Cloning of peptidase genes from Lactobacillus helveticus CNRZ 32

Lactic acid bacteria express a complex proteolytic enzyme system that is capable of hydrolyzing casein to amino acids. We have begun to examine the number and specificity of exopeptidases from Lactobacillus helveticus CNRZ 32. A genomic library of L. helveticus CNRZ 32 DNA fragments from a Sau3A partial digestion was constructed in Escherichia coli DH5 utilizing pJDC9. This library was screened for the presence of aminopeptidase, X-prolyl dipeptidyl aminopeptidase (X-PDAP), and dipeptidase activities by two methods. The first screening identified an aminopeptidase II (APII) and X-PDAP. The X-PDAP was determined to be present on four independent DNA inserts ranging in size from 3.5 to 7.7 kilobase pairs (kbp). EcoRI/EcoRV digests of these plasmids suggested that all inserts had 1.0 and 1.8 kbp fragments in common. The gene for APII was determined to be present on three independent DNA inserts ranging in size from 8.2 to 11.3 kbp. EcoRI digestion of these plasmids indicated that 1.2 and 1.8 kbp fragments were in common. The second screening identified an additional aminopeptidase (API), a di/tripeptidase (DTP) with prolinase activity, a broad-specificity dipeptidase (DPI), and a narrow-specificity dipeptidase (DPII). The insert size of clones expressing API, DTP, DPI, DPII were 4.8, 9.5, 5.8, and 6.3 kbp, respectively. Histochemical staining of native polyacrylamide gels from recombinant E. coli cultures demonstrated that the cloned peptidase co-migrated with native L. helveticus CNRZ 32 enzymes. Correspondence to: J. L. Steele     

Characterisation of a cell-envelope proteinase from Lactobacillus helveticus

The cell-envelope proteinase from Lactobacillus helveticus CRL 1062 was detected in the cell membrane fraction. The enzyme remained associated with the cells even after treatment with lysozyme and was not released from washed cells in absence of calcium. The proteinase was maximally active at pH 6.5–7.0 and 42°C and hydrolysed - and -caseins at different rates. Activity was inhibited (98%) by 1 mM PMSF, suggesting it was a serine-type protease.     

Plasmids from Lactobacillus helveticus: distribution and diversity among natural isolates

AIMS: To investigate the distribution and the level of diversity of extrachromosomal molecules in Lactobacillus helveticus strains in relation to their different ecological niches. METHODS AND RESULTS: The plasmid profile of 22 Lact. helveticus strains, isolated from five different Italian cheeses, was determined. Among the tested strains, there was a variable presence of plasmids: eight plasmid-free strains and the remaining with several plasmids that could be differentiated on the basis of number and molecular weight. The profiles showed between one and five plasmid bands, which size ranged between 2.3 and 31 kb. Four of these plasmids were further analyzed by restriction digestion and compared with the plasmids from Lact. helveticus ATCC 15009(T). Analyses and comparison of their primary structures and hybridization experiments revealed the presence of different DNA homology groups. CONCLUSIONS: This study indicates that within Lact. helveticus species, there is a high degree of variability in relation to the presence of plasmid molecules. Moreover, the structural diversity found among some of these plasmids allows to hypothesize the presence of different evolutionary lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on plasmid distribution and diversity should be considered as an essential component in a continuing effort to explore microbial diversity as well as to understand the real role of plasmids in the flow of genetic information in natural bacterial communities.     

l-glutamate transport in Lactobacillus helveticus

An energy source (glucose or lactose) was required for the transport of l-glutamic acid by Lactobacillus helveticus. Mg²⁺, K⁺ and Li⁺ increased its accumulation while Ca²⁺ and Na⁺ decreased it. It was inhibited by NaF, indicating that ATP may be involved in uptake. Optimum transport was at pH 7.3 and 45°C. l-Glutamic acid transport showed a high degree of stereospecificity, as neither d-glutamate nor d-aspartate were active. Proton-conducting uncouplers, like carbonyl cyanide-m-chlorophenylhydrazone, and ionophores (nigericin, monensin and gramicidin) were strongly inhibitory. These results indicate that a proton motive force may be involved in the transport of l-glutamic acid.The authors are with the Centro de Referencia para Lactobacilos, Chacabuco 145 4000 S.M. de Tucumán, Argentina and the Cátedra de Microbiologia Superior, Universidad Nacional de Tucumán, Argentina.     

Characterization of an arylesterase from Lactobacillus helveticus CNRZ32

An esterase gene (estA) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The estA gene consisted of a 558 bp open reading frame encoding a putative peptide of 21.3 kDa. Protein sequence homology searches using BLAST revealed that EstA had low amino acid sequence identity with the serine-dependent arylesterases TesI (24%) and EtpA (26%) from Escherichia coli and Vibrio mimicus, respectively. A recombinant EstA fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstA revealed that it was a serine-dependent enzyme having a monomeric Mr of 22.6-25.1 kDa. Optimum temperature, NaCl concentration and pH for EstA activity were determined to be 35-40 degrees C, 3.5% NaCl and 7.5-8.0, respectively. EstA had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). EstA hydrolysed a variety of ester compounds and preferred those with substituted phenyl alcohol and short-chain fatty acid groups. Site-directed mutagenesis suggested that the S10 and H164 residues were essential for EstA activity.     

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.     

Mapping of three plasmids from Lactobacillus helveticus ATCC 15009

A 1.4 kb DNA fragment from the chromosomal DNA of Penicillium nalgiovense was isolated which confers proteolytic activity to E. coli DH5α cells when cloned under the control of the E. coli lacZ promoter. The protein was excreted by the cells as was shown by the formation of a clearing zone in skim milk medium. A retransformation of the plasmid carrying the protease gene into P. nalgiovense leads to transformants with both increased and with nearly no proteolytic activity under neutral conditions. Southern blotting experiments revealed that the transforming plasmid had apparently integrated into the homologous locus and thereby inactivated the residual gene.     

Gene replacement in Lactobacillus helveticus.

An efficient method for gene replacement in Lactobacillus helveticus CNRZ32 was developed by utilizing pSA3 as an integration vector. This plasmid is stably maintained in CNRZ32 at 37 degrees C but is unstable at 45 degrees C. This method consisted of a two-step gene-targeting technique: (i) chromosomal integration of a plasmid carrying an internal deletion in the gene of interest via homologous recombination and (ii) excision of the vector and the wild-type gene via homologous recombination, resulting in gene replacement. By using this procedure, the chromosomal X-prolyl dipeptidyl aminopeptidase gene (pepXP) of CNRZ32 was successfully inactivated.     

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus

Abstract Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus . Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis . No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.     

Exopolysaccharide biosynthesis by Lactobacillus helveticus ATCC 15807

Exopolysaccharide (EPS) production and the activities of the enzymes involved in sugar nucleotide biosynthesis in Lactobacillus helveticus ATCC 15807 under controlled pH conditions were investigated. Batch fermentations using lactose as energy source showed higher EPS synthesis by L. helveticus ATCC 15807 at pH 4.5 with respect to pH 6.2, the enzyme -phosphoglucomutase (-PGM) being correlated with both total and specific EPS production. When glucose was used as carbon source instead of lactose, the lower EPS synthesis obtained was linked to a decrease in -PGM and galactose 1-phosphate-uridyltransferase (GalT) activities, the reduction of the latter being more pronounced. Higher EPS production by L. helveticus ATCC 15807 at the acidic constant pH of 4.5 requires that both -PGM and GalT activities are high. These enzymes are needed to synthesize UDP-glucose and UDP-galactose for supplying the corresponding monomers for EPS biosynthesis. Although differences are observed in EPS production by this strain regarding the energy source (lactose or glucose), the monomeric composition of the polymers produced is independent of the carbohydrate used. The obtained results contribute to a better understanding of the physiological factors that affect EPS biosynthesis by lactobacilli, which could help in the correct handling of the fermentation parameters within the fermented dairy industry.     

Characterization of a K+-ATPase from Lactobacillus helveticus ATCC 15009

Lactobacillus helveticus ATCC 15009 (wild-type) membrane preparations hydrolyzed Mg²⁺-ATP as a function of K⁺ concentration (2–200 mM). Mg²⁺-ATP hydrolysis by L. helveticus membranes was strongly inhibited in the absence of exogenous K⁺, while it amounted to 6 nmol ATP hydrolyzed min^–1 (mg membrane protein)^–1 at 50 mM KCl (saturating conditions) and pH 7.2. The K⁺-dependent ATPase of L. helveticus displayed a relatively high affinity for potassium ions (K _m = 800 μM) and was not affected by pretreatment of membranes with N,N’-dicyclohexylcarbodiimide. Membrane preparations were subjected to hypotonic shock to obtain a maximum yield of open profiles. The formation of a maximum level of enzyme-phosphate complex with a molecular mass of approximately 82 kDa was induced upon treatment of L. helveticus membrane preparations with low concentrations of [γ-³²P]ATP in the presence of K⁺ and La³⁺ ions and was visualized by acidic SDS-PAGE. It was concluded that L. helveticus membranes contain an inwardly directed K⁺ pump whose presence is discussed in terms of its putative role in cytoplasmic pH regulation. Received: 16 December 1996 / Accepted: 14 May 1997     

Purification and Characterization of a Dipeptidase from Lactobacillus helveticus SBT 2171

A metal-dependent dipeptidase was purified to homogeneity from a cell extract of Lactobacillus helveticus SBT 2171 by fast protein liquid chromatography. The enzyme was purified 237-fold from the extract, with a yield of 1.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 50,000. The dipeptidase hydrolyzes a range of only dipeptides. Dipeptides containing proline, glutamic acid, and aspartic acid are not hydrolyzed. The enzyme was shown to be a metalloenzyme with a pH optimum of 8.0 and a temperature optimum of 55(deg)C. Dithiol-reducing reagents exert strong inhibition on enzyme activity. Kinetic studies indicated that the enzyme has a relative average affinity for leucyl-leucine (K(infm), 0.5 mM). The negative immunoresponse of the purified enzyme with monoclonal antibodies raised against a dipeptidase from Lactococcus lactis subsp. cremoris Wg2 shows that both enzymes can be immunologically distinguished.     

Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32.

An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.     

Characterization of a Cell Membrane-Associated Proteinase from Lactobacillus helveticus CRL 581

The production of a proteinase from Lactobacillus helveticus CRL 581 was studied. The highest specific activity was found at the early exponential growth phase of cells cultured in milk. The lowest levels of proteinase were detected in MRS broth, while in the casein–yeast extract–glucose broth enzyme production increased gradually during the fermentation and reached maximal values at the stationary phase. The proteinase, found to be associated with the cell membrane fraction, hydrolyzed β-casein more rapidly than α-casein. The enzyme was not released from washed cells in the presence or absence of calcium, which suggests that the enzyme did not undergo self-digestion. Received: 28 January 1997 / Accepted: 8 March 1997