Metabolic engineering of acetaldehyde production by Streptococcus thermophilus.

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.

Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans.

A stable isotope dilution method is described that allows measurement of cholic acid (CA) kinetics, that is, pool size, fractional turnover rate (FTR), and synthesis rate in mice, rats, and humans. Decay of administered [2,2,4,4-2H4]CA enrichment was measured in time in 50-microl plasma samples by gas-liquid chromatography/electron capture negative chemical ionization-mass spectrometry, applying the pentafluorobenzyl-trimethylsilyl derivative. The kinetic data expressed species-dependent differences. The CA pool sizes were 16.8 +/- 2.1, 10.6 +/- 1.2, and 2.4 +/- 0.7 micromol/100 g body weight for mice, rats, and humans, respectively. The FTR values were 0.44 +/- 0.03, 0.88 +/- 0.10, and 0.46 +/- 0.14 per day for mice, rats, and humans. The corresponding synthesis rates were 7.3 +/- 1.6, 9.3 +/- 0.1, and 1.0 +/- 0.2 micromol/100 g body weight per day. The human data agreed well with literature data obtained by conventional isotope dilution techniques. For rats and mice these are the first reported isotope dilution data. The method was validated by confirmation of isotopic equilibrium between biliary CA and plasma CA in the rat. Its applicability was demonstrated by the observation of increased CA FTR and CA synthesis rate in rats fed cholestyramine, which is known to increase fecal bile acid excretion. The presented stable isotope dilution method enables the determination of CA kinetic parameters in small plasma samples. The method can be applied in unanesthetized rodents with an intact enterohepatic circulation and may also be valuable when studying the development of human neonatal bile acid kinetics.

Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli.

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization.

A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (bsh) in a member of the genus Bifidobacterium. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of B. longum BSH is between 5 and 7, and the temperature optimum is 40 degrees C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH of B. longum can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the K(m) values. The nucleotide sequence of bsh was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of Bacillus sphaericus were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that bsh is part of an operon containing at least two genes, bsh and glnE (GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from B. longum SBT2928 cultures and characterized. These mutants had point mutations that inactivated bsh by premature termination, frameshift, or amino acid exchange.

Purification and characterization of bovine pancreatic bile salt-activated lipase.

An enzyme with lipase and esterase activity was purified from bovine pancreas. Furthermore, a non-radioactive lipase assay was developed which is 100 times more sensitive than the conventional methods and allowed the characterization of the lipase activity of the enzyme. The lipase activity increased 42 times in the presence of 10 mM sodium taurocholate, which for the first time provides direct evidence that a bile salt-activated lipase (bp-BAL) was isolated from bovine pancreas. This conclusion is further supported by the fact that the N-terminal amino acid sequence of this lipase/esterase is 88% homologous to human milk BAL and human pancreatic BAL. Staining with various lectins showed that bp-BAL is a glycoprotein which contains fucose residues. Previously from bovine pancreas a lysophospholipase has been purified and a gene was cloned and sequenced encoding an enzyme with cholesterol esterase/lysophospholipase activity. Comparison of the N-terminal amino acid sequence of bp-BAL with the deduced amino acid sequence of the latter revealed that they are identical. Furthermore, the molecular weight of the purified bp-BAL of 63,000, as estimated by SDS-PAGE, is very similar to that of the purified lysophospholipase (65,000) and to the theoretical molecular weight of 65,147 of the cholesterol esterase/lysophospholipase. These data suggest that these three enzymes are one and the same.

Screening of lactic acid bacteria for bile salt hydrolase activity.

Bile salt hydrolysis is an important metabolic reaction in the bile salt metabolism of mammals. This reaction has a facilitating effect for bile salt excretion but can also be involved in various illnesses. In recent years interest has increased to use bile salt hydrolysis to influence the cholesterol metabolism of humans and farm animals. To understand the distribution and range of bile salt hydrolase activity in lactic acid bacteria, we screened more than 300 strains of the genera Bifidobacterium and Lactobacillus and the species Lactococcus lactis, Leuconostoc mesenteroides, and Streptococcus thermophilus. Results obtained for 273 strains showed that bile salt hydrolase activity is common in Bifidobacterium and Lactobacillus but absent in L. lactis, Leu. mesenteroides, and S. thermophilus. Nearly all bifidobacteria species and strains have bile salt hydrolase activity, whereas this activity can only be found in selected species of lactobacilli. A strong correlation can be observed between the habitat of a genus or species and the presence of bile salt hydrolase activity. Most often bile salt hydrolase activity is found in strains that have been isolated from the intestines or from feces from mammals--an environment rich in conjugated and unconjugated bile acids. Strains and species from other habitats like milk or vegetables--environments from which bile salts are absent--do normally not have bile salt hydrolase activity. In two independent assays, we established that bile salt hydrolase activity in bifidobacteria is, in general, much higher than in lactobacilli.

Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363.

The gene pepV, encoding a dipeptidase from Lactococcus lactis subsp. cremoris MG1363, was identified in a genomic library in pUC19 in a peptidase-deficient Escherichia coli strain and subsequently sequenced. PepV of L. lactis is enzymatically active in E. coli and hydrolyzes a broad range of dipeptides but no tri-, tetra-, or larger oligopeptides. Northern (RNA) and primer extension analyses indicate that pepV is a monocistronic transcriptional unit starting 24 bases upstream of the AUG translational start codon. The dipeptidase of L. lactis was shown to be similar to the dipeptidase encoded by pepV of L. delbrueckii subsp. lactis, with 46% identity in the deduced amino acid sequences. A PepV-negative mutant of L. lactis was constructed by single-crossover recombination. Growth of the mutant strain in milk was significantly slower than that of the wild type, but the strains ultimately reached the same final cell densities.

A general system for generating unlabelled gene replacements in bacterial chromosomes.

A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

The proteolytic systems of lactic acid bacteria.

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin).

A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus. The recombinant enzyme was expressed to high levels in Escherichia coli (approximately 10 mg/liter of culture) and purified to homogeneity in a two-step procedure. A number of peptides were studied as substrates for the enzyme. The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A-17, and alpha-neo-endorphin. In addition the enzyme hydrolyzes bradykinin, substance P, beta-endorphin, ACTH, and VIP. Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates. As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher kcat/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine. Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine. This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme. Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme. These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme.

Fate of peptides in peptidase mutants of Lactococcus lactis.

The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN, pepC, pepO, pepX and pepT were deleted. Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu-Gly-Gly, Gly-Phe-Leu, Leu-Gly-Pro, Ala-Pro-Leu and Gly-Leu-Gly-Leu, respectively, indicating that no other peptidases are present to release the essential amino acid Leu. In these mutants, peptides accumulate intracellularly, demonstrating that peptides are translocated as whole entities prior to degradation. The mutant lacking all five peptidases could still grow on Gly-Leu and Tyr-Gly-Gly-Phe-Leu, which confirmed the presence of a dipeptidase and led to the identification of an unknown PepO-like endopeptidase. These studies have also shown that the general aminopeptidases PepN, PepC and PepT have overlapping but not identical specificities and differ in their overall activity towards individual peptides. In contrast, PepX has an unique specificity, because it is the only enzyme which can efficiently degrade Ala-Pro-Leu. The concerted action of peptidases in the breakdown of particular peptides revealed how these substrates are utilized as sources of nitrogen.

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.

Novel insertion sequence-like element IS982 in lactococci.

A novel insertion sequence-like (IS) element, designated IS982, was found on the lactose plasmid, pSK11L, from Lactococcus lactis subsp. cremoris SK11 and was located between the origin of replication and the oligopeptide transport gene cluster. The 1003-base pair (bp) IS982 was flanked by 18-bp perfect inverted repeats. IS982 contained an open reading frame encoding a putative transposase of 296 amino acids. An almost identical IS-like element (99% DNA sequence identity) was cloned and partially sequenced from the chromosome of Lactococcus lactis subsp. cremoris Wg2 with 17-bp perfect inverted repeats. Southern analysis indicated that in 12 lactococcal strains examined, IS982 was present with copy numbers ranging from 1 to at least 20. IS982 displayed sequence homology to the putative IS element RSBst-alpha from Bacillus stearothermophilus CU21, IS982 and RSBst-alpha were not related to other known insertion sequences and may represent a new family of IS elements.

Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant.

The gene encoding a tripeptidase (pepT) of Lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the deduced amino acid sequences. L. lactis PepT was enzymatically active in Escherichia coli and allowed growth of a peptidase-negative leucine-auxotrophic E. coli strain by liberation of Leu from a tripeptide. Using a two-step integration-excision system, a pepT-negative mutant of L. lactis was constructed. No differences between the growth of the mutant and that of the wild-type strain in milk or in chemically defined medium with casein as the sole source of essential amino acids were observed.

Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase.

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.