Colicin V can be produced by lactic acid bacteria

Colicin V is a small, proteinaceous bacterial toxin, produced by many strains of Escherichia coli and other members of the Enterobacteriaceae, that fits the definition of class II bacteriocins of Gram-positive bacteria. Export of colicin V is dependent on specific ABC (ATP-binding cassette) secretion proteins which recognize a double-glycine-type leader peptide on the immature colicin V bacteriocin. Replacement of the colicin V leader peptide by a signal peptide from the signal sequence-dependent bacteriocin divergicin A allowed expression of colicin V in lactic acid bacteria. This system may serve as a model for the heterologous expression of other small bacteriocins active against Gram-negative bacteria and other antibacterial peptides from lactic acid bacteria.     

Sec-Mediated Secretion of Bacteriocin Enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Production of Pediocin PA-1 by Lactococcus lactis Using the Lactococcin A Secretory Apparatus

The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.Bacteriocins of lactic acid bacteria have received considerable attention in recent years due to their potential application in the food industry as natural preservatives. Most interest has focused on lantibiotics (class I bacteriocins), e.g., nisin, and small heat-stable non-lanthionine-containing bacteriocins (class II) (22, 23). A major subgroup of class II bacteriocins (IIa) has been given the generic name of pediocin family (28) after its most extensively studied member, pediocin PA-1. Members of this class have a number of features in common, including a very strong antimicrobial activity against Listeria species (28). The food-borne pathogen Listeria monocytogenes is a major concern in the dairy industry since it can grow in a variety of dairy products at low temperature and pH (13). Although a pediocin PA-1-producing Lactobacillus plantarum strain has recently been isolated (12), this bacteriocin is generally produced by Pediococcus acidilactici strains of meat origin (3, 16, 18, 29, 31). Because of its antilisterial activity, the expression of pediocin PA-1 in strains of dairy origin would be highly desirable.Pediocin PA-1 production, immunity, and secretion are determined by an operon containing four genes (26). The structural gene, pedA, encodes the pediocin PA-1 precursor, pedB specifies immunity, and the pedC and pedD gene products are membrane-bound proteins required for secretion of the active peptide (39). Homologs of these genes have been described for related peptides. Biosynthesis of the well-characterized class II bacteriocin, lactococcin A, produced by strains of Lactococcus lactis also involves four genes (20, 36, 40). In addition to the structural gene (lcnA) and immunity gene (lciA), there are two genes (lcnC and lcnD) whose products together form a transport system dedicated to the translocation of lactococcin through the host membrane. The LcnC protein belongs to the family of ATP-binding cassette transporter proteins (40), and LcnD acts as an accessory protein (14). These two proteins have considerable homology to PedD and PedC, respectively (39), suggesting that the latter proteins play a similar role in the transport of active pediocin. The two bacteriocins also share the double glycine-processing site found in many lactic acid bacteria class II bacteriocins, some lantibiotics, and the Escherichia coli bacteriocin, colicin V (17).Van Belkum et al. (38) have recently investigated the role of leader sequences of the class II bacteriocins in the recognition of the precursor peptide by the dedicated translocation machinery of the host organism. By constructing hybrid genes, they demonstrated that the leader peptides of leucocin A, lactococcin A, and colicin V, which are cleaved at the Gly-Gly (positions −2 and −1) site, can direct the secretion of the nonrelated bacteriocin divergicin A. Our studies have focused on the class II bacteriocins pediocin PA-1 and lactococcin A. Since these peptides have a number of features in common, it might be expected that a pediocin PA-1 precursor could be secreted and processed by using the lactococcin A translocation machinery. L. lactis IL1403 is a plasmid-free strain that does not produce bacteriocin but contains chromosomal copies of genes analogous to lcnC and lcnD (33, 40). In addition, the natural resistance of this strain to pediocin PA-1 (8) makes it an ideal candidate for a production host to investigate the expression of pediocin PA-1 in lactococci.This paper describes the development of an expression system geared to the production of heterologous peptides in L. lactis. Testing the system with pediocin PA-1 involved the construction of a vector containing an in-frame fusion between sequences encoding the lactococcin A leader and the structural part of mature pediocin PA-1. The hybrid genes were introduced into L. lactis IL1403, and the ability of these strains to produce and secrete pediocin PA-1 was investigated.     

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.     

Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15

Aim: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. Methods and Results: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A‐QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A‐QU 15 were identical to that of leucocin A‐UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C‐terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A‐QU 15, leucocin Q and leucocin N were obtained. Conclusions: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C‐terminal region of leucocin A‐QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A‐QU 15 was not. Significance and Impact of Study: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity.     

Cell-mediated killing of Listeria monocytogenes by leucocin C producing Escherichia coli

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Listeria in foods can be inhibited with bacteriocins or bacteriocin producing cultures. The aim of this study was to enhance the killing of L. monocytogenes by binding bacteriocin producing Escherichia coli cells to Listeria cells. Antilisterial E. coli was obtained by transferring leucocin C production from Leuconostoc carnosum 4010. For binding of E. coli cells to Listeria cells, the Listeria phage endolysin PlyP35 cell wall binding domain (CBD) was displayed on E. coli cell surface as FliC::CBD chimeric protein in flagella. CBD insertion in flagella was confirmed by Western analysis and enterokinase cleavage. By mixing isolated flagella with L. monocytogenes WSLC 1019 cells, the FliC::CBD flagella was shown to bind to Listeria cells. However, the wild type flagella also attached to Listeria cells masking putative additional binding mediated by the CBD. Yet, the cell-mediated leucocin C killing resulted in two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. Cells binding Listeria and secreting antilisterial peptides may have applications in protection against listeriosis as they kill Listeria better than free antilisterial peptides.     

Characterization, Production, and Purification of Leucocin H, a Two-Peptide Bacteriocin from Leuconostoc MF215B

Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin Hα and leucocin Hβ. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6°C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 10⁵-fold, and the final specific activity was 2 × 10⁷ BU/OD₂₈₀. Amino acid composition analyses of leucocin Hα and leucocin Hβ indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hβ did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin Hα, the sequence of 20 amino acids was obtained; from leucocin Hβ the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar. Received: 10 February 1999 / Accepted: 15 March 1999     

Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP,an enzyme involved in cell‐wall synthesis

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor‐mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two‐peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G‐resistant mutants and performed whole‐genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non‐sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.     

Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

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

Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.     

Nucleotide-dependent dimerization of the C-terminal domain of the ABC transporter CvaB in colicin V secretion

The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.     

Effects of colicin A and staphylococcin 1580 on amino acid uptake into membrane vesicles of Escherichia coli and Staphylococcus aureus

Staphylococcin 1580 increased the relative amount of diphosphatidylglycerol and decreased the amount of phosphatidylglycerol in cells of Staphlococcus aureus, while the amounts of lysylphosphatidylglycerol, phosphatidic acid and total phospholipid remained constant.Treatment of cells of Escherichia coli and S. aureus with colicin A and staphylococcin 1580, respectively, did not affect proton impermeability but subsequent addition of carbonylcyanide-m-chlorophenylhydrazone resulted in a rapid influx of protons into the cells.Bacteriocin-resistant and -tolerant mutants of E. coli and S. aureus were isolated. The bacteriocins caused leakage of amino acids preaccumulated into membrane vesicles of resistant mutants and had no significant effect on membrane vesicles of tolerant mutants.The uptake of amino acids into membrane vesicles was inhibited by both bacteriocins, irrespective of the electron donors applied. The bacteriocin inhibition was noncompetitive. The bacteriocins did not affect oxygen consumption and dehydrogenases in membrane vesicles.Both bacteriocins suppressed the decrease in the fluorescence of 1-anilino-8-naphthalene sulfonate caused by d-lactate or α-glycerol phosphate when added to membrane vesicles.It is concluded that the bacteriocins uncouple the transport function from the electron transport system.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export

Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so- called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions -1 and 2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.     

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing <Emphasis Type="Italic">Leuconostoc pseudomesenteroides</Emphasis> 607 Isolated from Persimmon

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C₁₈ cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH₂-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.     

Characterization and Structure Analysis of a Novel Bacteriocin,Lacticin Z,Produced by Lactococcus lactis QU 14

A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse’s intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BHT-B from Streptococcus rattus strain BHT. It exhibited a nanomolar range of MICs against various Gram-positive bacteria, and the activity was completely stable up to 100 °C. Unlike many of other LAB bacteriocins, the stability of lacticin Z was emphasized under alkaline conditions rather than acidic conditions. All the results indicated that lacticin Z belongs to a novel type of bacteriocin.     

Utilization of the leucocin A export system in Leuconostoc gelidum for production of a Lactobacillus bacteriocin

Abstract The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 (ATCC 11506) and is active against various lactobacilli and Enterococcus faecalis . The genetic determinants encoding the lactacin F peptides, LafA and LafX, are organized in a chromosomal operon comprised of genes lafA, lafX , and ORFZ. The lactacin F operon was introduced into Leuconostoc (Lc.) gelidum UAL187-22 which produces leucocin A. Leucocin A, a plasmid-encoded bacteriocin, inhibits E. faecalis, Listeria monocytogenes , and other lactic acid bacteria. The culture supernatant of the Leuconostoc transformant containing the lactacin F operon inhibited both lactacin F-and leucocin A-sensitive indicators. Concurrent expression of both bacteriocins did not alter the production of native leucocin A. Additive inhibitory effects due to the presence of both bacteriocins were not observed. An isogenic derivative of UAL187-22, which has lost the leucocin-encoding plasmid, was unable to produce active lactacin F when transformed with the appropriate recombinant plasmid. The ability of Lc. gelidum UAL187-22 to produce lactacin F demonstrates that the export system for leucocin A is capable of producing both bacteriocins simultaneously.     

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 P₃₂ 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.     

tRNA-targeting ribonucleases: molecular mechanisms and insights into their physiological roles

Most bacteria produce antibacterial proteins known as bacteriocins, which aid bacterial defence systems to provide a physiological advantage. To date, many kinds of bacteriocins have been characterized. Colicin has long been known as a plasmidborne bacteriocin that kills other Escherichia coli cells lacking the same plasmid. To defeat other cells, colicins exert specific activities such as ion-channel, DNase, and RNase activity. Colicin E5 and colicin D impair protein synthesis in sensitive E. coli cells; however, their physiological targets have not long been identified. This review describes our finding that colicins E5 and D are novel RNases targeting specific E. coli tRNAs and elucidates their enzymatic properties based on biochemical analyses and X-ray crystal structures. Moreover, tRNA cleavage mediates bacteriostasis, which depends on trans-translation. Based on these results and others, cell growth regulation depending on tRNA cleavage is also discussed.     

Simple method to identify bacteriocin induction peptides and to auto-induce bacteriocin production at low cell density

The production of some bacteriocins by lactic acid bacteria is regulated by induction peptides (IPs) that are secreted by a dedicated secretion system. The IP gene cbaX, for carnobacteriocin A production by Carnobacterium piscicola LV17A, and a presumptive IP gene (orf6), associated with the genetic locus for enterocin B production in Enterococcus faecium BFE 900, were fused to the signal peptide of the bacteriocin divergicin A from Carnobacterium divergens LV13 to access the general secretory pathway. The culture supernatants of C. piscicola UAL26 and Lactococcus lactis MG1363 containing either of these constructs were used to induce bacteriocin production by Bac(-) cultures of C. piscicola LV17A or E. faecium CTC492. The cbaX fusion product induced bacteriocin production by Bac(-) C. piscicola LV17A, but the orf6 fusion product did not induce bacteriocin production by E. faecium CTC492. This represents a relatively simple method of confirming the role of presumptive IPs. The transformation of C. piscicola LV17A with the CbaX gene under expression of the P32 promoter from L. lactis resulted in constitutive production of bacteriocin by either the dedicated transport apparatus or the general secretory pathway.