High throughput screening of technological and biopreservation traits of a large set of wild lactic acid bacteria from Brazilian artisanal cheeses.

This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).

Transcriptome Analysis of a Spray Drying-Resistant Subpopulation Reveals a Zinc-Dependent Mechanism for Robustness in L. lactis SK11.

The viability of starter cultures is essential for an adequate contribution to the fermentation process and end-product. Therefore, robustness during processing and storage is an important characteristic of starter culture strains. For instance, during spray drying cells are exposed to heat and oxidative stress, generally resulting in loss of viability. In this study, we exposed the industrially relevant but stress-sensitive Lactococcus lactis strain SK11 to two cycles of heat stress, with intermediate recovery and cultivation at moderate temperatures. After these two cycles of heat exposure, the abundance of robust derivatives was increased as compared with the original culture, which enabled isolation of heat-resistant subpopulations displaying up to 1,000-fold enhanced heat stress survival. Moreover, this heat-resistant subpopulation demonstrated an increased survival during spray drying. Derivatives from two independent lineages displayed different transcriptome changes as compared with the wild type strain, indicating that the increased robustness within these lineages was established by different adaptive strategies. Nevertheless, an overlap in differential gene expression in all five derivatives tested in both lineages included three genes in an operon involved in zinc transport. The link between zinc homeostasis and heat stress survival in L. lactis was experimentally established by culturing of the wild type strain SK11 in medium with various levels of zinc ions, which resulted in alterations in heat stress survival phenotypes. This study demonstrates that robust derivatives of a relatively sensitive L. lactis strain can be isolated by repeated exposure to heat stress. Moreover, this work demonstrates that transcriptome analysis of these robust derivatives can provide clues for improvement of the robustness of the original strain. This could boost the industrial application of strains with specific desirable traits but inadequate robustness characteristics.

Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis.

Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nanE and genes encoding transport proteins. The transcript levels of these genes can function as indicators of robustness and could aid in selection of fermentation parameters, potentially resulting in more optimal robustness during spray drying.

Fermentation-induced variation in heat and oxidative stress phenotypes of Lactococcus lactis MG1363 reveals transcriptome signatures for robustness.

BACKGROUND: Lactococcus lactis is industrially employed to manufacture various fermented dairy products. The most cost-effective method for the preservation of L. lactis starter cultures is spray drying, but during this process cultures encounter heat and oxidative stress, typically resulting in low survival rates. However, viability of starter cultures is essential for their adequate contribution to milk fermentation, supporting the ambition to better understand and improve their robustness phenotypes. RESULTS: This study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress. CONCLUSIONS: Overall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival.

Genotype-phenotype matching analysis of 38 Lactococcus lactis strains using random forest methods.

BACKGROUND: Lactococcus lactis is used in dairy food fermentation and for the efficient production of industrially relevant enzymes. The genome content and different phenotypes have been determined for multiple L. lactis strains in order to understand intra-species genotype and phenotype diversity and annotate gene functions. In this study, we identified relations between gene presence and a collection of 207 phenotypes across 38 L. lactis strains of dairy and plant origin. Gene occurrence and phenotype data were used in an iterative gene selection procedure, based on the Random Forest algorithm, to identify genotype-phenotype relations. RESULTS: A total of 1388 gene-phenotype relations were found, of which some confirmed known gene-phenotype relations, such as the importance of arabinose utilization genes only for strains of plant origin. We also identified a gene cluster related to growth on melibiose, a plant disaccharide; this cluster is present only in melibiose-positive strains and can be used as a genetic marker in trait improvement. Additionally, several novel gene-phenotype relations were uncovered, for instance, genes related to arsenite resistance or arginine metabolism. CONCLUSIONS: Our results indicate that genotype-phenotype matching by integrating large data sets provides the possibility to identify gene-phenotype relations, possibly improve gene function annotation and identified relations can be used for screening bacterial culture collections for desired phenotypes. In addition to all gene-phenotype relations, we also provide coherent phenotype data for 38 Lactococcus strains assessed in 207 different phenotyping experiments, which to our knowledge is the largest to date for the Lactococcus lactis species.

Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution.

Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and in dairy environments, and it is proposed that dairy strains originate from the plant niche. Here we investigate the adaptation of a L. lactis strain isolated from a plant to a dairy niche by propagating it for 1000 generations in milk. Two out of three independently evolved strains displayed significantly increased acidification rates and biomass yields in milk. Genome resequencing, revealed six, seven, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport and in the gene encoding MutL, which is involved in DNA mismatch repair. Two strains lost a conjugative transposon containing genes important in the plant niche but dispensable in milk. A plasmid carrying an extracellular protease was introduced by transformation. Although improving growth rate and growth yield significantly, the plasmid was rapidly lost. Comparative transcriptome and phenotypic analyses confirmed that major physiological changes associated with improved growth in milk relate to nitrogen metabolism and the loss or down-regulation of several pathways involved in the utilization of complex plant polymers. Reproducing the transition from the plant to the dairy niche through experimental evolution revealed several genome, transcriptome, and phenotype signatures that resemble those seen in strains isolated from either niche.

High-resolution amplified fragment length polymorphism typing of Lactococcus lactis strains enables identification of genetic markers for subspecies-related phenotypes.

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.

Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches.

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.

Regulatory phenotyping reveals important diversity within the species Lactococcus lactis.

The diversity in regulatory phenotypes among a collection of 84 Lactococcus lactis strains isolated from dairy and nondairy origin was explored. The specific activities of five enzymes were assessed in cell extracts of all strains grown in two different media, a nutritionally rich broth and a relatively poor chemically defined medium. The five investigated enzymes, branched chain aminotransferase (BcaT), aminopeptidase N (PepN), X-prolyl dipeptidyl peptidase (PepX), alpha-hydroxyisocaproic acid dehydrogenase (HicDH), and esterase, are involved in nitrogen and fatty acid metabolism and catalyze key steps in the production of important dairy flavor compounds. The investigated cultures comprise 75 L. lactis subsp. lactis isolates (including 7 L. lactis subsp. lactis biovar diacetylactis isolates) and 9 L. lactis subsp. cremoris isolates. All L. lactis subsp. cremoris and 22 L. lactis subsp. lactis (including 6 L. lactis subsp. lactis biovar diacetylactis) cultures originated from a dairy environment. All other cultures originated from (fermented) plant materials and were isolated at different geographic locations. Correlation analysis of specific enzyme activities revealed significantly different regulatory phenotypes for dairy and nondairy isolates. The enzyme activities in the two investigated media were in general poorly correlated and revealed a high degree of regulatory diversity within this collection of closely related strains. To the best of our knowledge, these results represent the most extensive diversity analysis of regulatory phenotypes within a single bacterial species to date. The presented findings underline the importance of the availability of screening procedures for, e.g., industrially relevant enzyme activities in models closely mimicking application conditions. Moreover, they corroborate the notion that regulatory changes are important drivers of evolution.

Genome-scale genotype-phenotype matching of two Lactococcus lactis isolates from plants identifies mechanisms of adaptation to the plant niche.

Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L. lactis plant isolates. Diagnostic sequencing of their genomes resulted in over 2.5 Mb of sequence for each strain. A high synteny was found with the genome of L. lactis IL-1403, which was used as a template for contig mapping and locating deletions and insertions in the plant L. lactis genomes. Numerous genes were identified that do not have homologs in the published genome sequences of dairy L. lactis strains. Adaptation to growth on substrates derived from plant cell walls is evident from the presence of gene sets for the degradation of complex plant polymers such as xylan, arabinan, glucans, and fructans but also for the uptake and conversion of typical plant cell wall degradation products such as alpha-galactosides, beta-glucosides, arabinose, xylose, galacturonate, glucuronate, and gluconate. Further niche-specific differences are found in genes for defense (nisin biosynthesis), stress response (nonribosomal peptide synthesis and various transporters), and exopolysaccharide biosynthesis, as well as the expected differences in various mobile elements such as prophages, plasmids, restriction-modification systems, and insertion sequence elements. Many of these genes were identified for the first time in Lactococcus lactis. In most cases good correspondence was found with the phenotypic characteristics of these two strains.

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

IS981-mediated adaptive evolution recovers lactate production by ldhB transcription activation in a lactate dehydrogenase-deficient strain of Lactococcus lactis.

Lactococcus lactis NZ9010 in which the las operon-encoded ldh gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin. However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol. Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations. This recovery was shown to be due to the transcriptional activation of a silent ldhB gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native las operon-encoded Ldh protein. Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation. The mechanism underlying the ldhB gene activation was primarily studied in a single-colony isolate of the recovered culture, designated L. lactis NZ9015. Integration of IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-derived -35 promoter region at the correct spacing with a natively present -10 region. Subsequently, analysis of 10 independently isolated lactate-producing derivatives of L. lactis NZ9010 confirmed that the ldhB gene is transcribed in all of them. Moreover, characterization of the upstream region of the ldhB gene in these derivatives indicated that site-specific and directional IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis.

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.