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.

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.

A sigma(54)-dependent PTS permease of the mannose family is responsible for sensitivity of Listeria monocytogenes to mesentericin Y105.

Sensitivity of Listeria monocytogenes to the bacteriocin mesentericin Y105 was previously shown to be dependent on the sigma(54) subunit of the RNA polymerase. This points towards expression of particular sigma(54)-dependent genes. The present study describes first, ManR, a new sigma(54)-associated activator, and second, Ell(t)(Man), a new sigma(54)-dependent PTS permease of the mannose family, both involved in sensitivity to mesentericin Y105, since interruption of their corresponding genes led to resistance of L. monocytogenes EGDe. Ell(t)(Man) is likely composed of three subunits encoded by the mpt operon (mptA, mptC and mptD genes). Interruption of either the proximal (mptA) or distal (mptD) gene led to resistance, supporting results obtained in Enterococcus faecalis. Accordingly, such PTS permeases of the mannose family should be involved in sensitivity of different target strains to mesentericin Y105. In L. monocytogenes, expression of the mpt operon is shown to be controlled by sigma(54) and ManR and to be induced by both glucose and mannose. The latter result indicates that these sugars are transported by the Ell(t)(Man) permease. Moreover, these sugars correlatively induce sensitivity of L. monocytogenes to mesentericin Y105, strongly favouring the primary role of Ell(t)(Man). MptD, a membrane subunit of Ell(t)(Man), presents an additional domain compared to most IID(Man) subunits described in data banks. An in-frame deletion of this domain in mptD led to resistance of L. monocytogenes, showing its connection with sensitivity and suggesting that it could be directly involved in the recognition of the target cell by mesentericin Y105. Taken together, the results of this work demonstrate that Ell(t)(Man) is prominent in sensitivity to mesentericin Y105 and could be a receptor for subclass IIa bacteriocins.

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.

Initiation factor 2 of Myxococcus xanthus, a large version of prokaryotic translation initiation factor 2.

We have isolated the structural gene for translation initiation factor IF2 (infB) from the myxobacterium Myxococcus xanthus. The gene (3.22 kb) encodes a 1,070-residue protein showing extensive homology within its G domain and C terminus to the equivalent regions of IF2 from Escherichia coli. The protein cross-reacts with antibodies raised against E. coli IF2 and was able to complement an E. coli infB mutant. The M. xanthus protein is the largest IF2 known to date. This is essentially due to a longer N-terminal region made up of two characteristic domains. The first comprises a 188-amino-acid sequence consisting essentially of alanine, proline, valine, and glutamic acid residues, similar to the APE domain observed in Stigmatella aurantiaca IF2. The second is unique to M. xanthus IF2, is located between the APE sequence and the GTP binding domain, and consists exclusively of glycine, proline, and arginine residues.

Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature.

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.

The rpoN gene of Enterococcus faecalis directs sensitivity to subclass IIa bacteriocins.

The final sigma 54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding final sigma 54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that final sigma 54 is especially involved in sensitivity to subclass IIa bacteriocins.

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.

Atomic force microscopy imaging of dried or living bacteria.

Atomic force microscopy (AFM) was used to obtain micrographs of dried bacteria in air, and of living ones in their culture medium. Images of dried bacteria were very similar to images obtained elsewhere by the much more complicated cryoetching preparation technique for transmission electron microscopy. Living bacteria were immobilized on a poly-L-lysine film, and directly observed in their culture medium at a resolution unattainable by any other technique applicable to living material. The images were similar to those obtained in scanning electron microscopy where the specimen must be fixed, dried and coated with conductive material, and as a result, no longer viable.

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.

Characterization of the mesB gene and expression of bacteriocins by Leuconostoc mesenteroides Y105.

Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105.

Characterisation of a new operon encoding a Zur-like protein and an associated ABC zinc permease in Listeria monocytogenes.

Metal ions uptake is mainly studied for iron, as it has often been implicated in bacterial virulence. Although Listeria monocytogenes virulence is expected to be controlled by the iron availability, little is known about such an uptake and its regulation. We describe the analysis of the first operon involved in metal ions uptake in L. monocytogenes. Its three ORFs encode respectively (1) an ABC protein, likely implicated in zinc uptake, (2) a hydrophobic membrane protein, generally associated with ABC proteins and (3) a ferric uptake regulator-like protein, that we named zinc uptake regulator, as it shows strong homologies with the zinc uptake regulator, a regulator of the zinc homeostasis in Bacillus subtilis. The expression of this operon is regulated by the zinc concentration.

In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling.

We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wild-type IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2.

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex.

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets. We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.

An improved method to obtain a single recombinant vasoactive intestinal peptide (VIP) analog.

The vasoactive intestinal peptide (VIP) is an ubiquitous peptide of great potential for applications. Development of new bioactive VIP analogs using production in recombinant E coli has been carried out in our laboratory. This work presents a new multimeric fusion protein expressing several VIP units separated by factor Xa cleavage site linkers. The steps leading from the affinity purification of the fusion protein and its processing by the factor Xa to the full characterization of the new bioactive improved VIP analog are also described.

The rpoN (sigma54) gene from Listeria monocytogenes is involved in resistance to mesentericin Y105, an antibacterial peptide from Leuconostoc mesenteroides.

To gain insight into the mode of action of mesentericin Y105, a bacteriocin bactericidal agent against Listeria monocytogenes, we undertook to identify the listerial factors mediating this susceptibility by using a genetic approach. Transposon mutants resistant to the bacteriocin were obtained. One of them corresponded to a transposon insertion in a gene (rpoN) encoding a putative protein (447 amino acids) with strong homologies to alternative transcriptional sigma54 factors, including that of Bacillus subtilis (38% identity). Complementation experiments with the wild-type rpoN gene demonstrated that the insertion in rpoN was responsible for the resistance phenotype in L. monocytogenes. Moreover, expression of the L. monocytogenes rpoN gene in an rpoN mutant strain of B. subtilis promoted transcription of a sigma54-dependent operon in the presence of the associated regulator. These results demonstrate that the L. monocytogenes rpoN gene encodes a new sigma54 factor.

Translation initiation factor IF2 of the myxobacterium Stigmatella aurantiaca: presence of a single species with an unusual N-terminal sequence.

The structural gene for translation initiation factor IF2 (infB) was isolated from the myxobacterium Stigmatella aurantiaca on a 5.18-kb BamHI genomic restriction fragment. The infB gene (ca. 3.16 kb) encodes a 1,054-residue polypeptide with extensive homology within its G domain and C terminus with the equivalent regions of IF2s from Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus, and Streptococcus faecium. The N-terminal region does not display any significant homology to other known proteins. The S. aurantiaca infB gene encodes a single protein which cross-reacted with antiserum to E. coli IF2 and was able to complement an E. coli infB mutant. The S. aurantiaca IF2 is distinguished from all other IF2s by a sequence of 160 residues near the N terminus that has an unusual composition, made up essentially of alanine, proline, valine, and glutamic acid. Within this sequence, the pattern PXXXAP is repeated nine times. Complete deletion of this sequence did not affect the factor's function in initiation of translation and even increased its capacity to complement the E. coli infB mutant.

Linear correlation between bacterial overexpression of recombinant peptides and cell light scatter.

Fusion of multiple copies of a test peptide leads to insoluble inclusion bodies. Their presence within bacteria increases either forward-angle light scattering or, to a lesser extent, right-angle light scattering. A linear correlation has been established between cell forward-angle scattering and the level of overexpression of atrial natriuretic peptide. The correlation is valid only for unlysed cells and is protein product specific.

Changes in Helicobacter pylori ultrastructure and antigens during conversion from the bacillary to the coccoid form.

In vitro, Helicobacter pylori converts from a bacillary to a full coccoid form via an intermediate U-shaped form. Organisms with a full coccoid form keep a double membrane system, a polar membrane, and invagination structures. Western blots (immunoblots) of sera from colonized patients show that some high-molecular-mass antigenic fractions are expressed only in coccoids. Conversely, fractions of 30 and 94 kDa were more intensively detected in the bacillary forms. These results suggest that (i) coccoid conversion is not a degenerative transformation and (ii) antigens specific to the coccoid forms are expressed in vivo.

Production, analysis and bioactivity of recombinant vasoactive intestinal peptide analogs.

Recombinant vasoactive intestinal polypeptide (VIP) analogs were expressed in Escherichia coli as a fusion protein containing tandemly repeated multiple copies of a synthetic VIP gene joined to glutathione S-transferase. The encoded protein contains VIP units separated by a linker peptide, potentially excisable by a double cleavage with endoprotease factor Xa and hydroxylamine. Expression of different polyVIP genes, from 1 to 32 units, was detected and the production of a 16 VIP polymer was performed. MonoVIP analogs appended by 5 or 10 amino acids at their C terminus were released by factor Xa from this polymerized product. They were then submitted to hydroxylamine cleavage to remove the linker sequence to finally obtain a recombinant VIP analog devoid of any amino acid extension. The biological activity of the recombinant polyVIP and VIP analogs was tested. Although less efficient than the natural neuropeptide, some of these components bound to VIP receptor, activated adenylate cyclase in human colonic adenocarcinoma cells and displayed a relaxation activity on guinea pig tracheal rings.