No abstract

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.

In this paper, we describe the structure of chitinase B from Serratia marcescens, which consists of a catalytic domain with a TIM-barrel fold and a 49-residue C-terminal chitin-binding domain. This chitinase is the first structure of a bacterial exochitinase, and it represents one of only a few examples of a glycosyl hydrolase structure having interacting catalytic and substrate-binding domains. The chitin-binding domain has exposed aromatic residues that contribute to a 55-Å long continuous aromatic stretch extending into the active site. Binding of chitin oligomers is blocked beyond the ؊3 subsite, which explains why the enzyme has chitotriosidase activity and degrades the chitin chain from the nonreducing end. Comparison of the chitinase B structure with that of chitinase A explains why these enzymes act synergistically in the degradation of chitin.T he degradation of abundant insoluble carbohydrate polymers such as cellulose and chitin is achieved in nature with the help of batteries of glycosyl hydrolases with different substrate preferences and product specificities. For example, the degradation of chitin, a linear polysaccharide of ␤(1, 4)-linked N-acetylglucosamine (GlcNAc) residues, by the soil bacterium S. marcescens involves at least four enzymes (the exo-and endochitinases ChiA, ChiB, and ChiC, and an N-acetylglucosaminidase) (1-4). In addition to a catalytic domain, most enzymes involved in cellulose and chitin degradation usually contain one or more domains that are involved in substrate binding (refs. 5-7; see also http:͞͞afmb.cnrsmrs.fr͞pedro͞DB͞ncmCBM12.html). Removal of such domains often results in enzymes that are still active but display severely impaired binding to polymeric substrates (see examples in refs. 7 and 8). For cellulases, there is abundant structural information for a variety of catalytic domains and for isolated carbohydrate-binding domains (6, 9), but there is only one available crystal structure of a catalytic domain together with a (143-residue) CeBD (10).Chitinases belong to families 18 and 19 of the glycosyl hydrolases (9). The catalytic domain of family 18 chitinases has a TIM-barrel fold (2, 11) and includes a conserved glutamate residue that presumably acts as an acid during catalysis (Glu144 in ChiB; Fig. 1 ; refs. 12-14). Catalysis proceeds with retention of the anomeric configuration, which is achieved by a mechanism in which the carbonyl oxygen of the N-acetyl group of the Ϫ1 sugar (nomenclature according to ref. 15) acts as nucleophile (12)(13)(14). Judged from their sequences, most family 18 chitinases, including ChiA, ChiB, and ChiC from S. marcescens, contain domains putatively involved in the interaction with chitin (5,7,8,16). Perrakis et al. (2,5) have determined the structure of complete ChiA, revealing the location of a 114-residue domain with a fibronectin III-like fold that most likely participates in chitin-binding (2,5,8). On the basis of sequence analyses, ChiB has been suggested to consist of a catalytic domain followed by a putative linker region an...

Research Institute, As, Norway 2 The Norwegian Crop Serratia marcescens produces several chit inol y t ic enzymes, including chit i nase A (ChiA) and chitinase B (ChiB). In this study, Chis was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of Chis and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and Chis shared several characteristics such as a broad pH optimum around pH 5-0, and a temperature optimum between 50 and 60 OC. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 "C, pH 61. Analyses of the degradation of various N-acetylglucosamine oligomers, f luorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc),] from (GlcNAc), and thus possess an exolN,N'-diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc), substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N',N''-triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with ~methylumbelliferyl-~D-N,N'-d iacet y Ich i to bioside, an analogue of (GlcN Ac), , showed cooperative kinetics for ChiA, whereas for Chis normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than Chis and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in S. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.

SummaryProduction of the bacteriocin sakacin P by Lactobacillus sake LTH673 is dependent on a secreted 19-residue peptide pheromone (IP-673). The gene encoding IP-673 (sppIP ) was identified and sequenced. SppIP was shown to be co-transcribed with genes encoding a histidine kinase (sppK ) and a response regulator (sppR ) typical for signal transduction in bacteria. Further sequencing and transcription studies have shown that IP-673 induces transcription of its own gene and of what are often considered to be all genes necessary for bacteriocin production and immunity. Studies with a reporter gene showed that the promoter in front of the sakacin P structural gene (sppA) is strictly regulated. The promoter in front of sppIP turned out to be less strictly regulated, and low basal promoter activity could be detected in uninduced cells. Bacteriocin production in Bac ¹ isolates of L. plantarum C11 could be induced by the non-cognate IP-673 only after the introduction of sppK, indicating that sppK encodes the pheromone receptor. These results show that bacteriocin production in lactobacilli is regulated using a short, strain-specific peptide pheromone. Growth conditions were shown to have considerable effects on the functionality of this regulatory mechanism.

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen Listeria monocytogenes was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibitingListeria, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active towardListeria but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria.

Background: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.Various lactic acid bacteria, such as members of the genera Lactococcus, Lactobacillus, and Pediococcus, are known to secrete antimicrobial peptides called bacteriocins (20,22). These peptides normally contain between 30 and 60 residues. They usually have a basic character, and parts of their sequences show amphiphilicity when projected onto a helical wheel. Some bacteriocins undergo posttranslational modification and are called lantibiotics (37). Bacteriocins are synthesized as precursor molecules containing a leader peptide. For almost all nonlantibiotics this leader peptide is of the so-called double-glycine type (10,11,14,15), which directs secretion mediated by a specific type of ABC transporter (9,14,16). In addition to the transporter, several other proteins are involved in bacteriocin production (1,5,8,(22)(23)(24)32). These include an immunity protein, a protein similar to HlyD from Escherichia coli, and proteins with homologies to the histidine kinases and response regulators that make up two-component regulatory systems (40).Little is known about the regulation of bacteriocin production. However, it is clear that regulatory mechanisms exist. Bacteriocin production is growth phase dependent (5,7,8,12,23), and genes encoding two-component regulatory systems were found to be essential for bacteriocin production in all cases tested (1,23,31,47).Many bacterial processes, such as the production of extracellular proteins and toxins and the development of natural competence, are growth phase dependent. In several cases it has been shown that the cognate regulatory mechanisms involve the secretion of signal molecules involved in cell-cell signalling (21). For example, such signal molecules include a modified peptide inducing competence in Bacillus subtilis (25), an unidentified proteinaceous factor inducing the agr system in Staphylococcus aureus (2), and N-acyl homoserine lactones involved in various regulatory mechanisms in gram-negative bacteria (18, 41). Transduction of these (and many other) bacterial signals often i...