Role of cystathionine beta-lyase in catabolism of amino acids to sulfur volatiles by genetic variants of Lactobacillus helveticus CNRZ 32

Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine beta-lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine.     

Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase

Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.     

Genetic Characterization and Physiological Role of Endopeptidase O from Lactobacillus helveticus CNRZ32

A previously identified insert expressing an endopeptidase from a Lactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) from Lactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticus CNRZ32 in amino acid defined or skim milk medium.     

Dissimilation of methionine in cell suspension cultures from Catharanthus roseus L.

Plant cell suspension cultures from Catharanthus roseus were investigated for their capability to dissimilate methionine or its analogs in order to reutilize the sulphane group for cysteine biosynthesis. Three steps have been described as prerequisites of this process: (a) oxidative degradation by the amino-acid oxidase of methionine giving rise to methanethiol production; (b) demethylation by methyltransferases leading to homocysteine and S-methylmethionine (c) replacement of the homocysteine sulphane sulphur by alkylthiol yielding methionine and free hydrogen sulphide. A reversal of the cystathionine pathway as a source of cysteine was ruled out because the cells lack cystathionine γ-lyase. The absence of this enzyme is compensated by the S-alkyl exchange of homocysteine with methylmercaptan. Hydrogen sulphide thus liberated is used for de novo synthesis of cysteine. The complete pathway can be catalyzed by the constitutive set of enzymes present in the higher plant.     

Pathways of Assimilative Sulfur Metabolism in Pseudomonas putida

Cysteine and methionine biosynthesis was studied in Pseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine gamma-synthase and cystathionine beta-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine gamma-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine gamma-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.     

Phenotypic and Genotypic Analysis of Amino Acid Auxotrophy in Lactobacillus helveticus CNRZ 32

The conversion of amino acids into volatile and nonvolatile compounds by lactic acid bacteria in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma. Because amino acid breakdown by microbes often entails the reversible action of enzymes involved in biosynthetic pathways, our group investigated the genetics of amino acid biosynthesis in Lactobacillus helveticus CNRZ 32, a commercial cheese flavor adjunct that reduces bitterness and intensifies flavor notes. Most lactic acid bacteria are auxotrophic for several amino acids, and L. helveticus CNRZ 32 requires 14 amino acids. The reconstruction of amino acid biosynthetic pathways from a draft-quality genome sequence for L. helveticus CNRZ 32 revealed that amino acid auxotrophy in this species was due primarily to gene absence rather than point mutations, insertions, or small deletions, with good agreement between gene content and phenotypic amino acid requirements. One exception involved the phenotypic requirement for Asp (or Asn), which genome predictions suggested could be alleviated by citrate catabolism. This prediction was confirmed by the growth of L. helveticus CNRZ 32 after the addition of citrate to a chemically defined medium that lacked Asp and Asn. Genome analysis also predicted that L. helveticus CNRZ 32 possessed ornithine decarboxylase activity and would therefore catalyze the conversion of ornithine to putrescine, a volatile biogenic amine. However, experiments to confirm ornithine decarboxylase activity in L. helveticus CNRZ 32 by the use of several methods were unsuccessful, which indicated that this bacterium likely does not contribute to putrescine production in cheese.     

Molecular and Functional Analyses of the metC Gene of Lactococcus lactis, Encoding Cystathionine β-Lyase

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via α,γ elimination to form volatile aroma compounds.     

Cloning,characterization and insertional inactivation of theLactobacillus helveticus D(−) lactate dehydrogenase gene

A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD⁺ oxidoreductase, EC 1.1.1.28] fromLactobacillus helveticus CNRZ32 was identified from a genomic library by complementation ofEscherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kbDraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-) LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci orLactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative ofL. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.     

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine

Aims:  To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma.
Methods and Results:  Probiotic strains ( Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H₂S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs ( P  < 0·05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses.
Conclusions:  Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species.
Significance and Importance of the Study:  This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.     

Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine beta-lyase

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine beta-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an alpha, gamma elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via alpha, gamma elimination to form volatile aroma compounds.     

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

Properties of Recombinant Staphylococcus haemolyticus Cystathionine β‐Lyase (metC) and Its Potential Role in the Generation of Volatile Thiols in Axillary Malodor

Enzymes implicated in cysteine and methionine metabolism such as cystathionine β‐lyase (CBL; EC 4.4.1.8), a pyridoxal‐5′‐phosphate (PLP)‐dependent carbon–sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC‐cystathionine β‐lyase from the axillary‐isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect β‐elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the β‐lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme.     

Comparative High-Density Microarray Analysis of Gene Expression during Growth of Lactobacillus helveticus in Milk versus Rich Culture Medium

Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.     

Metabolism of Sulfur Compounds in Bacillus subtilis during Sporulation

Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.

Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.

It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine.     

Methionine biosynthesis in higher plants: biochemical and molecular characterization of the transsulfuration pathway enzymes

In plants, the transfer of the sulfur atom between cysteine and homocysteine, the direct precursor of methionine, is ensured by two chloroplastic enzymes, cystathionine γ-synthase and cystathionine β-lyase. These proteins have been purified to homogeneity from spinach chloroplasts and their biochemical properties determined. Cystathionine γ-synthase and cystathionine β-lyase are tetramers and are typical pyridoxal 5′-phosphate-dependent proteins. These enzymes are targets for the potent inhibitors of methionine synthesis that are lethal for plants. An Arabidopsis thaliana cDNA encoding chloroplastic cystathionine β-lyase was isolated by functional complementation of a bacterial mutant and cloned in a pET expression vector in order to transform Escherichia coli cells. Preliminary observations of the active site of the purified recombinant enzyme have been performed by characterization of the interaction between i) pyridoxal 5′-phosphate and the polypeptide chain, and ii) the active site-directed inhibitor aminoethoxyvinylglycine and the bound cofactor. This study will be developed further by crystallographic analyses.     

Production of methanethiol from methionine by Brevibacterium linens CNRZ 918

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.     

YtjE from Lactococcus lactis IL1403 Is a C-S lyase with alpha, gamma-elimination activity toward methionine

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.     

Comparison of the effects of buthioninesulfoximine and phorone on the metabolism of sulfur-containing amino acids in rat liver

Alterations in hepatic transsulfuration reactions were determined in rats treated with a glutathione-depleting agent. A dose of l-buthionine-(S, R)-sulfoximine decreased hepatic methionine, cysteine, S-adenosylmethionine, and glutathione levels rapidly. Methionine adenosyltransferase and γ-glutamylcysteine lygase activities were decreased transiently, but significantly. The activity of cysteine dioxygenase was increased, resulting in an elevation of hypotaurine and taurine concentrations. Administration of phorone reduced hepatic glutathione and cysteine similarly, but S-adenosylmethionine concentrations were elevated for as long as 72 h. Hepatic methionine adenosyltransferase, cystathionine β-synthase, cystathionine γ-lyase, and γ-glutamylcysteine lygase activities were all increased but cysteine dioxygenase activity and taurine generation were markedly depressed. The results show that a decrease in hepatic GSH induces profound changes in sulfur amino acid metabolomics, which would subsequently influence various cellular processes. It is suggested that the change in hepatic levels of sulfur-containing substances and its physiological significance should be considered when a glutathione-depleting agent is utilized in biological experiments.     

Alterations in the metabolomics of sulfur-containing substances in rat kidney by betaine

Earlier studies have shown that betaine administration may modulate the metabolism of sulfur amino acids in the liver. In this study, we determined the changes in the metabolomics of sulfur-containing substances induced by betaine in the kidney, the other major organ actively involved in the transsulfuration reactions. Male rats received betaine (1 %) in drinking water for 2 weeks before killing. Betaine intake did not affect betaine–homocysteine methyltransferase activity or its protein expression in the renal tissue. Expression of methionine synthase was also unchanged. However, methionine levels were increased significantly both in plasma and kidney. Renal methionine adenosyltransferase activity and S-adenosylmethionine concentrations were increased, but there were no changes in S-adenosylhomocysteine, homocysteine, cysteine levels or cystathionine β-synthase expression. γ-Glutamylcysteine synthetase expression or glutathione levels were not altered, but cysteine dioxygenase and taurine levels were decreased significantly. In contrast, betaine administration induced cysteine sulfinate decarboxylase and its metabolic product, hypotaurine. These results indicate that the metabolomics of sulfur-containing substances in the kidney is altered extensively by betaine, although the renal capacity for methionine synthesis is unresponsive to this substance unlike that of the liver. It is suggested that the increased methionine availability due to an enhancement of its uptake from plasma may account for the alterations in the metabolomics of sulfur-containing substances in the kidney. Further studies need to be conducted to clarify the physiological/pharmacological significance of these findings.