Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano Reggiano cheese

AIMS: Lactobacillus helveticus is the dominant microflora of the natural whey starters used for Parmigiano Reggiano cheese making. The aim of this work was to study the biodiversity of different strains of Lact. helveticus present in six cultures and to compare them with strains of the same species previously isolated from natural whey cultures used for Grana Padano and Provolone cheeses. METHODS AND RESULTS: Twenty different biotypes of Lact. helveticus strains were identified combining the results deriving from SDS-PAGE of cell surface proteins and PCR fingerprinting using M13 as a primer. The biotypes were present in varying amounts in the six natural whey starters and the biodiversity was demonstrated not only within the whey cultures, but also between the whey cultures. CONCLUSIONS: Lact. helveticus strains isolated from Parmigiano Reggiano whey cultures analysed by PCR M13, SDS-PAGE and RFLP were distinguishable from Lact. helveticus strains of different dairy origin, namely Grana Padano and Provolone natural whey starters. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of different Lact. helveticus biotypes seems to be related to the specific ecosystem of cheese making and may be considered as one of the elements contributing to the typicality of Parmigiano Reggiano cheese.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep. thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep, thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Genetic diversity in Swiss cheese starter cultures assessed by pulsed field gel electrophoresis and arbitrarily primed PCR

AIMS: To assess intraspecific genetic heterogeneity among commercial Swiss cheese starter culture strains of Lactobacillus helveticus, Streptococcus thermophilus and Propionibacterium freudenreichii and to compare the efficacy of two genetic typing methods. METHODS AND RESULTS: Two genetic typing methods, pulsed field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR), were used. Nine Strep. thermophilus strains revealed eight PFGE and five AP-PCR genotypes. Seventeen Lactobacillus strains yielded 16 and five genotypes by PFGE and AP-PCR, respectively. Eleven Propionibacterium strains yielded 10 PFGE genotypes. Cluster analysis of PFGE profiles generated similarity coefficients for Strep. thermophilus, Lact. helveticus and Prop. freudenreichii strains of 29.5%, 60.3%, and 30.5%, respectively. Milk acidification rates for Strep. thermophilus and Lact. helveticus were determined. CONCLUSIONS: Pulsed field gel electrophoresis is more discriminatory than AP-PCR. The Lact. helveticus group is more homogeneous than the other species examined. Strains with > 87% similarity by PFGE consistently had the same acidification rate and AP-PCR profile. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strains sold for Swiss cheese manufacture in the United States are genetically diverse. Clustering of genetically related bacteria may be useful in identifying new strains with industrially relevant traits.     

Genetic (RAPD-PCR) and technological diversities among wild Lactobacillus helveticus strains

Diversity in 25 Lactobacillus helveticus strains isolated from natural whey cultures for Argentinian hard cheese production was studied by means of RAPD-PCR patterns and technological parameters (acidifying and proteolytic activities, salt tolerance, diacetyl, H₂O₂ and slime production, phage sensitivity). In the RAPD diversity study, 10 Lact. helveticus strains from the CNRZ collection were also included.
The clustering of RAPD patterns from the Argentinian strains revealed the existence of two Lact. helveticus biotypes. Cluster 1 contained 22 strains (15 wild and seven CNRZ collection strains), Cluster 2 grouped 10 wild strains and Cluster 3 contained only three CNRZ collection strains. RAPD groups could be related to specific cheese-making characteristics (cheese plants). Analysis of technological characteristics in the Argentinian strains showed the occurrence of different natural variants. According to their capacity for growing in milk, they were classified as 'fast', 'intermediate' and 'slow' variants. Among the strains, low salt tolerance and widespread phage resistance were demonstrated. The genetic diversity (RAPD-PCR clustering) did not show any clear relationship with phenotypical diversity. Study of genetic and technological diversity allows a better characterization of wild strains belonging to Lact. helveticus .     

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

Use of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheese

AIMS: This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis. METHODS AND RESULTS: Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli. RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact. helveticus at strain level. Thirteen different Lact. helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures. Besides Lact. helveticus, Lact. delbrueckii subsp. lactis was the main microbial species detected by TTGE. CONCLUSIONS: RAPD-PCR resulted very useful in studying Lact. helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures. IMPACT OF THE STUDY: By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time.     

Partial characterization of a bacteriocin produced by Lactobacillus helveticus

AIMS: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus. METHODS AND RESULTS: The culture supernatant fluid Lact. helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa. The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action. CONCLUSION: The antimicrobial activity expressed by Lact. helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on Lact. helveticus bacteriocins. The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process.     

Isolation of cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and molecular comparison with dairy-related Lactobacillus helveticus strains

Aims:  To isolate cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and compare them with dairy-related Lactobacillus helveticus strains using molecular typing methods.
Methods and Results:  The number of thermophilic bacteria in seven commercial cheeses manufactured with mesophilic starters was estimated to be <10 CFU g⁻¹. Implementation of an enumeration step in the isolation method made it possible to isolate one thermophilic strain from each of five of seven cheeses. Comparing repetitive sequence PCR (rep-PCR) profiles of the isolates with dairy-related Lact. helveticus strains indicated that one isolate was a Lact. helveticus . Partial sequencing of 16S rRNA confirmed this, and the remaining four strains were identified as Lactobacillus delbrueckii , Lactobacillus fermentum and Enterococcus faecium . The rep-PCR profile of the isolated Lact. helveticus was identical to the rep-PCR profile of the Lact. helveticus adjunct culture used in the specific cheese, but their pulsed field gel electrophoresis profiles differed slightly.
Conclusion:  It was possible to isolate cultivable thermophilic bacteria from ripened cheeses manufactured with mesophilic starter and thermophilic adjunct cultures by using an enumeration step.
Significance and Impact of the Study:  Isolation of cultivable thermophilic bacteria from ripened cheeses made with mesophilic starters offers an original source for new dairy-relevant cultures.     

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus

AIMS: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with alpha (s1)-casein (f 1-23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward alpha(s1)-casein (f 1-23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and alpha(s1)-casein (f 1-23) cleavage profiles. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: Using the alpha(s1)-CN (f 1-23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.     

Recombinant Lactococcus starters as a potential source of additional peptidolytic activity in cheese ripening

AIMS: The aim of this study was to modulate the lactococcal proteolytic system for enhancement of the cheese ripening process. METHODS AND RESULTS: The genes encoding PepN, PepC, PepX and PepI peptidases of a highly proteolytic Lactobacillus helveticus strain were transferred into Lactococcus lactis in a food-grade cloning system. A comparison of the relative peptidase activities from the transformants with those from the untransformed host, determined in the conditions of maturing cheese, showed that an increase in peptidase activity could be achieved by introducing a selected peptidase gene from Lact. helveticus into L. lactis. CONCLUSIONS: Recombinant L. lactis starter strains, carrying a peptidase gene from Lact. helveticus, may have an important contribution to the proteolysis of maturing cheese by producing an additional peptidolytic enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be of importance in shortening the ripening period and production of special cheeses (e.g. reduced-fat cheeses) with improved characteristics.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.     

Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds

AIMS: The ability of 31 Lactobacillus plantarum strains to adhere to biological matrixes was evaluated, and the molecules involved in adherence were studied. METHODS AND RESULTS: Mucin, basement membrane proteins and Caco-2 cells were used in adhesion tests. These in vitro assays, together with a yeast agglutination test, were found to be discriminative for screening Lact. plantarum strains for adhesion. Some strains, such as 299v, CBE, BMCM12, Col4S and T25, were shown to possess interesting adhesion properties in at least two models. The adhesion of these strains was strongly inhibited when the bacterial cells were pretreated with trypsin. Lithium chloride and methyl-alpha-D-mannoside also inhibited adhesion to a lower extent. CONCLUSIONS: The adhesion of Lact. plantarum depends on both the model and the strain used. The chemical and enzymatic pretreatments applied to the bacterial cells suggested that lectin-like adhesins and other proteinaceous cell-surface structures are involved in adhesion of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We found a great diversity in the adhesion properties between Lact. plantarum strains. Based upon the adhesive property of these strains interesting candidates were identified, that will undergo further study as potential probiotics.     

Cell-wall protein profiles of dairy thermophilic lactobacilli

SDS–PAGE fingerprinting of cell-wall proteins extracted from 119 strains belonging to different species of lactic acid bacteria have been compared. The method of extraction and electrophoretic separation utilized in this work was found to be a reliable and rapid way for characterizing thermophilic lactobacilli species and strains. A protein of approximately 50 kDa was found to be characteristic for all the Lact. helveticus strains, and two cell-wall proteins of about 20 and 30 kDa were typical of the species Lact. delbrueckii , but the discrimination between the subspecies lactis and bulgaricus was not possible by the electrophoretic technique used. The other thermophilic species studied in this work presented cell-wall protein patterns that permitted their differentiation from both Lact. helveticus and Lact. delbrueckii .     

Plasmids, Lactic Acid Production, and N-Acetyl-d-Glucosamine Fermentation in Lactobacillus helveticus subsp. jugurti

Two lactobacillus strains, Lactobacillus helveticus subsp. jugurti S13-8 and L. helveticus subsp. jugurti S36-2, were examined for the presence of plasmids. Plasmids of 16.45, 13.03, and 11.83 kilobases (kb) were found in the first, low lactic acid-producing strain; their function is not presently known. A single plasmid species of 13.17 kb was found in the second, high lactic acid-producing strain. This plasmid was found to be associated with lactic acid production and N-acetyl-d-glucosamine fermentation.     

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.     

Plasmid diversity within the genus Chlamydia

Examination of 12 Chlamydia psittaci strains recovered from nine different host species (three avian and six mammalian) revealed the presence of a 7.5 kb plasmid in all isolates except two ovine abortion strains, the human strain IOL207 and the Cal 10 strain. Restriction mapping analysis distinguished four different plasmids that were associated with avian, feline, equine and guinea-pig C. psittaci isolates, respectively. The restriction maps of these four C. psittaci plasmid types all differed from that of the plasmid recovered from C. trachomatis L2/434. Despite this plasmid diversity, which is likely to be of taxonomic importance, all four plasmids identified within the species C. psittaci were found to share some sequence homology, which was mapped to two separate regions in the plasmid molecules. One region, which showed a high degree of homology between C. psittaci plasmids and also detectable homology with the C. trachomatis plasmid, may represent a common replication control region for plasmids of this genus.     

Comparative evaluation of plasmid profiling and ribotyping in the analysis of Lactobacillus plantarum strain heterogeneity in silage

F. DUFFNER AND M. O'CONNELL. 1995. Seventy-two Lactobacillus plantarum isolates were recovered from six uninoculated grass silages for the purposes of firstly evaluating the usefulness of (1) restriction endonuclease digestion of total genomic DNA, (2) plasmid profiling and (3) ribotyping in Lact. plantarum strain differentiation and secondly, examining the strain heterogeneity in well preserved silage.
The three methods for differentiation were applied to 72 of the isolates and allowed at least 32 different strains to be identified. Twenty-five different plasmid profiles were detected (26 if the absence of plasmids is included as a profile). Ribotyping with Eco RI identified only 11 patterns among the silage isolates. A variety of restriction enzymes was screened to increase the sensitivity of ribotyping to detect strain differences and Bam HI was used successfully for this purpose, differentiating all of the strains tested.
Two dominant strains (I and II) were identified in one particular silage, comprising 47% and 17% respectively of the isolates, while strains III and V comprised 37% and 25% of the Lact. plantarum population isolated from another of the silages.     

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship

Abstract Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analyses and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80–90% identity between the different plasmids identified so far in L. helveticus . Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.     

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of Lactobacillus manihotivorans , a new Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of Lact. manihotivorans strains LMG 18010^T and LMG 18011 was measured and compared with electroporability of Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For Lact. manihotivorans strain LMG 18010^T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture; better electroporation was obtained without air. The presence of MgCl₂ in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with Lact. manihotivorans strain LMG 18010^T and the plasmids pLZ12 and pGK13, whereas Lact. manihotivorans strain LMG 18011 was transformable with plasmids pLP825 and pLZ12, with different electroporation conditions.