Background Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus , Lactobacillus helveticus and Lactobacillus delbrueckii . Results Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats. Conclusions Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities. Electronic supplementary material The online version of this article (10.1186/s12866-019-1500-0) contains supplementary material, which is available to authorized users.

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

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 ␤-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 ␣-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.Volatile sulfur compounds (VSC) are found in many cheese varieties and are important components of flavor (10). The sulfur compounds methanethiol, methional, and dimethyl trisulfide (DMTS) are key odorants of Swiss Gruyère cheese flavor and are presumably derived from the bacterial metabolism of methionine, which is present in caseins at higher concentrations than cysteine. Swiss Gruyère (place of designated origin) cheese is produced with thermophilic mixed-strain starters which usually contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. In addition, depending on the cheese factory, Lactobacillus helveticus can also be present. However, after ripening, Lactobacillus casei and Lactobacillus rhamnosus bacteria are found in high numbers (8). Interestingly, L. casei is also a constituent of the nonstarter lactic acid bacteria of many other cheese varieties (6). Therefore, this species is considered to play an important role during ripening and may be a key player in the formation of flavor compounds.The catabolic pathway(s) of methionine and cysteine in lactobacilli is not well characterized. Possible pathways involve transaminases, methionine ␥-lyases, cystathionine ␤-lyases (CBL), and cystathionine ␥-lyases (CGL) (13). The transamination of methionine by aminotransferases has been studied in lactococci (18,27,34). The results of these studies suggested that methionine is converted to 2-oxo-4-methylthiobutyric acid, which is then converted, either enzymatically or chemically, to methanethiol. In Brevibacterium linens, a methionine ␥-lyases which catalyzes the deamination of methionine and releases methanethiol has been identified and the corresponding gene has been cloned (2,14).CBL, which is encoded by the gene metC in Escherichia coli, is involved in the ␣,␤-elimination of cystathionine to form homocysteine, pyruvate, and ammonia. In comparison, CGL catalyzes the ␣,␥-elimination reaction of cystathionine, producing cysteine, ammonia, and ␣-ketobutyrate. CBL and CGL generally show catalytic promiscuity, which means that they catalyze different reactions. Thus, the CBL from Lactococcus lactis subsp. cremor...