Comparative Genome Analysis of Lactococcus lactis Indicates Niche Adaptation and Resolves Genotype/Phenotype Disparity

Lactococcus lactis is one of the most important micro-organisms in the dairy industry for the fermentation of cheese and buttermilk. Besides the conversion of lactose to lactate it is responsible for product properties such as flavor and texture, which are determined by volatile metabolites, proteolytic activity and exopolysaccharide production. While the species Lactococcus lactis consists of the two subspecies lactis and cremoris their taxonomic position is confused by a group of strains that, despite of a cremoris genotype, display a lactis phenotype. Here we compared and analyzed the (draft) genomes of 43 L. lactis strains, of which 19 are of dairy and 24 are of non-dairy origin. Machine-learning algorithms facilitated the identification of orthologous groups of protein sequences (OGs) that are predictors for either the taxonomic position or the source of isolation. This allowed the unambiguous categorization of the genotype/phenotype disparity of ssp. lactis and ssp. cremoris strains. A detailed analysis of phenotypic properties including plasmid-encoded genes indicates evolutionary changes during niche adaptations. The results are consistent with the hypothesis that dairy isolates evolved from plant isolates. The analysis further suggests that genomes of cremoris phenotype strains are so eroded that they are restricted to a dairy environment. Overall the genome comparison of a diverse set of strains allowed the identification of niche and subspecies specific genes. This explains evolutionary relationships and will aid the identification and selection of industrial starter cultures

MINOMICS:visualizing prokaryote transcriptomics and proteomics data in a genomic context

We have developed MINOMICS, a tool that allows facile and in-depth visualization of prokaryotic transcriptomic and proteomic data in conjunction with genomics data. MINOMICS generates interactive linear genome maps in which multiple experimental datasets are displayed together with operon, regulatory motif, transcriptional promoter and transcriptional terminator information

Novel Fructosyltransferases

The present invention describes two novel proteins having fructosyltransferase activity. Both enzymes are derived from lactobacilli, which are food-grade microorganisms with the Generally Recognized As Safe (GRAS) status. One of these proteins produces an inulin and fructo-oligosaccharides, while the other produces a levan. According to the invention lactobacilli capable of producing an inulin and/or a levan and/or fructo-oligosaccharides using one or both of the fructosyltransferases can be used as a probiotic or a symbiotic

Projector 2: contig mapping for efficient gap-closure of prokaryotic genome sequence assemblies

With genome sequencing efforts increasing exponentially, valuable information accumulates on genomic content of the various organisms sequenced. Projector 2 uses (un)finished genomic sequences of an organism as a template to infer linkage information for a genome sequence assembly of a related organism being sequenced. The remaining gaps between contigs for which no linkage information is present can subsequently be closed with direct PCR strategies. Compared with other implementations, Projector 2 has several distinctive features: a user-friendly web interface, automatic removal of repetitive elements (repeat-masking) and automated primer design for gap-closure purposes. Moreover, when using multiple fragments of a template genome, primers for multiplex PCR strategies can also be designed. Primer design takes into account that, in many cases, contig ends contain unreliable DNA sequences and repetitive sequences. Closing the remaining gaps in prokaryotic genome sequence assemblies is thereby made very efficient and virtually effortless. We demonstrate that the use of single or multiple fragments of a template genome (i.e. unfinished genome sequences) in combination with repeat-masking results in mapping success rates close to 100%. The web interface is freely accessible at

SIMAGE: SImulation of DNA-MicroArray Gene Expression data

Background: Simulation of DNA-microarray data serves at least three purposes: (i) optimizing the design of an intended DNA microarray experiment, (ii) comparing existing pre-processing and processing methods for best analysis of a given DNA microarray experiment, (iii) educating students, lab-workers and other researchers by making them aware of the many factors influencing DNA microarray experiments. Results: Our model has multiple layers of factors influencing the experiment. The relative influence of such factors can differ significantly between labs, experiments within labs, etc. Therefore, we have added a module to roughly estimate their parameters from a given data set. This guarantees that our simulated data mimics real data as closely as possible. Conclusions: We introduce a model for the simulation of dual-dye cDNA-microarray data closely resembling real data and coin the model and its software implementation SIMAGE which stands for simulation of microarray gene expression data. The software is freely accessible at: http://bioinformatics.biol.rug.nl/websoftware/simag

Transcriptional response of Lactococcus lactis during bacterial emulsification

Microbial surface properties are important for interactions with the environment in which cells reside. Surface properties of lactic acid bacteria significantly vary and some strains can form strong emulsions when mixed with a hydrocarbon. Lactococcus lactis NCDO712 forms oil-in-water emulsions upon mixing of a cell suspension with petroleum. In the emulsion the bacteria locate at the oil-water interphase which is consistent with Pickering stabilization. Cells of strain NCDO712 mixed with sunflower seed oil did not stabilize the oil droplets. This study shows that the addition of either ethanol or ammonium sulfate led to cell aggregation, which subsequently allowed stabilizing oil-in-water emulsions. From this, we conclude that bacterial cell aggregation is important for emulsion droplet stabilization. To determine how bacterial emulsification influences the microbial transcriptome RNAseq analysis was performed on lactococci taken from the oil-water interphase. In comparison to cells in suspension 72 genes were significantly differentially expressed with a more than 4-fold difference. The majority of these genes encode proteins involved in transport processes and the metabolism of amino acids, carbohydrates and ions. Especially the proportion of genes belonging to the CodY regulon was high. Our results also point out that in a complex environment such as food fermentations a heterogeneous response of microbes might be caused by microbe-matrix interactions. In addition, microdroplet technologies are increasingly used in research. The understanding of interactions between bacterial cells and oil-water interphases is of importance for conducting and interpreting such experiments

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