost9::ISS1 regions flanking the integration site were sequenced. For both types of mutants mutations were found in genes encoding Man-PTS components IIC and IID indicating that Man-PTS likely serves as the receptor for these bacteriocins as well. This was subsequently confirmed by deletion of the man-PTS operon in the bacteriocin-sensitive L. garvieae IBB3403, which resulted in resistant cells, and by heterologous expression of appropriate man-PTS genes in the resistant Lactococcus lactis strains, which resulted in sensitive cells. GarA, GarB, GarC and other Man-PTS-targeting bacteriocins differ in the amino acid sequence and activity spectrum, suggesting that they interact with the receptor through distinct binding patterns. Comparative analyses and genetic studies identified a previously unrecognized extracellular loop of Man-PTS subunit IID (γ+) implicated in the L. garvieae sensitivity to the bacteriocins studied here. Additionally, individual amino acids localized mostly in the sugar channel-forming transmembrane parts of subunit IIC or in the extracellular parts of IID likely involved in the interaction with each bacteriocin were specified. Finally, template-based 3D models of Man-PTS subunits IIC and IID were built to allow a deeper insight into the Man-PTS structure and functioning.
Highlights
Mannose phosphotransferase system (Man-PTS) is a major phosphoenolpyruvate (PEP)-dependent sugar transporting system for mannose uptake and its concurrent phosphorylation in Firmicutes and Gammaproteobacteria[1]
Garvicin A (GarA) and Garvicin B (GarB) showed a narrow spectrum of antimicrobial activity, being highly active only against L. garvieae strains; at a high concentration (1 mg/ml), GarA exhibited minimal activity against L. lactis strains and some strains from the genera Carnobacterium, Enterococcus, Lactobacillus, Leuconostoc and Pediococcus (Supplementary Table S2)
In this study we focused on three bacteriocins, GarA, GarB and Garvicin C (GarC), encoded by plasmids from L. garvieae 21881 isolated from the blood of a patient suffering from septicemia[48]
Summary
Mannose phosphotransferase system (Man-PTS) is a major phosphoenolpyruvate (PEP)-dependent sugar transporting system for mannose uptake and its concurrent phosphorylation in Firmicutes and Gammaproteobacteria[1]. A definitive conclusion that Man-PTS is a bacteriocin receptor came from a study by Diep et al.[4] Using immunoprecipitation, they showed that several subclass IIa bacteriocins and two subclass IId bacteriocins - lactococcin A (LcnA) and lactococcin B (LcnB) (LcnA-like bacteriocins; lactococcins) interact with the components of Man-PTS and that for self-protection, an immunity protein directly binds the receptor and the bacteriocin in a tripartite complex (receptor:bacteriocin:immunity). Subclass IIa bacteriocins are known to have a broad antimicrobial spectrum being active against bacteria from the genera Carnobacterium, Clostridium, Enterococcus, Lactobacillus, Leuconostoc, Listeria, Pediococcus and Streptococcus They are most active against Listeria and Enterococcus spp. that harbor the mptACD operon encoding Man-PTS containing regions α, β and γ. It has been proposed that the interaction of GarQ with its receptor may involve a few amino acids located in the N-terminal part and the extracellular loop (region γ) of subunit IID and in the transmembrane region of subunit IIC12
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