Massive gene presence-absence variation shapes an open pan-genome in the Mediterranean mussel.

BACKGROUND: The Mediterranean mussel Mytilus galloprovincialis is an ecologically and economically relevant edible marine bivalve, highly invasive and resilient to biotic and abiotic stressors causing recurrent massive mortalities in other bivalves. Although these traits have been recently linked with the maintenance of a high genetic variation within natural populations, the factors underlying the evolutionary success of this species remain unclear. RESULTS: Here, after the assembly of a 1.28-Gb reference genome and the resequencing of 14 individuals from two independent populations, we reveal a complex pan-genomic architecture in M. galloprovincialis, with a core set of 45,000 genes plus a strikingly high number of dispensable genes (20,000) subject to presence-absence variation, which may be entirely missing in several individuals. We show that dispensable genes are associated with hemizygous genomic regions affected by structural variants, which overall account for nearly 580 Mb of DNA sequence not included in the reference genome assembly. As such, this is the first study to report the widespread occurrence of gene presence-absence variation at a whole-genome scale in the animal kingdom. CONCLUSIONS: Dispensable genes usually belong to young and recently expanded gene families enriched in survival functions, which might be the key to explain the resilience and invasiveness of this species. This unique pan-genome architecture is characterized by dispensable genes in accessory genomic regions that exceed by orders of magnitude those observed in other metazoans, including humans, and closely mirror the open pan-genomes found in prokaryotes and in a few non-metazoan eukaryotes.

Population genomic footprints of environmental pollution pressure in natural populations of the Mediterranean mussel.

Bivalve molluscs of the genus Mytilus are considered a model organism in ecotoxicology and are known to be well adapted to marine ecosystems affected by multiple anthropogenic factors, including pollution. In order to assess whether pollution interferes with the reproductive success of Mytilus and affects the diversity within and between populations, we sequenced the transcriptomes of 72 individuals from 9 populations of Mytilus galloprovincialis collected along a ca. 130-km north-south transect on the Western coast of the Iberian Peninsula. We found that polluted areas are acting as a barrier to gene flow, potentially because of the detrimental effect of anthropogenic chemicals on larvae carried from more pristine environments. Furthermore, we observed an increase in genetic diversity in populations from polluted site, which could be indicative of higher mutagenicity driven by the environment. We propose that a microevolutionary perspective is essential for a full characterization of human activities on the dispersal of M. galloprovincialis and that it should be incorporated into management, and conservation plans and policies in the context of the effects of pollution on populations.

Genome sequence of Bradyrhizobium sp. LMTR 3, a diazotrophic symbiont of Lima bean (Phaseolus lunatus).

Bradyrhizobium sp. LMTR 3 is a representative strain of one of the geno(species) of diazotrophic symbionts associated with Lima bean (Phaseolus lunatus) in Peru. Its 7.83 Mb genome was sequenced using the Illumina technology and found to encode a complete set of genes required for nodulation and nitrogen fixation, and additional genes putatively involved in root colonization. Its draft genome sequence and annotation have been deposited at GenBank under the accession number MAXC00000000.

Draft genome sequence of Bradyrhizobium paxllaeri LMTR 21T isolated from Lima bean (Phaseolus lunatus) in Peru.

Bradyrhizobium paxllaeri is a prevalent species in root nodules of the Lima bean (Phaseolus lunatus) in Peru. LMTR 21T is the type strain of the species and was isolated from a root nodule collected in an agricultural field in the Peruvian central coast. Its 8.29 Mbp genome encoded 7635 CDS, 71 tRNAs and 3 rRNAs genes. All genes required to stablish a nitrogen-fixing symbiosis with its host were present. The draft genome sequence and annotation have been deposited at GenBank under the accession number MAXB00000000.

Genome of Rhizobium leucaenae strains CFN 299(T) and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici "group" are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian "Cerrados" soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299(T) and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions. RESULTS: The genomes of R. leucaenae strains CFN 299(T) and CPAO 29.8 were estimated at 6.7-6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299(T) is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different. CONCLUSION: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899(T) and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

Correction: A First Insight into the Genome of the Filter-Feeder Mussel Mytilus galloprovincialis.

[This corrects the article DOI: 10.1371/journal.pone.0151561.].

A First Insight into the Genome of the Filter-Feeder Mussel Mytilus galloprovincialis.

Mussels belong to the phylum Mollusca, one of the largest and most diverse taxa in the animal kingdom. Despite their importance in aquaculture and in biology in general, genomic resources from mussels are still scarce. To broaden and increase the genomic knowledge in this family, we carried out a whole-genome sequencing study of the cosmopolitan Mediterranean mussel (Mytilus galloprovincialis). We sequenced its genome (32X depth of coverage) on the Illumina platform using three pair-end libraries with different insert sizes. The large number of contigs obtained pointed out a highly complex genome of 1.6 Gb where repeated elements seem to be widespread (~30% of the genome), a feature that is also shared with other marine molluscs. Notwithstanding the limitations of our genome sequencing, we were able to reconstruct two mitochondrial genomes and predict 10,891 putative genes. A comparative analysis with other molluscs revealed a gene enrichment of gene ontology categories related to multixenobiotic resistance, glutamate biosynthetic process, and the maintenance of ciliary structures.

RNA-Seq in Mytilus galloprovincialis: comparative transcriptomics and expression profiles among different tissues.

BACKGROUND: The Mediterranean mussel (Mytilus galloprovincialis) is a cosmopolitan, cultured bivalve with worldwide commercial and ecological importance. However, there is a qualitative and quantitative lack of knowledge of the molecular mechanisms involved in the physiology and immune response of this mollusc. In order to start filling this gap, we have studied the transcriptome of mantle, muscle and gills from naïve Mediterranean mussels and hemocytes exposed to distinct stimuli. RESULTS: A total of 393,316 million raw RNA-Seq reads were obtained and assembled into 151,320 non-redundant transcripts with an average length of 570 bp. Only 55 % of the transcripts were shared across all tissues. Hemocyte and gill transcriptomes shared 60 % of the transcripts while mantle and muscle transcriptomes were most similar, with 77 % shared transcripts. Stimulated hemocytes showed abundant defense and immune-related proteins, in particular, an extremely high amount of antimicrobial peptides. Gills expressed many transcripts assigned to both structure and recognition of non-self patterns, while in mantle many transcripts were related to reproduction and shell formation. Moreover, this tissue presented additional and interesting hematopoietic, antifungal and sensorial functions. Finally, muscle expressed many myofibril and calcium-related proteins and was found to be unexpectedly associated with defense functions. In addition, many metabolic routes related to cancer were represented. CONCLUSIONS: Our analyses indicate that whereas the transcriptomes of these four tissues have characteristic expression profiles in agreement with their biological structures and expected functions, tissue-specific transcriptomes reveal a complex and specialized functions.

Mosaic-like sequences containing transposon, phage, and plasmid elements among Listeria monocytogenes plasmids.

Sequencing of plasmid pLM33 from the food isolate Listeria monocytogenes Lm1 revealed a molecule of 32,307 bp with a G+C content of 36.2%. The plasmid displays a mosaic pattern of identities common to several closely related L. monocytogenes plasmids isolated from food and clinical sources.

Comparative genomics and proteomics of Helicobacter mustelae, an ulcerogenic and carcinogenic gastric pathogen.

BACKGROUND: Helicobacter mustelae causes gastritis, ulcers and gastric cancer in ferrets and other mustelids. H. mustelae remains the only helicobacter other than H. pylori that causes gastric ulceration and cancer in its natural host. To improve understanding of H. mustelae pathogenesis, and the ulcerogenic and carcinogenic potential of helicobacters in general, we sequenced the H. mustelae genome, and identified 425 expressed proteins in the envelope and cytosolic proteome. RESULTS: The H. mustelae genome lacks orthologs of major H. pylori virulence factors including CagA, VacA, BabA, SabA and OipA. However, it encodes ten autotransporter surface proteins, seven of which were detected in the expressed proteome, and which, except for the Hsr protein, are of unknown function. There are 26 putative outer membrane proteins in H. mustelae, some of which are most similar to the Hof proteins of H. pylori. Although homologs of putative virulence determinants of H. pylori (NapA, plasminogen adhesin, collagenase) and Campylobacter jejuni (CiaB, Peb4a) are present in the H. mustelae genome, it also includes a distinct complement of virulence-related genes including a haemagglutinin/haemolysin protein, and a glycosyl transferase for producing blood group A/B on its lipopolysaccharide. The most highly expressed 264 proteins in the cytosolic proteome included many corresponding proteins from H. pylori, but the rank profile in H. mustelae was distinctive. Of 27 genes shown to be essential for H. pylori colonization of the gerbil, all but three had orthologs in H. mustelae, identifying a shared set of core proteins for gastric persistence. CONCLUSIONS: The determination of the genome sequence and expressed proteome of the ulcerogenic species H mustelae provides a comparative model for H. pylori to investigate bacterial gastric carcinogenesis in mammals, and to suggest ways whereby cag minus H. pylori strains might cause ulceration and cancer. The genome sequence was deposited in EMBL/GenBank/DDBJ under accession number FN555004.

The Bifidobacterium dentium Bd1 genome sequence reflects its genetic adaptation to the human oral cavity.

Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.

Microbial diversity in the human intestine and novel insights from metagenomics.

Bacterial communities reside in very different ecological niches on and within the human host, such as those associated with the alimentary tract. The human gastrointestinal tract is populated with as many as 100 trillion bacterial cells, whose collective genome likely reflects the co-evolution between the microbial community and its host. Recent progress has highlighted the intriguing diversity of these bacterial populations and their important contributions to human physiology. Thus, a thorough understanding of the autochthonous component of the intestinal microbiota is expected to provide crucial information not only on how to develop therapies for various gastrointestinal diseases but also on how to choose the next generation of probiotic bacteria as part of novel functional foods. Recently, novel culture-independent approaches such as metagenomics-based techniques were shown to be crucially important for the exploration of the biodiversity of the human intestinal microbiota.

Lactobacillus hordei sp. nov., a bacteriocinogenic strain isolated from malted barley.

A high-throughput screening effort, designed to isolate bacteriocin-producing lactic acid bacteria (LAB) from malted cereals, resulted in the isolation of four bacteriocin-producing strains that could not be assigned conclusively to recognized species. The four isolates (UCC128T, UCC125, UCC126 and UCC127) were found to share identical (100%) 16S rRNA gene sequences and were therefore deemed to belong to the same species. The strains were Gram-positive, catalase-negative, non-motile homofermentative LAB. The closest recognized relative to strain UCC128T identified based on comparative 16S rRNA gene sequence analysis was Lactobacillus mali DSM 20444T (97% similarity). The strains were characterized phenotypically to identify specific growth requirements. DNA-DNA hybridization between strain UCC128T and L. mali DSM 20444T revealed a level of relatedness of only 39.4%. This indicates that strain UCC128T does not belong to the species L. mali. The four bacteriocin-producing strains are therefore considered to represent a novel species of the genus Lactobacillus, for which the name Lactobacillus hordei sp. nov. is proposed. The type strain is UCC128T (=DSM 19519T=LMG 24241T).

Comparative analyses of prophage-like elements present in two Lactococcus lactis strains.

In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages.

Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum.

Actinobacteria constitute one of the largest phyla among bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.

GANNPhos: a new phosphorylation site predictor based on a genetic algorithm integrated neural network.

With the advance of modern molecular biology it has become increasingly clear that few cellular processes are unaffected by protein phosphorylation. Therefore, computational identification of phosphorylation sites is very helpful to accelerate the functional understanding of huge available protein sequences obtained from genomic and proteomic studies. Using a genetic algorithm integrated neural network (GANN), a new bioinformatics method named GANNPhos has been developed to predict phosphorylation sites in proteins. Aided by a genetic algorithm to optimize the weight values within the network, GANNPhos has demonstrated a high accuracy of 81.1, 76.7 and 73.3% in predicting phosphorylated S, T and Y sites, respectively. When benchmarked against Back-Propagation neural network and Support Vector Machine algorithms, GANNPhos gives better performance, suggesting the GANN program can be used for other prediction tasks in the field of protein bioinformatics.

Exploiting Bifidobacterium genomes: the molecular basis of stress response.

Bifidobacteria represent important human commensals because of their perceived contribution to the maintenance of a balanced gastro intestinal tract (GIT). In recent years bifidobacteria have drawn much scientific attention because of their use as live bacteria in numerous food preparations with various health-related claims. For such reasons these bacteria constitute a growing area of interest with respect to genomics, molecular biology and genetics. This review will discuss the current knowledge on the molecular players that allow bifidobacteria to contend with heat-, osmotic-, bile-and acidic stress. Here, we describe the principal molecular chaperones involved in such stresses, as well as their use as phylogenetic markers for gaining insight into the evolutionary history of high G+C Gram positive bacteria.

Distribution of megaplasmids in Lactobacillus salivarius and other lactobacilli.

The genome of Lactobacillus salivarius UCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains of L. salivarius isolated from humans and animals all harbor a megaplasmid, which hybridized with the repA and repE replication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118 repA probe were also found in some strains of L. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of the repE and groEL sequences of 28 L. salivarius strains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids in L. salivarius was highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six other Lactobacillus species from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids in L. salivarius, and restricted carriage by other Lactobacillus species, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology of Lactobacillus.

Molecular characterization of hsp20, encoding a small heat shock protein of bifidobacterium breve UCC2003.

Small heat shock proteins (sHSPs) are members of a diverse family of stress proteins that are important in cells to protect proteins under stressful conditions. Genome analysis of Bifidobacterium breve UCC2003 revealed a single sHSP-encoding gene, which was classified as a hsp20 gene by comparative analyses. Genomic surveillance of available genome sequences indicated that hsp20 homologs are not widely distributed in bacteria. In members of the genus Bifidobacterium, this gene appears to be present in only 7 of the 30 currently described species. Moreover, phylogenetic analysis using all available bacterial and eukaryotic sHSP sequences revealed a close relationship between bifidobacterial HSP20 and the class B sHSPs found in members of the division Firmicutes. The results of this comparative analysis and variation in codon usage content suggest that hsp20 was acquired by certain bifidobacteria through horizontal gene transfer. Analysis by slot blot, Northern blot, and primer extension experiments showed that transcription of hsp20 is strongly induced in response to severe heat shock regimens and by osmotic shock.

Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363.

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.