Functional analysis of promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus

Bacteriocin production in Lactobacillus sake LTH673 involves at least four operons: a regulatory operon (sppIPKR); two operons encoding bacteriocins and their immunity proteins (sppAiA and orfX); and an operon needed for secretion (sppTE). We show here that the response regulator encoded by sppR in L. sake LTH673, as well as the homologous response regulators encoded by plnC and plnD in Lactobacillus plantarum C11, bind to characteristic repeats found in the -80 to -40 regions of spp operons. The promoters controlling bacteriocin operons are strictly regulated, and their activity is increased more than 1000-fold upon activation. Constitutive expression for the regulatory and transport operons is driven, at least in part, by promoters upstream of the -80 to -40 regions. Peak promoter activity of the regulatory and transporter operons precedes that of the two bacteriocin operons. The results reveal how promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus differ in strength, leakiness and timing of their activity.     

Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity.

Sakacin A is an antilisterial bacteriocin produced by Lactobacillus sake Lb706. In order to identify genes involved in sakacin A production and immunity, the plasmid fraction of L. sake Lb706 was shotgun cloned directly into a sakacin A-nonproducing and -sensitive variant, L. sake Lb706-B, by using the broad-host-range vector pVS2. Two clones that produced sakacin A and were immune to the bacteriocin were obtained. A DNA fragment of approximately 1.8 kb, derived from a 60-kb plasmid of strain Lb706 and present in the inserts of both clones, was necessary for restoration of sakacin A production and immunity in strain Lb706-B. The sequence of the 1.8-kb fragment from one of the clones was determined. It contained one large open reading frame, designated sakB, potentially encoding a protein of 430 amino acid residues. Hybridization and nucleotide sequence analyses revealed that the cloned sakB complemented a mutated copy of sakB present in strain Lb706-B. The sakB gene mapped 1.6 kb from the previously cloned structural gene for sakacin A (sakA) on the 60-kb plasmid. The putative SakB protein shared 22% amino acid sequence identity (51% similarity if conservative changes are considered) to AgrB, the deduced amino acid sequence of the Staphylococcus aureus gene agrB. The polycistronic agr (accessory gene regulator) locus is involved in the regulation of exoprotein synthesis in S. aureus. Similar to the AgrB protein, SakB had some features in common with a family of transmembrane histidine protein kinases, involved in various adaptive response systems of bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of bacteriocin promoters for gene expression in Lactobacillus plantarum C11

AIMS: To exploit promoters involved in production of the bacteriocin sakacin P for regulated overexpression of genes in Lactobacillus plantarum C11. METHODS AND RESULTS: Production of sakacin P by Lact. sakei LTH673 is controlled by a peptide-based quorum sensing system that drives strong, regulated promoters. One of these promoters (PorfX) was used to establish regulated overexpression of genes encoding chloramphenicol acetyltransferase from Bacillus pumilus, aminopeptidase N from Lactococcus lactis or chitinase B from Serratia marcescens in Lact. plantarum C11, a strain that naturally possesses the regulatory machinery that is necessary for promoter activation. The expression levels obtained were highly dependent on which gene was used and on how the promoter was coupled to this gene. The highest expression levels (14% of total cellular protein) were obtained with the aminopeptidase N gene translationally fused to the regulated promoter. CONCLUSIONS: Sakacin promoters permit regulated expression of a variety of genes in Lact. plantarum C11. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the usefulness of regulated bacteriocin promoters for developing new gene expression systems for lactic acid bacteria, in particular lactobacilli.     

High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter

AIMS: To use promoters and regulatory genes involved in the production of the bacteriocin sakacin P to obtain high-level regulated gene expression in Lactobacillus plantarum. METHODS AND RESULTS: In a plasmid containing all three operons naturally involved in sakacin P production, the genes encoding sakacin P and its immunity protein were replaced by the aminopeptidase N gene from Lactococcus lactis (pepN) or the beta-glucuronidase gene from Escherichia coli (gusA). The new genes were precisely fused to the start codon of the sakacin P gene and the stop codon of the immunity gene. This set-up permitted regulated (external pheromone controlled) overexpression of both reporter genes in L. plantarum NC8. For PepN, production levels amounted to as much as 40% of total cellular protein. CONCLUSIONS: Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a system for controllable gene expression in lactobacilli, giving some of the highest expression levels reported so far in this genus.     

Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake Lb674

Abstract Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes , was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 ( sakR ) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.     

Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5

Lactobacillus sakei 5, isolated from malted barley, produces three bacteriocins. Genetic and functional analysis of the purified bacteriocins showed that this strain produces a plasmid-encoded bacteriocin that is identical to sakacin P, as well as two novel, chromosomally encoded bacteriocins, which were designated sakacin T and sakacin X. The structural genes specifying sakacin T and sakacin X are part of the sakacin TX locus, which consists of two adjacent but divergently oriented gene clusters. The first gene cluster includes stxP, stxR, stxK, and stxT, which, based on functional and comparative sequence analysis, are believed to encode an inducing peptide and proteins involved in regulation and secretion of these bacteriocins. The second gene cluster includes the structural and immunity genes for sakacin T, a class IIb two-peptide bacteriocin composed of SakTalpha and SakTbeta, and sakacin X, a class IIa bacteriocin. Interestingly, a so-called transport accessory protein was absent from the locus, and based on our results it appears that a dedicated accessory protein is not required for processing and transport of sakacin T and sakacin X.     

Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K

Sakacin K is an antilisterial bacteriocin produced by Lactobacillus sake CTC 494, a strain isolated from Spanish dry fermented sausages. The biokinetics of cell growth and bacteriocin production of L. sake CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth. The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior. The model was validated successfully for all components. However, the specific bacteriocin production rate seemed to have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity. Furthermore, cells grew well at 35 degrees C but no bacteriocin production could be detected at this temperature. L. sake CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K.     

Characterization of a New Bacteriocin Operon in Sakacin P-Producing Lactobacillus sakei, Showing Strong Translational Coupling between the Bacteriocin and Immunity Genes

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.     

Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M(r) of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.     

Sequencing and expression analysis of sakacin genes in Lactobacillus curvatus strains

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain.     

Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages

A Lactobacillus sakei strain, designated as I151 and isolated from naturally fermented sausages, was found to produce the sakacin P bacteriocin which is active against Listeria monocytogenes. In this study, we performed the sequencing of the gene cluster involved in the production of the sakacin P, and we followed the expression of the sppA gene, encoding for the bacteriocin, in vitro, using Rogosa–Sharpe medium, and in situ, inoculating the strain in fermented sausages as starter culture. The results obtained underlined the high similarity (>99%) of the entire sakacin P gene cluster from the L. sakei studied here with others present in strains of L. sakei already described. Moreover, from the expression experiments, it was shown that the gene is expressed during the exponential phase and that production procedures typical of fermented sausages are not turning off the expression of the gene encoding the bacteriocin. The capability of the strain studied to produce sakacin P during production is considered an advantage for its use as starter culture to improve the safety aspect of traditional fermented sausages produced in Italy.     

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Antibacterial activity of Lactobacillus sake isolated from meat

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei

AIMS: To evaluate the potential of sakacin P and sakacin P-producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food? METHODS AND RESULTS: Listeria monocytogenes, a Lact. sakei strain and/or the bacteriocin sakacin P were added to chicken cold cuts, vacuum packed and incubated at 4 or 10 degrees C for 4 weeks. Each of two isogenic Lact. sakei strains, one producing sakacin P and the other not, had an inhibiting effect on the growth of L. monocytogenes. The effect of these two isogenic strains on the growth of L. monocytogenes was indistinguishable, even though sakacin P was produced in the product by one of the two Lact. sakei strains. The addition of purified sakacin P had an inhibiting effect on the growth of L. monocytogenes. A high dosage of sakacin P (3.5 microg x g(-1)) had a bacteriostatic effect throughout the storage period of 4 weeks, while a low dosage (12 ng x g(-1)) permitted initial growth, but at a slow rate. After 4 weeks of storage, the number of L. monocytogenes in the samples with a low dosage of sakacin P was 2 logs below that in the untreated control. When using a high dosage of sakacin P, the bacteriocin was detected in samples stored for up to 6 weeks. CONCLUSIONS: (i) Sakacin P is produced by a Lact. sakei strain when growing on vacuum-packed chicken cold cuts. (ii) Inhibiting effects of Lact. sakei, other than sakacin P, are active in inhibiting the growth of L. monocytogenes growing on chicken cold cuts. (iii) Sakacin P is stable on chicken cold cuts over a period of 4 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: Both sakacin P and Lact. sakei were found to have potential for use in the control of L. monocytogenes in chicken cold cuts.     

Sakacin B, a bacteriocin produced by Lactobacillus sake isolated from Greek dry fermented sausages

When Lactobacillus sake 251, a strain isolated from naturally fermented Greek dry sausage was grown in MRS broth it excreted an antimicrobial factor that differed from organic acids and hydrogen peroxide. The substance was proteinaceous, heat stable and inhibitory towards various lactic acid bacteria of meat origin. This suggested that a narrow spectrum bacteriocin, designated sakacin B, was present in the broth. Sakacin B displayed a bactericidal mode of action on sensitive cells without causing cell lysis. It was secreted during late logarithmic phase and was stable within a pH range 2 to 9. In vitro production of sakacin B by the producer strain in a mixed culture caused a strong biocidal effect on growing indicator cells. Sakacin B was partially purified and found not to contain unusual amino acids. That it was a hydrophobic peptide was confirmed by SDS-PAGE electrophoresis. The molecular weight of sakacin B was estimated to be 6.3 kDa.     

Cloning and heterologous expression of a bacteriocin sakacin P from <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.     

Characterisation of an Antilisterial Bacteriocin Produced by Lactobacillus sakei CWBI-B1365 Isolated from Raw Poultry Meat and Determination of Factors Controlling its Production

Amongst 101 lactic acid bacteria isolated from meat and fish samples, strain CWBI-B1365, identified as Lactobacillus sakei, was found to produce the subclass IIa bacteriocin sakacin G. Partial sequencing of the gene involved in the biosynthetic pathways revealed an unusual gene organisation in that the accessory gene associated with bacteriocin transport did not occur immediately downstream of the gene encoding an ABC transporter, but upstream of the putative immunity gene and encoded on the opposite DNA strand. Sakacin G production was strongly regulated by pH, temperature and the carbon sources used in the growth medium, as well as the concentration of carbon and nitrogen sources. The condition of pH 5.5 and the temperature of 25°C appeared to be optimal for bacteriocin production. The use of sucrose during culturing and the fed batch addition of sucrose and meat extract greatly enhanced bacteriocin production.