Bioprospecting of a Metschnikowia pulcherrima Indigenous Strain for Chasselas Winemaking in 2022 Vintage.

Interest in Metschnikowia (M.) pulcherrima is growing in the world of winemaking. M. pulcherrima is used both to protect musts from microbial spoilage and to modulate the aromatic profile of wines. Here, we describe the isolation, characterization, and use of an autochthonous strain of M. pulcherrima in the vinification of Chasselas musts from the 2022 vintage. M. pulcherrima was used in co-fermentation with Saccharomyces cerevisiae at both laboratory and experimental cellar scales. Our results showed that M. pulcherrima does not ferment sugars but has high metabolic activity, as detected by flow cytometry. Furthermore, sensory analysis showed that M. pulcherrima contributed slightly to the aromatic profile when compared to the control vinifications. The overall results suggest that our bioprospecting strategy can guide the selection of microorganisms that can be effectively used in the winemaking process.

Selective enrichment of the raw milk microbiota in cheese production: Concept of a natural adjunct milk culture.

In cheese production, microorganisms are usually added at the beginning of the process as primary starters to drive curd acidification, while secondary microorganisms, with other pro-technological features important for cheese ripening, are added as selected cultures. This research aimed to investigate the possibilities of influencing and selecting the raw milk microbiota using artisanal traditional methods, providing a simple method to produce a natural supplementary culture. We investigated the production of an enriched raw milk whey culture (eRWC), a natural adjunct microbial culture produced from mixing an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment protocols were tested: heat treatment before incubation, heat treatment plus salt addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of 1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during cultures' preparation was evaluated through the determination of colony forming units on selective growth media, and next-generation sequencing (16S rRNA gene amplicon sequencing). The enrichment step increased the streptococci and lactobacilli but reduced microbial richness and diversity of the eRMs. Although the lactic acid bacteria viable count was not significantly different between the eRWCs, they harbored higher microbial richness and diversity than NWC. Natural adjunct cultures were then tested in cheese making trials, following the microbial development, and assessing the chemical quality of the 120 d ripened cheeses. The use of eRWCs slowed the curd acidification in the first hours of cheese making but the pH 24 h after production settled to equal values for all the cheeses. Although the use of diverse eRWCs contributed to having a richer and more diverse microbiota in the early stages of cheese making, their effect decreased over time during ripening, showing an inferior effect to the raw milk microbiota. Even if more research is needed, the optimization of such a tool could be an alternative to the practice of isolating, geno-pheno-typing, and formulating mixed-defined-strain adjunct cultures that require knowledge and facilities not always available for artisanal cheese makers.

Production of Putrescine and Cadaverine by Paucilactobacillus wasatchensis.

Lactic acid bacteria (LAB) play a key role in many food fermentations. However, some LAB species can also cause food spoilage, e.g., through the formation of biogenic amines. Paucilactobacillus wasatchensis is a LAB that causes late gas production in Cheddar cheese, the molecular causes of which are not fully understood. This study reports on the ability of P. wasatchensis WDC04 to produce cadaverine and putrescine in broth supplemented with lysine and ornithine, as well as in a model cheese. The raclette-type semi-hard cheese produced with P. wasatchensis as an adjunct culture contained 1,085 mg kg-1 of cadaverine and 304 mg kg-1 of putrescine after 120 days of ripening. We identified two ornithine decarboxylase genes (odc) and a putrescine-ornithine antiporter gene (potE) in the genome sequence of P. wasatchensis. We could show that the two odc genes, which are located on two contigs, are contiguous and form the genetic cluster odc2-odc1-potE. Alignment searches showed that similar gene clusters exist in the genomes of Levilactobacillus paucivorans DSMZ22467, Lentilactobacillus kribbianus YH-lac9, Levilactobacillus hunanensis 151-2B, and Levilactobacillus lindianensis 220-4. More amino acid sequence comparisons showed that Odc1 and Odc2 shared 72 and 69% identity with a lysine and ornithine decarboxylase from Ligilactobacillus saerimneri 30a, respectively. To clarify the catalytic activities of both enzymes, the odc-coding genes were cloned and heterologously expressed as His-tagged fusion protein. The purified Odc1 protein decarboxylated lysine into cadaverine, while the recombinant Odc2 protein preferentially produced putrescine from ornithine but also exhibited low lysine decarboxylating activity. Both enzymes were active at pH of 5.5, a value often found in cheese. To our knowledge, this is only the second lysine decarboxylase in LAB whose function has been verified. The tandem arrangement of the genes in a single cluster suggests a gene duplication, evolving the ability to metabolize more amino. Divergent substrate preferences highlight the necessity of verifying the functions of genes, in addition to automatic annotation based on sequence similarity. Acquiring new biochemical data allows better predictive models and, in this case, more accurate biogenic amine production potential for LAB strains and microbiomes.

High-throughput qPCR and 16S rRNA gene amplicon sequencing as complementary methods for the investigation of the cheese microbiota.

BACKGROUND: Next-generation sequencing (NGS) methods and especially 16S rRNA gene amplicon sequencing have become indispensable tools in microbial ecology. While they have opened up new possibilities for studying microbial communities, they also have one drawback, namely providing only relative abundances and thus compositional data. Quantitative PCR (qPCR) has been used for years for the quantification of bacteria. However, this method requires the development of specific primers and has a low throughput. The constraint of low throughput has recently been overcome by the development of high-throughput qPCR (HT-qPCR), which allows for the simultaneous detection of the most prevalent bacteria in moderately complex systems, such as cheese and other fermented dairy foods. In the present study, the performance of the two approaches, NGS and HT-qPCR, was compared by analyzing the same DNA samples from 21 Raclette du Valais protected designation of origin (PDO) cheeses. Based on the results obtained, the differences, accuracy, and usefulness of the two approaches were studied in detail. RESULTS: The results obtained using NGS (non-targeted) and HT-qPCR (targeted) show considerable agreement in determining the microbial composition of the cheese DNA samples studied, albeit the fundamentally different nature of these two approaches. A few inconsistencies in species detection were observed, particularly for less abundant ones. The detailed comparison of the results for 15 bacterial species/groups measured by both methods revealed a considerable bias for certain bacterial species in the measurements of the amplicon sequencing approach. We identified as probable origin to this PCR bias due to primer mismatches, variations in the number of copies for the 16S rRNA gene, and bias introduced in the bioinformatics analysis. CONCLUSION: As the normalized microbial composition results of NGS and HT-qPCR agreed for most of the 21 cheese samples analyzed, both methods can be considered as complementary and reliable for studying the microbial composition of cheese. Their combined application proved to be very helpful in identifying potential biases and overcoming methodological limitations in the quantitative analysis of the cheese microbiota.

Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures.

Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

Antimicrobial Susceptibility of Lactobacillus delbrueckii subsp. lactis from Milk Products and Other Habitats.

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either 'sensitive' or 'resistant' based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains' genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing.

Evaluation of a new culture medium for the enumeration and isolation of Streptococcus salivarius subsp. thermophilus from cheese.

Enumeration and isolation of Streptococcus salivarius subsp. thermophilus from cheese is challenging, due to the relatively high number of species it may host. We describe medium SPY9.3 for the cultivation of S. salivarius subsp. thermophilus from cheese. The medium and related incubation conditions (SPY) was compared with 2 other protocols, M17 and ST: sensitivity was assessed by parallel cultivation of 55 strains of S. salivarius subsp. thermophilus, and selectivity by (i) parallel cultivation of 60 strains belonging to 20 different non-target species and sub-species and (ii) isolating bacteria from 3 raw-milk cheeses. Colony counts were similar on SPY9.3 and M17 (mean difference 0.07 log(cfu/mL), p > 0.001) and significantly higher on ST than on M17 and SPY9.3 (mean differences 0.42 and 0.48 log(cfu/mL), respectively, p < 0.001). SPY was more specific than ST and M17, with respectively 20%, 40%, and 50% of the investigated non-target species able to grow. S. salivarius subsp. thermophilus, Enterococcus spp., and Staphylococcus aureus were indistinguishable using all 3 protocols. Only SPY avoided growth of Lactobacillus delbrueckii subsp. lactis. Finally, ST and SPY displayed higher recoveries of S. salivarius subsp. thermophilus colonies from cheese than M17 (5.6, 5.5, and 3.0 adjusted log(cfu/mL), respectively) and the lowest proportion of non-specific isolates. The protocol described here and based on SPY9.3 presents a promising alternative to existing protocols for the enumeration and isolation of S salivarius subsp. thermophilus from cheese or other complex fermented products.

Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.

Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI®) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

SpeciesPrimer: a bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species.

BACKGROUND: Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reagents. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in complex systems such as fermented foods (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. RESULTS: We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer, specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. CONCLUSION: In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

Development of a High-Throughput Microfluidic qPCR System for the Quantitative Determination of Quality-Relevant Bacteria in Cheese.

The composition of the cheese microbiome has an important impact on the sensorial quality and safety of cheese. Therefore, much effort has been made to investigate the microbial community composition of cheese. Quantitative real-time polymerase chain reaction (qPCR) is a well-established method for detecting and quantifying bacteria. High-throughput qPCR (HT-qPCR) using microfluidics brings further advantages by providing fast results and by decreasing the cost per sample. We have developed a HT-qPCR approach for the rapid and cost-efficient quantification of microbial species in cheese by designing qPCR assays targeting 24 species/subspecies commonly found in cheese. Primer pairs were evaluated on the Biomark (Fluidigm) microfluidic HT-qPCR system using DNA from single strains and from artificial mock communities. The qPCR assays worked efficiently under identical PCR conditions, and the validation showed satisfying inclusivity, exclusivity, and amplification efficiencies. Preliminary results obtained from the HT-qPCR analysis of DNA samples of model cheeses made with the addition of adjunct cultures confirmed the potential of the microfluidic HT-qPCR system to screen for selected bacterial species in the cheese microbiome. HT-qPCR data of DNA samples of two downgraded commercial cheeses showed that this approach provides valuable information that can help to identify the microbial origin of quality defects. This newly developed HT-qPCR system is a promising approach that will allow simultaneous monitoring of quality-relevant species in fermented foods with high bacterial diversity, thereby opening up new perspectives for the control and assurance of high product quality.

DAIRYdb: a manually curated reference database for improved taxonomy annotation of 16S rRNA gene sequences from dairy products.

BACKGROUND: Reads assignment to taxonomic units is a key step in microbiome analysis pipelines. To date, accurate taxonomy annotation of 16S reads, particularly at species rank, is still challenging due to the short size of read sequences and differently curated classification databases. The close phylogenetic relationship between species encountered in dairy products, however, makes it crucial to annotate species accurately to achieve sufficient phylogenetic resolution for further downstream ecological studies or for food diagnostics. Curated databases dedicated to the environment of interest are expected to improve the accuracy and resolution of taxonomy annotation. RESULTS: We provide a manually curated database composed of 10'290 full-length 16S rRNA gene sequences from prokaryotes tailored for dairy products analysis ( https://github.com/marcomeola/DAIRYdb ). The performance of the DAIRYdb was compared with the universal databases Silva, LTP, RDP and Greengenes. The DAIRYdb significantly outperformed all other databases independently of the classification algorithm by enabling higher accurate taxonomy annotation down to the species rank. The DAIRYdb accurately annotates over 90% of the sequences of either single or paired hypervariable regions automatically. The manually curated DAIRYdb strongly improves taxonomic annotation accuracy for microbiome studies in dairy environments. The DAIRYdb is a practical solution that enables automatization of this key step, thus facilitating the routine application of NGS microbiome analyses for microbial ecology studies and diagnostics in dairy products.

Conversion of Methionine to Cysteine in Lactobacillus paracasei Depends on the Highly Mobile cysK-ctl-cysE Gene Cluster.

Milk and dairy products are rich in nutrients and are therefore habitats for various microbiomes. However, the composition of nutrients can be quite diverse, in particular among the sulfur containing amino acids. In milk, methionine is present in a 25-fold higher abundance than cysteine. Interestingly, a fraction of strains of the species L. paracasei - a flavor-enhancing adjunct culture species - can grow in medium with methionine as the sole sulfur source. In this study, we focus on genomic and evolutionary aspects of sulfur dependence in L. paracasei strains. From 24 selected L. paracasei strains, 16 strains can grow in medium with methionine as sole sulfur source. We sequenced these strains to perform gene-trait matching. We found that one gene cluster - consisting of a cysteine synthase, a cystathionine lyase, and a serine acetyltransferase - is present in all strains that grow in medium with methionine as sole sulfur source. In contrast, strains that depend on other sulfur sources do not have this gene cluster. We expanded the study and searched for this gene cluster in other species and detected it in the genomes of many bacteria species used in the food production. The comparison to these species showed that two different versions of the gene cluster exist in L. paracasei which were likely gained in two distinct events of horizontal gene transfer. Additionally, the comparison of 62 L. paracasei genomes and the two versions of the gene cluster revealed that this gene cluster is mobile within the species.

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey.

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

Transcriptional Regulation of Cysteine and Methionine Metabolism in Lactobacillus paracasei FAM18149.

Lactobacillus paracasei is common in the non-starter lactic acid bacteria (LAB) community of raw milk cheeses. This species can significantly contribute to flavor formation through amino acid metabolism. In this study, the DNA and RNA of L. paracasei FAM18149 were sequenced using next-generation sequencing technologies to reconstruct the metabolism of the sulfur-containing amino acids cysteine and methionine. Twenty-three genes were found to be involved in cysteine biosynthesis, the conversion of cysteine to methionine and vice versa, the S-adenosylmethionine recycling pathway, and the transport of sulfur-containing amino acids. Additionally, six methionine-specific T-boxes and one cysteine-specific T-box were found. Five of these were located upstream of genes encoding transporter functions. RNA-seq analysis and reverse-transcription quantitative polymerase reaction assays showed that expression of genes located downstream of these T-boxes was affected by the absence of either cysteine or methionine. Remarkably, the cysK2-ctl1-cysE2 operon, which is associated with te methionine-to-cysteine conversion and is upregulated in the absence of cysteine, showed high read coverage in the 5'-untranslated region and an antisense-RNA in the 3'-untranslated region. This indicates that this operon is regulated by the combination of cis- and antisense-mediated regulation mechanisms. The results of this study may help in the selection of L. paracasei strains to control sulfuric flavor formation in cheese.

The Histidine Decarboxylase Gene Cluster of Lactobacillus parabuchneri Was Gained by Horizontal Gene Transfer and Is Mobile within the Species.

Histamine in food can cause intolerance reactions in consumers. Lactobacillus parabuchneri (L. parabuchneri) is one of the major causes of elevated histamine levels in cheese. Despite its significant economic impact and negative influence on human health, no genomic study has been published so far. We sequenced and analyzed 18 L. parabuchneri strains of which 12 were histamine positive and 6 were histamine negative. We determined the complete genome of the histamine positive strain FAM21731 with PacBio as well as Illumina and the genomes of the remaining 17 strains using the Illumina technology. We developed the synteny aware ortholog finding algorithm SynOrf to compare the genomes and we show that the histidine decarboxylase (HDC) gene cluster is located in a genomic island. It is very likely that the HDC gene cluster was transferred from other lactobacilli, as it is highly conserved within several lactobacilli species. Furthermore, we have evidence that the HDC gene cluster was transferred within the L. parabuchneri species.

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects.

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6-8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively.

Catabolism of serine by Pediococcus acidilactici and Pediococcus pentosaceus.

The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from L- and D-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity.

A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.