Comparative genomics of Lactobacillus sakei supports the development of starter strain combinations.

Strains of Lactobacillus sakei can be isolated from a variety of sources including meat, fermented sausages, sake, sourdough, sauerkraut or kimchi. Selected strains are widely used as starter cultures for sausage fermentation. Recently we have demonstrated that control about the lactic microbiota in fermenting sausages is achieved rather by pairs or strain sets than by single strains. In this work we characterized the pan genome of L. sakei to enable exploitation of the genomic diversity of L. sakei for the establishment of assertive starter strain sets. We have established the full genome sequences of nine L. sakei strains from different sources of isolation and included in the analysis the genome of L. sakei 23K. Comparative genomics revealed an accessory genome comprising about 50% of the pan genome and different lineages of strains with no relation to their source of isolation. Group and strain specific differences could be found, which namely referred to agmatine and citrate metabolism. The presence of genes encoding metabolic pathways for fructose, sucrose and trehalose as well as gluconate in all strains suggests a general adaptation to plant/sugary environments and a life in communities with other genera. Analysis of the plasmidome did not reveal any specific mechanisms of adaptation to a habitat. The predicted differences of metabolic settings enable prediction of partner strains, which can occupy the meat environment to a large extent and establish competitive exclusion of autochthonous microbiota. This may assist the development of a new generation of meat starter cultures containing L. sakei strains.

Lifestyle of Lactobacillus hordei isolated from water kefir based on genomic, proteomic and physiological characterization.

Water kefir is a traditional fermented beverage made from sucrose, water, kefir granules, dried or fresh fruits. In our water kefir granules, Lactobacillus (L.) hordei is one of the predominant lactic acid bacteria (LAB) species of this presumed symbiotic consortium. It faces abundant sucrose versus limitation of amino- and fatty acids in an acidic environment. Sequencing of the genome of L. hordei TMW 1.1822 revealed one chromosome plus three plasmids. The size of the chromosome was 2.42 Mbp with a GC content of 35% GC and 2461 predicted coding sequences. Furthermore, we identified 1474 proteins upon growth on water kefir medium. Metabolic prediction revealed all enzymes required for the glycolytic Embden-Meyerhof (EMP) and phosphoketolase (PKP) pathways. Genes encoding all enzymes involved in citrate, pyruvate and mannitol metabolism are present. Moreover, it was confirmed that L. hordei is prototrophic for 11 amino acids and auxotrophic for 6 amino acids when combining putative biosynthesis pathways for amino acids with physiological characterization. Still, for glycine, serine and methionine no sure auxotype could be determined. The OppABCDF peptide transport system is complete, and 13 genes encoding peptidases are present. The arginine deiminase system, was predicted to be complete except for carbamate kinase, thus enabling neutralization reactions via ammonium formation but no additional energy generation. Taken together our findings enable prediction of the L. hordei lifestyle in water kefir: Abundant sucrose is consumed directly via parallel EMP and PK pathways and is also extracellularly converted to dextran and fructose by a glucansucrase, leaving fructose as additional carbon source. Essential amino acids (in the form of peptides) and citrate are acquired from fruits. In the lack of FabB unsaturated fatty acids are synthesized by predicted alternative enzymes. Formation of acetoin and diacetyl as well as arginine conversion reactions enable acidification limitation. Other members of the water kefir consortium (yeasts, acetic acid bacteria) likely facilitate or support growth of L. hordei by delivering gluconate, mannitol, amino- and fatty acids and vitamins.

Comparative genomics of Lactobacillus curvatus enables prediction of traits relating to adaptation and strategies of assertiveness in sausage fermentation.

Lactobacillus (L.) curvatus reaches high numbers in a variety of habitats, which suggests a high (genomic) diversity within this species. Empirically selected strains are used as starter cultures in sausage fermentation. Determinants for the assertiveness of a strain in this environment are assumed to be multifactorial. We used comparative genomics and in silico proteomics of 10 L. curvatus strains, which were representative of its genetic and physiological biodiversity, to possibly derive genetic determinants for strain or group specific assertiveness in sausage fermentation. Their genome sizes ranged from 1.7 Mb up to 2.0 Mb. The estimated pan- and core genomes were 3.0 Mb and 1.4 Mb, respectively. The accessory genome, GC-content and coding density revealed a significant genomic diversity within this species. Plasmids were found, which were either closely related or unique in several strains. Putative assertiveness determinants including CRISPR/Cas systems, prophages, bacteriocin production, or specific metabolic settings were detected. Such traits of the accessory genome could not be correlated with the source of isolation. Pathways, which previously have been predicted for a relation with adaptation to meat of L. sakei, are part of the core genome of L curvatus. Intraspecies differences in the accessory genome of L. curvatus comprise ribose metabolism, enzymes involved in nucleotide metabolism (nucleoside phosphorylases, phosphopentomutase, adenosine deaminase, ribose transporters), and tyrosine decarboxylases, ornithine decarboxylases. One group of the strains encoded a phosphotransferase system (PTS) as ribose transporter, whereas the second group encoded an ATP binding cassette (ABC) transporter. Analysis of the ribose uptake by HPLC analysis revealed different efficiencies of both transporter systems. Except for bacteriocin formation, no strain specific traits were identified predicting assertiveness of single strains. This fits our previous observation that single strains of L. curvatus could not override others in a competitive setting. Rather, pairs or sets of strains, comprising metabolically synergistic or non-assertive partner strains were able to dominate the fermentation. Indeed, this work suggests that assertive partnerships can be predicted along their complementary accessory genomes.

Assertiveness of Lactobacillus sakei and Lactobacillus curvatus in a fermented sausage model.

Fresh meat harbors autochthonous microbiota with unknown risk potential, which is introduced in raw fermented sausages. Their growth can be limited by the use of safe, competitive starter strains. In the lack of time and cost-effective methods to track those starters at strain level, their assertiveness upon meat fermentation is widely unknown. Lactobacillus (L.) sakei and L. curvatus, which can be isolated from a variety of habitats, are frequently used as starter cultures. We monitored the assertiveness of 9 L. sakei and 9 L. curvatus strains in a model fermentation using MALDI-TOF-MS. An "in-house" MALDI-TOF-MS database with sub-proteome spectra of L. sakei and L. curvatus strains, as well as members of the autochthonous, spontaneously growing meat microbiota was established, validated and recognition rates were determined for each L. curvatus and L. sakei strain used. Competition studies were performed with standardized sausage batter, which was inoculated with a total of 106 cells of sets of 4-5 strains each of L. sakei and L. curvatus and 106Staphylococcus carnosus ssp. carnosus cells. The pH and redox potential were monitored continuously. On days 0, 2 and 5 samples were taken to determine the CfU/g and a total of 96 isolates per sample were identified via MALDI-TOF-MS. MALDI-TOF-MS generally proved suitable for identification of isolates on strain level within the starter sets employed, but the recognition rate varied depending on the strain. Competition studies revealed dominance or co-dominance of strains within each set. However, their assertiveness significantly depended on the composition of the strain sets. Still, co-dominance or cooperation appeared effective to outgrow other members of the autochthonous meat microbiota, rather than dominance of single strains. For the latter, the ability to produce bacteriocins suggested itself for a crucial role in the assertiveness of starter strains.

Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.

The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida's chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter-GFP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida.