Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis.

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp.

Resistance to vancomycin permitted detection, in a culture of Streptococcus cremoris 290PC, of a contaminant gram-positive coccus. Morphological and physiological characteristics indicated that this bacterium was a strain of Leuconostoc sp., designated PO184. This strain contained four plasmid species, which were distinct from those harbored by S. cremoris 290PC. Antibiotic disk susceptibility tests indicated that Leuconostoc sp. strain PO184 was also resistant to sulfathiazole and trimethoprim and susceptible to 17 other antimicrobials. The MIC of vancomycin for this strain was greater than 2,000 micrograms/ml, and resistance did not depend on drug inactivation. Leuconostoc sp. strain PO184 produced a substance which was inhibitory to S. cremoris U134, but not to S. lactis ATCC 11454. Five other leuconostocs produced substances with antibacterial activity. Of 18 strains of Leuconostoc sp., 14 were resistant to at least 500 micrograms of vancomycin per ml, including four L. oenos strains which harbored no plasmid DNA in the 1- to 76-megadalton range. Twelve Leuconostoc sp. strains contained at least one plasmid species in this mass range. These findings are discussed from the physiological, taxonomical, and ecological standpoints and with regard to their potential applications.     

Arginine metabolism in lactic streptococci.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.     

Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.     

The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes.

Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L. lactis used for industrial fermentations, L. lactis subsp. cremoris and subsp. lactis. Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1%. Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L. lactis subsp. cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese. Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes. However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements. Fourteen new L. lactis subsp. cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.     

Genetic diversity among dairy lactococcal strains investigated by polymerase chain reaction with three arbitrary primers

Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy. It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas. The patterns from 58 isolates were analysed by UPGMA dendrograms. At a similarity level of 50%, four RAPD clusters were distinguished. Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis. The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3. Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.     

Profiling the surfacome of Staphylococcus aureus

Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life‐threatening diseases. Especially for the colonization of human tissues and the development of invasiveness, surface‐exposed proteins are of major importance. In the present studies, we optimized a proteolytic shaving approach to identify those surface‐exposed protein domains – the surfacome – of S. aureus that are accessible to extracellular bio‐macromolecules, for example in the host milieu. Subsequently, this approach was applied to define the surfacomes of four strains with different genetic backgrounds. This resulted in the identification of 96 different proteins. Surprisingly, the overlap between the surfacomes of the four different strains was below 10% and each strain displayed its own characteristic set of surface‐exposed proteins. The data were also evaluated at the peptide level and here we observed a similar phenomenon. From 190 unique peptides only five were commonly found in the four strains. Besides well known cell wall proteins, we also identified some essential proteins, several yet uncharacterized exported proteins and predicted intracellular proteins. These results show for the first time that the cell surface of different S. aureus strains is not only highly variable, but also that the displayed proteins are very heterogeneous.     

Lysogeny in Lactic Streptococci Producing and Not Producing Nisin

Eighty-seven strains of lactic streptococci (46 of Streptococcus lactis, 24 of S. diacetilactis, and 17 of S. cremoris) were tested for lysogeny; 12 S. lactis strains produced nisin. Lysogeny was found in five S. lactis strains (two of them were nisin producers) and in two S. diacetilactis strains. Four S. lactis and two S. diacetilactis lysogens liberated phages both spontaneously and after ultraviolet treatment, and one S. lactis strain liberated phages spontaneously only. No lysogens were found among the S. cremoris strains tested. An initial characterization of the lysogens and their phages was made. The lytic spectrum of some of the examined phages was very narrow (homospecific), whereas that of others was wide, including strains of the three investigated species.     

Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli.

Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins. Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides. The NmpA protein and the NmpB protein appeared to be identical by these criteria. The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles. Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein. These results indicate that the various pore proteins of E. coli K-12 fall into four different classes.     

Identification of three different plasmid-encoded restriction/modification systems in Streptococcus lactis subsp. cremoris W56

Abstract Streptococcus lactis subsp. cremoris W56 ( S. cremoris W56) is a strain partially resistant to phage attack. Derivatives which had lost either plasmid pJW563 or pJW566 no longer expressed the restriction and modification systems encoded by these plasmids. Genetic evidence for the correlation between the plasmids and the R/M systems was obtained by transformation. In addition, a third R/M system was discovered among the transformants and was shown to be encoded by pJW565. Thus, genetic evidence for at least 3 distinct R/M systems encoded by plasmids in S. cremoris W56 is presented. One of the R/M-systems showed stronger restriction of the isometric phage p2 than of the prolate phage c2. The other two systems restricted both classes of phages with equal efficiencies.     

In Lactococcus lactis subsp. cremoris SK110 protein, instead of lipoteichoic acid, reacts with the group-N-specific antiserum

The reaction between cell-surface components, isolated from two Lactococcus lactis subsp. cremoris strains, with their Group-specific antiserum were studied. No reaction between purified lipoteichoic acid and the antiserum was observed. Both strains, however, did belong to the lactococci (Group-N streptococci), as was demonstrated by the positive reaction between the antiserum and an acid- (Lancefield) or alkaline-extract. Experiments with proteolytic enzymes demonstrated the involvement of protein in the antigenic material in the latter reaction.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Proteolytic activity of Staphylococcus xylosus strains on pork myofibrillar and sarcoplasmic proteins and use of selected strains in the production of "Naples type" salami

AIMS: The aim of this study was to determine the proteolytic activities of Staphylococcus xylosus strains on sarcoplasmic and myofibrillar proteins in order to evaluate the suitability of selected strains as starter cultures in the processing of a dry fermented pork sausage. METHODS AND RESULTS: The proteolytic activity of 27 strains of Staphylococcus xylosus on sarcoplasmic and myofibrillar proteins was determined by agar plate method, o-phtaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four strains were selected for the formulation of six starter cultures to use in the production of "Naples type" salami. The proteolytic contribution of starters was determined by SDS-PAGE, comparing the protein profile of inoculated sausages with that of uninoculated sausages after 0, 15 and 33 days of ripening. The results showed that the proteolytic activity of some strains, determined by the agar plate method, were not confirmed by electrophoretic and spectrophotometric assays. In fact, of 24 strains of Staphylococcus xylosus able to hydrolyse muscle protein extracts on agar plate, only 12 strains were shown to change SDS-PAGE profile of pork proteins. The SDS-PAGE profile of sarcoplasmic proteins extracted from all sausages showed that the major changes were produced with starters S3, S4 and S5 after 15 days of ripening. Also myofibrillar proteins undergo major changes after 15 days of ripening and the protein profiles showed the same pattern in all samples, except for the sausages produced with starter S4. CONCLUSIONS: The results of this work showed that the muscle protein extracts hydrolysis test is suitable for preliminary screening of Staphylococcus xylosus strains on the basis of their proteolytic activity. However, evaluation of muscle protein hydrolysis in a food model system could then be more appropriate for selecting micro-organisms for use as starter cultures for fermented sausages. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the formulation of starter cultures for the dry fermented sausages production.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk "Viili"

"Viili," a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes.

Lactococcus lactis subsp. cremoris is widely used in the manufacture of fermented milk products. Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful. Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L. lactis subsp. cremoris from related strains. These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures. A total of 170 strains of L. lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L. lactis. Fifty-nine of these isolates also hybridized to L. lactis subsp. cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L. lactis subsp. cremoris-specific probe had the L. lactis subsp. cremoris phenotype.