Dzung B. Diep

Biosynthesis of bacteriocins in lactic acid bacteria

SummaryA 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 transcripion of the regulated class II bacteriocin gene(s).The present review discusses recent findings concerning biosynthesis, genetics, and regulation of class II bacteriocins.

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.

Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication

Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being the main topic of this review. During the past decade many of these bacteriocins have been isolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have been unravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closely related bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for these narrow inhibitory spectra by producing several bacteriocins belonging to different classes and having different inhibitory spectra. The latter may also help in counteracting the possible development of resistance mechanisms in target organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using a secreted peptide-pheromone for quorum-sensing. The sensing of its own growth, which is likely to be comparable to that of related species, enables the producing organism to switch on bacteriocin production at times when competition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocins and the regulation of their production, several fundamental questions remain to be solved. These include questions regarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity.

Common mechanisms of target cell recognition and immunity for class II bacteriocins

The mechanisms of target cell recognition and producer cell self-protection (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.

Bacteriocin Diversity in Streptococcus and Enterococcus

Most bacteriocins in gram-positive bacteria are small and heat stable (peptide bacteriocins), and their antimicrobial activities are directed against a broader spectrum of bacteria than is seen for bacteriocins of gram-negative bacteria. Many excellent bacteriocin reviews have been published in recent years (10, 15, 16, 19, 27, 29, 77, 83). The heat-stable peptide bacteriocins from lactic acid bacteria have so far been grouped into two major classes: class I, the lantibiotics, and class II, the heat-stable nonlantibiotics. In addition, a third class of bacteriocins has been suggested which includes secreted heat-labile cell wall-degrading enzymes (71, 88), but classification of such enzymes as bacteriocins has recently been disputed (19, 49). Lantibiotics contain a number of posttranslational modifications that include dehydration of serine and threonine to form 2,3dehydroalanine (Dha) and 2,3-dehydrobutyrine (Dhb), respectively. Some of the dehydrated residues are covalently bound to the sulfur in neighboring cysteines, creating the characteristic lantionine and methyllantionine residues. It has also been shown that in a few cases the dehydroalanine can be converted to D-alanine (109, 118) and that additional modifications, such as lysinoalanine, 2-oxobutyrate, S-aminovinyl-D-cysteine, and S-aminovinyl-D-methylcysteine, are formed in some lantibiotics (59). Both class I and class II bacteriocins display great diversity with regard to their modes of action, structures, genetics, modes of secretion, choices of target organisms, etc. There is still lack of consensus on how to subdivide class I and II peptide bacteriocins further into subclasses. The lantibiotics have been divided into two subgroups, type A and type B, according to structural features (64). Type A lantibiotics (e.g., nisin, subtilin, and Pep5) are elongated molecules with a flexible structure in solution, while type B lantibiotics adapt a more rigid and globular structure (64). However, this picture is changing, since structural studies of the lantibiotic plantaricin C has been shown to hold structural elements of both type A and B lantibiotics (123). Also, nuclear magnetic resonance spectroscopy has shown that the peptides of the two-peptide lantibiotic lacticin 3247 are structurally different. While the peptide designated lacticin 3147 A1 has a specific lanthionine bridging pattern resembling the globular type B lantibiotic mersacidin, the A2 peptide is a member of the elongated type A lantibiotic subclass (80). In the present review, we refer to the A and B types of lantibiotics as one-peptide lantibiotics and mention specifically when a bacteriocin is a two-peptide lantibiotic. Lack of consensus also exists in the differentiation between subgroups of the nonlantibiotic class II peptide bacteriocins. In this review, we retain the pediocin-like bacteriocin in class IIa, the two-peptide bacteriocins in class IIb, and the leaderless peptide bacteriocins in class IIc, and finally, we define the circular bacteriocins as class IId. This overview will discuss the dissemination of the class I and II peptide bacteriocins in enterococci and streptococci and the possibility of identifying such bacteriocins in genome sequences. The lactic acid bacteria in fermented food have been the focus of bacteriocin research during the last 15 to 20 years. Numerous peptide bacteriocins have been characterized, and many have been used intentionally or unintentionally in food

A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11.

In this study, we show that bacteriocin production in Lactobacillus plantarum C11 is an inducible process triggered by a secreted protein factor produced by the bacteriocin producer itself. The induction factor was identified to be plantaricin A, a bacteriocin‐like peptide whose gene (plnA) is located in the same operon as a two‐component regulatory system (plnBCD). When L. plantarum C11 cultures were depleted for plantaricin A, either by growing individual colonies on agar plates or by starting a new culture with a highly diluted inoculum, no bacteriocin was produced during the following growth. When chemically synthesized plantaricin A or purified bacterially produced plantaricin A was added to non‐producing cultures, bacteriocin production was induced. Only 1 ng ml−1 plantaricin A is sufficient to induce the bacteriocin production in non‐producing L. plantarum C11, and bacteriocin activity appears in the growth medium approximately 150 min after induction. Northern analyses, using a plnA‐specific probe, demonstrated that plantaricin A is able to induce its own synthesis by transcription of the plnABCD operon, and this is observed approximately 15 min after adding plantaricin A. Furthermore, heterologous expression of the plnABCD operon in a Lactobacillus sake strain showed that the conditioned growth medium contained the active induction factor. Neither synthetic nor expressed plantaricin A from the heterologous system possesses any bacteriocin activity, suggesting that plantaricin A is primarily an induction factor and not a bacteriocin as claimed earlier.

Glycosylphosphatidylinositol Anchors of Membrane Glycoproteins Are Binding Determinants for the Channel-forming Toxin Aerolysin

Cells that are sensitive to the channel-forming toxin aerolysin contain surface glycoproteins that bind the toxin with high affinity. Here we show that a common feature of aerolysin receptors is the presence of a glycosylphosphatidylinositol anchor, and we present evidence that the anchor itself is an essential part of the toxin binding determinant. The glycosylphosphatidylinositol (GPI)-anchored T-lymphocyte protein Thy-1 is an example of a protein that acts as an aerolysin receptor. This protein retained its ability to bind aerolysin when it was expressed in Chinese hamster ovary cells, but could not bind the toxin when expressed in Escherichia coli, where the GPI anchor is absent. An unrelated GPI-anchored protein, the variant surface glycoprotein of trypanosomes, was shown to bind aerolysin with similar affinity to Thy-1, and this binding ability was significantly reduced when the anchor was removed chemically. Cathepsin D, a protein with no affinity for aerolysin, was converted to an aerolysin binding form when it was expressed as a GPI-anchored hybrid in COS cells. Not all GPI-anchored proteins bind aerolysin. In some cases this may be due to differences in the structure of the anchor itself. Thus the GPI-anchored proteins procyclin ofTrypanosoma congolense and gp63 of Leishmania major did not bind aerolysin, but when gp63 was expressed with a mammalian GPI anchor in Chinese hamster ovary cells, it bound the toxin.

Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance.

Because of the emergence of antibiotic‐resistant pathogens worldwide, a number of infectious diseases have become difficult to treat. This threatening situation is worsened by the fact that very limited progress has been made in developing new and potent antibiotics in recent years. However, a group of antimicrobials, the so‐called bacteriocins, have been much studied lately because they hold a great potential in controlling antibiotic‐resistant pathogens. Bacteriocins are small antimicrobial peptides (AMPs) produced by numerous bacteria. They often act toward species related to the producer with a very high potency (at pico‐ to nanomolar concentration) and specificity. The common mechanisms of killing by bacteriocins are destruction of target cells by pore formation and/or inhibition of cell wall synthesis. Several studies have revealed that bacteriocins display great potential in the medical sector as bacteriocinogenic probiotics and in the clinic as therapeutic agents. In this review, we discuss the emerging antibiotic resistance and strategies to control its dissemination, before we highlight the potential of AMPs from bacteria as a new genre of antimicrobial agents.

The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system

Sakacin A is a bacteriocin produced by Lactobacillus sakei Lb706. The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR. sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph). The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production. Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 degrees C, but production was reduced or absent at higher temperatures (33.5-35 degrees C). Production was restored by lowering the growth temperature to 30 degrees C, but at temperatures of 33-34 degrees C also by adding exogenous Sap-Ph to the growth medium. A knock-out mutation in orf4 abolished sakacin A production. Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production. Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph.

An overview of the mosaic bacteriocin pln loci from Lactobacillus plantarum

The pln locus responsible for bacteriocin biosynthesis in Lactobacillus plantarum C11 was first unraveled about 15 years ago and since then different strains of L. plantarum (NC8, WCFS1, J23 and J51) have been found to harbor mosaic pln loci in their genomes. Each locus is of 18-19kb and contains 22-25 genes organized into 5-6 operons. Together these strains produce four different class IIb two-peptide bacteriocins, plantaricins EF, JK, NC8 and J51 and a pheromone peptide plantaricin A with antimicrobial activity. Their production has been found to be regulated through a quorum-sensing based network consisting of a secreted peptide pheromone, a membrane-located sensor and one or two transcription regulators. The individual loci each contain a set of semi-conserved regulated promoters with subtle differences necessary for the regulators to regulate their promoter activity individually with respect to timing and strength. These subtle differences in the promoters are highly conserved across the different pln loci, in a functionally related manner. In this review we will discuss various aspects of these bacteriocin loci with special focus on their mosaic genetic composition, gene regulation and mode of action. We also present a novel pln locus containing a transposon of the MULE superfamily, a mobile element which has not been described in L. plantarum before.