No abstract

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at-85°C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 x 107 transformants per ,ug of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.

The mechanisms of target cell recognition and producer cell selfprotection (immunity) are both important yet poorly understood issues in the biology of peptide bacteriocins. In this report, we provide genetic and biochemical evidence that lactococcin A, a permeabilizing peptide-bacteriocin from Lactococcus lactis, uses components of the mannose phosphotransferase system (man-PTS) of susceptible cells as target/receptor. We present experimental evidence that the immunity protein LciA forms a strong complex with the receptor proteins and the bacteriocin, thereby preventing cells from being killed. Importantly, the complex between LciA and the man-PTS components (IIAB, IIC, and IID) appears to involve an on-off type mechanism that allows complex formation only in the presence of bacteriocin; otherwise no complexes were observed between LciA and the receptor proteins. Deletion of the man-PTS operon combined with biochemical studies revealed that the presence of the membrane-located components IIC and IID was sufficient for sensitivity to lactococcin A as well as complex formation with LciA. The cytoplasmic component of the man-PTS, IIAB, was not required for the biological sensitivity or for complex formation. Furthermore, heterologous expression of the lactococcal man-PTS operon rendered the insensitive Lactobacillus sakei susceptible to lactococcin A. We also provide evidence that, not only lactococcin A, but other class II peptide-bacteriocins including lactococcin B and some Listeria-active pediocin-like bacteriocins also target the man-PTS components IIC and IID on susceptible cells and that their immunity proteins involve a mechanism in producer cell self-protection similar to that observed for LciA.antimicrobial peptides ͉ Mannose-PTS ͉ receptor ͉ protein complex ͉ coprecipitation

A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, kcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as kcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli IcnA did not cause an LCN-A+ phenotype. L. lacfis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying kcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.A number of strains of Lactococcus lactis produce bacteriocins. In an extensive survey by Geis et al. (15), it was found that about 5% of the 280 lactococcal strains tested were bacteriocin producers. These workers divided the bacteriocins into eight different classes, but none of them was characterized in detail. Despite numerous reports of lactococcal bacteriocins, little is known about their chemical composition and structure, mode of action, or genetics. Nisin is the only bacteriocinlike compound from L. lactis that has been studied in detail. The molecular structure and genetic determinant of nisin have been identified and, to some extent, its mode of action has been elucidated (5,11,16,21,22,36). Another bacteriocin, termed diplococcin, produced by L. lactis subsp. cremoris, has also been purified and its amino acid composition has been determined (10). Davey (9) showed that the gene coding for diplococcin was located on a 54-MDa conjugative plasmid. Conjugal transfer of bacteriocin plasmids in L. lactis has also been observed by others (30,38). Harmon and McKay (19) identified a Bcll DNA fragment carrying a bacteriocin determinant from a conjugative plasmid. Recently, two bacteriocin genes from another conjugative lactococcal plasmid, previously described by Neve et al. (30), were cloned by van Belkum et al. (46). The clones also carried the immunity factors of the two bacteriocins. The inhibitory spectra of the different lactococcal bacteriocins described vary but are generally more narrow than that of nisin (15). It is therefore plausible that many of the lactococcal bacteriocins described are very different from nisin and thus do not belong to the lanthibiotic family (39) of bacteriocinlike compounds....

A large number of new bacteriocins in lactic acid bacteria (LAB) has been characterized in recent years. Most of the new bacteriocins belong to the class II bacteriocins which are small (30-100 amino acids) heat- stable and commonly not post-translationally modified. While most bacteriocin producers synthesize only one bacteriocin, it has been shown that several LAB produce multiple bacteriocins (2-3 bacteriocins). Based on common features, some of the class II bacteriocins can be divided into separate groups such as the pediocin-like and strong anti-listeria bacteriocins, the two-peptide bacteriocins, and bacteriocins with a sec-dependent signal sequence. With the exception of the very few bacteriocins containing a sec-dependent signal sequence, class II bacteriocins are synthesized in a preform containing an N-terminal double-glycine leader. The double-glycine leader-containing bacteriocins are processed concomitant with externalization by a dedicated ABC-transporter which has been shown to possess an N-terminal proteolytic domain. The production of some class II bacteriocins (plantaricins of Lactobacillus plantarum C11 and sakacin P of Lactobacillus sake) have been shown to be transcriptionally regulated through a signal transduction system which consists of three components: an induction factor (IF), histidine protein kinase (HK) and a response regulator (RR). An identical regulatory system is probably regulating the transcription of the sakacin A and carnobacteriocin B2 operons. The regulation of bacteriocin production is unique, since the IF is a bacteriocin-like peptide with a double-glycine leader processed and externalized most probably by the dedicated ABC-transporter associated with the bacteriocin. However, IF is not constituting the bacteriocin activity of the bacterium, IF is only activating the transcription of the regulated class II bacteriocin gene(s). The present review discusses recent findings concerning biosynthesis, genetics, and regulation of class II bacteriocins.

The strain Enterncoccus faeciurn T I 36 produces two bacteriocins, enterocin A, a member of the pediocin family of bacteriocins, and a new bacteriocin termed enterocin B. The N-terminal amino acid sequences of enterocins A and B were determined, and the gene encoding enterocin B was sequenced. The primary translation product was a 71 aa peptide containing a leader peptide of the double-glycine type which is cleaved off to give mature enterocin B of 53 aa. Enterocin B does not belong to the pediocin family of bacteriocins and shows strong homology to carnobacteriocin A. However, sequence similarities in their leader peptides and C-termini suggest that enterocin B and carnobacteriocin A are related to bacteriocins of the pediocin family. Enterocins A and B had only slightly different inhibitory spectra, and both were active against a wide range of Gram-positive bacteria, including listeriae, staphylococci and most lactic acid bacteria tested. Both had bactericidal activities, but survival at a frequency of 10-4-10-2 was observed when sensitive cultures were exposed to either bacteriocin. The number of survivors was drastically reduced when a mixture of the two bacteriocins was added to the cells.

A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL4B, and reverse-phase chromatography. The final yield was about 20%k, and nearty a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed al, a2, and 13, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, al and a2 were the same gene product. Differences in their configurations presumably resulted in a2 having a slightly lower affinity for the reverse-phase column than al and a reduced bacteriocin activity when combined with 13. Bacteriocin activity required the complementary action of both the a and the 1 peptides. When neither al nor 1 was in excess, about 0.3 nM al and 0.04 nM 13 induced 50%1 growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, a1has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 104 M-1 cm-', and a molecular weight of 4,346 (39 amino acid residues long). Similarly, 13 has an isoelectric point of 10.4, an extinction coefficient of 2.4x 104 M-1 cm'1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for al and (, respectively, were obtained by mass spectrometry. The N-terminal halves of both the a and the 13 peptides may form amphiphilic a-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism. The C-terminal halves of both peptides consist largely of polar amino acids.Bacteriocins are proteins that show bactericidal activity towards bacteria that are closely related to the bacteriocinproducing species (25). Because of their potential use as antibacterial agents, bacteriocins have been the subject of much research. In recent years, there has been considerable interest especially in bacteriocins from lactic acid bacteria (LAB), because of their potential as food and feed additives.The LAB bacteriocins appear to be structurally quite different from the colicins of Escherichia coli (7,14). LAB bacteriocins are usually small peptides, seldom containing more than 60 amino acids, while colicins are proteins of 300 to 800 amino acids. On the basis of their structure, LAB bacteriocins may be divided into two groups. The first group contains the so-called lantibiotics, which have been known for a long time (6, 11). Lantibiotics consist of a polypeptide chain that has been posttranslationally modified. The modified amino acids are lanthionine and methyllanthionine and their precursors dehydroalanine and dehydrobutyrine. Among the lantibiotics, nisin is by far the most studied (1, 4, 12), although three new LAB lantibiotics recently have been purified and characterized (15,16,21,24 which does not show any apparent amino acid sequence homology to other isolated LAB bacteri...

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 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°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.