Data mining and characterization of a novel pediocin-like bacteriocin system from the genome of Pediococcus pentosaceus ATCC 25745

Abstract

The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system. The gene cluster has an operon-like structure consisting of a putative pediocin-like bacteriocin gene (termed penA) and a potential immunity gene (termed peiA). Genetic determinants involved in bacteriocin transport and regulation are also found in proximity to penA and peiA but the so-called accessory gene involved in transport and the inducer gene involved in regulation are missing. Consequently, this bacterium is a poor bacteriocin producer. To analyse the potency of the putative bacteriocin operon, the two genes penA-peiA were heterologously expressed in a Lactobacillus sakei host that contains the complete apparatus for gene activation, maturation and externalization of bacteriocins. It was demonstrated that the heterologous host expressing penA and peiA produced a strong bacteriocin activity; in addition, the host became immune to its own bacteriocin, identifying the gene pair penA-peiA as a potent bacteriocin system. The novel pediocin-like bacteriocin, termed penocin A, has an isotopic mass [M+H] + of 4684?6 Da as determined by mass spectrometry; this value corresponds well to the expected size of the mature 42 aa peptide containing a disulfide bridge. The bacteriocin is heat-stable but protease-sensitive and has a calculated pI of 9?45. Penocin A has a relatively broad inhibition spectrum, including pathogenic Listeria and Clostridium species. Immediately upstream of the regulatory genes reside some features that resemble remnants of a disrupted inducer gene. This degenerate gene was restored and shown to encode a double-glycine leader-containing peptide. Furthermore, expression of the restored gene triggered high bacteriocin production in P. pentosaceus ATCC 25745, thus confirming its role as an inducer in the pen regulon.

Bacteriocin Diversity in Streptococcus and Enterococcus

“…Correction of a mutation in a gene required for bacteriocin synthesis has been used successfully to identify a new bacteriocin in lactic acid bacteria (23). The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system.…”

“…Deficiency in production of antimicrobial activity is often due to a dysfunctional genetic system. Occasionally, it has been observed that bacterial genomes encode only parts of the bacteriocin production system or that mutations have inactivated the functionality of the bacteriocin genes (23,85).…”

The Abi Proteins and Their Involvement in Bacteriocin Self-Immunity

“…1B). Abolished bacteriocin production due to defects in the regulatory genes has been reported for Pediococcus pentosaceus ATCC 25745, and when this bacterium was complemented with an intact gene by cloning, normal bacteriocin production was restored (11). Similarly, to investigate whether the lack of active bacteriocins could result from no or low gene expression, the bacteriocin locus (GenBank accession no.…”

“…This strategy has previously proven successful for heterologous production of both one-and two-peptide bacteriocins (5,11,29,36 Fig. 5A.…”

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…Peptides in subgroup 3 as well as enterocin SE-K4 and carnobacteriocin B2 in subgroup 4 exceptionally have neither the conserved tryptophan at the C-tails, nor the cysteine residues to form the C-terminal disulfide bridge (Table 2). Marrec et al 2000Metivier et al 1998Aymerich et al 1996Henderson et al 1992Marugg et al 1992Tichaczek et al 1994Kalmokoff et al 2001Birri et al 2010Feng 2009Kawamoto et al 2002Bennik et al 1998Yamazaki et al 2005Bhugaloo-Vial et al 1996Jack et al 1996Vaughan et al 2001Fimland et al 2002bHeng et al 2007 Subgroup 2 Kwaadsteniet et al 2006Quadri et al 1995Eguchi et al 2001Diep et al 2006 The YGNGV/L "pediocin box" is marked (bold), the leucine residue (L), odd, and uncertain amino acids (X) are marked as bold and red. Names of the six newly identified class IIa bacteriocins are in red (Rea et al 2011).…”