Streptococcus phocae subsp. phocae is a pathogen that affects different pinniped and mammalian species. This announcement reports the genome sequence of the type strain ATCC 51973 isolated in Norway from clinical specimens of harbor seal (Phoca vitulina), revealing interesting genes related to possible virulence factors. Streptococcus phocae is a beta-hemolytic Gram-positive bacterium and member of the pyogenic streptococcal group (Phoca vitulina)  farm  . This paitulina) –6, and iar) farm .S. phocae as S. phocae subsp. salmonis, thus becoming the only subspecies apart from S. phocae subsp. phocae to be isolated from seals. These two subspecies differ biochemically and physiologically, including in hemolytic capacities, range of growth temperature, and host specificity (S. phocae subsp. phocae is ATCC 51973 (A recent comparative polyphasic study analyzed strains from different host origins to clarify the taxonomic position of strains isolated from Atlantic salmon. This study reclassified cificity . The typCC 51973 .S. phocae subsp. phocae ATCC 51973T genome was sequenced from a DNA library using the Illumina Nextera XT library preparation protocol, according to the manufacturer’s instructions, and MiSeq technology with 50-fold coverage. The MiSeq reads were de novo assembled into 111 contigs using the CLC Genomics Workbench version 8.0.2, with 92 contigs >200 bp. The estimated length of the chromosome was 1,700,445 bp, with a G+C content of 39.5%, which is very close to that described by Skaar et al. (The T revealed gene sets related to information subsystems coding for binding factors, such as the fibronectin-binding protein, and toxins, such as streptolysin S, which is a recognized virulence factor of Streptococcus pyogenes (Streptococcus species, hyaluronidase influences pathogenicity and is directly associated with bacteria propagation during infection (S. phocae subsp. phocae.Whole-genome analysis of ATCC 51973pyogenes . Moreovenfection , 12. ThiLHQM00000000. The version described in this paper is the second version.The complete genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession no.


P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions.Outside of Africa, P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia . Measures of diversity, linkage disequilibrium (LD) and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species.Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given setting will have a major role in prioritising malaria control strategies, particularly against the relatively neglected non-falciparum species. Plasmodium vivax is a growing public health burden across the globe. Largely overshadowed by the more fatal P. falciparum parasite, increasing reports of anti-malarial drug resistance and life-threatening disease complications demand concerted efforts to eliminate P. vivax. Outside of Africa, P. vivax usually co-exists with P. falciparum. In these regions, malaria interventions have demonstrated greater success against falciparum. The authors genotyped P. vivax and P. falciparum parasites from 4 co-endemic sites in Indonesia, and used the data to gauge how frequently and how focally parasites were transmitted, and how much they spread between sites. Their results indicated that whilst the P. falciparum populations displayed evidence of low and unstable transmission, the P. vivax populations were more diverse and exhibited more stable transmission patterns, requiring different intervention approaches. Relative to P. falciparum, the P. vivax parasites demonstrated evidence of greater spread between populations, possibly facilitated by the dormant liver stage which enables P. vivax to be carried asymptomatically in patients for long periods of time. These findings highlight the need to reconcile the intervention requirements for non-falciparum malaria species in co-endemic regions, and the general utility of parasite genotyping to facilitate surveillance.The malaria parasite  Plasmodium vivax is a major global health burden, with an estimated 2.85 billion people living at risk of infection  [1-Σpi2], where n is the number of isolates analyzed and pi is the frequency of the ith allele in the population. The pairwise FST metric was used to gauge the genetic distance between populations. Calculations were undertaken using Arlequin software (version 3.5) [F’ST) were additionally calculated to adjust for high marker diversity [K (populations) from 1–10 using the model parameters of admixture with correlated allele frequencies. The most probable K was derived by applying the delta K method [distruct software version 1.1 [IAS) using the web-based LIAN 3.5 software [IAS estimates was assessed using 10,000 random permutations of the data. For each population, LD was assessed in 1) all samples, 2) samples with a maximum of one multi-allelic locus, and 3) with each unique haplotype represented just once. The genetic relatedness between sample pairs was assessed by measuring the proportion of alleles shared between multi-locus haplotype pairs (ps). Using (1-ps) as a measure of genetic distance [An infection was defined as polyclonal if more than one allele was observed at one or more loci. The Multiplicity of infection (MOI) for a given sample was defined as the maximum number of alleles observed at any of the loci investigated. With the exception of measures of polyclonality and MOI, only the predominant allele at each locus in each isolate was used for analysis . The expion 3.5) . Standariversity . Populativersity . Twenty K method . STRUCTUsion 1.1 . Multi-lsoftware . The sigdistance , an unrodistance  was genedistance . Mantel’distance .Statistical comparisons of patient gender ratio and infection polyclonality between sites and species were undertaken using Pearson’s Chi-square test. The significance of difference between sites and species in patient age, parasite density and expected heterozygosity were assessed using the Mann-Whitney U test. Statistical comparison of the MOI between sites and species was undertaken using the Kruskal-Wallis test and Mann-Whitney U test, respectively. All tests were performed using R software, with a significance threshold of 0.05.Ethical approval for this study was obtained from the Eijkman Institute Research Ethics Commission (EIREC) number 45/2011. Written informed consent was obtained from all adults or a parent or guardian for participants less than 18 years of age.P. falciparum or P. vivax  and P. falciparum patients  (P = 0.223). In Bangka, the median age of PCD patients with P. vivax  was 2 years older than the ACD patients  (P = 0.021). In the same population, a 5 year difference was observed in the median age of the P. falciparum PCD  versus ACD  patients but the difference was not significant (P = 0.389). In Sumba and West Timor, the patient demographic was younger, with a significant difference in age between P. vivax  and P. falciparum cases  (P = 3.08 x 10–5). In Sumba, the median age of PCD patients with P. vivax  was 25 years older than the ACD patients  (P = 3.7 x 10–4), although caution is advised in interpretation owing to the modest sample size of the former group (n = 9). The median age of P. falciparum PCD patients  was also higher than the ACD patients  but the difference was not significant (P = 0.060).In Bangka and Kalimantan, the majority of patients were young adults. No significant difference was observed in the median age between P = 1.78 x 10–5). In Bangka, the proportion of males was significantly higher amongst PCD versus ACD groups in both P. vivax  and P. falciparum . In Sumba, no significant difference was observed in gender proportions between PCD and ACD groups in either species (both P >0.05).Relative to the two western provinces , the representation of male patients was less skewed in Sumba and West Timor  (-1 blood) between P. falciparum  and P. vivax  (P = 0.057). In both species, parasite density was significantly lower in ACD versus PCD patients. In P. vivax, the median parasite density was 1,126 ul-1  in ACD versus 6,551 ul-1  in PCD (P = 1.25 x 10–5). In P. falciparum, the median parasite density was 1,412 ul-1  in ACD versus 21,160 ul-1  in PCD (P = 7.28 x 10–11). In P. vivax, no significant difference was observed between the sites in the parasite density in the PCD (P = 0.097) or ACD (P = 0.261) patients. In P. falciparum, no significant difference was observed between the sites in the parasite density in the PCD (P = 0.084) patients. A significant difference was observed amongst the P. falciparum ACD patients (P = 8.01 x 10–5), largely reflecting the low parasite density in West Timor (median = 480 ul-1). The various differences observed in parasite density justified the implementation of the 33% predominant peak height threshold for calling additional alleles to reduce potential bias in characterising polyclonal infections.No significant difference was observed in the median parasite density  and 3 (1.8%) isolates exhibited 1 and 3 genotyping failures, respectively. Whilst the TA109 and TA42 loci exhibited 9 (5.4%) and 8 (4.8%) genotyping failures, respectively, all other loci exhibited 3 or less (0–2%) failures. In P. vivax, 21 (12.5%) and 2 (1.2%) isolates exhibited 1 and 2 genotyping failures, respectively. With the exception of the MS5 locus, which exhibited 12 (7%) genotyping failures, all other markers exhibited 3 or less (0–1%) failures.A summary of the genotype calls for each of the The distribution of allele frequencies at each marker is illustrated in P. falciparum infections (0–20%) and mean MOI (1–1.23) across sites, although none of the differences reached statistical significance. In contrast, the variation in the polyclonality of P. vivax infections (23–79%) was highly significant (P = 0.0002), as was the variation in mean MOI (1.23–1.90); P = 0.0002. Across the sites, higher proportions of polyclonal infections (P = 1.042 x 10–12) and higher mean MOI (P = 4.39 x 10–13) were observed in P. vivax versus P. falciparum. In P. falciparum, 41.7% (10/24) of polyclonal infections were multi-allelic at just one of the nine loci. A similar proportion of the polyclonal P. vivax infections, 38% (33/87) were multi-allelic at just one locus. In both P. falciparum and P. vivax 95% or more polyclonal infections could be identified with the seven most diverse markers.A summary of polyclonality, MOI and population diversity is presented in P. falciparum population diversity varied between sites (HE = 0.397–0.715), with the highest diversity observed in Sumba. The differences between Sumba and each of Bangka (P = 0.031) and Kalimantan (P = 0.024) reached significance. Less variation was observed between the sites for P. vivax infections (HE = 0.806–0.856) . The diversity in P. vivax was consistently higher than in sympatric P. falciparum populations, reaching significance in Bangka, Kalimantan and West Timor  but not Sumba (P > 0.05).FST = 0.087; P <1 x10-5), the differentiation between the P. falciparum populations was high  (P. vivax (FST range: -0.003–0.066), with significance in all comparisons  with the exception of Kalimantan, possibly reflecting the limited sample size.With the exception of Sumba and West Timor (1 x10-5) . DiffereP. falciparum population  populations, and increasing sub-structure was observed within West Timor.STRUCTURE analysis demonstrated clear sub-structure in the pulation . The delkelihood . At K = P. vivax, the delta K method identified K = 5 as the most likely number of sub-populations . Relative to P. falciparum, little distinction was observed between the P. vivax infections from the other three sites. With the exception of the P. vivax population in West Timor, no significant correlation was observed between the distance in sampling date and the proportion of alleles shared between infections for P. falciparum or P. vivax.Fewer identical isolates were observed in P. falciparum, LD ranged from an IAS of 0.018 to 0.239   . HoweverP. vivax, significant LD was observed in Bangka, Sumba and West Timor , but not Kalimantan . Identical isolates were less frequent than in P. falciparum, and the IAS scores from the unique multi-locus data sets retained equivalent significance to the unfiltered data sets.In P. vivax and P. falciparum isolates in multiple settings. Using molecular approaches, we demonstrated striking contrasts in diversity and population structure, indicative of varying transmission patterns within and between the two species in different endemicities.The heterogeneous epidemiology of malaria in Indonesia provided an opportunity to investigate the comparative transmission dynamics of co-endemic P. falciparum diversity and structure across the sites. In all populations, polyclonal infection rates (0–20%) were comparable to rates observed in low endemic settings in Southeast Asia [HE = 0.397–0.715). The highest level of diversity was observed in Sumba, reflecting the higher prevalence in this population relative to the other three sites. Whilst the population diversity in Sumba was comparable to levels observed in hyper-holoendemic regions common to Africa [A range of patterns were observed in ast Asia ,23, Soutast Asia ,26 and past Asia ,30, suggo Africa ,27,30, Bo Africa ,26 and So Africa ,23,29. RP. falciparum samples collected from two regencies , as well as population sub-structure, one possible explanation for the significant LD observed in Bangka was admixture. However, STRUCTURE analysis demonstrated that the majority of isolates in Bangka exhibited predominant ancestry to a single population. In contrast, although sampled from a single administrative district, STRUCTURE analysis indicated that the West Timor population comprised isolates from at least two sub-populations. Thus, admixture may have contributed to the LD observed in West Timor. As demonstrated by the greater than two-fold decline in the index of association (IAS) after accounting for identical multi-locus haplotypes, epidemic expansions also appear to have contributed to the LD observed in West Timor, as well as in Bangka, and Kalimantan. In accordance with epidemic dynamics, clusters of isolates with identical multi-locus haplotypes were prevalent in these populations. Whilst it is possible that the modest number of loci reduced the ability to discriminate between different ‘strains‘, the high diversity observed in Sumba suggests that the resolving capacity was adequate.Owing to pooling of P. falciparum transmission in Bangka, Kalimantan and West Timor. These trends are consistent with Ministry of Health data on malaria incidence in these regions, where P. falciparum endemicity is characterised as unstable endemic to hypo-mesoendemic. Relative to Sumba, patients from Bangka, Kalimantan and West Timor were older (largely teenagers and young adults), with a higher proportion of males in Bangka and Kalimantan. These demographics might in part reflect at-risk populations such as particular occupational groups, and might explain some of the epidemic transmission dynamics. In contrast to the other populations, the high diversity and linkage equilibrium in Sumba indicated that local P. falciparum transmission remained disconcertingly high and stable in this region. In accordance, the endemicity in Sumba lay at the higher end of the hypo-mesoendemic spectrum, and the P. falciparum patient demographic largely reflected children, with limited gender bias.In concert, the observed trends in diversity and LD demonstrated a pattern of low, epidemic P. falciparum transmission across populations has important implications for elimination. Although Sumba and West Timor are grouped in the same target category for elimination by the national malaria elimination program (elimination by 2020), our data demonstrates that they would likely benefit from different intervention activities. The P. falciparum dynamics in Sumba demonstrate the need for total coverage intervention activities such as broad-ranging distribution of long lasting insecticide-treated bed nets. In contrast, more targeted approaches such as reactive case detection and treatment or indoor residual spraying targeted to households in transmission hot-spots or at-risk populations would be more cost-effective in West Timor [As discussed with the specific examples from our data in Indonesia, the heterogeneity in the dynamics of st Timor . We noteP. falciparum populations was generally high, comparable to levels observed in Malaysian Borneo, where declining transmission and limited migration led to a fragmented parasite population [Differentiation between the pulation . The decP. falciparum, variation was also observed between sites in P. vivax diversity and structure. Polyclonal infection rates ranged from 23 to 79%, in broad agreement with local API estimates, indicating potential utility in informing on transmission intensity. In the eastern provinces, where patient demographics largely reflected young children, within-host diversity was comparable to high transmission regions of the Pacific [P. falciparum populations, within-host diversity was consistently higher in P. vivax, in accordance with other studies [P. vivax likely explain these observations, and thus have implications for P. vivax control and elimination in other endemic populations.Albeit less marked than in  Pacific , Southea Pacific ,28 and t Pacific . In the  Pacific  and Mala Pacific . Relativ studies ,19,28. TP. vivax population diversity was high in all four sites (HE = 0.81–0.86), with no apparent correlation with endemicity. Furthermore, the P. vivax populations demonstrated consistently higher diversity than their sympatric P. falciparum populations. The factor(s) responsible for these trends remain unclear. A plausible explanation is that relapsing infections, by enhancing infection complexity, increase the probability that different clones are taken up in the mosquito blood meal, thus promoting the opportunity for new variants to be generated via recombination. However, in temperate regions such as Central China and South Korea, relapse rates are low (20%) and yet P. vivax population diversity remains comparably high [P. vivax populations [In contrast to within-host diversity, as demonstrated in other studies across a range of endemic sites across the globe ,25,33,50bly high ,25. Anotulations ,67.P. vivax populations with extensive within-host and population diversity presents a conundrum also encountered in other studies [The observation of significant LD in  studies ,28,34,67P. falciparum populations, the LD patterns in P. vivax did not demonstrate evidence of epidemic transmission in any of the populations investigated. In concert with the observed differences in within-host and population diversity, the differences in LD demonstrated that in several of the endemic settings investigated here, the P. vivax isolates exhibited more frequent and stable transmission than their P. falciparum counterparts. Hence different interventions may be optimal for the respective malaria species in certain settings; local intervention strategies will need to reconcile these differences to ensure adequate impact against all species of malaria. For example, in Bangka, it appears that ongoing interventions have effectively reduced the intensity of local P. falciparum transmission. The current dynamics are moderately unstable with frequent clonal outbreaks amenable to containment by targeted approaches such as reactive case detection and treatment, and indoor residual spraying of case and surrounding households where the predominant vector species behaviour is compatible, prioritizing cases with rapidly emerging outbreak strains. However, the available evidence indicates that targeted approaches would be less effective for the co-endemic P. vivax population. Likely owing to biological differences between the two species such as the hypnozoite stage or comparatively early development of gametocytes in P. vivax versus P. falciparum, the Bangka P. vivax population appeared to have been less impacted by ongoing interventions than the P. falciparum population, displaying more stable transmission with no evidence of clonal outbreaks. These patterns suggest that broad ranging interventions such as large-scale long lasting insecticide-treated bed net coverage are still needed against P. vivax. In addition, effective treatment of the dormant liver stage may be critical to the success of P. vivax intervention efforts.In contrast to the P. falciparum, genetic differentiation was generally low in P. vivax. This observation might reflect the enhanced ‘mobility’ of the parasite afforded by the asymptomatic hypnozoite reservoir, with important implications for the containment of infection, particularly with the rise of anti-malarial drug resistance in P. vivax infection [P. vivax population diversity and differentiation may in part reflect older, historical events [P. vivax populations will be critical to the successful elimination of this species.In contrast to nfection . Alternal events ,28. A beP. vivax infection if malaria elimination is to be successful in co-endemic regions. A better understanding of the local variation in how frequently parasites are transmitted, how focal these transmission events are in time and space, and the ‘mobility’, or potential for parasite spread, can facilitate more optimal malaria intervention strategies. Parasite genotyping can provide useful insights on these dynamics, providing a complementary surveillance tool to the more traditional epidemiological measures, particularly in regions where non-falciparum species circulate.Concerted efforts are required to target the major reservoirs of S1 Table1 2010 Census.2 Annual parasite incidence (API) expressed as cases per 1000 population of the administrative region examined (year of investigation and percentage of the population at risk examined are provided in parentheses). Details on the API were provided by Dr Iqbal Elyazar, Malaria Atlas Project.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(CSV)Click here for additional data file.S4 Table(CSV)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.


Lactobacillus rossiae is an obligately hetero-fermentative lactic acid bacterium, which can be isolated from a broad range of environments including sourdoughs, vegetables, fermented meat and flour, as well as the gastrointestinal tract of both humans and animals. In order to unravel distinctive genomic features of this particular species and investigate the phylogenetic positioning within the genus Lactobacillus, comparative genomics and phylogenomic approaches, followed by functional analyses were performed on L. rossiae DSM 15814T, showing how this type strain not only occupies an independent phylogenetic branch, but also possesses genomic features underscoring its biotechnological potential. This strain in fact represents one of a small number of bacteria known to encode a complete de novo biosynthetic pathway of vitamin B12 (in addition to other B vitamins such as folate and riboflavin). In addition, it possesses the capacity to utilize an extensive set of carbon sources, a characteristic that may contribute to environmental adaptation, perhaps enabling the strain's ability to populate different niches. Lactobacillus rossiae  among lactic acid bacteria, and contain a large number of regulatory and transport functions. Similar to L. plantarum, strains of L. rossiae exhibit remarkable ecological adaptability, genotypic and phenotypic diversity, and may be used to ferment a variety of foods Comparative genome analysis of http://www.ncbi.nlm.nih.gov/genome). Knowledge on the genomic features of lactobacilli will facilitate the identification of species and/or strain-specific traits, which may allow the rational design of strategies for improved fermentations or probiotic formulations for targeted food matrices or human applications The peculiarities of food fermentations mainly depend on the physiological and biochemical traits of the processing microbiota. Substantial progress has been achieved in genomic sequencing of lactic acid bacteria, especially for those species that are used for making dairy, sourdough, meat and vegetable foods, or those with promising probiotic traits L. rossiae DSM 15814T, in addition to phenotypic analyses to confirm some of the predicted functional features.For the above reasons, this study carried out the genomic annotation, comparative analyses, and metabolic pathway reconstruction of L. rossiae DSM 15814TLactobacillus genus In silico annotation assigned a function to 74% of these deduced proteins, while the remaining 26% were deemed to represent hypothetical proteins. A Venn diagram, displaying core gene families, and the unique genes of L. rossiae DSM 15814T and other representatives of the Lactobacillus genus, is shown in L. rossiae DSM 15814T appears to harbour a single plasmid , which contains genes predicted to encode a type I R/M system (LROS_2631-33), and proteins involved in conjugation (TraG/TraD/TraE), where the latter genes clustered together with genes specifying putative surface proteins (LROS_2616-25) and several hypothetical proteins (Gln gene (data not shown).Whole-genome shotgun sequencing results of proteins . In addiL. rossiae within the Lactobacillus genus, a supertree based on the concatenation of 96 orthologous genes was built, using relevant sequences from the genomes of L. rossiae DSM 15814T, of 20 other fully sequenced Lactobacillus genus, and of Bifidobacterium breve UCC2003 as an outgroup , accounting for 33% of the total of genes , confirming that this species appears to be quite different from the other members of this genus. It furthermore allowed the identification of a gene cluster (designated here as the pdu-cbi-cob-hem cluster) putatively involved in cobalamin (vitamin B12) de novo biosynthesis, as well as the ethanolamine utilization cluster and the presence of various mobile elements   .pdu-cbi-cob-hem cluster of L. rossiae DSM 15814T showed a high degree of homology with the previously characterized pdu-cbi-cob-hem cluster present in the genome of L. reuteri DSM 20026 The comparative analysis conducted on the here identified pdu-cbi-cob-hem cluster has been extensively described in L. reuteri DSM 20026, where cbi-cob-hem genes are involved in cobalamin production, while the pdu locus encodes propanediol and glycerol dehydratase activity which requires B12 as a cofactor L. reuteri DSM 20026 the acquisition of this locus through horizontal transfer has been proposed because of the presence of IS elements in its flanking regions, even though the mechanism of acquisition is still unclear L. rossiae DSM 15814T with that of L. reuteri DSM 20026 and the pathogen Salmonella enterica var. Typhimurium LT2 showed that gene synteny of these gene clusters is conserved. The only apparent difference is the relative position of the transcriptional regulator pocR, which is located at the beginning of the pdu-cbi-cob-hem gene cluster in L. reuteri (Lreu_1750) S. enterica and L. rossiae (L. reuteri DSM 20016 that the genetic transfer of the gene cluster occurred by means of two independent events eut) of DSM 15814T represents a so far unique feature within the genus Lactobacillus and that protein products of these genes exhibit 30–40% similarity to the corresponding proteins encoded by genes STM2452-70 of S. enterica var. Typhimurium LT2, supporting the notion that horizontal transfer of this region from a progenitor or close relative of S. enterica var. Typhimurium may have occurred in L. rossiae, and that the two species adopted a similar cobalamin-dependent ethanolamine-propanediol utilization strategy. Previous studies have indicated that ethanolamine utilization is a feature that is not uncommon among gut pathogens and gut commensals  de novo biosynthesis of the cofactor vitamin B12 occurs under anaerobic conditions. A recent comparative study conducted on the ethanolamine utilization cluster (eut) in bacteria highlighted that the ethanolamine-ammonia lyase-encoding genes (eutB and eutC) are the most conserved among species (in our case LROS_0819-20), while the accessory genes , homologues of which appear to be absent from the L. rossiae DSM 15814T genome, are believed to be involved in the formation of a metabolosome, yet are not strictly required as they merely seem to increase the substrate utilization efficiency Among lactobacilli evidence of a  rossiae , support12 production in L. rossiae DSM 15814T a bioassay was performed using Lactobacillus delbrueckii ATCC 7830, a strain known as being unable to grow in the absence of vitamin B12 cofactor (see L. delbrueckii ATCC 7830 was observed only following the addition of cytoplasmic extract (CE) obtained from L. rossiae or, alternatively, vitamin B12 to the medium, thus confirming that L. rossiae is capable of producing B12 and that this cofactor is present in its CE   , indicating that this biosynthetic pathway is incomplete or non-functional.te (GTP) . Indeed,IG broth . L. rossd (pABA) . In facten added . Nine geIn silico analyses also predicted that the L. rossiae DSM 15814T genome harbours a substantial number of genes involved in carbohydrate utilization. Based on previous studies, DSM 15814T is capable of metabolizing ribose, D-xylose, arabinose, galactose, glucose, fructose, mannose, N-acetyl glucosamine, maltose, turanose, D-gluconic acid, palatinose, α-hydroxy-butyric acid and D-L lactic acids, when grown in MRS medium T genome sequence revealed 17 complete phosphoenolpyruvate phospho-transferase systems (PEP-PTS) belonging to different families , but also putative ABC transporters involved in the uptake of various carbohydrates .L. rossiae DSM 15814T is also predicted to produce a number of extracellular or cell wall-associated polysaccharide-degrading enzymes, represented by a cyclomaltodextrinase , an α-amylase , a β-glucosidase , a mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase  and a neopullulanase . Furthermore, the L. rossiae DSM 15814T genome specifies putative enzymatic pathways predicted to degrade arabinose and xylose-containing poly- and/or oligosaccharides  are present, which convert pyruvate into D- and L-lactate, respectively  or small  genomes Regarding pyruvate metabolism, both D- and L-lactate dehydrogenase-encoding genes .Furthermore, it has previously been demonstrated that growth of hetero-fermentative lactic acid bacteria is enhanced when certain organic carbon sources (for example citrate) are used as external electron acceptors Lactobacillus genus L. rossiae DSM 15814T is predicted to contain genes encoding the enzymes that make up biosynthetic pathways for certain amino acids .The various biosynthetic pathways involved in amino acid synthesis are variably present or absent in the different members of the L. rossiae DSM 15814T genome is predicted to specify a complete arginine deaminase (ADI) pathway, glutamine synthase and glutamate decarboxylase enzymes  produced via this pathway enables the extrusion of cytoplasmic protons through the F0F1 ATPase, thus supporting cell survival following sugar depletion L. rossiae DSM 15814T was detected at phenotypic level (L. rossiae DSM 15814T did not differ (P>0.05) in the arginine-supplemented medium. However, an increase of cell survival was found after 48 h in the growth medium containing arginine compared to the same medium without arginine. Due to the production of NH3 from arginine metabolism, different kinetics of acidification were found, which at 24 h showed a substantial difference of pH 6.79 (arginine-containing medium) versus 4.32 (medium without arginine). The activity of all three ADI pathway enzymes was also found in the cytoplasmic extract of DSM 15814T . Similar data was previously found for L. sanfranciscensisThe ADI gene cluster, characterized in lactococci and lactobacilli, has been correlated with having a positive effect on the flavour of leavened baked goods ic level  by deterL. rossiae contains a gene that is predicted to encode a glutamate decarboxylase (EC 4.1.1.15), which converts glutamate into γ-amino butyric acid (GABA). In addition, the production of GABA was also confirmed phenotypically. The capacity for synthesizing GABA of L. rossiae was assayed in SDB medium. After 24 h of fermentation at 30°C, the concentration of GABA was 1.75 mg/kg. GABA, a four-carbon non-protein amino acid, has been reported to act as the major inhibitory neurotransmitter of the central nervous system, to possess anti-hypertensive, diuretic and tranquilizer effects, and to prevent diabetes Glutamate is inter-convertible to glutamine through the activity of the glutamine synthase . The genome of prt) genes were found in the genome of L. rossiae, which is in agreement with the fact that L. rossiae was shown to be unable to grow in milk without the addition of peptides or free amino acids. In addition, only a weak protease activity was found on albumin and globulin (data no shown).Except for two putative and uncharacterized zinc proteases  , no predicted extracellular proteinase (L. sanfranciscensisL. rossiae has more than 20 genes encoding putative intracellular peptidases with different specificities (e.g. aminopeptidase and oligo-/tri-/di-peptidase activities), which may confer this strain the ability of hydrolysing a broad range of peptides , some of which are involved in stress response , L. helveticus  or L. sanfranciscensis , while it is similar to that found for the highly versatile species L. casei , yet lower than L. plantarum . These findings are in agreement with the notion that large genomes specify a relative high number of regulatory proteins Regulatory proteins play an important role in the adaptation of bacteria to different environments and response . The numL. rossiae has been shown to be a highly versatile species capable of colonizing different environments, such as fermented cereals, legumes, fruits and meat, as well as being an inhabitant of the human and animal gut-intestinal tract. Analysis of L. rossiae DSM 15814T, a sourdough strain, revealed a minimum predicted genome size of 2,870,031 bp, encoding relatively high numbers of genes involved in carbohydrate and amino acid transport and metabolism, stress resistance, transcriptional regulation and signal transduction. Based on phylogeny, phylogenomic and comparative analyses, the L. rossiae taxon forms an independent phylogenetic branch, in close proximity to the L. reuteri, L. fermentum, L. plantarum and L. brevis groups, which suggests that these species all adopted a similar strategy for phylogenetic evolution and environmental adaptation. From a technological point of view, the genome of L. rossiae DSM 15814T shows some interesting features: (i) the de novo production of B12 cofactor and other B-complex vitamins (e.g. riboflavin and folate), which represents an important biotechnological potential for industrial applications; (ii) the synthesis of flavor-related compounds from the pyruvate metabolism, proteolysis and amino acid catabolism; and (iii) the capacity to use a substantial number of carbon sources, which provides metabolic flexibility in terms of nutrient availability. In addition, the high number of genes correlated to regulatory systems indicates that L. rossiae is a versatile species with a high potential for environmental colonization. The results of this study are expected to provide the foundation for further investigations to exploit the biotechnology potential of L. rossiae isolates.Since its isolation, L. rossiae DSM 15814T was obftained from the NCBI database (accession number: NZ_AKZK00000000) and the analysis focused on 57 large contigs (400 bp size cut-off). Open Reading Frame (ORF) prediction and functional assignment was automatically performed using the RAST webserver (http://rast.nmpdr.org/), and the generated information was integrated and assessed with the contribution of alternative databases as protein family (Pfam) (http://pfam.sanger.ac.uk), ENZYME database http://www.ncbi.nlm.nih.gov/Ftp/databases/COG), using a cut-off E-value of 0.0001 with at least 50% of identity across a minimum of 50% of a given protein length.The genome sequence of Lactobacillus species, including L. rossiae DSM 15814T, through the bi-directional BLASTP alignment BLAST-based comparative analyses at protein level were performed on 20 representative Lactobacillus species.Using Markov Cluster Algorithm (MCL) http://www.r-project.org), using as input the binary matrix computed with the above method. A heatmap displaying the clustering of organisms and related families was generated through the Multiple Experiment Viewer (MeV) tool v.4.8.1 (http://mev-tm4.sourceforge.net).Hierarchical clustering analysis was performed with the hclust package in R  and 96 selected orthologues. The relative phylogenetic relations were inferred using the JTT model in Phyml v3.0 http://evolution.genetics.washington.edu/phylip.html). In all cases, bootstrap assessment of the inferred relations was carried out by means of 1000 bootstrap iterations.First, phylogenetic analysis of L. rossiae DSM 15814T, which should guide experimental studies to investigate genotype–phenotype relationships. EC numbers were extracted from genome annotations. They were then automatically mapped onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways Metabolic reconstruction may provide insights regarding the metabolic potential of L. rossiae DSM 15814T was assayed in modified semi-defined medium (LDMIIIG) (pH 5.6) Growth of L. rossiae DSM 15814T, a bioassay was performed using Lactobacillus leichmannii ATCC 7830 (formerly Lactobacillus delbrueckii subsp. lactis ATCC 7830), a strain that requires B12 for growth  and amino acid analyses see .L. rossiae DSM 15814T was propagated for 24 h at 30°C in modified MRS broth  with the addition of fresh yeast extract  and 28 mM maltose to a final pH of approximately 5.6 et al. Figure S1Predicted plasmid of L. rossiaeT. DSM 15814 Genome atlas representing the organization of ORFs in the predicted plasmid pLR1 of L. rossiae DSM 15814T. Displayed are, from inner to outer circle: G+C skew, G+C content and ORFs organization. Each ORF is also classified based on the predicted function.(TIF)Click here for additional data file.Figure S2Utilization of arabinose and xylose-containing poly/oligosaccharides.(TIF)Click here for additional data file.Figure S3Pyruvate metabolic pathway of L. rossiaeT. DSM 15814(TIF)Click here for additional data file.Figure S4Locus map showing the organization of the arginine deaminase (ADI) cluster.L. rossiae DSM 15814T, L. amylovorus GRL 1112, L. acidophilus 30SC, L. fermentum IFO 3956, L. sakei subsp. sakei 23K, L. brevis ATCC 367, L. buchneri NRRL B-30929, L. kefiranofaciens ZW3.(TIF)Click here for additional data file.Figure S5Kinetics of growth (Log CFU/ml) (A) and pH of growth medium (B).L. rossiae DSM 15814T cells were cultivated in MAM broth with (empty triangles) and without (filled triangles) 17 mM arginine.(TIF)Click here for additional data file.Figure S6Peptidase activities of L. rossiaeT. DSM 15814(TIF)Click here for additional data file.Table S1Selected contig and accession number of the genome of Lactobacillus rossiaeT. DSM 15814(XLSX)Click here for additional data file.Table S2General features of representatives of the genus Lactobacillus analysed in this study.(DOCX)Click here for additional data file.Table S3In silico search of the genome of Lactobacillus rossiaeT for ORFs putatively involved in amino acid metabolism. DSM 15814(DOCX)Click here for additional data file.Table S4In silico analysis of the genome of Lactobacillus rossiaeT for ORFs encoding proteases and peptidases, and their cleavage specificities, gene names and relative abundance in the genome. DSM 15814(DOCX)Click here for additional data file.Table S5Predicted transcriptional regulators encoded by the Lactobacillus rossiaeT genome. DSM 15814(DOCX)Click here for additional data file.Text S1Results - Supporting Information.(DOCX)Click here for additional data file.Text S2Experimental Procedures - Supporting Information.(DOCX)Click here for additional data file.


The assembled genome size was 462.1 Mb, covering 75.2% of the genome (615.2Mb) and containing 32, 456 predicted protein-coding genes. Since the approximate divergence around 5.8 million years ago, the genomes of Musa itinerans and Musa acuminata have shown conserved collinearity. Gene family expansions and contractions enrichment analysis revealed that some pathways were associated with phenotypic or physiological innovations. These include a transition from wood to herbaceous in the ancestral Musaceae, intensification of cold and drought tolerances, and reduced diseases resistance genes for subtropical marginally distributed Musa species. Prevalent purifying selection and transposed duplications were found to facilitate the diversification of NBS-encoding gene families for two Musa species. The population genome history analysis of M. itinerans revealed that the fluctuated population sizes were caused by the Pleistocene climate oscillations, and that the formation of Qiongzhou Strait might facilitate the population downsizing on the isolated Hainan Island about 10.3 Kya. The qualified assembly of the M. itinerans genome provides deep insights into the lineage-specific diversification and also valuable resources for future banana breeding.Crop wild relatives are valuable resources for future genetic improvement. Here, we report the  Musa acuminata (A genome) and M. balbisiana (B genome) or hybrids between subspecies of M. acuminata. Originally, domesticated in Southeast Asia about 7000 years agoFusarium wilt disease, Tropical Race 4′  has caused devastating damages to this cultivar. There are about 60 species in the Musa genus, and some of them have been utilized in banana resistance improvement, for instance, a wild non-edible accession of M. acuminata ssp. burmannica was once extensively used in banana breeding for its resistances to black Sigatoka and some races of Fusarium wiltCrop wild relatives (CWRs) are composed of progenitors and more or less distantly related species of cropsM. acuminata ssp malaccensis (A genome), one progenitor of the cultivated banana, was sequenced and the genome evolution peculiarities of the Musaceae in the monocots were thus revealedM. balbisiana (B genome) has been releasedM. itinerans, a close relative to both banana progenitors with wide distribution across subtropical ChinaMusa itinerans var itinerans, known also as Yunnan banana, is native to south-east Asia and can be found in moist ravines to mountainous areas up to 2200 mMusa genusM. itinerans also displays interesting agronomic characteristic such as rapid fruit maturation . Moreover, the studies will facilitate the understanding of the lineage-specific genome evolution in the Musa genus.In 2012, the genome of M. itinerans collected from Baoting County on Hainan Island and transplanted in South China Botanical Garden was sequenced by whole-genome shotgun strategy on the Illumina Hiseq2000 platform   . Thus, wde novo predictions with RepeatModeler approaches, 38.95% (179.99 Mbp) of the Musa itinerans genome were predicted to contain repetitive elements, somewhat higher than that of M. acuminata   were predominant in M. itinerans, and the two most common subfamilies LTR/Copia (16.7%) and LTR/Gypsy (16.3%) were almost in equal proportions in M. itinerans. The two peaks around sequence divergence rate were 20% and 30%, indicating that LTRs families in M. itinerans underwent two rounds of expansion events , estimated by this study), suggestive of high turnover rates of retrotransposable elements in the Musa genomes. One recent burst of LTRs occurring at about 0.2 Mya in M. itinerans but not in M. acuminata, showed rapid dynamic evolution for these repeat elements  and hAT (0.90%) were the most common types  as queries against the repeat-masked assemblies, 32,456 protein-coding genes were predicted with an average coding sequence length of 1065 bp and 5.2 exons per gene (Using both homology-search against the RepBase library.59 Mbp) , and then events . Using telements . For M. on types . Using tper gene . For theper gene . In addiper gene .M. itinerans and other eight species, namely, M. acuminata, M. balbisiana, Phoenix dactylifera, Elaeis guineensis, Oryza sativa, Sorghum bicolor, Vitis vinifera, and Arabidopsis thaliana. Of the 28,524 predicted protein sequences (>100 amino-acids in length) in M. itinerans, 26,955 (94.5%) were assigned into 16,742 gene families with 1201 single-copy orthologs, and 177 gene families were found to be unique to M. itinerans  than with M. balbisiana (140 gene families), which were suggestive of higher similarity for the latter two species. The unique gene families of M. itinerans among the five species were significantly enriched for 40 GO terms and involved in some essential functional categories such as DNA polymerase activity , endoribonuclease activity , and ribonuclease activity   . Twelve ic test) , and it ic test) . OverallMusa species  based on four-fold degenerated sites (4DTv sites) of 1201 single-copy orthologs, and the ancestor of M. itinerans and M. acuminata diverged with M. balbisiana about 8.3 Mya , and Musaceae and Arecaceae coalesced to 120.9 Mya   between 1.2 Mya) .https://genomevolution.org/CoGe/), syntenic regions between M. itinerans and M. acuminata were generated. Based on the collinear blocks between M. itinerans and M. acuminata, 26,670 genes, comprising of 82.5% of the M. itinerans gene sets distributed on 1043 scaffolds, were oriented and anchored to the 11 linkage groups  of these syntenic orthologous and paralogous gene pairs, the speciation and paleoploidization events in the Musa genus were inferred. When following an average Ks of 3.47 per 109 years for nuclear genes in the ZingiberalesM. itinerans and M. acuminata diverged approximately about 5.8 million years ago with peak Ks around 0.04 , hence, the ancient ages of these duplicated NBS-encoding genes were not in accordance with the classic arm-race model, which predicted a high turnover rate and predominant young NBS-encoding genes within a genome30Ka/Ks (nonsynonymous to synonymous substitution ratio) values of most duplicated pairs  within both genomes were less than 1, indicating that purifying selection played an essential role in the maintenance of intra-genomic diversity of these NBS-encoding genes in Musa, as the cases in chestnutSolanumMusa, the NBS-encoding gene repertoire seemed to be associated with their specific habitat shift.Following the method described in D’Hont ectively . The dec species . With duMusa species were identified by blast against known transcription factor gene families of related species, as well as homology prediction using all the known transcription factor proteins to blast against the M. itinerans genome. In total, 3176 and 2898 putative transcription factor genes, from 58 families, were identified for M. itinerans and M. acuminata, respectively  genes for the two ectively . These nher taxa . SimilarMusa species. With the number of repeats in the N-terminal conserved MYB DNA-binding domain, the MYB super-family can been categorized into four classes, 1R-, R2R3-, 3R-, and 4R-MYB, and the largest one R2R3 MYB was plant specific and key regulators in metabolism and responses to biotic and abiotic stress, also in flowering or fruit developmentMYB genes were identified in the genomes of M. acuminata and M. itinerans after manual inspection, respectively  and rice (70.1%)M. itinerans and M. acuminata for these MYB gene pairs was observed  with median Ks = 0.57 and followed by transposed duplications with median Ks = 0.89 (about 97 gene pairs), both of which were prior to the ancient α/β whole genome duplication events (Ks = 0.40~0.42) in Musaceae (Ka/Ks < 1), followed by transposed duplications . Under this scenario, purifying selections were expected to wipe out deleterious mutations and stabilize the MYB TFs gene repertoire for their essential functions. Similar evolutionary patterns were observed in M. itinerans  (Ne) since the inverse relationship between the effective population size and coalescent rate. The population history analysis of each individual from one population depicted the nearly identical trajectory  was substantial less than either of the two central populations , and this spatial pattern of genetic variations was consistent with theoretical prediction40NeM. itinerans overrode that of the population bottleneck until the progressive disappearance of population heterogeneities at the Last Glacial Maximum . However, due to genetic deficiency or less frequent admixtures, the isolated HN population showed more rapid decay of heterogeneity and earlier shrinkage of Ne than either of the continental populations (To infer the demographic history of overage) . Using tajectory . Using aulations . The finMusa itinerans, one of the most disease-resistant wild banana species, provides invaluable genetic resources for future banana genetic improvement. The M. itinerans diverged from M. acuminata only 5.8 Mya, and exhibited extensive collinearity between them, but the two species occupy distinct habitats, which provides an unpreceded chance to unravel the diversification within the Musa genus. Resorting to lineage-specific gene family expansions and contractions approaches, a batch of candidate genes categorized into diverse pathways or GO terms were inferred to be associated with some peculiar traits. However, more delicate experiments were required to clarify their roles in the physiological or phenotypic innovations, as one of the most disease resistant and cold hardy species in the Musa genus, the gene repertoire space of M. itinerans should be mined in the future. The non-synonymous to synonymous ratio Ka/Ks of <1 for most NBS-encoding gene family revealed pervasive purifying selections, together with the ancient duplication ages, both in support of the trench-warfare model hypothesis and against the classic arms-race model. A similar evolution pattern was also observed in the MYB transcription factor family, in which the purifying selection wiped out deleterious mutations and stabilize the MYB TFs’ gene repertoire for their essential functions. Demographic history has a profound effect on the genetic diversity of extant species. The isolated HN population of M. itinerans was more prone to genetic loss during glacial and interglacial cycles than the counterpart mainland populations, in which admixture differentially buffered against genetic loss. Furthermore, the detailed scenarios of demographic history for this marginally distributed species will be revealed with more extensive sampling and population genome resequencing studies.Genome sequencing of the wild banana M. itinerans are distributed in subtropical China, and individual leaves of M. itinerans were harvested and immediately storied with sillica-gel in the field  were constructed following the manufacturer’s instructions. All the libraries were sequenced on the Illumina Hiseq2000 platform. Raw reads were subject to the removals of PCR duplicates, adaptor sequences, and contaminants of bacterial or viruses. The processed high-quality reads were assembled into contigs and scaffolds using SOAPdenovo2 packagehttp://soap.genomics.org.cn/soapdenovo.html). The reference genome size of M. itinerans was estimated based on the K-mer frequency distribution analysis  and homolog research for the Repbase databasede novo interspersed repeats were annotated using RepeatModeler (http://www.repeatmasker.org/RepeatModeler.html). Tandem repeats in the genome of M. itinerans were screened using Tandem Repeat Finder v4.04Musa acuminata v1 from the Banana Genome HubPhoenix dactylifera DPV01, Oryza sativa IRGSP-1.0, Sorghum bicolor v3.1, Zea mays, and Arabidopsis thaliana TAIR10 from Phytozome (https://phytozome.jgi.doe.gov) or related databases . All the raw short reads, genome assembly were deposited in NBCI’s Sequence Read Archive (SRA) database with accession number PRJNA312694, and the gene annotation used for the analyses were provided to the Banana Genome Hub (banana-genome-hub.southgreen.fr/organism/Musa_itinerans).For noncoding RNAs, tRNAs were identified using tRNAscan-SEhttps://genomevolution.org/CoGe/)M. itinerans and M. acuminata were generated, and scaffolds or contigs of M. itinerans were mapped or oriented on the 11 pseudo-chromosomes of M. acuminata. Reciprocal BLASTP between protein sequences of two Musa species with E-value cut-off of 1e-5, were implemented to identify orthologs between species, and self BLASTP for both species were used to obtain paralogs within species. After removing the self-matches of the BLASTP results, the syntenic blocks (≥5 genes per block) were determined with the package MCScanXUsing the web-based tool SynMap on the plant comparative genome platform CoGe  were calculated in the package MEGA−8 substitutions per site per year in riceThe 5′- and 3′-LTR sequences of the full-length LTR retrotransposons were aligned, and the Arabidopsis thaliana, Vitis vinifera, Oryza sativa, Sorghum bicolor, Phoenix dactylifera, Elaeis guineensis, M. acuminata, and M. balbisiana were downloaded .The protein sequences of eight plant species including http://hmmer.janelia.org/software), protein sequences of M. acuminata, M. itinerans, and M. balbisiana were aligned against the raw hidden Markov model (HMM) corresponding to the Pfam NBS (NB-ARC) family . Following the method described in D’ Hont et al.Musa specific model, we identified NBS- candidate proteins in the three Musa species respectively. TIR and LRR domains of these NBS-encoding amino acid sequences were identified with Pfam_scan. Coiled-Coil (CC) motif was identified using MARCOILUsing HMMER V3 . Second, de novo predicted TFs were obtained using all the TFs of the other five species to TBLASTN against the genome of M. itinerans and corresponding gene models were predicted using GenewiseM. itinerans were obtained. The duplication history of the MYB gene family was inferred using the programme MCScanXTranscription factors’ gene families (TFs) of the g) of one year, and neutral mutation rate μ of 1.30 × 10−8 substitution per site per year.One individual from each of the three populations was sequenced in high depth , and quaAccession codes: The whole Genome assembly sequences and raw reads have been deposited in GenBank DDBJ/ENA/GenBank under the BioProject ID PRJNA312694, with accession numbers SRR3180710, SRR3180652, SRR3180729, SRR3180738, and SRR3180744 for raw reads, and LVTN00000000 for genome assembly, and the version described in this paper is version LVTN01000000. The genome assembly and annotation files for Musa itinerans can also be accessed by The Banana Genome Hub (banana-genome-hub.southgreen.fr/organism/Musa_itinerans).How to cite this article: Wu, W. et al. Whole genome sequencing of a banana wild relative Musa itinerans provides insights into lineage-specific diversification of the Musa genus. Sci. Rep.6, 31586; doi: 10.1038/srep31586 (2016).


Medicago truncatula, Glycine max (soybean), Lotus japonicus, Phaseolus vulgaris (common bean), Cicer arietinum (chickpea) and Cajanus cajan (pigeon pea) and two outgroup reference species: Arabidopsis thaliana and Poplar trichocarpa. Moreover, the LegumeIP 2.0 features the following new data resources and bioinformatics tools: (i) an integrative gene expression atlas for four model legumes that include 550 array hybridizations from M. truncatula, 962 gene expression profiles of G. max, 276 array hybridizations from L. japonicas and 56 RNA-Seq-based gene expression profiles for C. arietinum. These datasets were manually curated and hierarchically organized based on Experimental Ontology and Plant Ontology so that users can browse, search, and retrieve data for their selected experiments. (ii) New functions/analytical tools to query, mine and visualize large-scale gene sequences, annotations and transcriptome profiles. Users may select a subset of expression experiments and visualize and compare expression profiles for multiple genes. The LegumeIP 2.0 database is freely available to the public at http://plantgrn.noble.org/LegumeIP/.The LegumeIP 2.0 database hosts large-scale genomics and transcriptomics data and provides integrative bioinformatics tools for the study of gene function and evolution in legumes. Our recent updates in LegumeIP 2.0 include gene and protein sequences, gene models and annotations, syntenic regions, protein families and phylogenetic trees for six legume species:  Legumes are the second most important class of crops as a source of food for humans, feed for livestock and raw materials for industry . LegumesMedicago truncatula, Glycine max and Lotus japonicus plus two reference plant species, Arabidopsis thaliana and Populus trichocarpa. The LegumeIP 1.0 database contained detailed gene annotations based on the UniProt , Cicer arietinum (chickpea) and Cajanus cajan (pigeon pea) , Arachis duranensis and Arachis ipaensis (the ancestors of cultivated peanut), Vigna radiata (mung bean) (Vigna angularis (adzuki bean) (Lupinus angustifolius (lupin)  –12, haveng bean) , Vigna aki bean)  and Lupi (lupin)  genomes L. japonicas    and P. vvulgaris . These lLegumeIP 2.0 - A Platform for the Study of Gene Function and Genome Evolution in Legumes’ with a focus on the two most important new features of the database. First, we integrated genomic sequences for six legume species. These include M. truncatula, G. max and L. japonicus that were in the LegumeIP 1.0, three newly sequenced crop legumes, P. vulgaris, C. arietinum and C. cajan, and two reference plant species, A. thaliana and P. trichocarpa. The updated LegumeIP 2.0 covers 391,107 protein-coding gene sequences in total. All sequences were annotated based on sequence similarities and domain features by referring to the UniProt TrEMBL, InterProScan, GO and KEGG databases. Second, we built a brand new integrative gene expression atlas, which consists of large-scale gene expression profiles from multiple technologies for four model legumes, including 550 array hybridizations from M. truncatula, 962 gene expression profiles from G. max, 276 array hybridizations from L. japonicas, and 56 RNA-Seq transcriptome profiles from C. arietinum. All expression profiles were manually curated, annotated using free text descriptions, keywords and further hierarchically organized following the Experimental Ontology (EO)  (http://www.plantontology.org/)  ,22 and Pgy.org/) , allowinM. truncatula, G. max, L. japonicus, P. vulgaris, C. arietinum, C. cajan and two reference plant species, A. thaliana and P. trichocarpa. LegumeIP 2.0 contains 391,107 protein-coding gene sequences in total from eight plant species. Table In LegumeIP 2.0, we have compiled and integrated gene sequences and comprehensive gene annotations for six model or crop legume genomes, including To annotate these genes, the sequences were searched against the reference sequences from the UniProt, TrEMBL, GO and KEGG databases using BLASTP with an expect value <1e-06. Furthermore, these sequences were searched for conserved domains for inclusion of functional annotation using the InterProScan program. All transcription factors (TFs) were predicted using our previously published method  for imprCa_11537, C.cajan_12295 and Phvul.002G143400.1) with the same functions could also be identified with high confidence in C. arietinumx, C. cajan and P. vulgaris based on micro synteny analysis across the six legume species.We performed systematic syntenic analysis and reconstructed cross-species gene family and phylogenic trees for the six legume genomes and the two reference plant genomes using the same methods that were used in the development of LegumeIP 1.0 . SupplemM. truncatula, G. max (soybean) and L. japonicus in public repositories, those datasets were collected, compiled and further curated for inclusion in LegumeIP 2.0. All of the expression profiles were downloaded from ArrayExpress (https://www.r-project.org/) software package. In addition, we included RNA-Seq-based gene expression profile data for chickpea from the NCBI SRA  using the newly developed EO terms and PO terms. The former is a popular ontology that defines experimental conditions based on the published literature and the latter defines plant anatomical structures, which is analogous to the GO . IncorpoExpression Atlas’, for exploring and searching the gene expression atlas of LegumeIP 2.0, in which users can select a subset of microarray or RNA-Seq experiments and further compare expression profiles for multiple genes through EO and PO treeview browsers , KEGG-based enzyme annotations, transcription factors and transporters. Furthermore, we developed user-friendly interfaces, which can be accessed under the menu ‘s Figure , comprehThe research in legume biology has progressed significantly. Using Affymetrix GeneChip and next-generation sequencing technologies have allowed researchers to generate large-scale genomic and transcriptomic data for both model and economically important crop legumes in recent years, which necessitated an important update to our LegumeIP database. We significantly improved the database by integrating more legume species based on the latest legume genome sequencing efforts. Such improvements make LegumeIP 2.0 a more useful tool for comparative genomic study utilizing its hosted syntenic regions, cross-species gene families and phylogenetic models. In this regard, we will continuingly enhance the LegumeIP database as more legume genomes become available.In LegumeIP 2.0, we mapped microarray and RNA-Seq experiments to the graph structures of both EO and PO. In computer science, ontology is the formal naming and definition of the types, properties and inter-relationships of the entities. Both EO and PO define terms and organize information about experiment design and plant samples in graph structures. The introduction of EO and PO can standardize the search of a transcriptomic dataset based on experimental conditions and plant samples and treatments used in the experiments, which are the main challenges in the development of large gene atlas. This development will also simplify our efforts toward continuously updating and expanding transcriptomic data in the LegumeIP system.https://www.coolseasonfoodlegume.org/), Legume Information System (http://phaseolusgenes.bioinformatics.ucdavis.edu/), SoyBase (The LegumeIP 2.0 database features an integrative gene-atlas of four legume species and comparative genomic data, such as synteny and protein family information for all hosted legume and reference genomes. We collected raw data from the genome sequencing projects of legumes Table  and trann System , Phaseol SoyBase  and Soyb SoyBase . In futuhttp://plantgrn.noble.org/LegumeIP/.LegumeIP 2.0 is free to the public and available at


Lactobacillus hokkaidonensis is an obligate heterofermentative lactic acid bacterium, which is isolated from Timothy grass silage in Hokkaido, a subarctic region of Japan. This bacterium is expected to be useful as a silage starter culture in cold regions because of its remarkable psychrotolerance; it can grow at temperatures as low as 4°C. To elucidate its genetic background, particularly in relation to the source of psychrotolerance, we constructed the complete genome sequence of L. hokkaidonensis LOOC260T using PacBio single-molecule real-time sequencing technology.T comprises one circular chromosome (2.28 Mbp) and two circular plasmids: pLOOC260-1 (81.6 kbp) and pLOOC260-2 (41.0 kbp). We identified diverse mobile genetic elements, such as prophages, integrated and conjugative elements, and conjugative plasmids, which may reflect adaptation to plant-associated niches. Comparative genome analysis also detected unique genomic features, such as genes involved in pentose assimilation and NADPH generation.The genome of LOOC260L. vaccinostercus group, which is poorly characterized, so the genomic information obtained in this study provides insight into the genetics and evolution of this group. We also found several factors that may contribute to the ability of L. hokkaidonensis to grow at cold temperatures. The results of this study will facilitate further investigation for the cold-tolerance mechanism of L. hokkaidonensis.This is the first complete genome in the The online version of this article (doi:10.1186/s12864-015-1435-2) contains supplementary material, which is available to authorized users. Silage fermentation is promoted mainly by the microbial activities of lactic acid bacteria (LAB). During the fermentation process, LAB produce lactic acid anaerobically as the major end product of central carbohydrate metabolism, which reduces the pH of the surrounding environment. These anaerobic and acidic conditions prevent the propagation of detrimental microorganisms such as listeria, clostridia, yeasts, and other fungi. However, the acid production level tends to be insufficient if silage is prepared in cold weather conditions because of the impaired activity of LAB, thereby yielding lower quality silage. Therefore, the inoculation of appropriate LAB as a silage additive is required to enhance silage fermentation in low-temperature environments.Lactobacillus species, Lactobacillus hokkaidonensis, from Timothy grass (Phleum pratense) silage in Hokkaido, a subarctic region of Japan [L. hokkaidonensis can grow at temperatures as low as 4°C , and its type strain LOOC260T was shown to decrease pH even in cold conditions when used to inoculate pilot-scale grass silage. Thus, L. hokkaidonensis is expected to be suitable for use as an effective silage inoculant in cold regions.Previously, we isolated a novel psychrotrophic of Japan . L. hokkL. hokkaidonensis is classified as an obligate heterofermentative LAB in the L. vaccinostercus group [L. vaccinostercus [L. suebicus [L. oligofermentans [L. nenjiangensis [L. hokkaidonensis) that form a clade distinct from the well-known heterofermentative clades, which include L. reuteri, L. brevis, and L. buchneri. They share common phenotypic features such as the presence of meso-diaminopimelic acid in their peptidoglycan cell walls and faster assimilation of pentoses compared with hexoses, but little is known about their genetic background or genomic information.us group , which iostercus , L. suebsuebicus , L. oligrmentans , L. nenjangensis , and L. L. hokkaidonensis LOOC260T and comparative genome analysis, where we focused on the unique gene repertoire of the L. vaccinostercus group. In addition, determining the complete genome may provide a better genome-wide understanding of mobile genetic elements, thereby highlighting how flexible genome rearrangements contribute to adaptation to various ecological niches. Thus, we aimed to provide insights into the genomic features of the L. vaccinostercus group, which is poorly characterized at present, as well as to clarify the silage fermentation mechanism from a genomic perspective, particularly in cold conditions.In the present study, we performed whole-genome sequencing of L. hokkaidonensis LOOC260T. De novo assembly using the hierarchical genome assembly process (HGAP) method [T comprises one circular chromosome  and two circular plasmids designated as pLOOC260-1  and pLOOC260-2 . Two prophage regions were predicted, which are described in detail in the following section. No clustered regularly interspaced short palindromic repeat (CRISPR) loci were detected in the genome. The general genomic features of L. hokkaidonensis LOOC260T and four other species in the L. vaccinostercus group are summarized in Table T as well as BLASTP alignment results with its four close relatives, as described above. Sharp transitions in the GC-skew value were observed at both the predicted oriC site (0°) and its opposite site (176°). In particular, genes involved in metabolism (indicated in red) were densely encoded in the region from 300° to 360°. Several genes in this region were missing from all or some of the members of the L. vaccinostercus group, which may reflect the adaptation to specific ecological niches during the diversification of this group. Similar position-specific features have also been reported in L. plantarum [L. casei [Whole-genome sequencing was conducted with the PacBio single-molecule real-time (SMRT) sequencing system to determine the genome sequence of ) method  generatee 2,277,95 bp and de novo assembly often yields collapsed and/or fragmented contigs for such regions. We used the PacBio sequencer to correctly assemble these repetitive regions with much longer reads (4 kbp on average), thereby obtaining a genome-wide perspective of mobile genetic elements such as plasmids and prophages.Bacterial genomes include several repetitive sequences such as multiple copies of ribosomal RNA operons and insertion sequences or transposases. These regions are generally difficult to reconstruct from relatively short sequencing reads, and thus L. reuteri), 75% with ISLrh2 (L. rhamnosus), and 60% with ISLre1 (L. reuteri), respectively.In total, 59 ORFs, including partial ORFs and pseudogenes, were annotated as putative insertion sequences within the genome. In particular, three types of insertion sequence elements were annotated, with 13, 6, and 3 copies that shared almost 100% identity, and these new insertion sequence elements were registered in the ISfinder database [The ratio of the mapped read number normalized against the sequence length for each replicon was approximately 1:1:4 (chromosome:pLOOC260-1:pLOOC260-2). Thus, the plasmid copy number in the cell was estimated as one for pLOOC260-1 and multiple for pLOOC260-2.L. plantarum, L. casei, L. brevis, and L. coryniformis, thereby indicating the occurrence of numerous rearrangements and recombination events during its evolution. The plasmid mobilization protein, Mob, gene was present, which probably facilitated the transmission of pLOOC260-1 in the presence of other conjugation mechanisms. Another interesting characteristic was the presence of a gene cluster related to fatty acid synthesis (LOOC260_200520–LOOC260_200630), which was absent from the chromosome. To the best of our knowledge, plasmid-encoded fatty acid synthesis genes have not been reported previously in other LAB species.The first plasmid, pLOOC260-1, had a composite structure that comprised regions from several LAB species, such as tra conjugation gene cluster, which shared high similarity and colinearity with the plasmid pWCFS103 from L. plantarum WCFS1, for which conjugative transfer was demonstrated experimentally [tra region is also observed in several plant-associated LAB, such as L. brevis KB290, isolated from a Japanese fermented vegetable [L. oryzae, isolated from fermented rice grains [L. coryniformis, frequently isolated from silage.The other plasmid, pLOOC260-2, was considered to be a conjugative plasmid. It possessed a mentally . A similegetable , L. oryze grains , and L. attR and attL). A contig obtained by de novo assembly contained a misassembled region, which was presumably derived from an excised circular phage DNA, and thus spontaneous excision of the prophage must have occurred in a fraction of the cells. To confirm this prediction by PCR, two sets of primers were designed for each prophage so the fragments could be amplified only when the prophages were excised from the chromosome  and φLH-2 . We also found 12-bp direct repeats (5′-TCACTCGCTTCA-3′) flanking φLH-1 and 22-bp direct repeats (5′-ACTTAGAAAAATAAAAACGCGT-3′) flanking φLH-2, which appeared to constitute the core regions of phage attachment sites , sometimes known as conjugative transposons, are self-transmissible mobile genetic elements, which can be integrated into or excised from the host chromosome . ICEs ofaracasei  and the livarius . No othelivarius  and Tn Nlivarius .T in the chromosome region 1,799–1,851 kbp (approximately 52 kbp), which was deposited as Tn6254 in the Tn Number Registry.We identified a putative ICE in the genome of LOOC260T were compared against all the sequences in the NCBI non-redundant protein database, and similar gene organizations with high levels of sequence identity (>90%) were found in four species of plant-related LAB: L. vini LMG 23202T (isolated from grape must), L. nodensis JCM 14932T (from rice bran), L. paracasei LPP49 , and L. coryniformis . The level of shared nucleotide identity was also high between them  and three ABC transporters . The gene repertoire of these transporters was identical to that of L. sakei, a psychrotrophic LAB, in which the accumulation of compatible solutes is considered to be a key factor during acclimation to cold and saline environments [L. hokkaidonensis, GshF shared high similarity with that in L. coryniformis, which was a predominant isolate when we screened for psychrotolerant LAB in Timothy grass silage  gene for regenerating NAD(P)+ in the latter stage of heterolactic fermentation [L. vaccinostercus group possess adhE, which suggests that another mechanism is active.The species in the  hexoses ,5,31. The medium . These centation . By contL. hokkaidonensis  indicates the ability to utilize xylooligosaccharide.As a starter culture for silage fermentation, the ability to assimilate pentoses is advantageous when utilizing substrates derived from plant cell walls. Hemicellulose is one of the major components of the plant cell wall, which is composed of a branched heteropolymer of saccharides . During L. vaccinostercus group LAB. L. hokkaidonensis, L. vaccinostercus, and L. suebicus possessed membrane-bound NAD(P) transhydrogenase PntAB, which mediates the transfer of a hydrogen from NADH to NADP+ to produce NADPH using the electrochemical proton gradient [L. vaccinostercus and L. suebicus possessed NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, GapN, which catalyzes the one-step conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate, with the concomitant reduction of NADP+ to NADPH [Unique mechanisms were found for NADPH generation in the gradient . In addito NADPH . In convStreptococcus mutans, which lacks the oxidative part of the pentose phosphate pathway, has been suggested to participate in NADPH generation [+ mainly functions as an electron acceptor in catabolic reactions. Therefore, both PntAB and GapN are favorable, particularly in the biosynthetic process because they produce a higher NADPH/NADP+ ratio and a lower NADH/NAD+ ratio.The major cellular source of NADPH is considered to be the oxidative branch of the pentose phosphate pathway, where hexoses are decarboxylated into a C5-moiety. However, pentoses are assimilated without passing through this branch; thus, these enzymes may provide an alternative route for generating NADPH. Analogously, GapN in neration . NADPH mL. oligofermentans, the L. vaccinostercus group LAB members encode a relatively high number of genes for amino acid biosynthesis. These NADPH generation systems may support the diverse biosynthetic abilities of L. hokkaidonensis and its close relatives and may reflect the optimized utilization of pentoses as growth substrates.With the exception of the meat-borne L. hokkaidonensis LOOC260T by whole-genome sequencing using the PacBio SMRT sequencing system and de novo assembly based on the HGAP method. We found that the complete genome of L. hokkaidonensis LOOC260T contained various previously unreported mobile genetic elements, which included three new types of insertion sequences, two prophage loci, one ICE, and two plasmids, one of which was considered to be a conjugative plasmid. ICE contained many genes related to heavy metal resistance and shared several components with other plant-associated LAB. The ICE may have mediated the dissemination of genes that contributed to niche adaptation in plant-associated LAB species. Our comparative genome analysis also provided insights into the characteristic gene repertoire of this group, such as preferential pentose assimilation. Although our study could not obtain direct evidence of psychrotolerance, we detected possible factors that may contribute to psychrotolerance in this species, such as the uptake of compatible solutes and the synthesis of glutathione. These findings merit further investigations, and the genomic information obtained in this study should facilitate the development of an appropriate silage inoculant for use in cold regions.In this study, we successfully reconstructed the complete genome of L. hokkaidonensis LOOC260T were cultured in MRS  broth (Difco) and were harvested in the mid-logarithmic phase. The genomic DNA was extracted and purified using Qiagen Genomic-tip 500/G and Qiagen Genomic DNA Buffer Set with lysozyme (Sigma) and proteinase K (Qiagen) according to the manufacturer’s instruction. PacBio SMRT whole-genome sequencing was performed using a PacBio RSII sequencer with P4-C2 chemistry. Four SMRT cells were used for sequencing, thereby yielding 163,376 adapter-trimmed reads (subreads) with an average read length of approximately 4 kbp, which corresponded to approximately 250-fold coverage. De novo assembly was conducted using the HGAP method based on the SMRT Analysis package 2.0, which yielded seven contigs. Independent genome sequencing using the 250-bp paired-end Illumina MiSeq system generated 5,942,620 reads, which were assembled into contigs using Platanus assembler ver 1.2 with the default settings [oriC) was predicted using the Automated Prediction Of Bacterial Replication Origin (APBRO) tool [dnaA gene in the oriC region.The cells of settings . The inisettings . As a reThe plasmid copy numbers were calculated based on the read depth mapped onto each replicon. The reads obtained by the MiSeq system were mapped onto the assembled genome sequences using BWA, and the number of reads mapped onto each replicon was normalized by dividing by its sequence length. The plasmid copy numbers were determined based on the ratio of normalized read numbers for the plasmids relative to that for the chromosome.The genome was annotated using the Microbial Genome Annotation Pipeline (MiGAP)  and somehttp://genome.annotation.jp/genomerefine/), which assists with the refinement of annotations and registration at the DNA Data Bank of Japan (DDBJ).The annotated genome was submitted to the GenomeRefine web service (L. suebicus KCTC 3549T was obtained from GenBank (accession no. BACO01000000). The genomic reads were downloaded from the DDBJ Sequence Read Archive for L. oligofermentans DSM 15707T, L. vaccinostercus DSM 20634T, and L. vaccinostercus DSM 15802 , which were assembled using the Platanus assembler. These genome sequences were annotated by MiGAP and KAAS in the same manner as L. hokkaidonensis. In addition, the genomic data were obtained for 13 representative species in the genus Lactobacillus from the NCBI Reference Sequence (RefSeq) database: Lactobacillus acidophilus NCFM (NC_006814), Lactobacillus helveticus DPC 4571 (NC_010080), Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (NC_008054), Lactobacillus gasseri ATCC 33323 (NC_008530), Lactobacillus reuteri JCM 1112 (NC_010609), Lactobacillus fermentum IFO 3956 (NC_010610), Lactobacillus buchneri CD034 , Lactobacillus brevis ATCC 367 , Lactobacillus casei ATCC 334 , Lactobacillus rhamnosus GG (NC_013198), Lactobacillus plantarum WCFS1 , Lactobacillus sakei subsp. sakei 23 K (NC_007576), and Lactobacillus coryniformis subsp. coryniformis CECT 5711 (NZ_AKFP00000000).The draft genome sequence of L. hokkaidonensis LOOC260T and each reference strain, and an ortholog table was constructed based on the bidirectional best hit among the BLAST results , AP014681 (plasmid pLOOC260-1), and AP014682 (plasmid pLOOC260-2). All of the sequencing data were deposited in the DDBJ Sequence Read Archive under accession numbers DRR024500 and DRR024501. The phylogenetic tree and associated data matrix for in Additional file http://purl.org/phylo/treebase/phylows/study/TB2:S17206).The complete genome sequence of