Carmen Herranz

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

Enterococcus faecium L50 grown at 16 to 32 degrees 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 degrees 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. Hâvarstein, 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 to entQ 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 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.

Review: Bacteriocins of Lactic Acid Bacteria

During the last few years, a large number of new bacteriocins produced by lactic acid bacteria (LAB) have been identified and characterized. LAB-bacteriocins comprise a heterogeneous group of physicochemically diverse ribosomally-synthesized peptides or proteins showing a narrow or broad antimicrobial activity spectrum against Gram-positive bacteria. Bacteriocins are classified into separate groups such as the lantibiotics (Class I); the small (<10 kDa) heat-stable postranslationally unmodified non-lantibiotics (Class II), further subdivided in the pediocin-like and anti Listeria bacteriocins (subclass IIa), the two-peptide bacteriocins (subclass IIb), and the sec-dependent bacteriocins (subclass IIc); and the large (>30 kDa) heat-labile non-lantibiotics (Class III). Most bacteriocins characterized to date belong to Class II and are synthesized as precursor peptides (preprobacteriocins) containing an N-terminal double-glycine leader peptide, which is cleaved off concomitantly with externalization of biologically active bacteriocins by a dedicated ABC-transporter and its accessory protein. However, the recently identified sec-dependent bacteriocins contain an N-terminal signal peptide that directs bacteriocin secretion through the general secretory pathway (GSP). Most LAB-bacteriocins act on sensitive cells by destabilization and permeabilization of the cytoplasmic membrane through the formation of transitory poration complexes or ionic channels that cause the reduction or dissipation of the proton motive force (PMF). Bacteriocin producing LAB strains protect themselves against the toxicity of their own bacteriocins by the expression of a specific immunity protein which is generally encoded in the bacteriocin operon. Bacteriocin production in LAB is frequently regulated by a three-component signal transduction system consisting of an induction factor (IF), and histidine protein kinase (HPK) and a response regulator (RR). This paper presents an updated review on the general knowledge about physicochemical properties, molecular mode of action, biosynthesis, regulation and genetics of LAB-bacteriocins.

Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture

BackgroundThe microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food.ResultsThese LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities.ConclusionsTo our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.

Characterization of Garvicin ML, a Novel Circular Bacteriocin Produced by Lactococcus garvieae DCC43, Isolated from Mallard Ducks (Anas platyrhynchos)

ABSTRACT Lactococcus garvieae DCC43 produces a bacteriocin, garvicin ML (GarML), with a molecular mass of 6,004.2 Da. Data from de novo amino acid sequencing by tandem mass spectrometry and nucleotide sequencing by reverse genetics suggested that the bacteriocin is synthesized as a 63-amino-acid precursor with a 3-amino-acid leader peptide that is removed by cleavage. Subsequently, a covalent linkage between the N and C termini forms the mature version of this novel 60-amino-acid circular bacteriocin.

Biochemical and genetic evidence of enterocin P production by two Enterococcus faecium-like strains isolated from fermented sausages.

Abstract. Two bacteriocin-producing Enterococcus faecium-like strains were independently isolated from fermented sausages. Bacteriocins were purified to homogeneity by ammonium sulfate precipitation, gel filtration, cationic exchange, hydrophobic interaction, and reverse-phase liquid chromatography. Two peptide inhibitory fractions were purified from each strain, denominated A and B for E. faecium AA13, and C and D for E. faecium G16. Fraction B was blocked for amino acid sequencing by Edman degradation, while the amino acid sequences obtained from peptides A, C, and D contained the YGNGV consensus motif in positions 5 to 9, and the ATRS sequence in positions 1 to 4. By use of PCR techniques and nucleotide sequencing, the structural gene of enterocin P was found both in E. faecium AA13 and E. faecium G16. Metabolic and genetic features of the two strains suggest that they are slightly different, they may produce more than one bacteriocin, and both produce enterocin P.

Cloning and Heterologous Production of Hiracin JM79, a Sec-Dependent Bacteriocin Produced by Enterococcus hirae DCH5, in Lactic Acid Bacteria and Pichia pastoris

ABSTRACT Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis, Lactobacillus sakei, Enterococcus faecium, Enterococcus faecalis, and Pichia pastoris. For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene (hirJM79), with or without the HirJM79 immunity gene (hiriJM79), was cloned into the plasmid pMG36c under the control of the constitutive promoter P32 and into the plasmid pNZ8048 under the control of the inducible PNisA promoter. For the production of HirJM79 in P. pastoris, the gene encoding the mature HirJM79 protein was cloned into the pPICZαA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris.

Enterocin P Causes Potassium Ion Efflux from Enterococcus faecium T136 Cells

ABSTRACT Enterocin P is a bacteriocin produced by Enterococcus faecium P13. We studied the mechanism of its bactericidal action using enterocin-P-sensitive E. faecium T136 cells. The bacteriocin is incapable of dissipating the transmembrane pH gradient. On the other hand, depending on the buffer used, enterocin P dissipates the transmembrane potential. Enterocin P efficiently elicits efflux of potassium ions, but not of intracellularly accumulated anions like phosphate and glutamate. Taken together, these data demonstrate that enterocin P forms specific, potassium ion-conducting pores in the cytoplasmic membrane of target cells.

Production of Enterocin P, an Antilisterial Pediocin-Like Bacteriocin from Enterococcus faecium P13, in Pichia pastoris

ABSTRACT The gene encoding mature enterocin P (EntP), an antimicrobial peptide from Enterococcus faecium P13, was cloned into the pPICZαA expression vector to generate plasmid pJC31. This plasmid was integrated into the genome of P. pastoris X-33, and EntP was heterologously secreted from the recombinant P. pastoris X-33t1 derivative at a higher production and antagonistic activity than from E. faecium P13.

Antimicrobial activity of Enterococcus faecium L50, a strain producing enterocins L50 (L50A and L50B), P and Q against beer-spoilage lactic acid bacteria in broth, wort (hopped and unhopped), and alcoholic and non-alcoholic lager beers

Enterococcus faecium L50 produces enterocin L50 (L50A and L50B) (EntL50, EntL50A and EntL50B), enterocin P (EntP) and enterocin Q (EntQ) and displays a broad antimicrobial spectrum against the most relevant beer-spoilage lactic acid bacteria (LAB) (i.e., Lactobacillus brevis and Pediococcus damnosus), which is mainly due to the production of EntL50 (EntL50A and EntL50B). Bacteriocin assays using in vitro-synthesized EntL50 (EntL50A and EntL50B) showed that both individual peptides possess antimicrobial activity on their own, EntL50A being the most active, but when the two peptides were combined a synergistic effect was observed. The only virulence genes detected in E. faecium L50 were efaAfm (cell wall adhesin) and ccf (sex pheromone), and this strain was susceptible to most clinically relevant antibiotics. E. faecium L50 survived but did not grow nor showed antimicrobial activity in hopped and unhopped wort, and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) commercial lager beers. However, when unhopped wort was supplemented with 50% (v/v) MRS broth, E. faecium L50 grew and exerted antimicrobial activity similarly as in MRS broth. The enterocins produced by this strain were bactericidal (5 log decrease) against P. damnosus and Lb. brevis in a dose- and substrate-dependent manner when challenged in MRS broth, wort (hopped and unhopped), and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) lager beers at 32 degrees C, and no bacterial resistances were detected even after incubation for 6-15 days. The enterocins in wort and lager beer (5% ethanol, v/v) withstood the heat treatments commonly employed in the brewing industry during mashing, wort boiling, fermentation, and pasteurization, and retained most of their antimicrobial activity in lager beer (5% ethanol, v/v) after long-term storage at 8 and 25 degrees C.

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria.

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS(entA)) by the signal peptides (SP) of the protein Usp45 (SP(usp45)), and the bacteriocins enterocin P (SP(entP)), and hiracin JM79 (SP(hirJM79)) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP(usp45), the SP(entP), and the SP(hirJM79) fused to mature EntA plus the EntA immunity genes (entA+entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P(nisA) promoter, and in pMG36c, under control of the constitutive P(32) promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp.