Biochemical and Genetic Evidence that Enterococcus faecium L50 Produces Enterocins L50A and L50B, thesec-Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q

ABSTRACT Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321–4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation toentQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above.

[1]  V. Eijsink,et al.  A C-Terminal Disulfide Bridge in Pediocin-Like Bacteriocins Renders Bacteriocin Activity Less Temperature Dependent and Is a Major Determinant of the Antimicrobial Spectrum , 2000, Journal of bacteriology.

[2]  J. M. Rodríguez,et al.  Biochemical and Genetic Evidence of Enterocin P Production by Two Enterococcus faecium-Like Strains Isolated from Fermented Sausages , 1999, Current Microbiology.

[3]  J. Suárez,et al.  Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-encoded bicistronic operon. , 1999, Microbiology.

[4]  J. Vederas,et al.  Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900 , 1999, Applied and Environmental Microbiology.

[5]  A. Driessen,et al.  Plantaricin A is an amphiphilic alpha-helical bacteriocin-like pheromone which exerts antimicrobial and pheromone activities through different mechanisms. , 1998, Biochemistry.

[6]  Erik L. L. Sonnhammer,et al.  A Hidden Markov Model for Predicting Transmembrane Helices in Protein Sequences , 1998, ISMB.

[7]  V. Eijsink,et al.  Antagonistic Activity of Lactobacillus plantarum C11: Two New Two-Peptide Bacteriocins, Plantaricins EF and JK, and the Induction Factor Plantaricin A , 1998, Applied and Environmental Microbiology.

[8]  P. Hernández,et al.  Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria. , 1998 .

[9]  I. Nes,et al.  Enterocins L50A and L50B, Two Novel Bacteriocins from Enterococcus faecium L50, Are Related to Staphylococcal Hemolysins , 1998, Journal of bacteriology.

[10]  I. Nes,et al.  Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum , 1997, Applied and environmental microbiology.

[11]  V. Eijsink,et al.  Pheromone‐induced production of antimicrobial peptides in Lactobacillus , 1997, Molecular microbiology.

[12]  Trine Nilsen,et al.  Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. , 1997, Microbiology.

[13]  I. Nes,et al.  Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action , 1997, Archives of Microbiology.

[14]  B. Devreese,et al.  Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139 , 1997, Applied and environmental microbiology.

[15]  D. Marion,et al.  Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific inhibitory activity , 1996, Applied and environmental microbiology.

[16]  D. Diep,et al.  Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11 , 1996, Journal of bacteriology.

[17]  A. Revol-Junelles,et al.  Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins , 1996, Letters in applied microbiology.

[18]  M. Qiao,et al.  Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in Lactoccocus lactis N8. , 1996, Microbiology.

[19]  G. Venemâ,et al.  Topology of LcnD, a protein implicated in the transport of bacteriocins from Lactococcus lactis , 1996, Journal of bacteriology.

[20]  W. D. de Vos,et al.  Autoregulation of Nisin Biosynthesis in Lactococcus lactis by Signal Transduction (*) , 1995, The Journal of Biological Chemistry.

[21]  K. Sletten,et al.  Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. , 1995, Applied and environmental microbiology.

[22]  R. Leer,et al.  Genetic analysis of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus. , 1995, Microbiology.

[23]  J. Vederas,et al.  A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens , 1995, Journal of bacteriology.

[24]  D. Diep,et al.  A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export , 1995, Molecular microbiology.

[25]  G. Giraffa.  Enterococcal bacteriocins: their potential as anti-Listeria factors in dairy technology , 1995 .

[26]  R D Appel,et al.  A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. , 1994, Trends in biochemical sciences.

[27]  J. Vederas,et al.  Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B. , 1994, The Journal of biological chemistry.

[28]  C. Wandersman.  The general secretory pathway in bacteria. , 1993, Trends in microbiology.

[29]  T. Klaenhammer,et al.  Genetics of bacteriocins produced by lactic acid bacteria. , 1993, FEMS microbiology reviews.

[30]  J. Piard,et al.  Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation , 1993, Applied and environmental microbiology.

[31]  A. Pugsley.  The complete general secretory pathway in gram-negative bacteria. , 1993, Microbiological reviews.

[32]  J. C. Lozano,et al.  Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. , 1992, Journal of general microbiology.

[33]  S. Lory.  Determinants of extracellular protein secretion in gram-negative bacteria , 1992, Journal of bacteriology.

[34]  P. Stanley,et al.  Mutational analysis supports a role for multiple structural features in the C‐terminal secretion signal of Escherichia coli haemolysin , 1991, Molecular microbiology.

[35]  I. Nes,et al.  Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene , 1991, Journal of bacteriology.

[36]  G. Venemâ,et al.  Organization and nucleotide sequences of two lactococcal bacteriocin operons , 1991, Applied and environmental microbiology.

[37]  K. Sletten,et al.  Evidence that the amyloid fibril protein in senile systemic amyloidosis is derived from normal prealbumin. , 1988, Biochemical and biophysical research communications.

[38]  L. Mckay,et al.  Simple and rapid method for isolating large plasmid DNA from lactic streptococci , 1983, Applied and environmental microbiology.

[39]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[40]  K. Sonomoto,et al.  Class IIa bacteriocins: biosynthesis, structure and activity. , 2000, FEMS microbiology reviews.

[41]  Gary M. Dunny,et al.  Cell-cell signaling in bacteria , 1999 .

[42]  J. Tagg,et al.  Copyright � 1995, American Society for Microbiology Bacteriocins of Gram-Positive Bacteria , 1995 .

[43]  L. Vuyst,et al.  Bacteriocins of Lactic Acid Bacteria , 1994 .

[44]  Christopher M Thomas,et al.  Active partitioning of bacterial plasmids. , 1992, Journal of general microbiology.

[45]  W. V. Wezel,et al.  The SEC-system , 2022 .