A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope.

The complete nucleotide sequence of a gene located immediately upstream of the Lactococcus lactis subsp. cremoris SK11 prtP gene encoding the cell envelope-attached proteinase was determined. This gene, designated prtM, was found to be transcribed from the same promotor region as was the proteinase gene but in the opposite direction. The prtM gene directed the expression in Escherichia coli of a protein with a size similar to the expected value of 33 kilodaltons, as deduced from the nucleotide sequence data. The derived amino acid sequence of the PrtM protein indicated the presence of a consensus lipoprotein signal sequence at the N terminus, which suggested that PrtM is a lipoprotein. Plasmids containing the prtM gene, the prtP gene, or both were constructed. Expression studies of L. lactis clones containing these plasmids showed that the prtM gene encodes a trans-acting activity involved in the maturation of cell envelope-located and -secreted forms of the SK11 proteinase.     

The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3–18

Aims:  The study of proteolytic activity and examination of proteinase gene region organization in proteolytically active Lactobacillus plantarum strains from different natural sources.
Methods and Results:  A set of 37 lactobacilli was distinguished by using multiplex PCR assay. Results showed that 34 strains were Lact. plantarum and three of them were Lact. paraplantarum . The examination of proteolytic activity revealed that 28 Lact.   plantarum and two Lact.   paraplantarum hydrolyse β-casein. Further analyses of all proteolytically active Lact. plantarum with primers specific for different types of CEPs demonstrated that strain BGSJ3–18 has prtP catalytic domain as well as prtP – prtM intergenic region showing more than 95% sequence identity with the same regions present in Lact. paracasei , Lact. casei and L. lactis . No presence of prtB , prtH or prtR proteinase genes was detected in any of tested Lact. plantarum strains.
Conclusions:  One out of 28 analysed Lact. plantarum strains harbours the prtP -like gene. The other proteolytically active Lact. plantarum probably possesses a different type of extracellular proteinase(s).
Significance and Impact of the Study:  It is the first report about the presence of the prtP –like gene in Lact. plantarum , which illustrates the mobility of this gene and its presence in different species.     

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid. The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA. These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.     

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.     

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. I. Localization of the pyocin R2 gene cluster between the trpCD and trpE genes

Thirty-seven mutants defective in pyocin R2 production in the P. aeruginosa PAO strain were subjected to fine mapping of pyocin R2 genes by transduction with phage F116L. Sixteen complementation groups (designated prtA through prtP) involved in pyocin R2 production were tentatively identified by complementation tests using phage F116L. Their linkages to trpC and trpE markers and fine mapping by three point crosses demonstrated that most of the mutations (prtA through prtN) were located in between trpC and trpE, and that the prtP mutation was localized outside this major prt cluster but in the proximity of the rifA and strA region.     

Lactococcal proteinase maturation protein PrtM is a lipoprotein.

The production of enzymatically active proteinase by lactococci requires the joint presence of a proteinase gene, prtP, and a gene encoding a maturation protein, prtM. A 32-kDa protein produced by Escherichia coli upon expression of the prtM gene under the direction of the T7 RNA polymerase promoter was purified and used to obtain PrtM-specific antibodies. With these antibodies, immunogold labeling of lactococcal cells revealed that PrtM was associated with the lactococcal cell envelope. Western blot (immunoblot) analysis of whole lactococcal cells and isolated membrane vesicles indicated that PrtM was a membrane-associated protein. Radiolabeling of Lactococcus lactis with [3H]palmitic acid showed that PrtM was a lipoprotein. Partial secretion of PrtM into the culture medium was observed after Cys-24, the target residue for lipid modification, was replaced by an Ala residue by means of site-directed mutagenesis. This mutation did not affect proteinase activity.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.     

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid. The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA. These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.     

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.     

Monitoring and survival of Lactobacillus paracasei LTH 2579 in food and the human intestinal tract

A PCR-based detection system specific for Lactobacillus paracasei LTH 2579 was developed and applied to follow the fate of the strain in complex ecosystems. This strain was isolated from fruit mash and was characterised as being highly resistant to low pH and bile at concentrations as they occur in the human digestive tract. The application of the subtraction hybridisation technique permitted to identify a 235 bp chromosomal DNA fragment of strain LTH 2579. Based on this target sequence a specific PCR system was developed and combined with the species-specific PCR system for L. paracasei. This combination of PCR based detection systems was successfully applied to monitor L. paracasei LTH 2579 in fermented sausages which were inoculated with this strain (2.0 x 10(7) CFU/g) together with the strongly competitive L. sakei LTH 681 (1.0 x 10(6) CFU/g). At the time of consumption of the sausages the respective counts were 1.8 x 10(7) and 1.4 x 10(8) CFU/g. After consumption of the sausages by three volunteers L. paracasei LTH 2579 was recovered from fecal samples. The counts determined for the strain ranged between 1.2 x 10(7) and 1.5 x 10(8) CFU/g of feces. The fortuitous lactobacilli constituted a share of 5-12% of the lactobacilli in the fecal flora.     

Improved stress tolerance of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52 degrees C for 15 min). Subsequently, the groESL operon of L. paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred into Lactococcus lactis(pGRO1) and L. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54 degrees C) and lactobacilli (at 60 degrees C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.