Pediococcus damnosus LMG 28219 is a lactic acid bacterium dominating the maturation phase of Flemish acid beer productions. It proved to be capable of growing in beer, thereby resisting this environment, which is unfavorable for microbial growth. The molecular mechanisms underlying its metabolic capabilities and niche adaptations were unknown up to now. In the present study, whole-genome sequencing and comparative genome analysis were used to investigate this strain’s mechanisms to reside in the beer niche, with special focus on not only stress and hop resistances but also folate biosynthesis and exopolysaccharide (EPS) production.P. damnosus LMG 28219 harbored 183 contigs, including an intact prophage region and several coding sequences involved in plasmid replication. The annotation of 2178 coding sequences revealed the presence of many transporters and transcriptional regulators and several genes involved in oxidative stress response, hop resistance, de novo folate biosynthesis, and EPS production. Comparative genome analysis of P. damnosus LMG 28219 with Pediococcus claussenii ATCC BAA-344T (beer origin) and Pediococcus pentosaceus ATCC 25745 (plant origin) revealed that various hop resistance genes and genes involved in de novo folate biosynthesis were unique to the strains isolated from beer. This contrasted with the genes related to osmotic stress responses, which were shared between the strains compared. Furthermore, transcriptional regulators were enriched in the genomes of bacteria capable of growth in beer, suggesting that those cause rapid up- or down-regulation of gene expression.The draft genome sequence of P. damnosus LMG 28219 provided insights into the underlying mechanisms of its adaptation to the beer niche. The results presented will enable analysis of the transcriptome and proteome of P. damnosus LMG 28219, which will result in additional knowledge on its metabolic activities.Genome sequence analysis of The online version of this article (doi:10.1186/s12864-015-1438-z) contains supplementary material, which is available to authorized users. Depending on the beer type produced, lactate production may or may not be desired  beer at the end of its maturation phase. A microbial diversity analysis of this mature beer proved that this strain was present in high numbers and is therefore able to replicate in this environment (unpublished results). Flemish acid beers are produced by a mixed-culture fermentation and represent culturally important products, for which microbial activities play critical roles in beer production and quality formation , consisting of 69 large  and 114 small  contigs. This genome hence represents an intermediate size among the LAB [P. claussenii ATCC BAA-344T chromosomal DNA and were ordered accordingly  revealed many similarities towards plasmids of different bacterial species (see below). Many of these contigs , whereas the other six plasmids of P. claussenii ATCC BAA-344T range from 16 to 36 kb and contribute to roughly 7% of the strain’s coding capacity [P. claussenii ATCC BAA-344T, respectively. A total of 19 contigs did not show high similarities towards the plasmids of P. claussenii ATCC BAA-344T. Contigs 16 and 57 were similar to plasmids pBM400 and WSH-002_p1 of Bacillus megaterium strains QM B1551 and WSH-002, respectively, whereas others were similar to plasmids of L. brevis strains 925A, KB290, and ATCC 367 , Lactobacillus plantarum WCFS1 (contig 37), Lactobacillus buchneri strains CD034 and NRRL B-30929 , or other Lactobacillus species . In addition, several CDSs involved in plasmid replication were found, which encoded replication initiation proteins .A BLAST search of the remaining non-chromosomally encoded contigs against a plasmid-specific database  software resulted in a total gene count of 2266, among which 96.12% were CDSs. A total of 79.08% of the CDSs could be assigned to a protein, among which 23.39% and 12.80% were predicted to be enzymes and transporters, respectively. This agrees with the property of LAB to encode a broad repertoire of transporters for efficient nutrient uptake and reflects their limited biosynthetic capabilities [Lactobacillales and defined a core of Lactobacillales-specific clusters of orthologous groups (LaCOGs). The functional distribution of the conserved core of 567 LaCOGs showed that the majority encodes components of the information processing systems , which are likely to perform essential functions. With the exception of 9 LaCOGs , all core LaCOGs were found in the draft genome of P. damnosus LMG 28219. Genome closure could possibly lead to the identification of these missing core functions, although an update of the core LaCOGs is mandatory, because only 12 Lactobacillales species out of more than 500 species of this order were included; in addition, only one of these 12 species belonged to the genus Pediococcus.Gene finding and annotation of the bilities . Additiobilities , who anaP. damnosus LMG 28219 comprised 50 predicted CDSs that were associated with virulence, disease, and defense, including three multidrug resistance efflux pumps. Also, 42 CDSs related to stress response were detected, seven of which were involved in oxidative stress response. Four of the non-core LaCOGs present in the P. damnosus LMG 28219 draft genome comprised 12 or more CDSs in a COG, namely arabinose efflux permeases (COG2814), transcriptional regulators (COG1309), predicted transcriptional regulators (COG0789), and transposases and their inactivated derivatives (COG2826). Arabinose efflux permeases belong to the major facilitator superfamily, a large and diverse group of secondary transporters that includes uniporters, symporters, and antiporters [IS30 family. The presence of IS elements may lead to genomic instability due to its role in gene loss and gene gain [Next to these core functions, the draft genome sequence of iporters . A totalene gain .P. damnosus LMG 28219 was compared with the complete genome sequences of P. pentosaceus ATCC 25745 and P. claussenii ATCC BAA-344T. The number of CDSs predicted in the draft genome of P. damnosus LMG 28219 was higher compared to those in the reference genomes , P. claussenii ATCC BAA-344T and P. damnosus LMG 28219 (both of beer origin), whereas 25, 30, and 30 orthologs were unique to P. pentosaceus ATCC 25745, P. claussenii ATCC BAA-344T, and P. damnosus LMG 28219, respectively  production, and the presence or absence of complete or partial metabolic pathways may lead to the strain’s ability to grow in beer. Furthermore, the functions enriched in the genomes of bacteria capable of growing in beer are likely to contribute to their specific phenotypic traits.The predicted proteome of horA definitively correlates with LAB growth in beer, with the presence of horA together with hitA and/or horC allowing fast bacterial growth. A recent transcriptome study of P. claussenii ATCC BAA-344T showed increased transcript levels of several genes during growth in beer compared to growth in MRS-B medium [P. damnosus LMG 28219  and in the complete genome of P. pentosaceus ATCC 25745 (YP_805121), which all encoded ABC-type multidrug transporters. In depth analysis of these CDSs revealed that only AH70_02075 showed high (99%) amino acid sequence homology towards the horA gene product of P. claussenii ATCC BAA-344T; the remaining CDSs showed less than 70% homology. Possibly, these horA orthologs are descendants of a common ancestor that evolved towards a different functionality, with the horA gene in P. claussenii ATCC BAA-344T and AH70_02075 in P. damnosus LMG 28219 being specialized in the extrusion of iso-alpha acids.Research on hop resistance genes have suggested that these genes are typically acquired via horizontal gene transfer -29. AlthB medium . This wahorA gene is consistently surrounded by the same set of genes in bacteria growing in beer, suggesting that these entire regions are acquired via horizontal gene transfer [horA-surrounding genes are involved in phospholipid or cell wall biosynthesis. Orthologs of PECL_1950, PECL_1952, and PECL_1954 flanking the horA gene were found in the genome of P. damnosus LMG 28219  but not in the P. pentosaceus ATCC 25745 genome sequence. AH70_09615 and AH70_02080 were annotated as an acyl-phosphate glycerol 3-phospate and a glycosyl transferase, respectively. Both PECL_1950 and PECL_1952 are members of the lysophospholipid acyltransferase superfamily, which contains acyltransferases of de novo and remodeling pathways of glycerophospholipid biosynthesis. Finally, AH70_02070 contained a family 8 glycosyl transferase domain that catalyzes the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds.Interestingly, several studies have revealed that the transfer ,28 and chorA gene, the hitA gene encodes a divalent cation transporter and confers hop resistance by importing manganese and thereby counteracting proton gradient dissipation [hitA gene of L. brevis L5784 (AB035808) were found in the genomes of P. damnosus LMG 28219 (AH70_05540), P. claussenii ATCC BAA-344T (AEV94625.1), and P. pentosaceus ATCC 25745 (YP_805164). The reason for the presence of the hitA gene in P. pentosaceus ATCC 25745 of plant origin remains unclear. Furthermore, HorC has been suggested to be a proton motive force-dependent multidrug transporter, whose gene expression is under control of the HorB transcriptional regulator [horC gene of Lactobacillus backii LMG 23555 (BAF56899.1) were found on contig 18 (AH70_03090) in the P. damnosus LMG 28219 and P. claussenii ATCC BAA-344T genome sequences (AEV95756.1), but not in that of P. pentosaceus ATCC 25745. Finally, horB, bsrA, and bsrB homologs were not found in the draft genome sequence of P. damnosus LMG 28219 [In addition to the sipation . Ortholoegulator . OrtholoMG 28219 .P. damnosus LMG 28219 and P. claussenii ATCC BAA-344T contained a set of genes involved in folate biosynthesis that were absent in the genome of P. pentosaceus ATCC 25745 (plant origin). These included a 5,10-methylenetetrahydrofolate reductase , a dihydropteroate synthase , a 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase , a GTP cyclohydrolase I type 1 , and a dihydroneopterin aldolase .The beer isolates de novo, starting from GTP and chorismate [de novo folate biosynthesis is GTP cyclohydrolase I that catalyzes a complex reaction, in which the five-membered imidazole ring of GTP is opened and a six-membered dihydropyrazine ring is formed  gene. This gene is located on plasmid pPECL-7, which is not essential for growth in beer [dps) in P. damnosus IOEB8801, originating from wine, that produces a linear backbone of 3-β-D-glucose-1 moieties. Surprisingly, no homologies towards the gtf and dps genes were found in the draft genome sequence of P. damnosus LMG 28219. Yet, contig 56 harbored a CDS (AH70_07835), containing a glycosyl transferase (group 2) domain, which may be involved in EPS production. OrthoMCL analysis indicated that this CDS was unique to P. damnosus LMG 28219. Other CDS candidates involved in EPS production by P. damnosus LMG 28219 were AH70_01405, AH70_01410, and AH70_01415, which were all predicted to be present on contig 122. These proteins did not show amino acid sequence homologies of more than 70% towards the proteomes of P. claussenii ATCC BAA-344T and P. pentosaceus ATCC 25745. AH70_01405 is an EPS biosynthesis protein, consisting of an AAA domain containing a P-loop, and showed similarities towards the capsular EPS family protein (EHO53752) found in the genome of Lactobacillus kisonensis F0435, originating from the human oral cavity [L. buchneri ATCC 11577, isolated from the human oral cavity [L. buchneri ATCC 11577 and a biofilm regulatory protein of L. kisonensis F0435 (see above). Besides the presence of proteins potentially involved in EPS production in P. damnosus LMG 28219 (as discussed above), the mechanism of EPS production in P. damnosus LMG 28219 remains unclear.Laboratory experiments indicated that ormation , thereby in beer  reported AH70_078, containl cavity . One of l cavity . AH70_01P. claussenii ATCC BAA-344T during growth in beer as a response to the oxidative stress imposed by hops. These genes are manganese transport proteins, methionine sulfoxide reductases MsrA and MsrB as well as other metal transport and homeostasis proteins. Orthologs of these genes were found in the genomes of P. damnosus LMG 28219 and P. pentosaceus ATCC 25745, indicating that the presence of these genes is not unique to strains capable of growing in beer. These results indicate that growth in beer is a multifactorial reaction of the cell towards a challenging environment, not only involving the presence of specific genes but also the up-and down-regulation of specific sets of genes.Pittet and coworkers  found a P. damnosus LMG 28219 draft genome were also present in the P. claussenii ATCC BAA-344T and P. pentosaceus ATCC 25745 genomes, but were enriched only in the genomes of the bacterial strains originating from beer   in the P. damnosus LMG 28219 and its comparative analysis with the genomes of other pediococci provided insights into the adaptation of this strain to the beer environment. These adaptations included the presence of the horA gene and its surrounding genes, genes involved in de novo folate biosynthesis, genes involved in the production of EPS, and the enrichment of functions related to transcriptional regulation. Furthermore, the results presented in this study will enable future transcriptome analysis of P. damnosus LMG 28219, which can provide additional insights into its metabolic activities.The draft genome of pheS) genes [P. damnosus LMG 28219 was present in large numbers in the mature brew sample (data not shown) and was used for genome sequencing. To obtain cell pellets, P. damnosus LMG 28219 was propagated in 750 ml de Man-Rogosa-Sharpe broth  at 28°C for 3 days, followed by microcentrifugation .A mature Belgian red-brown acidic ale brew sample was serially diluted in 0.86% (wt/vol) saline and 50 μL of each dilution was plated on de Man-Rogosa-Sharpe  agar medium, supplemented with 5 ppm amphotericin B (Sigma-Aldrich) and 200 ppm cycloheximide (Sigma-Aldrich) to favor the growth of LAB and inhibit yeast growth. Agar plates were incubated at 28°C in an aerobic atmosphere for 4–6 days. LAB isolates were identified through sequence analysis of the 16S rRNA and phenylalanyl-tRNA synthase alpha subunit (S) genes . Strain Total DNA was extracted using a procedure applied by Gevers and colleagues , with seerror = 10Q/-10). Next, for every base a new value was calculated (limit – perror). This value was negative for low-quality bases. For every base, the CLC workbench calculated the running sum of this value. If the sum dropped below zero, it was set to zero. The part of the sequence not trimmed was the region ending at the highest value of the running sum and started at the last zero value before this highest score. Everything in front and behind this region was trimmed. With the ambiguous trim set to 2, the algorithm found the maximum length region containing 2 or fewer ambiguities and then trimmed away the ends not included in this region. Trimmed reads shorter than 15 bp were discarded. After trimming, an initial de novo assembly was performed in CLC Genomics Workbench v6.5.1, using the quality-trimmed paired reads. All contigs shorter than 500 bp were discarded. Contigs were ordered automatically with MAUVE [P. damnosus LMG 28219 with the complete chromosomal DNA of P. claussenii ATCC BAA-344T. Contigs that did not show similarities towards the P. claussenii ATCC BAA-344T chromosomal DNA were blasted against a plasmid and phage database, using the PATRIC (Pathosystems Resource Integration Center) [Library preparation and genome sequencing was performed by BaseClear BV . A paired-end DNA library with a mean gap length size between 230 and 360 bp was sequenced, with average reads of 100 bp, on an Illumina HiSeq2500 apparatus . Quality trimmed reads were obtained by quality trimming in CLC Genomics Workbench v6.5.1  based on a quality score limit of 0.05 and a maximum number of ambiguous nucleotides of 2. Quality trimming was based on the Phred quality scores (Q) from the fastq read files as follows. The Q value was used to calculate an error probability (pth MAUVE  by compa Center)  and PHAS Center)  websiteshttp://www.uniprot.org/), and InterProScan [Functional annotation and metabolic reconstruction were performed with (i) the Rapid Annotation Subsystem Technology (RAST) server , using GrProScan .P. damnosus LMG 28219 was performed using the available complete genome sequences of P. claussenii ATCC BAA-344T and P. pentosaceus ATCC 25745. The average nucleotide identities (ANIs) between the draft genome (P. damnosus LMG 28219) and both reference genomes were calculated using the in silico DNA-DNA hybridization method implemented in the Jspecies software [P. damnosus LMG 28219, P. claussenii ATCC BAA-344T, and P. pentosaceus ATCC 25745 (http://orthomcl.org/). The Kyoto encyclopedia of genes and genomes (KEGG) database was used for the reconstruction of the folate biosynthesis metabolic map (http://www.genome.jp/kegg/).Comparative genomics of the draft genome sequence of software , using Bsoftware . The OrtThe raw sequence data received from BaseClear BV were deposited at the Short Read Archive (SRA) of GenBank (accession number SRP035530). This Whole Genome Shotgun project was deposited at DDBJ/EMBL/GenBank under the accession number JANK00000000 after automatic annotation by PGAAP and manual curation of the genes of interest.


Triatoma, subdivided in several complexes and subcomplexes according to morphology, ecology and genetic features. One of them is the sordida subcomplex, involving four species: Triatoma sordida, T. guasayana, T. garciabesi and T. patagonica. Given the great morphological similarity of these species, their taxonomic identification, evolutionary relationships and population differentiation have been controversial for many years and even today remain under discussion.Chagas disease vectors (Hemiptera-Reduviidae) comprise more than 140 blood-sucking insect species of the Triatominae subfamily. The largest genus is We simultaneously analyzed two chromosomal markers, C-heterochromatin distribution and 45S ribosomal genes chromosomal position, of 139 specimens from several sordida subcomplex populations from Argentina, Bolivia, Brazil and Paraguay, collected both in nature and from several established insectaries. Our results were compared with COI sequences deposited in GenBank.We recognized five chromosomal taxa with putative hybrids, which each differ in at least one chromosome marker. Most of them present significant differences in their mtDNA sequences.The chromosomal taxa here show a significant chromosome differentiation involving changes in the C-heterochromatin content and in the ribosomal clusters position. This paper identifies several erroneously classified populations by morphological methods, delimits the geographical distribution of each taxon and proposes the existence of a new cryptic species, widely distributed in Argentina. We also suggest that sordida sibling species involve closely related as well as evolutionary distant species. Taxonomic status of each chromosomal taxon is discussed considering phenotypic and genetic results previously published. Trypanosoma cruzi, causative agent of Chagas disease or American trypanosomiasis. Of these, the most conspicuous genus is Triatoma with 80 species, grouped in 8 complexes and 8 subcomplexes according to morphology, habitat, ecology and genetic analyses [Triatoma sordida (Stål 1859), T. patagonica Del Ponte 1929, T. guasayana Wygodzinsky & Abalos 1949 and T. garciabesi Carcavallo, Cichero, Martínez, Prosen & Ronderos 1967. Triatoma sordida was the first described species in 1859 and it presents the most extensive distribution in parts of central Argentina, Bolivia, Brazil, Paraguay, and Uruguay. T. guasayana is found in Argentina, Bolivia and Paraguay, while T. garciabesi and T. patagonica are recorded only from Argentina [The subfamily Triatominae  comprises more than 140 blood-sucking insect species – most of them vectors of the protozoan rgentina .T. sordida, have been controversial for many years and even today remain under discussion.The taxonomic validity of sordida subcomplex species, their evolutionary relationships, and especially population differentiation within T. sordida populations appeared in 1951 [T. garciabesi based on morphological differences from T. sordida and T. guasayana [T. sordida [T. sordida populations from Argentina and Brazil suggested the existence of at least two distinct forms, one from Brazil and other from Argentina [T. garciabesi was revalidated as a species, according to differences in their morphology , isoenzymes (10 diagnostic loci) and chromosomal characteristics (C-heterochromatin distribution) [T. sordida populations, named group 1 and group 2, differentiated by their Idh-2 and Mdh-2 loci [T. sordida populations in Bolivia were of group 1, although this form could also be found in peridomestic and sylvatic environments [T. sordida group 2 is predominantly sylvatic and seems to be restricted to the Chaco region. In the Bolivian Chaco, both T. sordida groups and T. guasayana occurred in sympatry, including putative hybrids [T. garciabesi and T. sordida, and also show significant differences between T. sordida populations from Argentina and Brazil [T. sordida from Paraguay revealed ecological, genetic and morphometric differences between specimens from Western (Chaco region) and Eastern Paraguay [The first report regarding morphological variation among  in 1951 . These auasayana , but thi sordida . Severalrgentina . Based oibution) . In Boli hybrids . Morphomd Brazil , 13. MorParaguay .Karyotypic information for more than 80 species of Triatominae is currently available, showing a highly conserved diploid chromosome number ranging from 21 to 25 chromosomes in males . In spitT. sordida karyotypic information was described for Brazilian specimens [T. guasayana and T. garciabesi did not present autosomal C-heterochromatin while T. sordida (Brazilian populations) and T. patagonica presented C-blocks comprising around the 30 % of their autosomes [T. sordida population from Brazil and one T. garciabesi population. In both species 45S ribosomal DNA clusters are located on X chromosome [The four sordida subcomplex species exhibit the same diploid number: 22 chromosomes composed of 20 autosomes plus XY/XX  sex chromosomes , 8. The pecimens . With C-utosomes . Currentromosome .T. patagonica because it does not present morphological identification problems with the other species of the subcomplex [T. infestans [In the current work, we analyzed both chromosomal markers simultaneously on specimens from Argentina, Bolivia, Brazil and Paraguay. This study does not include bcomplex . The appnfestans . Both manfestans , 17, 21,T. sordida subcomplex from Argentina, Bolivia, Brazil and Paraguay, including field-caught and insectary specimens. The geographic origin of each population, habitat, and a priori species determination are given in Tables T. sordida, named here T. sordida sensu stricto, we considered as topotypes the specimens from Minas Gerais, Brazil, because the holotype locality is not specified. For T. garciabesi we analyzed the same individuals used in their redescription [T. sordida Argentina, was differentiated for specimens from San Luis del Palmar , and a further chromosomal taxon was differentiated from domestic populations from La Paz (Bolivia) provisionally named T. sordida La Paz.We carried out chromosomal analyses of 139 insects of the cription  and chrocription , 9. A neFor chromosome preparations, testes were removed from freshly killed adults, and fixed in an ethanol–glacial acetic acid mixture . Subsequently, we performed C-banding to establish the diploid chromosome number (2n) and the C-heterochromatin distribution . We alsoT. sordida, T. garciabesi and T. guasayana partial COI gene sequences deposited in GenBank were employed for phylogenetic analyses, plus two T. rubrovaria sequences used as outgroup. Other sequences of this subcomplex available in GenBank were excluded from this study since their geographical origin was unknown. All sequence accession numbers are specified in Fig. Fourteen Male specimens from all populations showed the same diploid number (2n = 22) constituted by 20 autosomes plus two sex chromosomes (XY). All individuals, including putative hybrids, presented normal meiosis without irregularities in the chromosome behavior. With C-banding, all specimens had a C-heterochromatic Y chromosome. Different C-banding patterns are observed according to the number of autosomes with C-regions. The ribosomal cluster has one or two chromosome loci per haploid genome, showing 4 location patterns: one autosomal pair, X chromosome, both sex chromosomes or on an autosomal pair and one sex chromosome simultaneously.Analysis of each individual with both chromosomal markers allowed us to identify five different chromosomal taxa, which are described below. Tables T. guasayana  and 1 specimen identified as T. guasayana (Bolivia); Clade 2 or T. sordida Argentina: include 2 samples identified as T. sordida from Corrientes (Argentina); Clade 3 or T. garciabesi: Include 1 T. garciabesi individual from Argentina  and 2 specimens identified as T. sordida from Bolivian Chaco (Romerillo); Clade 4 or T. guasayana: involve 2 T. guasayana individuals from Argentina (Santiago del Estero).The maximum likelihood tree was obtained using Tamura 3-parameters plus Gamma distribution (T92 + G). The resulting ML tree topology , since they grouped within a clade formed by individuals from another species  between the four clades reflect significant differences between them (from 5.3 to 14.9 %) Table . ConsideTriatoma groups such as the brasiliensis, dimidiata, phyllosoma and sordida subcomplexes, as well as in several species of Rhodnius and Panstrongylus .T. patagonica are morphologically very similar, with overlapping geographical distribution and even sympatric in many regions with putative hybrids, which significantly increase the taxonomic confusion. Although there are phenotypic and genetic markers able to differentiate the species, its recognition requires high expertise which greatly hinders their application by vector control staff– leading to mistaken determination even within insectary material   clearly differentiated bcomplex , 23–25.  % Table . In fact%) Table , as suggT. garciabesi: Until now, this species can be only described in Central and Northern Argentina [T. garciabesi geographical distribution to other Chaco regions previously not described: Bolivian Chaco (Santa Cruz), western Paraguay (Departments of Boquerón and Presidente Hayes) and the Argentine provinces of Tucumán and Santiago del Estero  which were originally identified as T. sordida, presented the chromosomal characteristics of T. garciabesi  and Eastern regions of Paraguay reveal striking differences in the feeding patterns, random amplified polymorphic DNA profiles (RAPD), and head and wing morphometrics [T. sordida populations in Paraguay involve at least three taxa. In the Chaco region (Western Paraguay) we only found T. garciabesi, while in the Eastern region it appears that T. sordida Argentina and T. sordida sensu stricto coexist in sympatry. The ecological differentiation and distinct feeding patterns described by [T. garciabesi and T. guasayana [Despite the genetic and phenotypic differentiation, taxonomic problems still persist in terms of distinguishing si Table . Hence, ida Fig. . Recentlometrics . These aribed by  support uasayana , also otT. sordida sensu stricto: This species is the most geographically widespread species of the sordida subcomplex, found in large parts of Argentina, Brazil, Bolivia (Santa Cruz) and Paraguay  [T. sordida group 1 in Bolivia by isoenzymes [T. sordida is forming abundant colonies in peridomestic habitats (particularly chicken coops), with the ability to invade and colonize human habitations in Brazil [T. sordida sensu stricto would be the most significant in terms of Chagas disease transmission [Eastern) . Surprisoenzymes –11 belonoenzymes –11. Histn Brazil , 29, Boln Brazil  and Paran Brazil . Considesmission , being tsmission .T. sordida Argentina: For this chromosomal taxon, we considered as topotypes the individuals from San Luis del Palmar . Chromosomal characteristics  were determined in most individuals from Argentina and some from Bolivia and Paraguay, all of them initially identified as T. sordida   Table . These iers Fig. . T. sordnfestans , 17. Forvia Fig.  could plT. sordida populations and with T. guasayana have been made by many researchers, showing either fertility or F1 sterility [T. guasayana and T. sordida group 1 and group 2) coexist in sympatry, together with the putative hybrids. Here, in Izozog we also identified three species  and putative hybrids, similar to isoenzymes [Experimental crosses between terility , 37–39. oenzymes , 21, theBy chromosomal markers, we recognize five chromosomal taxa and putative hybrids within the sordida subcomplex species in Argentina, Bolivia, Brazil and Paraguay. These morphologically similar taxa exhibit a striking karyological differentiation involving changes in the heterochromatin content and genome reorganization in the ribosomal clusters chromosomal position. In several regions, some of these taxa are sympatric and putative hybrids are detected. This paper identifies several erroneously classified populations, delimits the geographical distribution of each taxon and proposes the existence of a new cryptic species, widely distributed in Argentina. Most extensive population analyses (particularly from La Paz) and the application of other genetic techniques could resolve the taxonomic status of each chromosomal taxon. Considering the different epidemiological importance of these species, a morphological recognition key should be implemented for the selection of appropriate strategies for vector control.


Aspergillus oryzae is widely used for the industrial production of enzymes. In A. oryzae metabolism, transporters appear to play crucial roles in controlling the flux of molecules for energy generation, nutrients delivery, and waste elimination in the cell. While the A. oryzae genome sequence is available, transporter annotation remains limited and thus the connectivity of metabolic networks is incomplete. In this study, we developed a metabolic annotation strategy to understand the relationship between the sequence, structure, and function for annotation of A. oryzae metabolic transporters. Sequence-based analysis with manual curation showed that 58 genes of 12,096 total genes in the A. oryzae genome encoded metabolic transporters. Under consensus integrative databases, 55 unambiguous metabolic transporter genes were distributed into channels and pores (7 genes), electrochemical potential-driven transporters (33 genes), and primary active transporters (15 genes). To reveal the transporter functional role, a combination of homology modeling and molecular dynamics simulation was implemented to assess the relationship between sequence to structure and structure to function. As in the energy metabolism of A. oryzae, the H+-ATPase encoded by the AO090005000842 gene was selected as a representative case study of multilevel linkage annotation. Our developed strategy can be used for enhancing metabolic network reconstruction. Aspergillus oryzae belongs to a group of filamentous fungi that has long been used for the commercial production of different industrial enzymes, such as alpha-amylases [ A. oryzae produce various biological compounds, but also it has beneficial features, such as acting as a robust host system with high production yields and acclimatization to environmental and nutritional duress [ A. oryzae strain RIB40 was sequenced and annotated [ A. oryzae was enhanced using expressed sequence tags data [amylases , proteasamylases , glucoamamylases , xylanasamylases , other hamylases , and orgamylases . Not onll duress . In 2005nnotated . Very rennotated  and Illunnotated . Moreoveags data . Clusterags data , 13 and ags data . A. oryzae was reconstructed based on annotated genomic data, which contains 1,314 enzyme-encoding genes including 53 metabolic transporter-associated genes [ A. oryzae has been used to evaluate fungal biological processes and cellular physiology. However, the connectivity of metabolic networks remains incomplete because of the poor annotation of transporter genes. Among the 161 unique transport reactions, only 33% of annotated genes were identified and used in the network [Using a systems biology approach, a genome-scale metabolic network ofed genes . Modelin network . In meta network , 16. Add network . Aspergillus nidulans. In particular, use of the ClustalW program [ Saccharomyces cerevisiae was determined using electron cryomicroscopy wherein the conformational changes for three functional states were observed during proton translocation [ Piriformospora indica was determined using X-ray crystallography, suggesting both proton and phosphate exit pathways and the mechanism of phosphate transport [ A. nidulans, the relationship between the structure and function of the subfamily of urea/H+ membrane transporter for the UreA gene was studied [+-ATPase [The techniques used for transporter annotation are often performed by sequence-based analysis using pairwise and multiple sequence alignment. Many studies of fungal transporters have relied on similarity searching between orthologous sequences using the BLASTP algorithm , such as program  allowed  program . This st program . However program . To addrlocation . Recentlransport . However studied . Homolog studied , 26 as t studied . However studied  and puri studied ). Moreov+-ATPase , 30. A. oryzae, very few reports involved in metabolic transporters, such as maltose permease [4-dicarboxylate transporter [ A. oryzae genome. Sequence-based analysis is used to predict transporter genes. Next, candidate transporter genes were subjected to functional classification. The transporters involved in metabolic process were manually curated by integrative analysis . In addition, the combination of homology modeling and MD simulation was used to determine the relationship between sequence to structure and structure to function. This proposed metabolic annotation strategy can be used to improve the genome-scale metabolic network of A. oryzae and relevant fungi.As described above, current approaches can only be performed manually and specifically and cannot be used to describe the relationship between sequence, structure, and function for annotating high-throughput data of transporters. Based on experimental data ofpermease , 32, sulpermease , malic apermease , C4-dicansporter , and urinsporter , existed A. oryzae genome [+) [To identify all possible candidate transporter genes, 12,096 protein sequences frome genome  were seae genome  and Trane genome  using BLnome [+)  under binome [+)  as shownnome [+) . In contnome [+) .For functional classification, the candidate transporter genes obtained were submitted as dataset queries using the BlastKOALA and GhostKOALA annotation tools  as shownCandidate transporter genes categorized into the metabolic process were manually curated functions using integrative databases, including TCDB , KEGG 4, 43, and A. oryzae protein sequences belonging to the unambiguous function group were submitted as queries to the SWISS-MODEL [Protein structure is more evolutionarily conserved than amino acid sequence. Therefore, the analysis of 3D structures is a promising method for the functional annotation of transporters. Homology modeling was performed as shown in SS-MODEL  for searSS-MODEL . Next, aSS-MODEL . RemodelSS-MODEL . ProteinSS-MODEL  and solvSS-MODEL . To remoSS-MODEL –61. PeriSS-MODEL , 63 withSS-MODEL –66. All SS-MODEL . System SS-MODEL . The strUsing our developed metabolic annotation strategy for transporters, we achieved four main results as described in the following. First, we describe the assessment of candidate transporter genes. Next, we present the classified functions of candidate transporter genes. Focusing on metabolic process category, we describe the manually curated transporters associated in metabolism. To this end, the structure and function relationship assessment of unambiguous metabolic transporter is discussed. A. oryzae against protein sequences in TCDB and TransportDB. We identified 129 and 23 protein sequences with one-to-one homologous relationship by bidirectional best-hit analysis in TCDB and TransportDB, respectively. These results were subsequently subjected to sensitivity analysis by varying the E-values as cut-offs. The E-values of 6E − 09 and 5E − 04 were selected as the suitable estimated cut-off values. Hereby, we obtained 112 and 18 possible transporter genes from TCDB and TransportDB, respectively. All possible transporter genes under statistical significance were overlapped and removed duplicate data. Consequently, 123 candidate transporter genes of A. oryzae were obtained as presented in http://dx.doi.org/10.1155/2016/8124636.Candidate transporter genes were identified by sequence alignment analysis using 12,096 protein sequences ofA total of 123 candidate transporter genes were submitted as dataset queries to the BlastKOALA and GhostKOALA annotation tools. Based on the KEGG database results, 87 of the 123 submitted queries were assigned K numbers, which were manually classified into the metabolic process and nonmetabolic process categories (Table S2). As shown in Initially, 65 candidate metabolic transporter genes were manually curated to determine their functions using integrative databases, including TCDB, KEGG, and PFAM (Table S4). The results showed that the transporter functions were classified into three assigned function groups, namely, unambiguous, hypothetical, and ambiguous functions.VRG4) in S. cerevisiae caused a loss of mannosylation in vrg4 mutants, leading to cell death [ VRG4 identified in Aspergillus fumigatus [ A. nidulans [ A. oryzae [ A. oryzae [ A. oryzae towards pollutant metals. For the other remaining manually curated genes, 15 of 55 transporter genes were functionally assigned for the primary active transporters (class 3). As seen in class 3, observably most of the transporter function utilized energy from ATP hydrolysis to transport ions through cellular membranes against a concentration gradient [ Aspergillus species in terms of gene sequence and expression [For the unambiguous function group, 55 of the 65 transporter genes were manually curated and found to be overlapped among the integration of three databases as summarized in ll death . For genumigatus  and A. nnidulans , they we. oryzae . Interes. oryzae . Accordigradient  Table 2VRG4 in Spression .For the hypothetical function group, 3 of the 65 transporter genes  were manually curated for individual transporter function by either phylogenetic, protein domain, or transporter component analysis, respectively. A. oryzae and oligosaccharyl transferase (OST3) in S. cerevisiae, showed a closer evolutionary relationship than magnesium transporter (MAGT1) in Homo sapiens as illustrated in  S. cerevisiae [ A. oryzae alpha-amylase overproducing strain [ A. oryzae. Full details of horizontal cladogram can be seen in Figure S1.Performing phylogenetic analysis, the hypothetical metabolic transporter gene, for example, AO090001000747 inrevisiae . Previourevisiae  and it crevisiae . TherefoConsidering protein domain analysis, it is an alternative way for manual curation of transporter function. Once HMMER  and MEME A. oryzae with the highest sequence identity and coverage from sequence alignment analysis between the query  and the well-known structure and function of transporter in PDB. Among the unambiguous metabolictransporters, favorably AO090005000842 gene encoding for H+-ATPase was selected as a representative case study of multilevel linkage annotation due to the highest sequence identity and percent coverage between AO090005000842 gene and the well-known structure and function of the H+-ATPase of Neurospora crassa. To elaborate, AO090005000842 gene was initially submitted as a query onto the SWISS-MODEL for template searching against the PDB. According to the highest quality results among the top 10 identified templates (Table S5), the electron crystallography structure of H+-ATPase in N. crassa (PDB ID: 1MHS) [ A. oryzae H+-ATPase. Thus, the model was generated with detailed sequence alignment between H+-ATPase in A. oryzae and N. crassa   showed t Figures  and S2. +-ATPase models were created under the GROMOS96 force field and solvated in a simple point charge water model without constraints. These systems were then subjected to MD simulation for 100 ns while monitoring equilibration by examining the stability of the geometrical property (RMSD) of the H+-ATPase models. Subsequently, the RMSD and RMSF were calculated using the trajectories to quantify the stability and the fluctuation of the protein. The RMSD of global structures of the H+-ATPase in A. oryzae and N. crassa reached equilibrium after 50 ns using the quantities as shown in Figure S3. Indeed, all five principal domains in the A. oryzae and N. crassa H+-ATPases shared the same average RMSD over the equilibrium which indicated that the dynamic behavior of functional domains was conserved among these species (Table S7).In addition to the analysis of static structures by homology modeling, MD simulation was carried out in order to evaluate structural stability during dynamics simulation and the changes in the stability of proton-transporting regions compared with template structures. The dynamics systems of both H+-ATPase is embedded in membrane environment. Therefore, M-domain of A. oryzae H+-ATPase embedding in palmitoyl oleoylphosphatidylcholine (POPC) lipid bilayer was conducted using the MD simulation. The insertion of M-domain into membrane was done as followed by Kandt et al. [xy-plane and the z-direction  with a time constant of 3.0 ps and a compressibility of 4.5 × 10−5 bar−1 [ A. oryzae, H+-ATPase was 0.398 ± 0.007 nm . This RMSD result supported that the M-domain embedding in POPC of A. oryzae H+-ATPase was consistently preserved with the corresponding regions in the initial structure of N. crassa H+-ATPase.In fact, the proton-transport region (M-domain) of Ht et al. , 80 , acidic side chains , and polar side chains , were expected to show RMSF values comparable to those of the N. crassa H+-ATPase . For instance, the acidic side chain Arg705 and the basic side chain Asp740 in the A. oryzae H+-ATPase fluctuated with the RMSF by approximately 0.0737 nm and 0.1530 nm, respectively, which are the corresponding sites in the N. crassa H+-ATPase, Arg695 (0.0770 nm), and Asp730 (0.1118 nm).In addition, the proton-transporting unit of the HM-domain . Such mulyze ATP . Consistn cavity . Similarn cavity . Thus, t+-ATPase . The RMS A. oryzae and N. crassa H+-ATPase models were substantiated for their structural conservation at the dynamic level. Taken together, the integrative results derived from homology modeling and MD simulation supported that the proton-transporting role along the proton-transporting path in transmembrane domain was structurally conserved between H+-ATPases in A. oryzae and N. crassa, where functional conservation for the proton transporter is expected.In accordance with the overall comparable geometrical properties, the A. oryzae genome, our strategy could be used to identify 58 metabolic transporter genes. Under consensus integrative databases, 55 unambiguous metabolic transporter genes were distributed into channels and pores (7 genes), electrochemical potential-driven transporters (33 genes), and primary active transporters (15 genes). The remaining 3 hypothetical metabolic transporter genes were manually curated transporter functions by phylogenetic, protein domain, and transporter component analysis. Among the unambiguous metabolic transporter genes, the H+-ATPase or proton pump encoded by the AO090005000842 gene was selected as a representative case study of multilevel linkage annotation in order to reveal the transporter functional role in A. oryzae metabolism. Our metabolic annotation strategy can be used for improving functional annotation and enhancing cellular metabolic network and modeling in A. oryzae and relevant fungi.For the integrative multilevel annotation of metabolic transporters, we propose a metabolic annotation and assessment strategy based on sequence, structure, and function relationship as a platform for increasing the functional efficiency of transporter annotation. Of 12,096 total genes in theAspergillus oryzae are included in the Supplementary Material.The detailed information of metabolic transporter genes and functions achieved from sequence- and structure-based annotation and assessment in


Minostrobus chaohuensis is one of the earliest monosporangiate-strobilate isoetaleans. Based on new material of this plant, the vegetative axis and microsporangiate strobilus are studied in detail, and the whole plant knowledge is summarized. The vegetative axis is isotomously branched. The stem is up to 55 mm in diameter with helically arranged leaf cushions. Stems and thick branches bear long fusiform leaf cushions and interareas with vertical linear ornamentations. A ligule pit, oblanceolate leaf scar, and vascular bundle scar appear on the leaf cushion. Distal axes have persistent lanceolate leaves and rhombic leaf bases. The microsporangiate strobilus is cylindrical in shape, possesses sporophyll with alate pedicel and long triangular lamina, uniseriate sporangial wall, subarchesporial pad inside the sporangium, and microspore with cingulum. Based on comparisons with other isoetaleans, the usage of the terms “leaf cushion” and “leaf base” is discussed, and Minostrobus chaohuensis is considered as a tree-like lycopsid. It suggests that arborescent isoetaleans with monosporangiate strobili had appeared and diversified in the Late Devonian. The multi-dichotomous branching system of Minostrobus provides new data on the evolution of growth architecture in rhizomorphic lycopsids.Late Devonian  Su. Susensuth China –5. In thMinostrobus chaohuensis Wang was reported from the Upper Devonian Wutong (Wutung) Formation of Chaohu City, Anhui Province, South China, and was established on the basis of strobili containing megaspores [M. chaohuensis and its evolutionary significance.gaspores . Subsequgaspores , 8. AlthNew material was collected from the Fenghuangshan Section, about 3.0 km north of Chaohu City, Anhui Province, China. The specific location of this section (31°37′51″N and 117°50′54″E) was illustrated by Meng et al. . SpecimeThe plant is mostly preserved as impressions and compressions in black-gray mudstone, with some strobili permineralized by limonite. We used steel needles to expose the morphology. The permineralized microsporangiate strobili were embedded in epoxy resin, sectioned, grinded, and polished to make slides for examination under light microscopy (LM). Before embedding, we selected masses of microspores for observation under scanning electron microscopy (SEM). All specimens and slides are housed at Department of Geology, Peking University, Beijing, China.The stems are 25–55 mm wide , and braLycospora (Ibrahim) Schopf et al. in size and existence of cingulum [The permineralized strobili are cylindrical in shape and slightly curved, with both ends missing . The megcingulum .Class: Lycopsida Pichi-Sermolli 1958sensu lato Meyen 1987Order: Isoёtales Suborder: Dichostrobiles DiMichele et Bateman 1996Family: Incertae sedisMinostrobus Wang 2001 emend.Genus: Emended diagnosis: Arborescent lycopsid with monosporangiate-strobilus, possibly monoecious. Aerial axes multi-dichotomous. Leaf with single mid-vein abscised from stem and thick branch. Long fusiform leaf cushion with ligule pit and oblanceolate leaf scar. Among leaf cushion with evident interareas possessing linear ornamentations. Axis with solid exarch primary xylem. Protoxylem confined to ridges at periphery of primary xylem strands. Metaxylem tracheids bearing Williamson’s striations. Single spherical to elliptical sporangium inserted to adaxial side of sporophyll with smooth margin, pointed apex and alate pedicel. Sporangial wall comprising one layer of columnar cells. Subarchesporial pad along inner surface of sporangial wall. Megasporangium connected to sporophyll by narrow attachment, with sporangial base surrounded by alations. Each megasporangium with four megaspores.Minostrobus chaohuensis Wang 2001 emend.Type species: Holotype: PB18751 Type locality: The south slope of Beishan hill, Shizikou Section, 3 km north of Chaohu City, Anhui Province, China.Repository: Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing, China.Stratigraphy: Leigutai Member of the Wutong Formation.Specimens examined herein: PKUB12101, PKUB12124, PKUB12135, PKUB12137–PKUB12139, PKUB12145, PKUB12148, PKUB12159, PKUB12160, PKUB12163, PKUB12171, PKUB12188 and PKUB12189 .Lycospora-type, about 20–30 μm in diameter, with circular amb, inner body and narrow cingulum. Microspore exospore with two layers. Megasporangiate strobilus about 5.0 mm wide. Megasporophyll trace arising from stele at acute angles. Megasporophylls arranged in 2/9 helix, with angles between parastichies and horizontal line being about 35°. Pedicel about 1.8 mm long, proximally “V” form in transverse view, distally expanding to form alations. Lamina long-triangular in face view. Smooth megasporangium about 2.0 mm long, 1.5 mm wide and 1.5 mm high. Attachment between megasporangium and megasporophyll about 1.0 mm long and 0.2 mm wide. Megaspores Lagenicula-type, from about 370 μm to about 1490 μm in diameter, with distinct gula and spiny ornamentation, more or less circular amb, pear shape in equatorial view. Spiny ornamentation about 20 μm wide at base. Megaspore wall with thin inner exospore and spongy outer exospore.Emended diagnosis: As for generic diagnosis. Aerial axes dichotomizing at least eight times. Lanceolate sterile leaves with smooth margin. Long fusiform leaf cushions about 6.0–9.0 mm long and 1.0–1.6 mm wide, with oblanceolate leaf scar located at the middle part and occupying half the length and almost all the width of the cushion. Ligule pit located on the top of leaf scar. Axis with single, solid exarch primary xylem. Twelve protoxylem ridges with annular tracheids around the metaxylem core. Polygonal metaxylem tracheids about 20–40 μm in diameter, with scalariform and reticulate thickenings. Cortical cells rectangular in outline. Separate monosporangiate strobili attached at apices of the axes. Microsporangiate strobilus over 80 mm long and about 6.0 mm wide. Microsporophyll pedicel about 2.5 mm long, with alations up to 1.7 mm wide. Long triangular upturned lamina about 5.0 mm long and 3.0 mm wide at the base. Smooth microsporangium about 2.5 mm long and 1.5 mm high. Microspores Minostrobus chaohuensis, and similar with M. chaohuensis described by Wang et al. [M. chaohuensis. However, some important characters of vegetative axis and microsporangiate strobilus are emended  of South China is a small arborescent lycopsid [Minostrobus chaohuensis.lycopsid . It has Leptophloeum rhombicum Dawson has been widely reported from the Upper Devonian, and considered as a lycopsid tree with thick trunk and developed secondary tissue [Minostrobus chaohuensis, L. rhombicum is believed to possess a ligule. Nevertheless, this plant has rhombic or fan-shaped leaf cushions, small ovate leaf scars, and thicker strobili (20–30 mm in diameter) [y tissue –14. As iSublepidodendron with monosporangiate strobili was widespread from the Late Devonian to the Early Carboniferous [Sublepidodendron songziense Chen and Sublepidodendron grabaui (Sze) Wang and Xu, occur in the Upper Devonian of South China [Minostrobus chaohuensis, both of these two species possess fusiform leaf cushions/bases and vertical ornamentations on the interareas among leaf cushion/bases. S. songziense also has Lycospora-type microspores and Lagenicula-type megaspores. However, persistent vegetative leaves of Sublepidodendron are linear in shape, neither ligule nor ligule pit is found, and no leaf scar presents. Different with the microsporangiate strobilus of M. chaohuensis, that of S. songziense is thicker (8.0–12 mm wide), and S. grabaui has elongate sporangia (4.0 mm long and 0.8 mm high).Arborescent lycopsid niferous –17. Two th China , 18, 19.Changxingia longifolia Wang et al. from the Upper Devonian (Famennian) of South China is an isoetalean with monosporangiate strobili possessing four Lagenicula-type megaspores in each megasporangium [Minostrobus chaohuensis are similar in shape and both bear ligule pit on the top of leaf scar. However, the leaf scar of C. longifolia is oval-oblanceolate in shape, the vegetative leaf is linear, the megasporangiate strobilus is shorter (20–50 mm long), the megasporophyll is reflexed, and the megasporangium is not surrounded by sporophyll.orangium . Leaf cuLepidostrobus (Brongniart) Brack-Hanes and Thomas represents a group of microsporangiate strobili bearing Lycospora-type spores [Lepidostrobus xinjiangensis Wang—was described from the Upper Devonian of Northwest China [Minostrobus chaohuensis meets the definition of Lepidostrobus in many respects, including phyllotaxy, anatomy of axis, alations  and microspore type. Nevertheless, some characteristics of Lepidostrobus such as heel, abaxial keel of pedicel and microsporangium attachment are not clear in M. chaohuensis.e spores . Speciesst China . The micLepidodendron is one of the representative arborescent lycopsids that are widely distributed in the Carboniferous floras and persisted into the Late Permian in China [Lepidodendron species clearly show vertically elongated leaf cushions (height-width ratio > 1) with evident leaf scar and ligule pit, and possess Lycospora-type microspores [Minostrobus, the leaf cushion is more complex (with two or four parichnos scars), and the ligule pit may be located at some distance from the leaf scar [in China , 23. Thiin China . Lepidodrospores . In conteaf scar .Diaphorodendron DiMichele and Synchysidendron DiMichele and Bateman once included within the genus Lepidodendron have been separated and assigned to the Diaphorodendraceae DiMichele and Bateman [Minostrobus in the vertically elongate leaf cushion. In some species, e.g., Diaphorodendron selerotecum (Pannell) DiMichele [Synchysidendron dicentricum (Felix) DiMichele and Bateman [Achlamydocarpon Schumacker-Lambry type microsporangiate strobilus with Granasporites Alpern microspore. This kind of microsporangiate strobilus is thicker (about 13 mm in diameter) than that of Minostrobus, has heeled sporophyll and elongated sporangium [Species of  Bateman , 27. TheiMichele , 26 and  Bateman , the apeorangium .Archaeosigillaria Kidston, Tomiodendron (Radczenko) Meyen, and Eskdalia Kidston (Thomas) [Minostrobus chaohuensis, we have not found any indication of persistent leaves or the basal part of leaf blades, but have observed consistent oblanceolate scars (not slit) on leaf cushions and abscised leaves in the matrix. Therefore, we believe that leaves on these axes of M. chaohuensis had abscised, forming leaf scars on leaf cushions.Characters of the leaf cushion  play an important role in the classification of lycopsids, and the generally accepted definition of the term “leaf cushion” is “the lowermost part of the leaf which is usually widened and remaining on the stem after leaf abscission” , 32. Cor(Thomas) –35. Shap(Thomas) . The fal(Thomas) . The thiMinostrobus (this study) and Changxingia Wang [Although the terms “leaf cushion” and “leaf scar” have been precisely defined, they are still used indiscriminately at times. Especially, the term “leaf cushion” has been frequently used to describe species or specimens with persistent leaves , 37, 38.gia Wang . We propMinostrobus chaohuensis has been interpreted as a small herbaceous lycopsid [Sublepidodendron songziense (55–70 mm) [lycopsid , or regalycopsid . In thislycopsid , which hlycopsid . Therefo5–70 mm) .M. chaohuensis, this structure at least occurred on stems and branches thicker than 10 mm, represents the distribution of secondary tissue. Different with this plant, distribution of secondary tissue in pseudoherbaceous and shrubby lycopsids are always very restricted [Oxroadia Alvin [Paurodendron Fry [Chaloneria Pigg and Rothwell [Wrinkles on the interareas among leaf cushions are generally interpreted as the result of secondary growth of axes , 42. In stricted , e.g., iia Alvin –45, in rdron Fry –48 and CRothwell .M. chaohuensis is estimated to be 88.7%, exceeding the value of pseudoherbaceous lycopsid Paurodendron (75.2%—80.0%), but approaching that of arborescent Sublepidodendron (90.7%) [In arborescent lycopsids, the secondary xylem is not as thick as that in some euphyllophyte trees, but the secondary cortex (periderm) provides the major mechanical support , 2, 50.  (90.7%) .Minostrobus chaohuensis is similar to arborescent lycopsids rather than pseudoherbaceous and shrubby species in stem size, secondary tissue and cortex proportion. Therefore, M. chaohuensis is probably an arborescent lycopsid.The above evidences suggest that Minostrobus chaohuensis has been studied several times [Sublepidodendron and Lepidodendron [M. chaohuensis is suggested to be a tree-like lycopsid, with a stem at least 55 mm in diameter and multi-dichotomous branching system. The plant is possibly monoecious, with mega- and micro-sporangiate strobili attached to the apex of vegetative axes, bearing Lagenicula-type megaspores and Lycospora-type microspores [Till now, al times –8. Morphodendron , 52. M. rospores , 8.Lepidostrobus xinjiangensis, Changxingia longifolia, Sublepidodendron songziense and Minostrobus chaohuensis. The latter two species are comparatively more completely known plants and have been proved to be arborescent. The presence of Sublepidodendron and Minostrobus demonstrates that tree-like Dichostrobiles members already diversified from the bisporangiate-strobilate ancestor and diversified in the Late Devonian. Among rhizomorphic lycopsids, the multi-dichotomous branching system of Minostrobus also occurred in the highly derived group Lepidodendraceae and primitive pseudoherbs Oxroadia and Paurodendron. As DiMichele et al. proposed, the branches of these two groups are possibly nonhomologous because they are separated in the phylogenetic tree by lycopsids with lateral branches on the trunk [Minostrobus [Oxroadia and Paurodendron. This study of Late Devonian Minostrobus provides new data on the evolution of growth architecture in rhizomorphic lycopsids.The earliest isoetaleans with monosporangiate strobili have been described from the Late Devonian: he trunk . In viewostrobus , we supp


The capacity for pathogenic fungi to change morphology during their lifecycle is widespread. However, relatively few fungi are considered dimorphic, which refers to the ability to switch between two morphologies, yeast and hyphae . These pHistoplasma capsulatum and Coccidioides spp. are estimated to infect 500,000 and 150,000 persons annually, respectively [Worldwide, the thermally dimorphic fungi cause several million human infections each year. In the United States, ectively . The incectively . Althougectively . Immunosectively .Ophiostoma ulmi, which caused the first Dutch elm disease epidemic, has been replaced by a more virulent species, Ophiostoma novo-ulmi, which has destroyed millions of elm trees in the US and Europe [Taphrina deformans, the etiologic agent of peach leaf curl, results in economic losses of $2.5–3 million in the US [Ustilago maydis, which causes corn smut, is not a major agricultural threat, the galls  caused by infection are eaten as a delicacy [Phytopathogenic dimorphic fungi have had a major impact on urban landscapes and agriculture. d Europe . Taphrinn the US . Althougdelicacy . Entomopdelicacy ,9.Coccidioides) to cause pneumonia. Although temperature is the major stimulus, other factors such as CO2, cysteine, and estradiol influence conversion and growth at 37°C [Coccidioides spherules and inhibits the phase transition of Paracoccidioides brasiliensis at 37°C [The morphologic shift between hyphae and yeast is critical for the pathogenesis, virulence, and lifecycle of the dimorphic fungi. In the soil (22–25°C), the thermally dimorphic fungi grow as mycelia that produce infectious conidia . Following soil disruption, aerosolized conidia and hyphal fragments inhaled into the lungs of a mammalian host (37°C) convert to yeast  [Cokeromyces recurvatus grows as budding yeast in humans and has been misidentified as Paracoccidioides [Malassezia furfur, the cause of pityriasis versicolor, converts from yeast to hyphae in the presence of L-DOPA [Hortaea werneckii, which serves as a model for halotolerance, grows as yeast at ambient temperature and converts to hyphae to cause black skin discoloration known as tinea nigra [U. maydis, but are dispensable for T. deformans [Ophiostoma spp. [Varied stimuli influence the morphologic switch for non-thermally dimorphic fungi.  tissue) . In contidioides . Malassef L-DOPA . Hortaeaea nigra . Mating eformans ,7. Nitrooma spp. ,15. The oma spp. .M. furfur and H. werneckii only penetrate the superficial epidermal layers by growth as hyphae, not yeast [H. capsulatum conidia are enriched for transcripts involved with stress resistance, DNA replication, cell signaling, and transcriptional regulation  [To cause invasive infection in humans , fungi must overcome structural, thermal, and immunologic barriers. Intact skin is resistant to invasive infection and cutaneous pathogens such as ot yeast . The smaot yeast ,17. Pref., RYP1) .Blastomyces dermatitidis and H. capsulatum, a hybrid histidine kinase encoded by DRK1 (dimorphism-regulating kinase-1) is important for the transition to yeast at 37°C and virulence [DRK1 results in B. dermatitidis and H. capsulatum cells that grow as hyphae (instead of yeast) at 37°C and renders these pathogens avirulent [DRK1 in Penicillium marneffei, DRKA, is critical for germinating conidia to develop into yeast within macrophages at 37°C [DRK1, transcription factors RYP1–4 (required for yeast phase) in H. capsulatum promote the transition to yeast [RYP1–4 results in hyphal growth at 37°C and a failure to up-regulate yeast-phase genes critical for virulence [RYP1–4 knockdown strains at 37°C is similar to wild-type H. capsulatum mycelia [During the past decade, there have been substantial advances in understanding the transition to yeast. In irulence . Deletiovirulent . This prvirulent . Moreove at 37°C . In addito yeast –21. Knocirulence –21. The BAD1, SOWgp, CBP1, and YPS3, are transcribed only in the yeast phase. B. dermatitidis BAD1 (formerly WI-1) is an adhesin that attaches yeast to host cells by binding heparan sulfate, CR3, and CD14 [Coccidioides spherule outerwall glycoprotein (SOWgp) and P. brasiliensis glycoprotein 43 (gp43) facilitate adhesion of yeast to extracellular matrix proteins and contribute to virulence [H. capsulatum, alpha--glucan, which contributes to virulence in chemotype II strains, is located in the outermost layer of the cell wall, and blocks β-glucan recognition by immune cells [H. capsulatum CBP1  is a secreted, saposin-like protein that binds calcium (and possibly lipids) and is essential for survival within macrophages [H. capsulatum to the liver and spleen [BAD1 and CBP1 renders yeast avirulent without affecting the phase transition [During the transition to yeast, genes critical for immune evasion, intracellular survival, and dissemination are up-regulated. Many of these genes, including and CD14 . BAD1 prand CD14 . Coccidiirulence ,24. In Hne cells . H. capsrophages . YPS3 , yeast-like cells invade the muscles and possibly the central nervous system and secrete toxic metabolites to alter the behavior of the infected Camponotus and Polyrhachis ants [Insect and plant pathogenic fungi must also penetrate structural defenses and avoid immune defenses to cause invasive disease. Once inside insect hemocele, the invading filamentous form switches to yeast-like growth. These yeast-like cells, which are also known as blastospores, replicate by budding, evade host immune defenses, promote dissemination, and alter host behavior. The MAD1 adhesion in ormation . MAD1 deirulence . Beauverdefenses . In Ophihis ants , 28. Infhis ants ,28.U. maydis, diploid spores on the plant surface germinate to form haploid, budding yeast [a and b mating-type loci) fuse to form a filamentous dikaryon that penetrates the plant cuticle and invades tissue [T. deformans also switches from budding yeast on the leaf surface to filamentous growth to invade plant tissue; however, mating is not required for the dimorphic switch [O. ulmi and O. novo-ulmi, yeast-like blastospores disseminate in the tree using the xylem network, whereas hyphae facilitate spread between vascular structures [The importance of the yeast phase for plant pathogens is species specific. In ng yeast . Yeast cs tissue . T. defoc switch . For O. ructures .Coccidioides posadasii to infect healthy hosts and survive in the soil has contributed to long-range geographic dispersal [Coccidioides isolates indicated that South American C. posadasii strains originated in Texas, US [C. posadasii corresponded with human migration  into South America [Hyphal growth coupled with conidiogenesis promotes environmental survival, transmission to new hosts, and genetic diversity via mating. The ability of ispersal ,29. AnalB. dermatitidis and H. capsulatum with either mating-type loci can infect humans, but how mating contributes to virulence for these fungi is speculative [C. posadasii genes to Coccidioides immitis includes genes that are postulated to facilitate immune evasion [O. novo-ulmi, introgression of O. ulmi MAT1 and vegetative incompatibility genes has allowed the pandemic strain to mate, which, in turn, eliminates d-factor viruses  [For the thermally dimorphic fungi, sexual reproduction occurs when hyphae with opposite mating-type loci  fuse and form cleistothecia, specialized structures that produce spores by meiosis . B. dermculative . Introgr evasion . For O. urvival) .B. dermatitidis SREB and H. capsulatum SRE1 homologs encode GATA transcription factors that regulate siderophore-mediated iron assimilation and the temperature-dependent conversion to mold [SREB null mutants and SRE1 knockdown strains fail to complete the conversion from yeast to mold at 22–25°C independent of exogenous iron concentrations [SREB and SRE1 in Cryptococcus neoformans, CIR1, regulates iron uptake and thermotolerance [SRE1, a vacuolar ATPase, VMA1, involved with iron homeostasis in H. capsulatum, affects the mycelial transition at ambient temperature [Penicillium marneffeii, HGRA  and TUPA facilitate conversion and maintenance of mycelial growth at 25°C, respectively [H. capsulatum and B. dermatitidis mycelial conversion is accelerated by N-acetylglucosamine (GlcNAc), which is mediated by NGT1 and NGT2 transmembrane transporters [Several advances have illuminated the molecular biology underlying the yeast-to-hyphal transition.  to mold ,34. SREBtrations ,34. A hoolerance . This superature . In Peniectively ,38. The sporters . CollectMucor circinelloides, an agent of mucormycosis, the calcineurin regulatory unit encoded by CNBR is essential for the transition to pathogenic mycelia [CNBR null mutants are locked in the yeast phase and have attenuated virulence [M. circinelloides to FK506, a calcineurin inhibitor, results in yeast growth; this may explain why transplant recipients treated with FK506 have lower rates of mucormycosis [U. maydis has been extensively investigated and involves the interaction of two homeodomain transcription factors  along with MAPK and cAMP-PKA signaling cascades [U. maydis proliferates, secretes proteins to suppress host defenses, and induces tumor  growth [For non-thermally dimorphic fungi, mycelial growth is important for superficial mycoses , invasive mucormycosis, plant infections, and exit of entomopathogens from their insect host. In  mycelia . CNBR nuirulence . Similarrmycosis . The myccascades . Once in) growth . The genThe morphologic switch is essential for the pathogenesis of dimorphic fungi. For mammalian and insect fungi, the transition to a yeast or yeast-like growth results in altered cell wall composition as well as production of proteins to evade immune defenses or toxins to alter host behavior. Growth as yeast coupled with thermotolerance allows for replication within mammalian phagocytic cells, which promotes dissemination. For phytopathogens, the role of the yeast phase is species specific; it can be involved with sexual reproduction or dissemination within vascular tissue. Growth as mycelia promotes transmission to new hosts via conidia, and for the thermally dimorphic fungi has contributed to long-range geographic dispersal. Sexual reproduction occurs in the mycelial phase for most dimorphic fungi and has facilitated gene introgression to enhance survival and virulence. For non-thermally dimorphic fungi, mycelial growth is essential for invasive disease. Although a number of genes that govern the phase transition are known, how these genes fit into a larger network of regulated genes remains to be fully answered.


Yersinia entomophaga type strain MH96T. The genome shows 93.8% nucleotide sequence identity to that of Yersinia nurmii type strain APN3a-cT, and comprises a single chromosome of approximately 4,275,531 bp. In silico analysis identified that, in addition to the previously documented Y. entomophaga Yen-TC gene cluster, the genome encodes a diverse array of toxins, including two type III secretion systems, and five rhs-associated gene clusters. As well as these multicomponent systems, several orthologs of known insect toxins, such as VIP2 toxin and the binary toxin PirAB, and distant orthologs of some mammalian toxins, including repeats-in-toxin, a cytolethal distending toxin, hemolysin-like genes and an adenylate cyclase were identified. The genome also contains a large number of hypothetical proteins and orthologs of known effector proteins, such as LopT, as well as genes encoding a wide range of proteolytic determinants, including metalloproteases and pathogen fitness determinants, such as genes involved in iron metabolism. The bioinformatic data derived from the current in silico analysis, along with previous information on the pathobiology of Y. entomophaga against its insect hosts, suggests that a number of these virulence systems are required for survival in the hemocoel and incapacitation of the insect host.Here we report the draft genome of  Yersinia is a genus of Gram-negative facultative anaerobes belonging to the family Enterobacteriaceae, of the gamma subdivision of Proteobacteria [Yersinia species such as Y. pestis, the causative agent of bubonic plague [Y. ruckeri, which causes enteric redmouth disease in salmonid fish [Y. aldovae [Y. entomophaga MH96T is isogenic to Y. nurmii type strain APN3a-cT [Y. ruckeri, form a discrete clade away from other Yersinia species [Y. ruckeri within the genus Yersinia is still a matter of conjecture [bacteria . The Yerc plague , and Y. nid fish , along w aldovae , being iAPN3a-cT , which w species ,8. The pnjecture . Y. entomophaga was originally isolated from the cadaver of a New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae). Y. entomophaga is consistently pathogenic by per os challenge to this host, as well as a wide range of lepidopteran, coleopteran, and orthopteran species, with death of the insect typically occurring within 2–5 days of ingestion [. Aside from hemocytes, there are no obvious sites of Y. entomophaga colonization in the insect [Y. entomophaga were observed both in the greater wax moth Galleria mellonella  larval hemocoel and in vitro in Luria-Bertani broth. Electron microscopy of these in vitro cell aggregates revealed the presence of polar fimbriae [ngestion ,12. Aside insect . At 37 °fimbriae .Y. entomophaga is an insect-active toxin complex (TC) derivative termed the Yen-TC, the genes encoding which are located on a pathogenicity island designated PAIYe96 [N-terminal region and a divergent C-terminal “tip” region of 100–200 amino acids [The main virulence determinant of  PAIYe96 . The Yen PAIYe96 . Rhs prono acids  that canno acids ,18,19. Tno acids ,20. Y. entomophaga is likely to be confronted by a wide range of proteolytic gut enzymes, the endogenous microbial community, and physiological variables such as pH, ionic strength, and redox potential of the gut [Once ingested,  the gut . The epi the gut ,22. Iron the gut  unsuitab the gut ,25. In i the gut . Y. entomophaga or its Yen-TC deletion derivative Y. entomophaga ΔTC towards G. mellonella is approximately three cells [Photorhabdus cells required to cause lethality. Once released into the hemocoelic cavity, the nematode-vectored Photorhabdus and Xenorhabdus species produce a range of toxins active in the hemoceol and antimicrobial compounds [Photorhabdus insect-related binary toxins (PirAB), which are active when injected into the hemocoelic cavity of G. mellonella [P. luminescens-derived Mcf (makes caterpillars floppy) protein [P. luminescens and Xenorhabdus nematophila secrete hydroxystilbene and benzylideneacetone, respectively, which limit the growth of competing microorganisms and inhibit phenol oxidase produced by phagocytic hemocytes of the insect [The 4-day intra-hemocoelically-injected median lethal dose of either ee cells . This loompounds ,27. Thesompounds , called llonella ,30, and  protein ,32. In ae insect ,34. Y. entomophaga cells required to cause an effect post-hemocoelic injection leads us to suggest that, similar to Photorhabdus and Xenorhabdus species, Y. entomophaga encodes and produces a wide range of factors that suppress the host immune system, and possibly limit the growth of the endemic microbial community [Y. entomophaga genome was assembled, allowing the in silico-based identification of possible virulence determinants. The low number of ommunity . To helpYersinia entomophaga genome sequence comprises one scaffold consisting of six contigs joined by PCR. The contig boundaries are indicated in The draft Y. entomophaga draft genome sequence comprises 4,275,531 bp, with a G+C content of 48.58%, with seven 16S rRNA ribosomal-associated regions. Analysis of the functional categories and genome-wide distribution of all unique genes with assigned clusters of orthologous groups (COGs) revealed a total of 4225 proteins. In total, 325 hypothetical proteins and 703 proteins not represented in the COG database, comprising 24.2% of the Y. entomophaga genome, were identified . No region with significant DNA identity to the Y. entomophaga PAIYe96 Yen-TC virulence-associated region was identified in the Y. nurmii APN3a-cT genome sequence. Alignment of the Y. entomophaga ORFeome with the Yersinia species Y. ruckeri 29473 and Y. enterocolitica 8081 identified several genomic regions greater than 10 kb in size that were either unique or had an atypical G+C content compared with the rest of the Y. entomophaga genome sequence , and to TccC3 from Vibrio parahemolyticus, where the putative C-terminus effector is similar (Escherichia coli inhibitor of vertebrate lysozyme protein (Ivy) [Y. entomophaga ortholog (PL78_18760) of the Yersinia pekkanenii and P. luminescens LopT type III secretion system (T3SS) effector proteins (Including the YenC1 and YenC2 subcomponents of the Yen-TC (Rhs-associated Region 1), the  element . Encoded element , which s similar . Locatedin (Ivy) , a lysozin (Ivy) . A regioproteins .P. luminescens pmt genomic island, which encodes an E. coli macrophage toxin-like (mt-like) protein [Y. entomophaga genome, several similarly oriented loci, some with partial homology to the Rhs core domain were identified 3ʹ of the Rhs element (PL78_00990) , located within Rhs-associated Region 3, shares approximately 70%–72% DNA identity with the S. fonticola strain DSM 4576 genome sequence (gb|CP011254.1). Rhs-associated Region 3 contains a large gene cluster comprising loci with similarity to several type VI secretion system (T6SS) inner membrane protein (Imp) components, IcmF (PL78_00970), and a VgR locus (PL78_00980) . A subse protein . Though 8_00990) . The N terfamily . Anothererfamily . BlastX erfamily . Of inteYersinia pseudotuberculosis RhsA wapA_1 protein (Xanthomonas campestris pv. musacearum NCPPB 4379 [Rhs-associated Region 4 encodes an ortholog (PL78_12135) of the  protein . DownstrPPB 4379 . SimilarPseudomonas fluorescens , to which the NusG regulator binds, within 15 kb of Y. entomophaga PL78_15075. Of note, the G+C content of Rhs Region 5  was significantly divergent to that of the Y. entomophaga genome as a whole  (Rhs-associated Region 5 encodes the largest Rhs element (PL78_15070), consisting of 1,584 amino acids with high similarity to type IV secretion system protein RhsA from orescens . Several78_15070 . A NusG-b2 locus  was iden, 48.6%) , and hadY. entomophaga genome encodes two T3SSs, designated T3SSYE1 and T3SSYE2  present in biotype IB strains of Y. enterocolitica [Y. enterocolitica Ysa T3SS [prgH (PL78_14530) to orgB (PL78_14505), are oriented in the opposing direction  with similarity to the T2SS of several related species, including Y. ruckeri (The type II secretion system (T2SS) is an alternate delivery system enabling the transport of folded macromolecules such as toxins. T2SS’s comprise a composite of proteins that span both the inner and outer bacterial cell membranes ,46. We i ruckeri . We noteIn silico analysis of the Y. entomophaga sequence identified the presence of several other virulence determinants with orthologs in both insect and mammalian pathogens. The Y. entomophaga loci PL78_09590 and PL78_09595 share high amino acid similarity with the P. luminescens PirA and PirB binary toxins [y toxins , respectY. entomophaga locus, PL78_16910, encodes a 4660-amino acid repeats-in-toxin (RTX) protein   . Typicalve toxin ,47. Inte peptide ,49, onlyC-terminal region of PL78_16145 showed partial amino acid similarity to the ADP ribosyltransferase domain of the VIP2 superfamily , each encoding a serralysin metalloprotease with at least 68% amino acid sequence similarity to each other, was identified within Region 1 . The Y. entomophaga ORF PL78_03965 is truncated by 17 N-terminal amino acid residues  with a high level of amino acid similarity to hemolysins from Y. ruckeri  with orthologs in Y. intermedia and Y. ruckeri, respectively, were also identified. The first, PL78_05310, is positioned upstream of PL78_05315 and PL78_05320, the respective orthologs of which (srfA and srfB) are found in the S.typhimurium pathogenicity island SPI-2. Transcription of srfA and srfB is upregulated in epithelial and macrophage cells [Aside from the previously documented Yen-TC Chi1 and Chi2 proteins ,17, a thge cells . The locIn silico analysis of the Y. entomophaga genome identified several fimbrial gene clusters and sub-component fimbrial gene clusters  with Y. intermedia strain Y228 (gb|CP009801.1). This region was designated fimbrial-associated island FAIYE96. Similar to iron clusters, many of these fimbrial gene clusters are co-located with virulence-associated genes  gene cluster, PL78_00685-PL78_00780 , in the clusters . Of noteproteins . Y. entomophaga genome encodes several loci that are likely to assist in the survival of the bacterium in the insect host. The genome encodes a colicin V protein (PL78_16525), two non-ribosomal peptide synthases (PL78_04165 and PL78_15615) that are likely to direct the production of microbial- and or host-active metabolites , Y. entomophaga encodes two T3SSs. Y. entomophaga T3SSYE1 has a gene order similar to that of S.typhimurium T3SS SPI1, which is required for entry into intestinal epithelial cells. Once internalized, the effectors from the second S.typhimurium T3SS, SPI2, are released. This leads to the formation of a specialized Salmonella-containing vacuole where the bacterium can reside and replicate [Y. entomophaga T3SSYE1 is associated with an intimin protein, which in other systems facilitates the attachment of the pathogen to the intestinal epithelial cells [Y. entomophaga initimin BlastP orthologs were in non-Yersinia species, including insect-associated bacteria such as Xenorhabdus bovienii and Sodalis praecaptivus (Y. enterocolitica Ysa system (T3SS), which has a gene order resembling that of T3SSYE2, is implicated in the initial colonization of the animal intestinal ileum [Y. enterocolitica in Drosophila melanogaster S2 cells, with the authors suggesting that Y. enterocolitica has an insect host [P. luminescens and Pseudomonas aeruginosa, they are active against hemocytes [Y. entomophaga has previously been observed within insect hemocytes [The eplicate . We noteal cells . In thiscaptivus . The Y. al ileum  and has ect host . In instemocytes . In thisemocytes . in silico analysis, are likely to play a significant role in virulence. The most prominent of these is Rhs-associated Region 3, which encodes components of a T6SS and several loci PL78_00995, PL78_01005, and PL78_01040 with similarity to different DNA-associating proteins, suggesting that these proteins are likely to interact with host cell DNA and a requirement for their delivery to a target cell. Although there are several prokaryotic orthologs in the current databases , VIP2 (PL78_16145), Rhs (PL78_18780), LopT (PL78_18760), and the adenylate cyclase toxin (PL78_08395), are not accompanied by their associated transport module. It is plausible that some of these putative toxin effectors are co-opted by existing transport modules, such as the T3SSs or T6SS’s, enabling their delivery. The P. luminescens LopT effector, which in Spodoptera littoralis and Locusta migratoria is activated at sites of cellular defense to prevent phagocytosis, complements the Y. pseudotuberculosis T3SS, allowing its delivery into mammalian cells [Y. entomophaga toxins may be released directly into the eukaryotic cell cytosol by a yet to be determined mechanism. This could include the Y. entomophaga T2SS, whose role in other systems has yet to be fully elucidated, but has been implicated in the secretion of cell surface-located toxins and adhesins [an cells . The Rhs PL78_184, VIP2  causes melaninization of G. mellonella and D. melanogaster larvae [Serratia marcescens induces hemolymph bleeding in the silkworm Bombyx mori, possibly through the degradation of host-derived factors required for coagulation [et al. [Xenorhabdus species with a high degree of amino acid similarity differ in their relative proteolytic efficiency in the presence of different chelation factors. This data would account for the presence of three co-located Y. entomophaga serralysin loci, each of which may be tailored to a specific host system. Based in the aforementioned scenarios the co-location of putative toxin, adhesion or lytic enzymes within Region 1 and Region 2 may reflect a subtle interplay between the proteins throughout the virulence process.Host cell lysis by proteolytic enzymes or toxins results in the release of iron. We noted that three serralysin metalloprotease loci were co-located with a Region 2 B. Intra-r larvae . A serragulation . Massaou [et al.  determinSeveral host defense systems identified in the current study, including Ivy, ROS defense systems, siderophores, and antimicrobial factors, have been documented in other systems ,27. AlthY. entomophaga genome did not identify any orthologs of known Y. pestis or Y. pseudotuberculosis virulence determinants. Those orthologs of known mammalian toxins that were identified, including hemolysin and adenylate cyclase toxin, had either low amino acid similarity or represented a sub-component of a composite toxin cluster. In this context, the ability of a toxin to traverse the mammalian mucosal intestinal lining or cross the chitinous insect intestinal tract relies on different delivery vehicles, which, when docked, enable the transfer of conserved effector toxin molecules that typically act to cause a similar subcellular effect [C-terminus, thought to be transported by the YenA and YenB components [Photorhadus virulence cassette [E. coli-derived Cnf harbors its own amino terminal receptor-targeting domain [In recent years, it has become apparent that many plant- and insect-active toxins have distant mammalian orthologs, with both plant and invertebrate pathogens proposed as the progenitors of mammalian pathogens ,75,76,77r effect ,79. In tmponents , are veccassette . Howeverg domain .In silico analysis of the Y. entomophaga draft genome sequence confirmed that the bacterium encodes a diverse range of putative effector toxins and proteolytic enzymes, which along with complex export machinery and iron-sequestering factors, reflects a finely tuned pathogen capable of incapacitating a wide range of insect species. The wide variety of toxins produced by P. luminescens and Xenorhabdus species, and encoded by Y. entomophaga, is likely to reflect the broad and non-discriminant nature of their associated nematode [Y. entomophaga, the broad host range of the Yen-TC. For these bacteria, the associated nematode or the Yen-TC mediate the entry of the pathogen into the hemocoelic cavity, from where infection can occur. The Y. entomophaga genome also encodes a large number of hypothetical proteins, the functions of which have yet to be defined. To gain greater insight into the full functional potential of the genome, a range of in vitro and in vivo transcriptomic and proteomic studies will need to be undertaken. nematode ,82 and, Genomic DNA was prepared using a Bioline Isolate II Genomic DNA kit (BIO-52066), mini-spin column isolation. The yield and purity of the DNA was determined for 5-μL samples by electrophoresis on 0.8% agarose gel and by spectrophotometry on a NanoDrop 1000 apparatus . Genome sequencing was undertaken at the University of Liverpool Centre for Genomic Research, Liverpool, United Kingdom. Eight kilobase paired-end libraries were generated using the Roche “Paired End Rapid Library Preparation Method Manual, 20 kb and 8 kb Span, April 2012” according to manufacturers instructions. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyzer. The resultant libraries were further quantified by qPCR using a Roche LightCycler 480 and Kapa 454 library quantification kit. Medium volume emPCRs were performed for each library with 0.6 copies per bead of input material and recovery levels of beads confirmed using a Beckman Coulter Counter. Beads from emPCR reactions for each library were pooled and sequencing performed on half a picotiter plate according to manufacturers instructions using xlr70 (Titanium) chemistry. The genome sequencing gaps were closed in-house using PCR-based amplicon sequencing. PCR primer sets were manually designed or designed using Primer3 version 0.4.0  . p = 0.05, then normalized down to 30× coverage using the khmer digital normalization package [Y. entomophaga MH96 genome sequence is deposited in Genbank, as accession number: gb|CP010029; BioProject: PRJNA267025 and BioSample: SAMN03177402.For assembly, a combination of illumina single-end and Roche 454 pair-end data was used. Illumina data was trimmed for quality using SolexaQA version 2 software , using DynamicTrim , with pa package , and the package , run in  package , and assThe genome was automatically annotated using the NCBI pipeline . The COGhttp://www.cbs.dtu.dk/services/gwBrowser/) [Y. enterocolitica 8081, and a custom Y. ruckeri ATCC 29473 Blast database. Deduced amino acid sequences of the Y. entomophaga ORFeome were compared against the respective database using gapped BlastP  [Y. entomophaga MH96 genome sequence was compared by Latz  [Y. nurmii type strain APN3a-c whole genome shotgun sequencing project (NZ_CPYD00000000).The assembled draft genome was annotated using an updated version of the GAMOLA software suite  and resurowser/)  and in-hA, 2010) . The draA, 2010)  against