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Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive Listeria monocytogenes (mpt) and the nonsensitive Lactococcus lactis (ptn) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins. doi: 10.1128/JB.00777-10

Colicin release provides producer strains with a competitive advantage under certain circumstances. We found that propagation of M13 bacteriophage in cells producing colicin E? is impaired, without alteration in the efficiency of bacteriophage adsorption, as compared with non-producing cells. In contrast to the protective effect of the colicin against M13 bacteriophage infection, the endogenously expressed colicin does not confer limited protection against transfection with M13 bacteriophage DNA. Furthermore, it was found that the translocation-receptor-binding domain and toxicity domain of the colicin are able to interact with the M13 major coat protein, g8p, during bacteriophage infection. Based on these observations, we propose that interaction between colicin E7 and g8p during infection interferes with g8p depolymerizing into the cytoplasmic membrane during bacteriophage DNA penetration, thus resulting in the limited protection against M13 bacteriophage infection. DOI 10.1099/mic.0.040592-0

Pseudomonas aeruginesa (Pa) and Burkholderia cepacia complex (Bcc) lung infections are responsible for much of the mortality in cystic fibrosis (CF). However, little is known about the ecological interactions between these two, often co-infecting, species. This study provides what is believed to be the first report of the intra- and interspecies bacteriocin-like inhibition potential of Pa and Bcc strains recovered from CF patients. A total of 66 strains were screened, and shown to possess bacteriocin-like inhibitory activity (97% of Pa strains and 68 % of Bcc strains showed inhibitory activity), much of which acted across species boundaries. Further phenotypic and molecular-based assays revealed that the source of this inhibition differs for the two species. In Pa, much of the inhibitory activity is due to the well-known S and RF pyocins. In contrast, Bcc inhibition is due to unknown mechanisms, although RF-like toxins were implicated in some strains. These data suggest that bacteriocin-based inhibition may play a role in governing Pa and Bcc interactions in the CF lung and may, therefore, offer a novel approach to mediating these often fatal infections. DOI: 10.1099/mic.0.036848-0

The importance of 3D structuring in the N-and C-terminal ends of the two peptides (39-mer LcnG-[alpha] and 35-mer LcnG-[beta]) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding D-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-[alpha] with four D-residues in its C-terminal region and LcnG-[beta] with four D-residues in either its N- or its C-terminal region were relatively active (two- to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-[alpha] and the C- and N-terminal regions in LcnG-[beta] is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-[alpha] may be more important, as LcnG-[alpha] with four N-terminal D-residues was the least active of these four peptides (10- to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-[alpha] and -[beta] form a transmembrane parallel helix-helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-[alpha] and at about residue 13 in LcnG-[beta]. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these D-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the D-residue-containing peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N- and C-terminal residues with D-residues. DOI 10.1099/mic.0.038430-0

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-[alpha] and Trn-[beta], that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [[sup.13]C, [sup.15]N] Trn-[alpha] and [[sup.13]C, [sup.15]N]Trn-[beta] were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the [alpha]-carbons of the modified residues, forming three sulfur to [alpha]-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S'-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to [alpha]-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora. two-component bacteriocin | posttranslational modifications | Clostridium difficile-associated disease | peptide | NMR doi: 10.1073/pnas.0913554107

Mierocin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the [alpha]-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of [tRNA.sup.Asp] by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell. doi: 10.1128/JB.00829-09

Membrane-located proteins (IIC and IID) of the mannose-phosphotransferase system (man-PTS) have previously been shown to serve as target receptors for several bacteriocins. Although many bacteria contain at least one such man-PTS in their genome, most bacteriocins display a narrow inhibitory spectrum, targeting predominantly bacteria closely related to the producers. In the present study we have analysed the receptor spectrum of one-peptide bacteriocins of class II. A phylogenetic analysis of 86 man-PTSs from a wide range of bacterial genera grouped the man-PTSs into three main clusters (groups I-III). Fourteen man-PTSs distributed across the phylogenetic tree were selected for experimental analysis in a heterologous host. Only members of group I could serve as receptors for class IIa bacteriocins, and the receptor efficiencies varied in a pattern directly related to their phylogenetic position. A multiple sequence alignment of IIC and IID proteins revealed three sequence regions (two in IIC and one in IID) that distinguish members of the bacteriocin-susceptible group from those of the other groups, suggesting that these amino acid regions confer the specific bacteriocin receptor function. Moreover, we demonstrated that variation in sensitivity might also exist within the same species due to differential expression levels of the receptor, since three strains of Lactobacillus sakei harbouring identical man-PTSs were shown to display different expression levels of a man-PTS gene that corresponded to the variation in bacteriocin sensitivity. Together, the results of our study show that the level of bacteriocin susceptibility for a bacterial cell is primarily determined by differences in its man-PTS proteins, although the expression levels of the corresponding genes also play an important role.

Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit [Mg.sup.2+]-dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.

The aim of this study was to investigate in vitro the effects of pediocin A [a bacteriocin produced by lactic acid bacteria (LAB) Pediococcus pentosaceus FBB61] on microbial metabolism in the small and large intestine of pigs. Pedioein A was partially purified by ion exchange chromatography and added to an in vitro fermentation system. The intestihal inoculum was collected from pigs immediately after slaughter, diluted with a buffer, and dispensed into fermentation syringes and vessels of the 2 experimental groups: 1)Bac+ = cecal liquor + predigested diet + pediocin A (final concentration 160 activity units/mL); 2) Bac- = cecal liquor + predigested diet + partially purified supernatant of P. pentosaceus FBB61-2. Intestinal microbial growth was monitored using the cumulative gas production technique; the kinetics of fermentation, bacterial counts, VFA, ammonia, polyamines, and p-cresol production were analyzed. Pediocin A had almost no effects on small intestine fermentation parameters, whereas in the cecum pediocin A decreased gas production (-16%; P < 0.05), ammonia, and VFA production (-52 and -21%, respectively, after 24 h; P < 0.001) compared with the control group. Significant inhibition of clostridia and LAB occurred in cecal fermentations: the Bac+ group yielded a decreased number of clostridia and LAB in cecal fermentations (8.19 and 7.80 cfu/mL, respectively) compared with Bac-(9.32 and 8.95 cfu/mL, respectively; P < 0.001). The low clostridia counts in the pediocin-treated group may also explain the reduced concentration of the carcinogenic compound p-cresol (-88%; P < 0.01). Our results suggest that pediocin A could be an alternative to replace antibiotic growth promoters for the prophylaxis of enteric diseases and to improve production of farm animals. Key words: antibiotic growth promoter, bacteriocin, cresol, intestinal fermentation, pig, polyamine

Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gin-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 ([ColE9.sup.s-s]) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in [ColE9.sup.s-s], we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 ([Im9.sup.AF]) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.

The determination and comparison of characteristics of lacticin Q (Lactococcus lactis QU 5 isolated from corn) to those of nisin A revealed that lacticin Q belongs to a new family of class II bacteriocins and it could be employed as an alternative to or in combination with nicin A.

The analysis of two peptides (Lcn-[alpha] and Lcn-[beta]) of the two-peptide bacteriocin lactococcin G (Lcn) by stepwise site-directed mutagenesis into the corresponding peptides (Ent-[alpha] and Ent-[beta]) of the two-peptide bacteriocin enterocin 1071 is presented.

The mechanisms by which probiotic strains enhance the health of the host remain largely uncharacterized. Here we demonstrate that Lactobacillus salivarius UCC118, a recently sequenced and genetically tractable probiotic strain of human origin, produces a bacteriocin in vivo that can significantly protect mice against infection with the invasive foodborne pathogen Listeria monocytogenes. A stable mutant of Lb. salivarius UCC118 that is unable to produce the Abp118 bacteriocin also failed to protect mice against infection with two strains of L. monocytogenes, EGDe and LO28, confirming that bacteriocin production is the primary mediator of protection against this organism. Furthermore, Lb. salivarius UCC118 did not offer any protection when mice were infected with a strain of L. monocytogenes expressing the cognate Abp118 immunity protein AbpIM, confirming that the antimicrobial effect is a result of direct antagonista between Lb. salivarius and the pathogen, mediated by the bacteriocin Abp118. infection | Listeria | probiotic

The characteristics of Weissella cibaria 110 bacteriocin, which is isolated from the Thai fermented fish product plaa-som, are analyzed. The bacteriocin is defined as a new bacteriocin and termed as weissellicin 110 as it has no similarities with any other known 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 liD 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

The production of streptocins ST[H.sub.1] and ST[H.sub.2] by Streptococcus gordonii DL1 (Challis) is directly controlled by the competence regulon, which requires intact comR and comAB loci. The streptocin (sth) locus comprises two functional genes, sthA and sthB. Whereas ST[H.sub.1] activity requires sthA alone, ST[H.sub.2] activity depends on both genes.

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp, carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp, carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin $3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.

The immunity protein of plantaricin 423 and the complete sequence of plasmid pPLA4 are described. Plataricin 423 has shared high sequence similarity with the pediocin PA-1 operon, but no cross-reactivity is recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.

Bacteria utilize quorum-sensing systems to modulate environmental stress responses. The quorum-sensing system of Streptococcus mutans is mediated by the competence-stimulating peptide (CSP), whose precursor is encoded by the comC gene. A comC mutant of strain GS5 exhibited enhanced antimicrobial sensitivity to a wide variety of different agents. Since the addition of exogenous CSP did not complement this phenotype, it was determined that the increased tetracycline, penicillin, and triclosan sensitivities resulted from repression of the putative bacteriocin immunity protein gene, bip, which is located immediately upstream from comC. We further demonstrated that the inactivation of bip or smbG, another bacteriocin immunity protein gene present within the smb operon in S. mutans GS5, affected sensitivity to a variety of antimicrobial agents. Furthermore, both the bip and smbG genes were upregulated in the presence of low concentrations of antibiotics and were induced during biofilm formation relative to in planktonic cells. These results suggest, for the first time, that the antimicrobial sensitivity of a bacterium can be modulated by some of the putative bacteriocin immunity proteins expressed by the organism. The implications of these observations for the evolution of bacteriocin immunity protein genes as well as for potential new chemotherapeutic strategies are discussed.

A collection of 266 faecal isolates of Escherichia coli from humans was assayed for the production of mitomycin C-inducible bacteriocins and screened using a PCR-based method for the presence of eleven colicins and seven microcins. Eight different colicins were detected and all seven microcins. Of the strains examined, 38 % produced a bacteriocin, 24% produced a colicin and 20% produced a microcin. Of the 102 bacteriocin-producing strains, 42% produced one type of bacteriocin, 41% produced two, 16% produced three and one strain was found to produce four different bacteriocins. Strains producing more than one bacteriocin were more likely to be members of E. coli genetic group B2 and less likely to belong to genetic groups A or D. Several of the bacteriocins were found to co-occur in a strain more often than would be expected by chance: microcins H47 and M; colicin la and microcin V; colicins B and M; colicins E1 and M; colicins E1 and la. No bacteriocins released as a consequence of cell lysis were found to co-associate more often than expected by chance. Three non-mutually exclusive hypotheses are presented that might explain the high frequency of multiple bacteriocin production in E. coli strains: (1) expanded killing range, (2) expanded receptor repertoire and (3) fitness benefits in different environments.

The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system. The gene cluster has an operon-like structure consisting of a putative pediocin-like bacteriocin gene (termed penA) and a potential immunity gene (termed peiA). Genetic determinants involved in bacteriocin transport and regulation are also found in proximity to penA and peiA but the so-called accessory gene involved in transport and the inducer gene involved in regulation are missing. Consequently, this bacterium is a poor bacteriocin producer. To analyse the potency of the putative bacteriocin operon, the two genes penA-peiA were heterologously expressed in a Lactobacillus sakei host that contains the complete apparatus for gene activation, maturation and externalization of bacteriocins. It was demonstrated that the heterologous host expressing penA and peiA produced a strong bacteriocin activity; in addition, the host became immune to its own bacteriocin, identifying the gene pair penA-peiA as a potent bacteriocin system. The novel pediocin-like bacteriocin, termed penocin A, has an isotopic mass [[M + H].sup.+] of 4684.6 Da as determined by mass spectrometry; this value corresponds well to the expected size of the mature 42 aa peptide containing a disulfide bridge. The bacteriocin is heat-stable but protease-sensitive and has a calculated pl of 9.45. Penocin A has a relatively broad inhibition spectrum, including pathogenic Listeria and Clostridium species. Immediately upstream of the regulatory genes reside some features that resemble remnants of a disrupted inducer gene. This degenerate gene was restored and shown to encode a double-glycine leader-containing peptide. Furthermore, expression of the restored gene triggered high bacteriocin production in P. pentosaceus ATCC 25745, thus confirming its role as an inducer in the pen regulon.

Inter-strain and inter-species inhibition mediated by a bacteriocin-like inhibitory substance (BLIS) from a pathogenic Vibrio harveyi strain VIB 571 was demonstrated against four isolates of the same species, and one culture each of a Vibrio sp., Vibrio fischeri, Vibrio gazogenes and Vibrio parahaernolyticus. The crude BLIS, which was obtained by ammonium-sulphate precipitation of the cell-free supernatant of a 72 h broth culture of strain VIB 571, was inactivated by lipase, proteinase K, pepsin, trypsin, pronase E, SDS and incubation at [greater than or equal to] 60 [degrees]C for 10 min. The activity was stable between pH 2-11 for at least 5 h. Anion-exchange chromatography, gel filtration, SDS-PAGE and two-dimensional gel electrophoresis revealed the presence of a single major peak, comprising a protein with a pl of ~5.4 and a molecular mass of ~32 kDa. The N-terminal amino acid sequence of the protein comprised Asp-Glu-Tyr-Ile-Ser-X-Asn-Lys-X-Ser-Ser-Ala-Asp-Ile (with X representing cysteine or modified amino acid residues). A similarity search based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) generated peptide masses and the N-terminal sequence did not yield any significant matches.

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB'-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB'-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.

An investigation on the mechanism of secretion of the heterologously produced enterococcal bacteriocin P (EntP) His in Lactococcus lactis is conducted. EntP belongs to the class II bacteriocins that are synthesized as precursors with an N-terminal extension that demonstrated the typical tripartite structure of Sec-dependent signal peptides.

Sensitivity to class Ila bacteriocins from lactic acid bacteria was recently associated with the mannose phosphotransferase system (PTS) permease, [Ell.sup.Man.sub.t], in Listeria monocytogenes. To assess the involvement of this protein complex in class Ila bacteriocin activity, the mptACD operon, encoding [Ell.sup.Man.sub.t], was heterologously expressed in an insensitive species, namely Lactococcus lactis, using the NICE double plasmid system. Upon induction of the cloned operon, the recombinant Lc. lactis became sensitive to leucocin A. Pediocin PA-1 and enterocin A also showed inhibitory activity against Lc. lactis cultures expressing mptACD. Furthermore, the role of the three genes of the mptACD operon was investigated. Derivative plasmids containing various combinations of these three genes were made from the parental mptACD plasmid by divergent PCR. The results showed that expression of mptC alone is sufficient to confer sensitivity to class Ila bacteriocins in Lc. lactis.

The tryptophan fluorescence and circular dichroism (CD) analyses of MesY105 derivatives obtained by randomized mutagenesis or chemical synthesis is presented. The role of specific amino acids in the binding to phospholipid vesicles, in an attempt to determine the structure and mode of action of bacteriocins are investigated.

Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 [micro]g per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm.

The inhibitory effects of different sodium chloride concentrations on the production of curvacin A, which is generated by Lactobacillus curvatus strain LTH 1174 and originates from fermented sausage were studied. The low concentration of sodium chloride probably will not affect the bacterial growth but high salt concentrations may result in an increase in the inhibition.

Immunoassays that can be used for the detection and quantification of lacticin RM and the influence of temperature and surfactant addition (Tween 80) on lactin RM expression and activity are studied. The lactin RM expression is temperature dependent and the lactin RM-producing strain could be used as a protective culture in dairy products.

Mutacin MT6223 from Streptococcus sobrinus MT6223 was purified through sulfate precipitation, chromatofocusing and column chromatography. The molecular mass of the mutacin was determined to be about 6.5 kilodaltons. Papain and ficin inactivated the bacteriocin while alpha-chymotrypsin exhibitedonly partial inhibition. S. mutans streptococcalstrains c, e and f were selectively inhibited by mutans MT6223 both in vivo and in vitro. These properties indicate a potential use of this bacteriocin in the prevention of dental caries.

A study was conducted demonstrate the potential of two-component salivaricin P-like bacteriocins as antimicrobials to control infections in the gastrointestinal tract (GIT). The results demonstrated the potential of engineering bacteriocin variants which exhibit resistance to specific protease action but retain significant antimicrobial activity.

The expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33 is reported. The study describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by yeast.

The combinations of wild-type peptides and hybrid peptides from the two bacteriocins (lactococcin G and enterocin 1071) are assayed against strains expressing either of the two immunity proteins in order to determine which peptide or parts of the peptides of each bacteriocin that are recognized by the cognate immunity protein. The regions important for recognition have included the N-terminal end of Lcn-[alpha] and the C-terminal part of Lcn-[beta].

Fecal enterococci are isolated from infants and screened for bacteriocin production, where bacteriocin-producing Enterococcus avium isolates are obtained and a new pediocin-like bacteriocin is purified and characterized. This bacteriocin has displayed strong antimicrobial activity against many species of Gram-positive bacteria and avicin A locus is the first bacteriocin locus identified in Enterococcus avium to be studied at the molecular level.

Several studies are conducted to explain the various phenotypic, as well as genotypic responses exhibited by Listeria monocytogens strains that are exposed to sakacin P, a class IIa bacteriocin. The presence of different resistance mechanisms in the different strains is clearly demonstrated by the results.

A species-independent functional gene microarray was developed to target 406 genes that play significant role in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics and in the stress response. The wide ranges in intensity and high reproducibility between microarrays were confirmed by validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides.

Ultrafiltration and cation-exchange and reversed-phase chromatographies were used for homogeneous purification of Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity. The amino acid sequence identified as enterocin 96 belonging to class II bacteriocins covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.

An attenuated mutant of Xanthomonas perforans is used that has colonized leaf tissue more effectively than nonpathogenic strains and has provided more effective colonization and has increased the likelihood for interaction between Xanthomonas perforans and Xanthomonas euvesicatoria, tomato plant pathogen. Attenuated pathogenic strains are deployed as biological control agents in order to improve disease control of foliar plant pathogens.

Byline: Anjana. Sharma, Virendra. Patel, Animesh. Chaturvedi Objectives: Screening of the medicinal plants and determination of minimum inhibitory concentration (MIC) against Vibrio cholerae and Vibrio parahaemolyticus. Materials and Methods: A [...]

A site-directed mutagenesis was used to produce eight variants in which a single amino acid replacement was specifically introduced into the sequence to understand the relationship between the structure of divercin V41 (DvnRV41). The variants produced provide insight into the structure-activity relationship of DvnRV41, a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41.