Detection and classification of the integrative conjugative elements of Lactococcus lactis.

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

Prediction and validation of novel SigB regulon members in Bacillus subtilis and regulon structure comparison to Bacillales members.

BACKGROUND: Sigma factor B (SigB) is the central regulator of the general stress response in Bacillus subtilis and regulates a group of genes in response to various stressors, known as the SigB regulon members. Genes that are directly regulated by SigB contain a promotor binding motif (PBM) with a previously identified consensus sequence. RESULTS: In this study, refined SigB PBMs were derived and different spacer compositions and lengths (N12-N17) were taken into account. These were used to identify putative SigB-regulated genes in the B. subtilis genome, revealing 255 genes: 99 had been described in the literature and 156 genes were newly identified, increasing the number of SigB putative regulon members (with and without a SigB PBM) to > 500 in B. subtilis. The 255 genes were assigned to five categories (I-V) based on their similarity to the original SigB consensus sequences. The functionalities of selected representatives per category were assessed using promoter-reporter fusions in wt and ΔsigB mutants upon exposure to heat, ethanol, and salt stress. The activity of the PrsbV (I) positive control was induced upon exposure to all three stressors. PytoQ (II) showed SigB-dependent activity only upon exposure to ethanol, whereas PpucI (II) with a N17 spacer and PylaL (III) with a N16 spacer showed mild induction regardless of heat/ethanol/salt stress. PywzA (III) and PyaaI (IV) displayed ethanol-specific SigB-dependent activities despite a lower-level conserved - 10 binding motif. PgtaB (V) was SigB-induced under ethanol and salt stress while lacking a conserved - 10 binding region. The activities of PygaO and PykaA (III) did not show evident changes under the conditions tested despite having a SigB PBM that highly resembled the consensus. The identified extended SigB regulon candidates in B. subtilis are mainly involved in coping with stress but are also engaged in other cellular processes. Orthologs of SigB regulon candidates with SigB PBMs were identified in other Bacillales genomes, but not all showed a SigB PBM. Additionally, genes involved in the integration of stress signals to activate SigB were predicted in these genomes, indicating that SigB signaling and regulon genes are species-specific. CONCLUSION: The entire SigB regulatory network is sophisticated and not yet fully understood even for the well-characterized organism B. subtilis 168. Knowledge and information gained in this study can be used in further SigB studies to uncover a complete picture of the role of SigB in B. subtilis and other species.

Dietary Inulin Increases Lactiplantibacillus plantarum Strain Lp900 Persistence in Rats Depending on the Dietary-Calcium Level.

Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit health benefits in the consumer. Lactiplantibacillus plantarum strains display high survival during transit through the mammalian gastrointestinal tract and were shown to have health-promoting properties. Growth on the fructose polysaccharide inulin is relatively uncommon in L. plantarum, and in this study we describe FosE, a plasmid-encoded ß-fructosidase of L. plantarum strain Lp900 which has inulin-hydrolyzing properties. FosE contains an LPxTG-like motif involved in sortase-dependent cell wall anchoring but is also (partially) released in the culture supernatant. In addition, we examined the effect of diet supplementation with inulin on the intestinal persistence of Lp900 in adult male Wistar rats in diets with distinct calcium levels. Inulin supplementation in high-dietary-calcium diets significantly increased the intestinal persistence of L. plantarum Lp900, whereas this effect was not observed upon inulin supplementation of the low-calcium diet. Moreover, intestinal persistence of L. plantarum Lp900 was determined when provided as a probiotic (by itself) or as a synbiotic (i.e., in an inulin suspension) in rats that were fed unsupplemented diets containing the different calcium levels, revealing that the synbiotic administration increased bacterial survival and led to higher abundance of L. plantarum Lp900 in rats, particularly in a low-calcium-diet context. Our findings demonstrate that inulin supplementation can significantly enhance the intestinal delivery of L. plantarum Lp900 but that this effect strongly depends on calcium levels in the diet.IMPORTANCE Synbiotics combine probiotics with prebiotics to synergistically elicit a health benefit in the consumer. Previous studies have shown that prebiotics can selectively stimulate the growth in the intestine of specific bacterial strains. In synbiotic supplementations the prebiotics constituent could increase the intestinal persistence and survival of accompanying probiotic strain(s) and/or modulate the endogenous host microbiota to contribute to the synergistic enhancement of the health-promoting effects of the synbiotic constituents. Our study establishes a profound effect of dietary-calcium-dependent inulin supplementation on the intestinal persistence of inulin-utilizing L. plantarum Lp900 in rats. We also show that in rats on a low-dietary-calcium regime, the survival and intestinal abundance of L. plantarum Lp900 are significantly increased by administering it as an inulin-containing synbiotic. This study demonstrates that prebiotics can enhance the intestinal delivery of specific probiotics and that the prebiotic effect is profoundly influenced by the calcium content of the diet.

Microbial bioinformatics for food safety and production.

In the production of fermented foods, microbes play an important role. Optimization of fermentation processes or starter culture production traditionally was a trial-and-error approach inspired by expert knowledge of the fermentation process. Current developments in high-throughput 'omics' technologies allow developing more rational approaches to improve fermentation processes both from the food functionality as well as from the food safety perspective. Here, the authors thematically review typical bioinformatics techniques and approaches to improve various aspects of the microbial production of fermented food products and food safety.

Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX.

In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies.IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Nomadic lifestyle of Lactobacillus plantarum revealed by comparative genomics of 54 strains isolated from different habitats.

The ability of bacteria to adapt to diverse environmental conditions is well-known. The process of bacterial adaptation to a niche has been linked to large changes in the genome content, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse habitats that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of habitat. With the aim of unravelling the link between evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Comparative genome analysis identified a high level of genomic diversity and plasticity among the strains analysed. Phylogenomic and functional divergence studies coupled with gene-trait matching analyses revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings revealed the absence of specific genomic signatures marking adaptations of L. plantarum towards the diverse habitats it is associated with. This suggests fundamentally similar trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species.

Data mining in the Life Sciences with Random Forest: a walk in the park or lost in the jungle?

In the Life Sciences 'omics' data is increasingly generated by different high-throughput technologies. Often only the integration of these data allows uncovering biological insights that can be experimentally validated or mechanistically modelled, i.e. sophisticated computational approaches are required to extract the complex non-linear trends present in omics data. Classification techniques allow training a model based on variables (e.g. SNPs in genetic association studies) to separate different classes (e.g. healthy subjects versus patients). Random Forest (RF) is a versatile classification algorithm suited for the analysis of these large data sets. In the Life Sciences, RF is popular because RF classification models have a high-prediction accuracy and provide information on importance of variables for classification. For omics data, variables or conditional relations between variables are typically important for a subset of samples of the same class. For example: within a class of cancer patients certain SNP combinations may be important for a subset of patients that have a specific subtype of cancer, but not important for a different subset of patients. These conditional relationships can in principle be uncovered from the data with RF as these are implicitly taken into account by the algorithm during the creation of the classification model. This review details some of the to the best of our knowledge rarely or never used RF properties that allow maximizing the biological insights that can be extracted from complex omics data sets using RF.

Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth rates.

This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation.

Genome-wide transcriptional responses to carbon starvation in nongrowing Lactococcus lactis.

This paper describes the transcriptional adaptations of nongrowing, retentostat cultures of Lactococcus lactis to starvation. Near-zero-growth cultures (µ = 0.0001 h(-1)) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence in L. lactis KF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conserved cis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation in L. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells.

Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling.

Lactobacillus rhamnosus is a bacterial species commonly colonizing the gastrointestinal (GI) tract of humans and also frequently used in food products. While some strains have been studied extensively, physiological variability among isolates of the species found in healthy humans or their diet is largely unexplored. The aim of this study was to characterize the diversity of carbohydrate utilization capabilities of human isolates and food-derived strains of L. rhamnosus in relation to their niche of isolation and genotype. We investigated the genotypic and phenotypic diversity of 25 out of 65 L. rhamnosus strains from various niches, mainly human feces and fermented dairy products. Genetic fingerprinting of the strains by amplified fragment length polymorphism (AFLP) identified 11 distinct subgroups at 70% similarity and suggested niche enrichment within particular genetic clades. High-resolution carbohydrate utilization profiling (OmniLog) identified 14 carbon sources that could be used by all of the strains tested for growth, while the utilization of 58 carbon sources differed significantly between strains, enabling the stratification of L. rhamnosus strains into three metabolic clusters that partially correlate with the genotypic clades but appear uncorrelated with the strain's origin of isolation. Draft genome sequences of 8 strains were generated and employed in a gene-trait matching (GTM) analysis together with the publicly available genomes of L. rhamnosus GG (ATCC 53103) and HN001 for several carbohydrates that were distinct for the different metabolic clusters: l-rhamnose, cellobiose, l-sorbose, and α-methyl-d-glucoside. From the analysis, candidate genes were identified that correlate with l-sorbose and α-methyl-d-glucoside utilization, and the proposed function of these genes could be confirmed by heterologous expression in a strain lacking the genes. This study expands our insight into the phenotypic and genotypic diversity of the species L. rhamnosus and explores the relationships between specific carbohydrate utilization capacities and genotype and/or niche adaptation of this species.

Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation.

BACKGROUND: To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. RESULTS: While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33 ± 0.02 h(-1) and 0.34 ± 0.03 h(-1), respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. CONCLUSIONS: Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium.

Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome.

There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.

Comparative analysis of Lactobacillus plantarum WCFS1 transcriptomes by using DNA microarray and next-generation sequencing technologies.

RNA sequencing is starting to compete with the use of DNA microarrays for transcription analysis in eukaryotes as well as in prokaryotes. The application of RNA sequencing in prokaryotes requires additional steps in the RNA preparation procedure to increase the relative abundance of mRNA and cannot employ the poly(T)-primed approach in cDNA synthesis. In this study, we aimed to validate the use of RNA sequencing (direct cDNA sequencing and 3'-untranslated region [UTR] sequencing) using Lactobacillus plantarum WCFS1 as a model organism, employing its established microarray platform as a reference. A limited effect of mRNA enrichment on genome-wide transcript quantification was observed, and comparative transcriptome analyses were performed for L. plantarum WCFS1 grown in two different laboratory media. Microarray analyses and both RNA sequencing methods resulted in similar depths of analysis and generated similar fold-change ratios of differentially expressed genes. The highest overall correlation was found between microarray and direct cDNA sequencing-derived transcriptomes, while the 3'-UTR sequencing-derived transcriptome appeared to deviate the most. Overall, a high similarity between patterns of transcript abundance and fold-change levels of differentially expressed genes was detected by all three methods, indicating that the biological conclusions drawn from the transcriptome data were consistent among the three technologies.

Lactobacillus plantarum possesses the capability for wall teichoic acid backbone alditol switching.

BACKGROUND: Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. RESULTS: Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type. CONCLUSIONS: The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.

Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling.

BACKGROUND: Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. RESULTS: The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (Δcps1A-I, Δcps2A-J, Δcps3A-J and Δcps4A-J) or combinations of cps clusters (Δcps1A-3J and Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The Δcps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the Δcps1A-I and Δcps3A-J mutants but appeared slightly increased after stimulation with the Δcps2A-J and Δcps4A-J mutants, while the Δcps1A-3J and Δcps1A-3J, Δcps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. CONCLUSIONS: Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.

From transcriptional landscapes to the identification of biomarkers for robustness.

The ability of microorganisms to adapt to changing environments and gain cell robustness, challenges the prediction of their history-dependent behaviour. Using our model organism Bacillus cereus, a notorious Gram-positive food spoilage and pathogenic spore-forming bacterium, a strategy will be described that allows for identification of biomarkers for robustness. First an overview will be presented of its two-component systems that generally include a transmembrane sensor histidine kinase and its cognate response regulator, allowing rapid and robust responses to fluctuations in the environment. The role of the multisensor hybrid kinase RsbK and the PP2C-type phosphatase RsbY system in activation of the general stress sigma factor σB is highlighted. An extensive comparative analysis of transcriptional landscapes derived from B. cereus exposed to mild stress conditions such as heat, acid, salt and oxidative stress, revealed that, amongst others σB regulated genes were induced in most conditions tested. The information derived from the transcriptome data was subsequently implemented in a framework for identifying and selecting cellular biomarkers at their mRNA, protein and/or activity level, for mild stressinduced microbial robustness towards lethal stresses. Exposure of unstressed and mild stress-adapted cells to subsequent lethal stress conditions (heat, acid and oxidative stress) allowed for quantification of the robustness advantage provided by mild stress pretreatment using the plate-count method. The induction levels of the selected candidate-biomarkers, σB protein, catalase activity and transcripts of certain proteases upon mild stress treatment, were significantly correlated to mild stress-induced enhanced robustness towards lethal thermal, oxidative and acid stresses, and were therefore suitable to predict these adaptive traits. Cellular biomarkers that are quantitatively correlated to adaptive behavior will facilitate our ability to predict the impact of adaptive behavior on cell robustness and will allow to control and/or exploit these adaptive traits. Extrapolation to other species and genera is discussed such as avenues towards mechanism-based design of microbial fitness and robustness.

Complete Closed Genome Sequence of the Inulin-Utilizing Lactiplantibacillus plantarum Strain Lp900, Obtained Using a Hybrid Nanopore and Illumina Assembly.

Lactiplantibacillus plantarum is a genetically and phenotypically diverse lactic acid bacterium species. We announce the hybrid de novo assembly of Oxford Nanopore Technologies and Illumina DNA sequence reads, producing a closed circular chromosome of 3,206,992 bp and six plasmids of the inulin-utilizing L. plantarum strain Lp900.

Complete genome sequence of Lactococcus lactis subsp. lactis KF147, a plant-associated lactic acid bacterium.

Lactococcus lactis is a lactic acid bacterium used in the production of many fermented dairy products. We report the complete genome sequence of L. lactis subsp. lactis KF147, a nondairy strain isolated from mung bean sprouts. The circular chromosome of 2,598,144 bp, the largest among the sequenced lactococcal strains, encodes many properties related to adaptation to the plant environment.

LAB-Secretome: a genome-scale comparative analysis of the predicted extracellular and surface-associated proteins of Lactic Acid Bacteria.

BACKGROUND: In Lactic Acid Bacteria (LAB), the extracellular and surface-associated proteins can be involved in processes such as cell wall metabolism, degradation and uptake of nutrients, communication and binding to substrates or hosts. A genome-scale comparative study of these proteins (secretomes) can provide vast information towards the understanding of the molecular evolution, diversity, function and adaptation of LAB to their specific environmental niches. RESULTS: We have performed an extensive prediction and comparison of the secretomes from 26 sequenced LAB genomes. A new approach to detect homolog clusters of secretome proteins (LaCOGs) was designed by integrating protein subcellular location prediction and homology clustering methods. The initial clusters were further adjusted semi-manually based on multiple sequence alignments, domain compositions, pseudogene analysis and biological function of the proteins. Ubiquitous protein families were identified, as well as species-specific, strain-specific, and niche-specific LaCOGs. Comparative analysis of protein subfamilies has shown that the distribution and functional specificity of LaCOGs could be used to explain many niche-specific phenotypes.A comprehensive and user-friendly database LAB-Secretome was constructed to store, visualize and update the extracellular proteins and LaCOGs http://www.cmbi.ru.nl/lab_secretome/. This database will be updated regularly when new bacterial genomes become available. CONCLUSIONS: The LAB-Secretome database could be used to understand the evolution and adaptation of lactic acid bacteria to their environmental niches, to improve protein functional annotation and to serve as basis for targeted experimental studies.

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.