Development of a new method for the detection of lactic acid bacteria capable of protecting ham against Enterobacteriaceae.

AIMS: Challenge trials seem to be the best assessment approach to evaluate the potency of food protective cultures. However, this method is time consuming and often difficult to implement. Here, we describe the development of the 'sequential culturing method', a new method for the screening of strains as protective cultures. METHODS AND RESULTS: The sequential culturing method is based on the simulation, in a meat simulation medium (named BHI5L200), of the inhibition of Enterobacteriaceae by Lactobacillus, observed previously in situ. Results obtained with this sequential culturing method were in good agreement with those of the challenge test on sliced cooked ham and confirmed the antagonistic potency of Lactobacillus. The results obtained from the screening of 187 lactic acid bacteria (LAB) indicated that Lactobacillus sakei, Lactococcus lactis diacetylactis and Carnobacterium spp. were strong inhibitors of Enterobacteriaceae whereas Pediococcus spp., Leuconostoc spp., Weisselia spp. and other species of Lactobacillus and Lactococcus, did not possess the same inhibitory capacity. CONCLUSIONS: Sequential culturing method appeared to be a useful tool to rapidly select LAB cultures which are good candidates for bioprotection of meat. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequential culturing method and simulating media could efficiently mimic challenge test experiments in the selection of potential protective culture for all types of food, on the condition to have the appropriate simulating media, corresponding to the food for which protective cultures were searched.

Genetic organization and sequence of the region encoding integrative functions from Lactobacillus gasseri temperate bacteriophage phi adh.

A 2.0-kb fragment from the Lactobacillus gasseri temperate bacteriophage phi adh contained the essential genetic determinants for site-specific integration. The nucleotide sequence of this fragment was determined. An open reading frame (intG), which adjoined the phage attachment site (attP), encoded a deduced protein related to the integrase family. The organization of this region was comparable to other phage site-specific recombination systems.

Nucleotide sequence and characterization of peb4A encoding an antigenic protein in Campylobacter jejuni.

The 29-kDa protein PEB4, a major antigen of Campylobacter jejuni, is present in all C. jejuni strains tested and elicits an antibody response in infected patients. By screening a lambda gt11 library of chromosomal DNA fragments of C. jejuni strain 81-176 in Escherichia coli Y1090 cells with antibody raised against purified PEB4, a recombinant phage with a 2-kb insert expressing an immunoreactive protein of 29 kDa was isolated. DNA sequence analysis revealed that the insert contains two complete open reading frames ORF-A and ORF-B. ORF-A (peb4A) encodes a 273-residue protein with a calculated molecular mass of 30,460 daltons. The deduced amino acid sequence, composition and pl of the recombinant mature protein are similar to those determined for purified PEB4. The first 21 residues resemble a signal peptide. Gene bank searches indicated 33.7% identity with protein export protein PrsA of Bacillus subtilis and 23.8% identity with protease maturation protein precursor PrtM of Lactococcus lactis. PCR experiments indicate that peb4A is highly conserved among C. jejuni strains. ORF-B begins 2 bp after the last codon of peb4A and encodes a putative protein of 353 residues with 63.4% identity with E. coli fructose 1,6-biphosphate aldolase. The sequence arrangement suggests that these two genes form an operon.

Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp. mesenteroides FR52, and its use as a food grade vector.

A 1.8-kb cryptic plasmid pFR18 was isolated from Leuconostoc mesenteroides ssp. mesenteroides FR52 and characterized. The identification of single-stranded DNA intermediate (ssDNA) in Leuconostoc demonstrated that the replication of pFR18 is directed by a rolling-circle mechanism (RCR). Sequence analysis revealed a single open reading frame (rep18) encoding a putative 335-amino acid protein homologous to the pT181 replicase. Furthermore, a putative double strand origin similar to that of the pT181 plasmid family was identified. A cloning vector was developed on the basis of the pFR18 replicon by inserting an erythromycin resistance cassette within a non-essential region of the plasmid. The resulting construction was able to transform Lactobacillus sake and various species of Leuconostoc. It was stable in L. mesenteroides, however, the segregational stability of a pFR18 derivative containing large Escherichia coli DNA fragments was affected. Nevertheless, the new RCR plasmid pFR18 may be useful for the construction of food grade vectors.

Mesentericin Y105 gene clusters in Leuconostoc mesenteroides Y105.

Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb DraII fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.

Molecular analysis of the lactacin F operon.

Lactacin F is a nonlantibiotic, heat-stable, peptide bacteriocin produced by Lactobacillus johnsonii VPI11088. Molecular analysis of the lactacin F DNA region characterized a small operon that codes for three open reading frames, designated lafA, lafX, and ORFZ. The peptide encoded by lafA, the lactacin F structural gene, was compared with various peptide bacteriocins from lactic acid bacteria, and similarities were identified in the amino and carboxy termini of the propeptides. Site-directed mutagenesis of the LafA precursor at the two glycine residues in positions -1 and -2 defined an essential motif for processing of mature lactacin F. The involvement of the peptides encoded by lafX and ORFZ in bacteriocin expression was investigated by subcloning various fragments from the lactacin F region into the shuttle vector pGKV210. In addition to lafA, expression of lafX is essential to lactacin F activity. The lactacin F operon resembles the genetic organization of lactococcin M. Although no function has been assigned to ORFZ by genetic analysis, both peptide Z and the lactococcin M immunity protein are predicted to be integral membrane proteins with four putative transmembrane segments. Lactacin F activity, defined by bactericidal action on Lactobacillus delbrueckii, is dependent on the expression of two genes, lafA and lafX.

Sequence analysis of Leuconostoc oenos DNA: organization of pLo13, a cryptic plasmid.

A Leuconostoc oenos plasmid, pLo13, was studied to analyze its genetic organization and to define its functions. The nucleotide sequence (3948 bp) revealed three major open reading frames. Features commonly found in plasmids that replicate via a rolling-circle mechanism were identified within pLo13: first, a sequence coding for a protein with an amino acid sequence homologous to the plasmid recombination enzymes (Pre), but for which a specific target site similar to those previously described was not found; second, a sequence probably encoding a replication protein (Rep). The putative pLo13 Rep protein amino acid sequence is divergent from the pC194-pUB110 family Rep proteins. However, the consensus sequence of the Rep protein active site was found, as well as the Rep protein consensus target site. No sequence similar to the previously described minus-origins (SSOs) are present in pLo13; nevertheless, a 200-bp sequence rich in imperfect palindromes may be involved in the minus-strand replication. These overall differences are in agreement with the previously reported important phylogenetic distance existing between Ln. oenos and other lactic acid bacteria.

Method for rapid purification of class IIa bacteriocins and comparison of their activities.

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared.

Genetic and molecular analysis of the tRNA-tufB operon of the myxobacterium Stigmatella aurantiaca.

The tufB gene, encoding elongation factor Tu (EF-Tu), from the myxobacterium Stigmatella aurantiaca was cloned and sequenced. It is preceded by four tRNA genes, the first ever described in myxobacteria. The tRNA synthesized from these genes and the general organization of the locus seem identical to that of Escherichia coli, but differences of potential importance were found in the tRNA sequences and in the intergenic regions. The primary structure of EF-Tu was deduced from the tufB DNA sequence. The factor is composed of 396 amino acids, with a predicted molecular mass of 43.4 kDa, which was confirmed by expression of tufB in maxicells. Sequence comparisons between S.aurantiaca EF-Tu and other bacterial homologues from E.coli, Salmonella typhimurium and Thermus thermophilus displayed extensive homologies (75.9%). Among the variable positions, two Cys residues probably involved in the temperature sensitivity of E.coli and S.typhimurium EF-Tu are replaced in T.thermophilus and S.aurantiaca EF-Tu. Since two or even three tuf genes have been described in other bacterial species, the presence of multiple tuf genes was sought for. Southern and Northern analysis are consistent with two tuf genes in the genome of S.aurantiaca. Primer extension experiments indicate that the four tRNA genes and tufB are organized in a single operon.

Expansion of bacteriocin activity and host range upon complementation of two peptides encoded within the lactacin F operon.

Lactacin F is a membrane-active bacteriocin produced by Lactobacillus johnsonii VPI11088 (Laf+). The genetic determinants encoding lactacin F are organized in a 1-kb polycistronic operon composed of a promoter (P(laf)), three genes (lafA, lafX, and ORFZ), and a functional rho-independent transcription terminator. Two Laf- derivatives of VPI11088, designated NCK64 and NCK65, were characterized. NCK64 contained a frameshift mutation in the lafA gene causing premature termination of translation. NCK65 harbored a 10-kb chromosomal deletion covering the laf operon. When the lafA gene was cloned independently and expressed in NCK65, bacteriocin activity was limited to L. helveticus 87, only one of the six known lactacin F-sensitive (Lafs) indicators. When lafX was introduced into NCK65, no bacteriocin activity against any of the sensitive strains was detected. Genetic combination of lafA and lafX, in cis or in trans, restored bacteriocin activity against all Lafs indicators. When two NCK65 clones containing either lafA or lafX were plated slightly apart on agar plates, fully active lactacin F was present in the intervening area where the two excreted gene products, LafA and LafX, diffused together. The genetic analysis revealed that the interaction of two bacteriocinogenic peptides encoded within the laf operon is likely to participate in the formation of poration complexes in the membranes of susceptible bacteria.

Luminescent method for the detection of antibacterial activities.

A new rapid and sensitive method for the detection of antibacterial activities was based on luminescent indicator strains. Listeria innocua 8811 and Enterococcus faecalis 32 were transformed with plasmid carrying bacterial luciferase genes. Subsequent strains became capable to emit light during the exponential growth phase. The addition of bacteriocin containing culture supernatants to such cultures induced a drop of their light emission which was correlated to the combined antibacterial activity of acid stress and bacteriocin. The detection of antagonistic activity is independent of its mode of action, i.e. bactericidal or bacteriostatic. This method allowed to directly visualize the antagonistic activity of bacteriocin producer strains toward target strains in coculture experiments. However, a control co-culture with non-producing bacteriocin mutant was necessary in order to distinguish between nutrients competition and bacteriocin activity. Finally, five class IIa bacteriocins were purified from culture supernatants of eight strains detected in 3 days from a 120 lactic acid bacteria collection.

Sakacin g, a new type of antilisterial bacteriocin.

Sakacin G is a 37-amino-acid-residue-long class IIa bacteriocin produced by Lactobacillus sake 2512, which is encoded by the duplicated structural genes skgA1 and skgA2. Sakacin G appears to be unique and seems to be an intermediate between pediocin-like bacteriocins, according to its double-disulfide bridges required for antimicrobial activity, and mesentericin-like bacteriocins in terms of sequence homologies, inhibition spectrum, and specific activity.

Effect of the metabolism of urea on the acidifying activity of Streptococcus thermophilus.

One of the main functions of Streptococcus thermophilus strains used in the dairy industry is the production of lactic acid. In cheese and fermented milk manufacturing processes, the pH evolution kinetics must be reproducible in order to ensure the good quality of the final products. The objective of the present study was to investigate the effect of the metabolism of urea on the acidifying activity of fast- and slow-acidifying strains of S. thermophilus. Milk treatment with a purified urease and utilization of the urease inhibitor flurofamide revealed that urea metabolism by S. thermophilus influences the pH evolution kinetics through 2 distinct means. First, ammonia production from urea tends to increase the pH. This effect is greater when lactic acid concentration is low due to a lower buffering capacity of milk. Second, urea metabolism also modifies growth and lactic acid production by S. thermophilus. Depending on the strains and the growth stage of the cultures, consumption of urea induces either a faster or a slower pH decrease. For the slow-acidifying strain RD678, suppression of urea metabolism by adding flurofamide decreased the time necessary to reach pH 6 by 195 min. This effect was less pronounced for the 2 fast-acidifying strains RD674 and RD677. These results show that urea metabolism may have a considerable influence on the acidifying properties of S. thermophilus strains.

Selection and properties of Streptococcus thermophilus mutants deficient in urease.

Natural variations of the urea content of milk have a detrimental effect on the regularity of acidification by Streptococcus thermophilus strains used in dairy processes. The aim of the present study was to select urease-deficient mutants of S. thermophilus and to investigate their properties. Using an improved screening medium on agar plates, mutants were selected from 4 different parent strains after mutagen treatment and by spontaneous mutation. Most mutants were stable and had a phage sensitivity profile similar to that of their parent strain. Some of them contained detrimental secondary mutations, as their acidifying activity was lower than that of the parent strain cultivated in the presence of the urease inhibitor flurofamide. The proportion of this type of mutant was much lower among spontaneous mutants than among mutants selected after mutagen treatment. Utilization of urease-deficient mutants in dairy processes may have several advantages, such as an increase in acidification, an improved regularity of acidification, and a lower production of ammonia in whey.

Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites.

The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.

The complete sequence of a 10.8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae.

The complete nucleotide sequence of the D10H fragment (10850 bp) was determined. The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene. Six open reading frames (ORFs) larger than 300 bp were found. One of them is the CIT2 gene encoding the cytoplasmic citrate synthase. The others are new putative genes and show no significant similarity with any known gene. In addition two tRNA genes (Asn and Pro) and a solo delta element were identified. Two ORFs were disrupted; no peculiar phenotype was observed.