Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains.Since the beginning of viral gastroenteritis outbreak surveillance in the early 1990s, noroviruses have become recognized as the major cause of reported outbreaks of acute viral gastroenteritis worldwide. Noroviruses form a genus within the family Caliciviridae and are genetically and antigenically highly variable. Currently, five distinct genogroups (GGs) are recognized. Strains belonging to GGI, GGII, and GGIV are known to cause infections in humans. The GGs have been subdivided further into genotypes, defined by a minimum amino acid sequence identity over the complete capsid sequence of 80% (1).The strains most commonly identified as the cause of outbreaks belong to genotype GGII.4. In The Netherlands, this was the case for 68% of all norovirus outbreaks that were characterized during 12 years of surveillance and for up to 81% of all health care-related outbreaks. Since their first detection in The Netherlands in January 1995, the GGII.4 strains have consistently been present in the Dutch population (46). These observations are in agreement with those of other surveillance studies worldwide (3,4,15,17,29,36,55).During the past 15 years, four epidemic norovirus seasons have occurred, in the winters of 1995-1996, 2002-2003, 2004-2005, and 2006-2007. These worldwide epidemics were invariantly caused by the predominant genotype, GGII.4, and were attributed to the emergence of new variant lineages of this genotype (4,3...

BackgroundLactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains.ResultsWe performed a genome-wide comparative genomics analysis of proteolytic system components, including cell-wall bound proteinase, peptide transporters and peptidases, in 22 sequenced LAB strains. The peptidase families PepP/PepQ/PepM, PepD and PepI/PepR/PepL are described as examples of our in silico approach to refine the distinction of subfamilies with different enzymatic activities. Comparison of protein 3D structures of proline peptidases PepI/PepR/PepL and esterase A allowed identification of a conserved core structure, which was then used to improve phylogenetic analysis and functional annotation within this protein superfamily.The diversity of proteolytic system components in 39 Lactococcus lactis strains was explored using pangenome comparative genome hybridization analysis. Variations were observed in the proteinase PrtP and its maturation protein PrtM, in one of the Opp transport systems and in several peptidases between strains from different Lactococcus subspecies or from different origin.ConclusionsThe improved functional annotation of the proteolytic system components provides an excellent framework for future experimental validations of predicted enzymatic activities. The genome sequence data can be coupled to other "omics" data e.g. transcriptomics and metabolomics for prediction of proteolytic and flavor-forming potential of LAB strains. Such an integrated approach can be used to tune the strain selection process in food fermentations.

Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent D-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted ␣-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these "adaptation" genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer.

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 ␣-galactosides, ␤-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, restrictionmodification 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.Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products. Because of its tremendous industrial importance, numerous studies have been dedicated to the elucidation of the physiology and molecular biology of many traits relevant for industrial applications, including the production of flavor compounds, vitamins and other nutraceuticals, and exopolysaccharides relevant for texture development (33,60,68). Moreover, due to the availability of a vast molecular toolbox for genetic engineering and recent genome sequencing efforts, L. lactis has gained a strong position as a model organism for low-GC gram-positive bacteria and lactic acid bacteria in particular (7,39,43). Much of the biochemical and genetic research has been conducted with a limited number of strains, mainly the plasmid-cured strains IL-1403 and MG1363, which originate from dairy fermentations.(Fermenting) plant material is a second important ecosystem occupied by L. lactis, where it typically occurs as an early colonizer that is later replaced by species that are more tolerant of low pH values (30, 31). Most plant-associated strains belong to Lactococcus lactis subsp. lactis, whereas Lactococcus lactis subsp. cremoris is typically found in dairy fermentations (30, 31). Fermenting ...