Gene_locus Report for: lache-pepx

Mobile genetic elements are major contributing factors to the generation of genetic diversity in prokaryotic organisms. For example, insertion sequence (IS) elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements. The complete genome sequence of the cheese culture Lactobacillus helveticus DPC 4571 was determined and revealed significant conservation compared to three nondairy gut lactobacilli. Despite originating from significantly different environments, 65 to 75% of the genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. However, the primary distinguishing feature was 213 IS elements in the DPC 4571 genome, 10 times more than for the other lactobacilli. Moreover, genome alignments revealed an unprecedented level of genome stability between these four Lactobacillus species, considering the number of IS elements in the L. helveticus genome. Comparative analysis also indicated that the IS elements were not the primary agents of niche adaptation for the L. helveticus genome. A clear bias toward the loss of genes reported to be important for gut colonization was observed for the cheese culture, but there was no clear evidence of IS-associated gene deletion and decay for the majority of genes lost. Furthermore, an extraordinary level of sequence diversity exists between copies of certain IS elements in the DPC 4571 genome, indicating they may represent an ancient component of the L. helveticus genome. These data suggest a special unobtrusive relationship between the DPC 4571 genome and its mobile DNA complement.

Lactobacillus helveticus CNRZ32 possesses an Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designated pepX, from Lb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bp pepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88,111 Da. The gene shows significant sequence identity with sequenced pepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction of pepX into Lactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) or pIL253 (a high-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction of pepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that the pepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative. No difference in growth rate or acid production was observed between CNRZ32 and its PepX-negative derivative in MRS. However, the CNRZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.

The X-prolyl dipeptidyl aminopeptidase gene (pepX) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 90.6 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with delta G of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 90.6 kDa protein showed 49.3, 49.4 and 77.7% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lactobacillus delbrueckii subsp. lactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6.5 and 45 degrees C. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding DTT, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.

Prolyl aminodipeptidase (PepX) is an enzyme that hydrolyzes peptide bonds from the N-terminus of substrates when the penultimate amino-acid residue is a proline. Prolyl peptidases are of particular interest owing to their ability to hydrolyze food allergens that contain a high percentage of proline residues. PepX from Lactobacillus helveticus was cloned and expressed in Escherichia coli as an N-terminally His-tagged recombinant construct and was crystallized by hanging-drop vapor diffusion in a phosphate buffer using PEG 3350 as a precipitant. The structure was determined at 2.0 A resolution by molecular replacement using the structure of PepX from Lactococcus lactis (PDB entry 1lns) as the starting model. Notable differences between the L. helveticus PepX structure and PDB entry 1lns include a cysteine instead of a phenylalanine at the substrate-binding site in the position which confers exopeptidase activity and the presence of a calcium ion coordinated by a calcium-binding motif with the consensus sequence DX(DN)XDG.

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 microkat(H-Ala-Pro-pNA) L(-1), which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 microkat(H-Ala-pNA) L(-1). After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50 degrees C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.

Lactobacillus amylovorus is a common member of the normal gastrointestinal tract (GIT) microbiota in pigs. Here, we report the genome sequence of L. amylovorus GRL1112, a porcine feces isolate displaying strong adherence to the pig intestinal epithelial cells. The strain is of interest, as it is a potential probiotic bacterium.

Lactobacillus amylovorus is a common member of the beneficial microbiota present in the pig gastrointestinal tract. Here, we report the genome sequence of the surface layer (S-layer) protein-carrying and potentially probiotic strain L. amylovorus GRL1118, which was isolated from porcine ileum and which shows strong adherence to pig intestinal epithelial cells.

Lactobacillus acidophilus 30SC has been isolated from swine intestines and considered a probiotic strain for dairy products because of its ability to assimilate cholesterol and produce bacteriocins. Here, we report the complete genome sequence of Lactobacillus acidophilus 30SC (2,078,001 bp) exhibiting strong acid resistance and enhanced bile tolerance.

Lactobacillus helveticus strain H10 was isolated from traditional fermented milk in Tibet, China. We sequenced the whole genome of strain H10 and compared it to the published genome sequence of Lactobacillus helveticus DPC4571.

Mobile genetic elements are major contributing factors to the generation of genetic diversity in prokaryotic organisms. For example, insertion sequence (IS) elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements. The complete genome sequence of the cheese culture Lactobacillus helveticus DPC 4571 was determined and revealed significant conservation compared to three nondairy gut lactobacilli. Despite originating from significantly different environments, 65 to 75% of the genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. However, the primary distinguishing feature was 213 IS elements in the DPC 4571 genome, 10 times more than for the other lactobacilli. Moreover, genome alignments revealed an unprecedented level of genome stability between these four Lactobacillus species, considering the number of IS elements in the L. helveticus genome. Comparative analysis also indicated that the IS elements were not the primary agents of niche adaptation for the L. helveticus genome. A clear bias toward the loss of genes reported to be important for gut colonization was observed for the cheese culture, but there was no clear evidence of IS-associated gene deletion and decay for the majority of genes lost. Furthermore, an extraordinary level of sequence diversity exists between copies of certain IS elements in the DPC 4571 genome, indicating they may represent an ancient component of the L. helveticus genome. These data suggest a special unobtrusive relationship between the DPC 4571 genome and its mobile DNA complement.

Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.

AIMS: The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression. METHODS AND RESULTS: The activities were evaluated by the hydrolysis of beta-naphthylamide substrates for both whole and mechanically disrupted cells of L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, collected from both the exponential and the stationary growth phase. In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells. The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg-betaNa, Lys-betaNa and Leu-betaNa. The lowest activity was observed for Pro-betaNa. CONCLUSIONS: It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity. The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences of peptidase activities in L. helveticus and L. delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening.

To evaluate the contribution of intracellular peptidases to the growth of the 14-amino-acid (aa) auxotroph Lactobacillus helveticus CNRZ32, single- and multiple-peptidase-deletion mutants were constructed. Two broad-specificity aminopeptidases (PepC and PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) were inactivated through successive cycles of chromosomal gene replacement mutagenesis. The inactivation of all three peptidases in JLS247 ((Delta)pepC (Delta)pepN (Delta)pepX) did not affect the growth rate in amino acid-defined medium. However, the peptidase mutants generally had decreased specific growth rates when acquisition of amino acids required hydrolysis of the proteins in milk, the most significant result being a 73% increase in generation time for JLS247. The growth rate deficiencies in milk were overcome by amino acid supplements with some specificity to each of the peptidase mutants. For example, milk supplementation with Pro resulted in the most significant growth rate increase for (Delta)pepX strains and a 7-aa supplement (Asn, Cys, Ile, Pro, Ser, Thr, and Val) resulted in a JLS247 growth rate indistinguishable from that of the wild type. Our results show that characterization of the activities of the broad-specificity aminopeptidases had little predictive value regarding the amino acid supplements found to enhance the milk growth rates of the peptidase mutant strains. These results represent the first determination of the physiological roles with respect to specific amino acid requirements for peptidase mutants grown in milk.

X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactobacillus helveticus IF03809 expressed nearly full activity under high salt conditions, such as 2 M NaCl. We cloned and sequenced the pepX gene for X-PDAP. The calculated M, of deduced X-PDAP (803 amino acids) was 90,847 and the protein was distantly related (35 to 44% identity) to known X-PDAPs of Lactobacillus sp. including L. helveticus CNRZ32 (40% identity). Native and recombinant X-PDAP were purified to homogeneity from both L. helveticus IF03809 and Escherichia coli DH5alpha harboring the pepX gene on a plasmid, respectively. The native enzyme appeared to be a dimer of 220 kDa, as estimated by gel filtration column chromatography. It hydrolyzed an X-prolyl-linkage, but not prolyl- or X-prolyl-X-peptide bonds, and tolerated up to 2 M NaCl as well as some other chlorides of monovalent cations. Determination of the flanking sequences revealed two divergent genes. The upstream region of the pepX gene encodes oppA gene for a putative oligopeptide permease, while the downstream region encodes tnp gene specifying a possible transposase of the IS3 family. The oppA gene shares a 176 bp-promoter region with pepX in the intergenic region, implying a relationship between this oligopeptide transport system and X-PDAP.

Lactobacillus helveticus CNRZ32 possesses an Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designated pepX, from Lb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bp pepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88,111 Da. The gene shows significant sequence identity with sequenced pepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction of pepX into Lactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) or pIL253 (a high-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction of pepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that the pepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative. No difference in growth rate or acid production was observed between CNRZ32 and its PepX-negative derivative in MRS. However, the CNRZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.

The X-prolyl dipeptidyl aminopeptidase gene (pepX) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 90.6 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with delta G of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 90.6 kDa protein showed 49.3, 49.4 and 77.7% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lactobacillus delbrueckii subsp. lactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6.5 and 45 degrees C. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding DTT, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.