Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency.Coffee is one of the world's most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories.Assembling the expressed sequence tags (ESTs) of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance.We present the first comprehensive genome-wide transcript profile study of  Coffee is the most important agricultural commodity in the world and is responsible for nearly half of the total exports of tropical products . Indeed,Coffea are responsible for almost all coffee bean production: C. arabica and C. canephora . C. arabica is an autogamous allotetraploid  species originating from a relatively recent cross (≅1 mya) between C. canephora (or a canephoroide-related species) and C. eugenioides, which occurred in the plateaus of Central Ethiopia [C. canephora is a diploid (2n = 2× = 22), allogamous and more polymorphic Coffea species. In contrast to C. arabica, which is grown in highland environments, C. canephora is better adapted to warm and humid equatorial lowlands. C. arabica is regarded as having a better cup quality, which seems to depend on the quality and amount of compounds stored in the seed endosperm during bean maturation [C. canephora is considered more resistant to diseases and pests and has a higher caffeine content than C. arabica [C. canephora blossoms earlier, its fruit maturation is delayed in comparison to C. arabica [C. arabica [Two species of the genus turation -7. Conve arabica . Other i arabica . Improve arabica  and the Expressed sequence tags (ESTs) provide a source for the discovery of new genes and for comparative analyses between organisms. Many EST sequencing efforts have successfully provided insights into crop plants development -18. EST C. arabica, C. canephora and C. racemosa, a coffee species used in breeding programs for the introgression of resistance against coffee leaf miner. Concomitantly, an initiative from the Nestlé Research Center and the Department of Plant Biology at Cornell University sequenced ESTs from C. canephora farm-grown in east Java, Indonesia. This research group compared the EST repertoires of C. canephora, Solanum lycopersicum (tomato) and Arabidopsis thaliana [C. canephora and tomato have a similar assembly of genes, which is in agreement with their similar genome size, chromosome karyotype, and chromosome architecture [http://sgn.cornell.edu), a genomics information resource for the Solanaceae family and related families in the Asterid clade, such as Coffea spp. and other Rubiaceae species [Research on coffee genomics and transcriptomics has gained increasing attention recently. A Brazilian consortium   was devethaliana ,23. Baseitecture . In addiCoffea spp. prompted us to perform a wide bioinformatics analysis. In this report, we surveyed the coffee transcriptome by analyzing ESTs from C. arabica and C. canephora. Resources developed in this project provide genetic and genomic tools for Coffea spp. evolution studies and for comparative analyses between C. arabica and C. canephora, regarding gene families' expansion and gene ontology. We also identified Coffea-specific/prominent gene families using automatic orthology analysis. Additionally, we describe the annotation of differentially expressed genes according to in silico analysis of EST frequencies.The availability of EST data from both of the commercially most important Coffea spp. we collected 187,412 ESTs derived from 43 cDNA libraries produced by the Brazilian Coffee Genome Project initiative [C. arabica libraries represent diverse organs, plant developmental stages and stress treatments from Mundo Novo and Catuaí cultivars, excluding germinating seeds (cv Rubi) , 135,876 C. arabica ESTs were assembled into 17,443 contigs and 17,710 singlets  from C. canephora  and 663 bp , respectively  have only two ESTs, each one from a different EST library. Due to the limited depth of sequencing and the variety of tissue samples used to construct the C. arabica libraries, a smoother distribution of contigs per library was observed in comparison with C. canephora  in our dataset using the QualitySNP program tools [C. arabica (8%) and 518 contigs in C. canephora  of  species , as is tC. arabica and C. canephora contigs. In the case of C. arabica, we selected contigs containing at least four reads, which in theory provide two copies for each allele, yielding 8,514 C. arabica and 3,832 C. canephora contigs. Approximately 53%  of the C. arabica contigs and 52%  of the C. canephora contigs were found to contain SNPs . Similarly, Lashermes et al. [C. canephora. The majority of polymorphisms found in both species was bi-allelic (99.8% for C. arabica and 99.5% for C. canephora), with a low percentage of tri-allelic and no tetra-allelic SNPs  available at AutoFACT were inspected to evaluate the similarity of Coffea clusters with proteins deposited in GenBank. Approximately 68% of C. arabica and 71% of C. canephora clusters have significant sequence similarity  with genes in the databank. The remaining clusters represented sequences with lower E-value scores  designated as "no-hits"  would be the most similar to Coffea sequences. However, the majority of Coffea clusters have higher similarity with Vitis vinifera sequences (~40%), a species from the Rosids clade, followed by the other rosids Arabidopsis (~5.5%) and Populus trichocarpa (~3.5%). The top hits of Coffee sequences with Solanaceae range from 1 to 2% , followed by V. vinifera (10.34 and 10.03%), S. lycopersicum (6.5 and 5%) and S. tuberosum . We believe that the most parsimonious hypothesis for these results is related to phylogenetic issues. Grape is basal to the rosids clade and did not undergo whole genome duplication (WGD) events, such as Arabidopsis, thus being theoretically more similar to the rosids paleohexaploid ancestor [Mimulus guttatus) revealed extensive synteny with grape, suggesting that paleohexaploidy antedates the divergence of the rosid and asterid clades [Coffea and V. vinifera genomic regions [We next used AutoFACT  to evalus" Table . Becausehocarpa ~.5%. The ancestor ,33. Anal regions . IntensiCoffea species analyzed, we estimated the rates of synonymous  and non-synonymous  substitutions generated by QualitySNP analysis, and performed the KA/KS test for positive selection of each hypothetical gene. KA/KS is a good indicator of selective pressure at the sequence level. Theoretically, a KA/KS >1 indicates that the rate of evolution is higher than the neutral rate. Conversely, a gene with KA/KS < 1 has a rate of evolution less than the neutral rate [C. arabica and C. canephora appear to be under purifying selection (KA/KS < 1), indicating that the majority of protein-coding genes are conserved over time as a result of selection against deleterious variants.To gain insight into the molecular evolution of protein coding genes in the two ral rate . As in oral rate ,38, mostThe correlation between AutoFACT annotations with KA/KS analysis allowed the detection of genes with low KA/KS ratios, such as those encoding proteins involved in photosynthesis, morphogenetic development and translation , because that two enzymatic reactions in C. arabica may be annotated as only one. The data for the fully annotated pathways are available at the website http://www.lge.ibi.unicamp.br/coffea.We constructed hypothetical metabolic maps for both g BioCyc . After mC. arabica and C. canephora gene clusters with the CDD-PFAM databank to catalog the protein domains present in the Coffea EST datasets. The submission of the clusters to RPS-BLAST resulted in 30%  of C. arabica and 32%  of C. canephora clusters containing an assigned domain. To compare the prevalence of protein domains in Coffea species, the number of clusters assigned to each domain was normalized by dividing by the total number of clusters containing a domain. Serine threonine kinases (Pfam00069), cytochrome P450 monooxygenases (Pfam00067), tyrosine kinases (Pfam07714) and proteins containing RNA recognition motifs  are among the top 20 PFAM families in Coffea species  than in C. arabica (0.02%). This domain is found in LTR-retrotransposons, the most widespread transposable element (TE) family in plants [Coffea species harbor fewer TE-cassettes (> 0.04%) than would be expected from the translation of TE-containing transcripts (0.23%). These authors hypothesized that such incongruence may either be a consequence of the exonization/exaptation of TE fragments or an indication of the tolerance of alternatively spliced "TE-invaded" mRNAs that do not encode functional proteins. A more detailed investigation is in progress to explore the diversity and differences between Coffea spp. TEs .One noteworthy difference between domains is the greater percentage of proteins containing the retrotransposon gag protein domain (Pfam03732) in n plants . Lopes en plants  found thC. arabica and 49% of C. canephora clusters were mapped with a biological process, and 43 and 55% were mapped with a molecular function. These differences reflect the greater amount of C. arabica ESTs in the libraries compared to C. canephora and are likely related to the fact that some tissues used in C. arabica libraries  were not extensively studied, resulting in genes with unassigned ontologies. To compare the gene ontologies, the amount of sequences associated with each term was normalized (see methods), and then hypergeometric statistics were applied [C. arabica has a greater amount of transcripts coding for proteins with catalytic activity, transferase activity and transporter activity than C. canephora  in fruits of Arabica plants, one of the traits that provides a better cup quality (see below). In contrast to C. arabica, C. canephora has more proteins annotated as containing binding activity, which is extended for the binding activity branch child terms of nucleic acid binding, DNA and RNA binding activities, transcription regulation and transcription factor activities  structures . Approxi applied . To compCoffea spp. in comparison to other species, we applied OrthoMCL, a graph-clustering algorithm designed to identify homologous proteins based on sequence similarity [A. thaliana, V. vinifera, Oryza sativa, Ricinus communis and Glycine max and ii) the proteins predicted by FrameDP software [C. arabica, C. canephora and S. lycopersicum. Based on the fact that some genes are not picked in EST libraries, the evaluation of Coffea spp. gene family retraction was not performed .To identify proteins that are hypothetically specific or at least prominent in milarity ,47. Two software  from theCoffea spp. are: i) a family composed of serine/threonine kinases (family 1), ii) pentatricopeptide repeat-containing proteins (family 2) and iii) cytochrome P450 monooxygenases , C. canephora-specific invertase inhibitors (family 14814), small secreted glycine-rich proteins (family 11055), Ty3 Gypsy-like retrotransposons (family 10952), kelch repeat phosphatases (family 14392), 2 S albumin storage proteins (family 14392), etc. . The other three include proteins similar to rapid alkalinization factor , GTP binding proteins (family 9023) and proline-rich extensins  and a heme-containing peroxidase (245 ESTs), both distributed among 29 libraries  encodes a putative VTC2 protein, a GDP-D-glucose phosphorylase involved in ascorbic acid biosynthesis [C. canephora, which is likely used as an antioxidant and as a cofactor for dioxygenases.We correlated the AutoFACT annotation results with the distribution of contigs in the  Arabica -52, whic Arabica . The lacynthesis , suggestCoffea libraries also revealed the contigs containing the most redundant (most highly expressed) ESTs  are small, cationic, Cys-rich proteins structurally and functionally related to biocide defensins previously characterized in mammals and insects . All ESTic Table , perhapsC. arabica, a PR-10 was found to be highly expressed in an incompatible reaction against the causative agent of coffee leaf rust, the biotrophic fungus Hemileia vastatrix [C. arabica (CaContig15067) was predicted to be more expressed in suspension cells treated with aluminum  and thaumatins (six contigs) in C. arabica and C. canephora, respectively. In addition, some CC-NBS-LRR families were prevalent in C. arabica  and in C. canephora , with similarity to the nematode resistance potato proteins Gro1 [Coffea spp. TIR-NBS-LRR proteins is similar to that described in cereals and sugar beet [Most of the disease resistance genes (R genes) in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. They are engaged in the recognition of pathogens, being considered specific determinants of the plant immune response ,73. Uponubfamily . Neverthins Gro1 . The extgar beet ,78 and lgar beet ,77,78. TGenes related to abiotic stresses are potentially important in the recent scenario of harsh environmental changes, such as the increase of extreme temperatures and drought periods. Coffee plantations are threatened by global warming due to coffee's susceptibility to high temperatures and drought when these stresses occurs during flowering and fruit development . The undC. arabica drought stressed plants (SH2) in comparison to well-watered plants conditions (SS1). Genes encoding RuBisCo activases (CaContig 5581 and 14729), a putative photosystem II type I chlorophyll a/b-binding (CAB) protein (CaContig5621) and a PSI-E subunit of photosystem I (CaContig5564) were preferentially expressed in the SH2 library  is similar to early light-induced proteins (ELIPs), thylakoid-target proteins that are similar to light harvesting complex (LHC) proteins . In addition, we also detected another putative auxin receptor in C. arabica, ABP1 (CaContig16576), a cupin-like protein that is implicated in early auxin responses [Plant hormones (phytohormones) are crucial for a series of developmental mechanisms, such as organ initiation and development, resistance to stress and reproduction. Auxins are the most studied class of phytohormones, being implicated in cell division, cell elongation and cell differentiation . Using OC. arabica (two members) and C. canephora (five members). However, the evaluation of OrthoMCL families revealed that coffee has a particular family of small peptides slightly similar to RALFs  were found to be differentially expressed in plantlets and leaves treated with arachidonic acid  superfamily is a large complex of plant proteins that share the presence of glycine-rich domains arranged in (Gly)n-X repeats . GeneralC. arabica and six members in C. canephora (Table Brassica oleracea (ABD64972). Two C. canephora members from those families (CcContigs 7844 and 3825) were highly expressed in leaves from water deficit stressed plants  are 12- to 16-kDa plant proteinaceous inhibitors of Cys-proteases of the papain C1A family ,100. Allra Table . ProteinPin2 genes preferentially expressed in EST libraries of stressed plants, predicted coffee Pin2 proteins contain signal peptides and, additionally, have 30-40% identity with a Pin2 protein of tobacco that confers tolerance to NaCl and resistance against herbivorous insects in transgenic plants [Members of the Potato type II (PotII) inhibitors (Pin2) family are PIs restricted to plants that belong to the MEROPS inhibitor family I20, clan IA . Severalc plants . In addiCoffee cup quality is a complex trait that is being unraveled. The components of coffee endosperm are the source of the precursors of aroma and flavor after roasting. The degradation of sucrose and cell wall polysaccharides generate reducing sugars, which react with amino acids during roasting through Maillard glycation reactions. This reaction gives rise to aromatic products, such as pyrazines, furans and aliphatic acids, which are associated with pleasant flavor and aroma . ConversC. arabica in relation to C. canephora .Due to the importance of the amount and composition of carbohydrates to the final quality of the coffee beverage, the study of coffee bean carbohydrate synthesis and degradation is intense ,108-112.a Figure . CesA prslA-CslH . It was slA-CslH ,116. TheC. arabica and C. canephora is related to low molecular weight carbohydrate content, especially sucrose [C. canephora grains (4-5%). Though sucrose is almost completely degraded during coffee bean roasting (0.4-2.8% dry weight), sucrose remains are thought to improve coffee sweetness and cup quality [C. arabica grains than in C. canephora, and invertase activity was lower in Arabica, likely due to the higher expression of invertase inhibitors in this species, justifying the higher sucrose content in C. arabica beans. Based on BLAST and OrthoMCL analysis, we found that Invertase Inhibitor 3 (InvI3) is part of a Coffea spp.-specific protein family . Therefore, raffinose may exert dual functions in coffee: galactose reservoirs in coffee grains and protective roles in vegetative development.The raffinose family oligosaccharides (RFOs) are soluble galactosyl-sucrose carbohydrates such as raffinose, stachyose and verbascose. Their participation in coffee seed development was assessed by Joet et al. , who inda Figure . Such da Arabica ,123. A m drought ,125. IndC. canephora seed development  activity. We identified three α-Gal-encoding genes as more expressed in the late stages of ee beans . Togethend α-Gal ,126, theC. canephora seed maturation  in the endosperm and the relationship between proteinases and their inhibitors during seed development are all factors that determine the amino acid content in mature beans. Examining the expression profile of the SE2 library, we found a series of ribosomal proteins expressed in this stage of seed maturation Figure , indicatC. arabica fruit libraries    and is pC. arabica has more trigoneline and sucrose, and C. canephora contains more CGA and caffeine [C. arabica and C. canephora plants.Other precursors of flavor and aroma in coffee are secondary metabolites, such as alkaloids (caffeine and trigoneline) and phenylpropanoid chlorogenic acid (CGA). These three components, together with sucrose, seem to be the main factors influencing coffee quality, because sucrose and trigoneline enhance coffee quality, while CGA and caffeine confer bitter taste ,130-133.caffeine . DespiteC. arabica and C. canephora and applied a diverse array of bioinformatics tools to extract information about gene content features, transcriptome changes and novel genes and gene families. The results concerning the prevalence of proteins related to sugar metabolism in C. arabica and signal transduction in C. canephora can be correlated with agronomical characteristics of each species due to the better cup quality of C. arabica and the high tolerance to specific stresses in C. canephora plants. Despite knowing that comparisons between these Coffea species data should be carefully inspected, our initiative established possible transcriptomic elements that could guide the coffee scientific community in unraveling the molecular mechanisms that distinguish these two extremely important Coffea species. In addition, the annotation of coffee-specific/prominent genes adds new elements to genomic initiatives that are searching for traits that could differentiate coffee from other Asteridae species. In a recent report, Vidal et al. [C. arabica displays differential expression of homeologous genes and suggested that C. arabica ancestral subgenomes encode proteins involved in different physiological mechanisms, adding a new element of investigation concerning gene expression regulation in coffee plants.We assembled ESTs from l et al.  showed thttp://www.lge.ibi.unicamp.br/coffea. We believe that such data are a valuable aid to the interpretation of coffee development, providing insights that could help coffee breeding programs and indicating potential targets for functional analysis and biotechnology products of such socially and economically important species.All data presented here are available at C. arabica  and C. canephora  were derived from 43 libraries collected by the BCGP and from 8 libraries of C. canephora EST sequencing initiative of the Nestlé Research Center (8). The Brazilian project sources were mainly two C. arabica genotypes  and one C. canephora genotype (Conillon). The Cornell-Nestlé project EST sources were five different varieties of C. canephora [E. coli contaminants [-40 and the percent of identical nucleotides > 80% were considered bacterial contamination. In addition, hits against NR with a percent of identity > 30% and all of the hits against bacteria were considered bacterial contamination. All of the BCGP ESTs were submitted to GenBank with accession numbers GT640310-GT640366, GT669291-GT734396, GW427076 - GW492625 (C. arabica) and GT645618-GT658452 (C. canephora).ESTs from anephora . Sequencaminants . EST assC. arabica and C. canephora. QualitySNP uses three quality filters for the identification of reliable SNPs. The first filter screens for all potential SNPs. False SNPs caused by sequencing errors are identified by the chromatogram quality given by Phred. The second filter is the core filter, which uses a haplotype-based strategy to detect reliable SNPs. The clusters with potential paralogs are identified using the differences in SNP number between potential haplotypes of the same contig. All potential haplotypes consisting of only one sequence are removed, and singleton SNPs that are not linked to other polymorphisms are not considered. This may lead to an underestimation of nucleotide diversity but assures that false positives will be discarded. The last filter screens SNPs by calculating a confidence score based on sequence redundancy and base quality. To label each polymorphism as synonymous or non-synonymous, the correct open reading frame (ORF) of each sequence was identified by looking for similarity calculated with the FASTA algorithm against the Uniprot databank http://www.uniprot.org using an E-value threshold of -05. The alignments were analyzed with QualitySNP script GetnonsySNPfasty, which corrects frame shifts and attempts to expand the 3' end until the next stop codon and the 5' end until the next ATG codon. This script identifies if the polymorphism changes the amino acid, labeling each polymorphism as non-synonymous (KA) or synonymous (KS). This information was used to calculate KA/KS ratios for positive selection using kaks calculator software [C. arabica and C. canephora. A total of 1,380 full length sequences > 200 bp of Arabidopsis thaliana were extracted from Genbank. Sequences of Solanum lycopersicum were also randomly retrieved from the Kazusa http://www.kazusa.or.jp/jsol/microtom/indexj.html and SGN databanks [QualitySNP  was usedsoftware . All of atabanks . Total GC. arabica and C. canephora were automatically annotated using the AutoFACT program [C. arabica and C. canephora metabolic maps were compared with PlantCyc, which contains curated information about pathways present in > 250 plant species. The divergence among the maps was manually annotated to eliminate false positives. To evaluate protein domains, ESTs were submitted to similarity searches against the CDD-PFAM database using RPS-BLAST . Data were normalized by dividing the number of clusters from each CDD-PFAM by the total number of hits from each species against CDD-PFAM.The complete set of ESTs from  program . AutoFACCoffee datasets were annotated and mapped for the gene ontologies "Biological Process" and "Molecular Function" (only level 3) by Blast2go . Blast2gA. thaliana , O. sativa , Ricinus communis  and Glycine max  and ii) the proteins predicted by FrameDP software [C. arabica , C. canephora  and S. lycopersicum . All proteins were compared  using BLASTP, and a score for each pair of proteins  with significant BLAST hits was assigned . Based on these scores, the MCL algorithm was applied to find clusters in this graph. The protocol used is described at http://lge.ibi.unicamp.br/Ortho_MCL_UserGuide.txt.The Ortho-MCL algorithm  was applsoftware  from thein silico expression analysis, contig and singlet frequencies across the libraries were obtained from the dataset derived from the CAP3 assembly. The frequency of a contig over a library represents its transcript abundance. Only contigs containing more than two ESTs were used for transcript profiling. Differentially expressed contigs were identified using two statistical tests, R [For tests, R  and AC [tests, R , with thtests, R . In R stIn an attempt to cluster elements that are similar (in some sense), hierarchical clustering  of the dJMCM: ESTs and cluster re-annotations, conception of bioinformatics analyses, evaluation and interpretation of GC content, Ortho-MCL, CDD-PFAM, Gene Ontology and Hierarchical clustering data, conception and writing of the manuscript; ROV: EST assembly, conception of bioinformatics analyses, GC content and SNP analyses and evaluation, Gene Ontology and Ortho-MCL analysis; MFC: EST assembly, conception of bioinformatics analyses and CDD-PFAM analysis; EKT: Hierarchical clustering analysis; LPP: AutoFACT and Metabolic Pathways analysis; GGLC: EST assembly and Ortho-MCL analysis; LFPP: SNP evaluation and revision of the manuscript; ACA: Coordination of EMBRAPA EST libraries and revision of the manuscript; CAC: Coordination of AEG/FAPESP EST libraries and revision of the manuscript; LGEV: Coordination of the EST consortium and revision of the manuscript; GAGP: coordination of the bioinformatics group and elaboration of the final manuscript. All authors read and approved the final manuscript.Description of Brazilian Coffee Genome Project ESTs and cDNA libraries. Word file describing the methods used for the production of Brazilian coffee ESTs libraries, the tissues and experimental conditions used.Click here for fileNumber of contigs composed from sequence originated from one or more libraries. TIF file containing a figure depicting the correlation between number of contigs vs number of libraries.Click here for fileC. arabica and C. canephora EST datasetsGlobal data of SNP detection in . Word file containing an overall analysis of SNPs from C. arabica and C. canephora contigs.Click here for fileCoffea spp. contigsAnnotation of KA/KS ratio in . Word file containing the annotation of Top 20 C. arabica contigs with highest and lowest KA/KS ratio (A); Annotation of Top 20 C. canephora contigs with highest and lowest KA/KS ratio (B). ID: Contig number; KS: rate of synonymous substitutions, KA: rate of non-synonymous substitutions; KA/KS: KA to KS ratio; First Hit (BLASTX-NR): Most similar sequence in GenBank; E-value: E-value of most similar sequence; Annotation: automatic annotation based in AutoFACT results.Click here for fileCoffea spp. PFAM familiesTop 20 . Word file containing the ranking of PFAM families in C. arabica and C. canephora.Click here for fileCoffea spp. cDNA librariesAnnotation of Top 20 genes with the widest distribution among . Word file containing the ranking of genes distributed throughout coffee EST libraries. ID: Contig number; #: number of libraries represented in each contig; #ESTs: number of ESTs that compose each contig; First Hit (BLASTX-NR): Most similar sequence in GenBank; E-value: E-value of most similar sequence; Annotation: automatic annotation based in AutoFACT results.Click here for fileCoffea spp. cDNA librariesAnnotation of 20 genes with the highest expression among . Word file containing the ranking of genes more expressed in coffee EST libraries. ID: Contig number; #: number of libraries represente in each contig; #ESTs: number of ESTs that compose each contig; First Hit (BLASTX-NR): Most similar sequence in GenBank; E-value: E-value of most similar sequence; Annotation: automatic annotation based in AutoFACT results.Click here for fileAnnotation of selected differentially expressed genes in coffee EST libraries according to hierarchical clustering analysis. Excel file containing the annotation of genes with differential expression profile in coffee EST libraries according to hierarchical clustering analysis. Worksheet CA: C. arabica contigs; Worksheet CC: C. canephora contigs. Libraries: Tissues and organs used in the libraries construction; Nomenclature: code of the library; Contig ID: Contig number; Annotation: automatic annotation based in AutoFACT results.Click here for fileResults concerning some genes related with drought stress . Word file describing results and a brief discussion about dehydrins, LEAs and Metallothioneins expressed in coffee EST libraries.Click here for fileRALF and RALF-like peptides. Word file containing the sequences of RALF and RALF-like peptides expressed in coffee. In magenta: dibasic sites; in yellow: cysteine residues.Click here for fileOrthoMCL families of Glycine Rich Proteins (GRP). Word file containing the sequences of Glycine Rich Proteins expressed in coffee. In yellow: cysteine residues; Underlined: signal peptide for secretion.Click here for fileOrthoMCL families of Cystatins. Word file containing the sequences of Cystatins expressed in coffee. In green: variation of LARFAV motif; in yellow: new motif GG-X-YY; in blue: QVVAG motif.Click here for fileOrthoMCL families of PinII Serine Proteinase Inhibitors. Word file containing the sequences of PinII Serine Proteinase Inhibitors in coffee.Click here for file


Sarcocystis species: Sarcocystis tenella, Sarcocystis gigantea, Sarcocystis arieticanis, and Sarcocystis medusiformis The purpose of this study was to perform a molecular identification of the macroscopic and microscopic cysts of Sarcocystis in sheep. In this investigation, the macroscopic and microscopic cysts of Sarcocystis were assessed in slaughtered sheep. The digestion method was used for bradyzoites observation in heart, liver, diaphragm and muscle samples. PCR analysis was conducted on macroscopic and microscopic cysts and also all other samples. Sequencing was performed for ten PCR products. Genotypes were identified by BLAST search and homology analysis. Macrocysts were seen in two muscle tissues. Digestion method and PCR analysis revealed positive results in all samples taken from heart, liver, diaphragm, and muscle. Genotyping of ten tissue samples proved that the genotype of macroscopic belonged to Sarcocystis gigantea and microscopic cysts to Sarcocystis tenella. Microscopic cysts are more prevalent than macroscopic cysts and they can cause enormous economic losses.Sarcocystis is an obligatory intracellular protozoan parasite which can infect humans and animals. Sheep are intermediate hosts of four  Sarcocystis species are intracellular protozoan parasites infecting a wide range of livestocks. Some of Sarcocystis genus are pathogenic for animals such as sheep and cattle which cause enormous economic losses , liver (N= 40), diaphragm (N= 40) and muscle (N= 40) from 40 slaughtered sheep  aged between 3 months to 3 years in the slaughterhouses of North Khorasan province.Tissue digestion  Sarcocystis. For observing bradyzoite in macroscopic cysts; macroscopic cysts were separated from intact tissues and then, both macroscopic cyst and intact tissues were digested, separately.Tissue digestion method was used for observing bradyzoite in tissue samples. Seventy grams of each tissue were ground and digested in 1.5% HCL and 0.5% Pepsin at 29°C overnight. The digested samples were filtered through mesh and centrifuged at 1500 rpm for 10 minutes. Then the supernatant was discarded and the pellet was stained by Giemsa and examined microscopically to detect bradyzoites ofDNA extraction and PCR amplification For DNA extraction, a small piece of each sample was selected, and DNAs were extracted using tissue DNA extraction kit (Cinnagen company). PCR analysis was performed on all tissue samples using Sar primers including Sar-F1 Forward 5'GCACTTGATGAATTCTGGCA3' and Sar-R1 Reverse 5'CACCACCCATAGAATCAAG 3' . PCR reaSequencing and genotyping of isolates 10 PCR products  were purified. using column-based purification kit and then sequenced through automatic sequencer of the Korean Macrogen Company.Sarcocystis DNA sequences in the GenBank based on sequence analysis of 18s rRNA region.The obtained sequences were edited using Bioedit software program. Genotype identification was performed by comparing with available Sarcocystis  were infected with ystisspp , 17-19. nfection , 19.Sacocystis were isolated from different animals (S. tenella from the sheep in Brazil (S. moulei from the sheep in Saudi Arabia (Gjerde isolated and characterized S. grueneri from reindeer based on molecular method (Sarcocystis hominis and Sarcocystis cruzi were studied in cattle (S.gigantea and S. arieticanis from the sheep by PCR-RFLP method in Qazvin province, Iran (S. miescheriana from boar (S. tenella from sheep in Iran (S. gigantea and S. tenella caused macroscopic and microscopic cysts (S. gigantea and S. tenella can cause macroscopic and microscopic cysts, respectively. The results of this investigation was in accordance with Anja and Astrid's study who reported that S. gigantea and S. tenella can cause macroscopic and microscopic cysts, respectively (In previous studies throughout the world, species of  animals , 14-15. i Arabia . Moreoven cattle , 16. Dalce, Iran . Furtherrom boar  and S. t in Iran . Additioic cysts . Using dic cysts . Kargar ic cysts . MoleculS. tenella is among the pathogenic species and can induce microscopic cysts ( S. tenella can lead to acute sarcocystosis in uninfected sheep (S. tenella caused symptoms such as inflammation, hepatitis and myocarditis in sheep inoculated with S. tenella sporocysts from canine feces (ic cysts . The sevic cysts , 25-26. ed sheep . Non-speed sheep -29. In ped sheep , 29-30. ne feces .S. tenella was isolated from the heart, diaphragm, muscle and liver of all tested samples. As noted above, S. tenella can cause severe clinical signs and dysfunction of organs in sheep, such as tachycardia and neurological disease (Sarcocystis tenella produces microscopic cyst which are neglected by veterinarians due to its invisible nature. Therefore, improvement of disease control and prevention strategies in sheep would be necessary.In this study,  disease . FurtherS.tenella produces microscopic cyst and which are neglected by veterinarians due to its invisible nature. While S.gigantea which causes macroscopic cysts and is non-pathogenic are easily noted. For this reason, macroscopic cysts are removed, but, microscopic cysts do remain. Subsequently, the life cycle of Sarcocystis tenella is repeated and it causes enormous economical losses.


Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported.Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes.P. xylostella larval genes following parasitization, DsIV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction.This study provides detailed information on differential expression of  Endoparasitoids of the insect order Hymenoptera inject their eggs inside a host insect where they hatch and subsequently feed on the host until its death. For successful parasitism, endoparasitoids bring about a change in their hosts' conditions in favour of the developing parasitoid larvae. For this purpose, female wasps introduce secretions such as venom or ovary fluids, which may contain symbiotic viruses (polydnavirus (PDV) and/or virus-like particles), and other maternal factors, into the host . In addiDiadegma semiclausum Hellén is an ichneumonid endoparasitoid that carries a PDV with a circular, double-stranded and segmented DNA genome encoding proteins that suppress the host immune response and cause developmental arrest/delay [st/delay . PDVs arst/delay , replicast/delay . No virust/delay -15.Ichnovirus containing ichnoviruses (IVs) and Bracovirus containing bracoviruses (BVs) [et al. suggested that IVs originated from ascoviruses by lateral transfer of ascoviral genes into wasp genomes [PDVs are classified into two genera based on their host wasp families; es (BVs) ,16. Theses (BVs) . Recentl genomes . Interes genomes . It has  genomes . Most of genomes . IV geno genomes .Research so far has concentrated mainly on individual genes or small defined groups of host or PDV genes, to explore their function or differential expression following parasitization. Deep sequencing data can provide extensive information about host-parasitoid interactions at the transcriptome level. The large amount of sequencing data that can readily be produced by next-generation sequencing platforms, such as the Illumina GAII, reduces the need for prior sequence knowledge for gene expression profiling and are now making direct sequencing approaches the method of choice for whole transcriptome analysis in many species -26. The D. semiclausum parasitization on its host, Diamondback moth Plutella xylostella L. , a notorious pest of cruciferous plants. P. xylostella has developed resistance to many groups of chemical insecticides and also Bacillus thuringiensis endotoxin [P. xylostella one of the world's most destructive insect pests and the estimated global cost of controlling this insect is around US$1 billion annually [P. xylostella larvae, revealing a significant number of D. semiclausum ichnovirus genes (DsIV) which have not been reported previously. This type of study may facilitate new controls for pest larvae by identifying molecules that are crucial for larval immune defence, development, pesticide resistance and other important metabolic regulatory functions.In this study, we used deep sequencing to explore the impact of ndotoxin ,29. Thisannually . This emA transcriptome is the complete set of expressed RNA transcripts in one or more cells. Transcriptome profiling of organisms under stress or parasitization challenge helps us to obtain a better understanding of subsequent related cellular activities in organisms including growth, development, and immune defence. Recently, the newly developed deep sequencing approaches have significantly changed how the functional complexity of the transcriptome can be investigated ,25,30.P. xylostella host larvae following parasitization by D. semiclausum, RNA samples isolated from the whole host larvae at various time points after parasitization were pooled together prior to sequencing. This may lead to bias in the results (e.g. a gene may first be up-regulated and down-regulated subsequently); however, we aimed at obtaining an overview of what occurs during parasitism and generating a transcriptome of P. xylostella which has not been previously available and to isolate as many DsIV genes as possible. Illumina GAII RNAseq deep sequencing analysis produced approximately 26.6 and 27.1 million single-end reads from RNA extracted from the whole body of non-parasitized (control) and parasitized larvae of P. xylostella, respectively. De novo assembly using the CLC Genomic Workbench produced 172,660 contigs with a minimum contig size of 100 bp; of these, 66% were between 100-199 bp and only 59,255 (34%) of the contigs were above 200 bp (Table Tribolium castaneum (Coleoptera) genes with a minimum E-value of 1e-06. Bombyx mori (silkworm) was another species with which a range of P. xylostella genes showed high levels of homology. One reason for the higher number of hits against the beetle genome is that three times more T. castaneum genes than B. mori genes are reported in databases.To analyse the transcriptome of Polistes metricus, were matched to the honeybee (Apis mellifera) genome sequence and EST resources, for annotation [http://www.blast2go.org through UniProt KB/TrEMBL and other available databases. GO-annotated consensus sequences were assigned to biological process clusters such as cellular component and molecular function, and distributed among various sub-categories such as metabolism, growth, development, apoptosis, immune defense, molecular processing, signal transduction, transcription regulator activity, catalytic activity etc.  with those of silkworm http://www.silkdb.org showed high similarity in the distribution of genes across GO categories indicating that the transcriptome analysed was not biased towards particular categories  suggested that a significant proportion of transcript signatures detected outside predicted genes represent regulatory non-coding RNAs, because these large numbers of non-coding RNA can be antisense, intergenic or overlapping with protein-coding genes [Overall, from the assembled contigs of over 200 bp in length, 44% showed similarity with genes or proteins in the NCBI database. The rest may represent unknown genes, non-coding RNA or misassembled contigs that are expected due to the presence of large repetitive or duplicated regions. David ng genes .P. xylostella larvae. The 'Oases' package was used to assemble data for differential expression analysis. Initially, reads from the parasitized and non-parasitized larvae were cleaned and combined, before de novo transcriptome assembly was carried out using Oases 0.1.18. [D. semiclausum parasitization, are displayed in Tables P. xylostella genes following D. semiclausum parasitization may not be consistent with proteomic observations in other host-parasitoid systems previously reported (e.g. [Campoletis sonorensis IV (CsIV) [Heliothis virescens it was shown that lysozyme activity declined after parasitization by C. sonorensis or injection of CsIV; however, the transcript levels of the gene increased after parasitization. This suggested that CsIV may regulate host cell gene expression at the translation level. We also found that lysozyme transcript levels increased following parasitization (Table P. xylostella larvae parasitized by Cotesia plutellae was decreased [P. xylostella was inhibited by two BV genes (CpBV15α and CpBV15β). In another study, it was shown that expression of antimicrobial peptides  were either unchanged or minimally induced in parasitized Drosophila melanogaster larvae by Leptopilina boulardi [Our sequencing data analyses indicate that parasitism has a significant impact on the transcriptome profile of  0.1.18. . The inded (e.g. ), it is V (CsIV) . Accordion Table . Anotherecreased ; howeverecreased  showed tboulardi . The autSpodoptera frugiperda larvae injected with Hyposoter didymator ichnovirus (HdIV) or Microplitis demolitor bracovirus (MdBV), the differentially expressed transcripts in hemocytes and fat body largely differed depending on the PDV injected suggesting that the tissues responded differently to the different viruses [HdIV or MdBV. Based on this study, HdIV affected transcript levels in both hemocytes and fat body, whereas MdBV mostly affected gene expression in the fat body.In addition to post-transcriptional effects of PDV infection, there may also be variations in different host-parasitoid interactions with regard to host transcripts affected by parasitization/PDV infection. In a very recent study, it was shown that in  viruses . HoweverP. xylostella larvae relative to other antimicrobial peptides  [et al. measured pxCecropin expression levels by quantitative RT-PCR (qRT-PCR) in parasitized P. xylostella larvae and showed that pxCecropin was suppressed by C. plutellae BV [P. xylostella [D. semiclausum attack.In our study, we found that antimicrobial peptides such as gloverin, moricin, lysozyme II and cecropin were up-regulated after parasitoid attack, compared to transcription levels in control unparasitized larvae. Among these, lysozyme II and gloverin had the highest transcription levels in parasitized s Figure ; Table 299 fold) . Barandoellae BV . Some anellae BV ,38. Recelostella . Our datP. xylostella larval plasma during parasitism by C. plutellae [et al. demonstrated that the ovarian calyx fluid of the ichneumonid endoparasitoid Venturia canescens has the potential to suppress the host immune system due to a putative serpin activity [P. xylostella. Thus, the induction of serine protease inhibitors upon immune challenge in parasitized larvae could be part of an endoparasitoid immune suppressive strategy. Many insect protease inhibitors are known to inactivate enzymes isolated from entomopathogenic fungi, and their involvement in insect-pathogen interactions has been widely postulated [et al. reported that five serine proteases involved in a single metabolic cascade were up-regulated in Anopheles gambiae upon microbial challenge, but they suggested a role for the proteases in protecting the mosquito from detrimental effects of an uncontrolled spread of immune reaction [Other related genes which were up-regulated in parasitized larvae included a serine protease and serine protease inhibitor (pxSerpin 3) Table . Serine lutellae . Beck etactivity . Here, wstulated . Aguilarreaction .P. xylostella. In a genome-wide microarray study of D. melanogaster, several genes encoding enzymes of the melanization cascade were found to be up-regulated by L. boulardi parasitization [et al. (2003) reported that venom protein (Vn50) from Cotesia rubecula is homologous to serine protease homologs [S. frugiperda hemocytes and fat body 24 hours after injection of HdIV revealed differential expression of several host genes [S. frugiperda larvae injected with HdIV; however, in the same host injected with MdBV, proPO transcript levels declined [Serine proteases also play a significant role in the activation of the prophenoloxidase (proPO) cascade. In insects, proPO is activated upon injury or invasion, which results in localized melanization of the wound area and/or melanotic capsules capturing invading microorganisms and parasites . In the tization , consisthomologs . It is lst genes . Among tst genes . A recendeclined  which suD. melanogaster larvae parasitized by Asobara tabida or L. boulardi, components of the Toll/Imd (Immune deficiency) pathways were up-regulated, and antimicrobial peptide expression was increased [Drosophila, Toll and Imd pathways are required for activation and stimulation of NF-κBs signal transduction and also responsible for innate immune response in parasitized Drosophila [Transcripts of most proteins, which are involved in the Toll pathway such as Relish, Dorsal, Pelle, Cactus and Toll receptor, were found in our deep sequencing analysis, but only transcription levels of proteins that showed similarity to the Toll receptor were up-regulated  reported that CsIV vankyrin genes also encode proteins with sequence homology to the inhibitory domains of NF-κB transcription factor inhibitors [It has been suggested that PDV-expressed vankyrin proteins may interfere with NF-κB-mediated signalling during immune response and development in parasitized larvae ,50. Fathhibitors .P. xylostella larvae [The results of our transcriptome analysis also indicated that genes known to be involved in insecticide resistance/detoxification are up-regulated following parasitism Table . In agrea larvae . Takeda a larvae .D. semiclausum. However, only a small group affected by parasitoid attack were found to be altered enough to be statistically biologically relevant (i.e. showing greater than two-fold change).Our analyses also detected a considerable number of other immune-related genes whose transcription levels altered after parasitization by P. xylostella larvae parasitized by D. semiclausum, development is arrested at the prepupal stage [Generally, larval endoparasitoids lay their eggs into the host hemocoel, and their progenies develop by consuming host hemolymph and tissues. As a consequence, the parasitoid's larval growth also fully depends on the host's development ,51,52. Ial stage . Developal stage ,54-56. Ial stage -61.D. semiclausum leads to down-regulation of genes associated with ecdysteroid activities; for example, the transcription level of ecdysteroid regulated protein was down-regulated more than 26 times in parasitized larvae  may protect JH from non-specific degradation and adsorption by preventing exposure of JH to epoxide hydration by JH epoxide hydrolase (JHEH), which generates the hormonally inactive JH diol . In agreAs indicated above, previous studies have shown that PDVs may inhibit translation of specific storage or growth-associated proteins despite up-regulation (or steady-state) of transcript levels of the encoding genes, following parasitism or injection of PDVs -71. In tP. xylostella genes were selected from immune- and development-related genes , the infected host larvae (class II) or both (class III) . Among tatabases ,72,74. SDsIV genes were detected in parasitized larvae. In our analysis, 19 unique sequences were identified from five PDV gene families including vankyrin, viral innexin, repeat elements, cysteine-rich motif, and polar residue rich protein families  using a neighbour-joining algorithm, classified BVs and IVs into two major groups Figure . All fouP. xylostella genes, including immune-related genes, upon parasitization by D. semiclausum. In addition, although presence of DsIV particles has been reported in parasitized larvae, our results provide evidence for expression of 19 DsIV genes expressed in the host, which have not been previously reported. Analysis of these sequences indicated the presence of conserved genes that belong to major IV class II genes. The transcriptome profiling data sets obtained in this study provide a basis for future research in this under-explored host-parasitoid interaction. In addition, the identified immune-, development- and detoxification-related genes may be targets for P. xylostella control and allow manipulation of host-parasite interactions.Overall, this study provides the first comprehensive analysis of the impact of a parasitoid wasp on its host at the transcriptomic level, using RNA deep sequencing technique. The results showed differential expression of a large number of P. xylostella and the parasitoid wasp (D. semiclausum) were raised on cabbage plants and host larvae, respectively, at 25°C. Twenty five 3rd and 4th instar P. xylostella larvae each were exposed to wasps until parasitization was observed. Individual larvae that had been attacked by the parasitoid were collected and fed on fresh cabbage leaves. Larval samples were taken at four different time intervals after parasitization  and the samples were kept at -80°C until RNA isolation. The same numbers of mixed larval instars (3rd and 4th) of non-parasitized larvae were collected as the control treatment. It is worth mentioning that P. xylostella larvae parasitized at 3rd instar continue to develop to 4th instar.Total RNA was extracted from all larval samples using Tri-Reagent™ (Molecular Research Center Inc.). RNA extracted from larvae at various time points post-parasitization were pooled and therefore temporal expression data was lost. This was also performed for non-parasitized samples. The pooled RNA sample concentrations were measured using a spectrophotometer and integrity was ensured through analysis on a 1% (w/v) agarose gel. The samples with total concentration of 3.9 and 4.1 μg/μl for parasitized and non-parasitized larvae, respectively, were used for cDNA library production.de novo sequence assembly was used to assemble contigs from a pooling of all the short-read data, using default parameters .The cDNA library was prepared by using 5 μg of starting material for the Illumina mRNA Sequencing Sample preparation procedure (kit RS-930-1001). This involved purification and fragmentation of mRNA, first strand cDNA synthesis, second strand cDNA synthesis, end repair, addition of "A" bases to 3' ends, ligation of adapters, purification of ligated products, and PCR amplification to enrich cDNA templates. The library was validated, quantified and subjected to deep sequencing using a Genome Analyzer IIx Next generation sequencer on a 66 cycle single-end sequencing run, following the supplier's instructions . The GAII analyzer data were output as sequence tags of 65 bases. Sequence.txt files (in FASTQ format) were generated using Illumina Pipeline version 1.5.1. The CLC Genome Workbench (version 4.0.2)  algorithde novo assembly were then used as a reference set of transcripts for RNAseq analysis. Short-read sequence data from parasitized and non-parasitized larvae were separately mapped against the reference set of assembled transcripts using the CLC Genome Workbench RNAseq function . The relative transcript levels were output as RPKM (Reads Per Kilobase of exon model per Million mapped reads) values, which take into account the relative size of the transcripts and only uses the mapped-read datasets (i.e. excludes the non-mapped reads), to determine relative transcript abundance. In this way, the output for each dataset can be directly compared as the number of mapped reads per dataset and transcript size has already been taken into account.The contigs arising from the de novo transcriptome assembly was carried out using Oases 0.1.18 [Reads from parasitized and non-parasitized larvae were cleaned and combined, before s 0.1.18 . The inds 0.1.18 . The aves 0.1.18 . The tra-6 was applied to the National Centre for Biotechnology Information (NCBI) non-redundant protein sequence database, to determine the homology of sequences with known genes. In the absence of P. xylostella and D. semiclausum genome sequences, we discarded annotations that showed similarity to hymenopteran genes and tried to use annotations that showed the highest similarity to lepidopteran genes. Gene ontology and annotation were performed on all assembled contigs greater than 200 bp length by BLAST2GO software http://www.blast2go.org[BLASTX algorithm  with an st2go.org. For genst2go.org. BLAST2Gst2go.org, enzyme D. semiclausum ichnovirus (DsIV) genes, ORFs were predicted and identified by using ORF finder at NCBI http://www.ncbi.nlm.nih.gov/gorf/gorf.html. Predicted ORFs with highest BLASTp E-values in internal comparisons involving other IV genes, were accepted for further analyses.For some of the identified nd instar P. xylostella larvae. For each time point after parasitization, a pool of 10 larvae was used. The RNA samples were extracted from larvae at 16, 24 and 48 hrs after parasitization.Quantitative RT-PCR technique was used on the same RNA samples which were used for transcriptome profiling to verify deep sequencing results using three replicates, each obtained from a pool of 10 larvae. In addition, to observe gene expression levels at different time points after parasitization for a selected group of genes, another experiment was performed by parasitizing 2First strand cDNA was synthesized from 1 μg of RNA using M-MuLV reverse transcriptase (New England BioLabs). The qPCR reaction consisted of 2 μL of diluted cDNA (10 ng), 5 μL of Platinum SYBR Green SuperMix-UDG with ROX (Invitrogen), and 1 μM of each primer (Table KE performed experiments, analysed data and wrote the manuscript. RP, DS, LN analysed data and contributed in writing the manuscript. RVG reviewed the manuscript and provided insect resources. SA conceived the study, analysed data and wrote the manuscript. All authors read and approved the final manuscript.


Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria.The opportunistic enterobacterium, Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes.The 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.This is the first genome sequence of  Morganella morganii is the only species in the genus Morganella, which belongs to the tribe Proteeae of the family Enterobacteriaceae. The other genera in the tribe Proteeae are Proteus and Providencia. Species belonging to Morganella, Proteus, and Providencia are found in the environment and as part of the normal flora of humans. They are also important opportunistic pathogens, which cause a wide variety of nosocomial infections following surgery. Reports of individual cases of infection and nosocomial-outbreaks have revealed that infection with M. morganii can lead to major clinical problems, which are usually associated with common causes of catheter-associated bacteriuria, infections of the urinary and hepatobiliary tracts [M. morganii that were associated with a high mortality rate following bacteraemia sepsis and/or nosocomial infection have also been reported, although most of such infections respond well to appropriate antibiotic therapy [The Gram-negative anaerobic rod y tracts -5, woundy tracts -9. A few therapy ,10-13.M. morganii was formerly classified as Proteus morganii [Morganella on the basis of DNA-DNA hybridisation results [Although morganii , it was  results . Members results .Enterobacteriaceae, M. morganii has a natural resistance to β-lactam antibiotics [M. morganii are resistant to the drugs cefazolin, cefixime, cefpodoxime, and ampicillin [Proteeae, which include Proteus, Providencia and M. morganii share homologous genes acquired from horizontal gene transfer via conjugative integration or mobile transposition [M. morganii was introduced via extra genetic elements [blaCTX-M gene are capable of producing β-lactamases [Like other members of the ibiotics . Many stpicillin ,2,18,19.position -25. The elements ,27 and/oelements ,24. The ctamases , which cctamases .M. morganii, Escherichia coli, P. mirabilis, Providencia stuartii, Klebsiella pneumoniae, and Pseudomonas aeruginosa frequently cause urinary tract infections [P. mirabilis, M. morganii is motile, with peritrichous flagellar. The flagella-encoding genes are located in a contiguous manner in a single locus of the P. mirabilis genome [Proteus-like (MR/P) fimbriae, uroepithelial cell adhesin , type-3 fimbriae, and P. mirabilis fimbriae .Complicated urinary tract infections, especially those associated with long-term catheterisation may be caused by polymicrobes, as well as biofilm formation. In addition to fections ,30,31. Ls genome . Besidess genome -37. TheyThe production of urease has a fitness factor that influences bacterial growth and biofilm formation during urinary tract infections. Other virulence factors may include iron acquisition systems, type-3 secretion system (T3SS), two-component systems (TCS), proteins that function in immune evasion (IgA protease), and haemolysins .The environment found in the guts of nematodes or insects may be an important determinant of bacterial pathogenicity . EthanolP. mirabilis is the closest relative of M. morganii. Only one complete Protues genome sequence and four draft sequences of Providencia spp. are available.Phylogenetic assessment of 16S rRNA sequences indicates that M. morganii and the results of its bioinformatic analysis to enhance understanding of M. morganii biology. Comparative analysis of the sequences with the sequences of other Proteeae and Enterobacteriaceae genomes identifies potential virulence determinants, which may provide new drug targets.Here we report the draft genome sequence of a clinical isolate of morganii and 3,503 (4.23%) were positive for Proteus spp. As shown in Table M. morganii was ranked between the eighth and fourteenth most prevalent Gram-negative bacterial species isolated from the hospital over 12 consecutive 6-month intervals during the 6 years of the study.Over a 6-year period 2006-2011), samples were collected from all patients at Changhua Christian Hospital, Taiwan, who presented with symptoms of Gram-negative bacterial infections. Of 82,861 samples, 1,219 (1.47%) were positive for M. 1, sampleM. morganii was isolated from the blood of a patient who developed sepsis during postoperative care. The KT strain was found to be susceptible to amikacin, ertapenem, gentamicin, meropenem, and cefepime but resistant to ampicillin, amoxicillin-clavulanate, cefazolin, cefuroxime, cefmetazole, flomoxef, and cefotaxime.The KT strain of M. morganii strain KT, which carries no plasmids, was assembled de novo into 58 contigs (each >200 bp long), which together comprised 3,826,919 bp with a GC content of 51.15%. The largest contig is 410,849 bp long, and the N50 statistic (the minimum contig length of at least 50% of the contigs) is 240,446 bp, with pair-end short read sequencing coverage >1,150-fold. Seven small contigs (each <13 kb) had low-depth reads (0.36- to 0.66-fold), whereas two pairs and one triad shared high sequence identity with minor differences at their ends  and mioC (MM01686) .As shown in Table http://www.cbs.dtu.dk/services/RNAmmer/) . Among them, 2,870 CDSs could be placed into clusters of orthologous groups with assigned biological functions Figure . The proM. morganii genome, 2 prophages (MM3229 through MM3271 and MM2276 through MM2290) and 12 degenerate prophages together comprise 236 prophage-related genes  and two tRNA PheV sites , are present in the strain KT genome.Neither the integrated conjugative element ICEirabilis ) nor ICEirabilis ) was fouM. morganii has been cloned [ampC (MM3167) and ampR (MM3166) genes, which are flanked by the hybF and orf-1 genes [ampC and ampR genes produce both broad-spectrum and the ampC β-lactamases or only the ampC β-lactamase [Providencia spp., the production of metallo-β-lactamases has been associated with resistance to carbapenems [M. morganii KT strain also contains the gene encoding β-lactamase, which confers resistance to aminopenicillins. Genes that encode three metallo-β-lactamases  were found. Moreover, the M. morganii genome also encodes the tetracycline resistant protein TetAJ (MM3521) and the chloramphenicol acetyltransferase CatA2 (MM3053).A chromosomally encoded β-lactamase from n cloned . The cli-1 genes . The ampactamase . In Provbapenems ,44. The bcr (MM2780), a catB3-like putative acetyltransferase gene (MM0469), and a kasugamycin resistance gene ksgA (MM2040). The tellurite resistance ter operon was also identified in P. mirabilis HI4320, P. stuartii ATCC# 25827, K. pneumoniae NTUH K2044, E. coli O157:H7, and E. coli APEC O1. The genes involved in multidrug efflux and the CDSs predicted to confer antimicrobial resistance are listed in Additional file Other drug resistance genes include a tellurite resistance operon (MM0993 through MM1004), the bicyclomycin (sulphonamide) resistance gene mrp operon immediately adjacent to mrpJ was designated mrp'  and hofB (MM3191) genes, four genes, namely ppdA (MM2057), ppdB (MM2058), ppdC (MM2060), and ppdD (MM3192), are involved in the assembly of type-IV pili. Two genes that encode putative trimeric autotransporter adhesins (MM2011 and MM2242) are homologues of proteins found in P. stuartii ATCC# 25827 and Y. enterocolitica 8081.Together with the cheA, cheW, cheD, tap, cheR, cheB, cheY, and cheZ) are contained in the M. morganii genome. With the exception of the genes that encode two LysR family transcriptional regulators (MM1739 and 1765), the camphor resistance protein CrcB and related genes (MM1743 through MM1749), a short-chain dehydrogenase/reductase SDR (MM1764), and the three insecticidal toxin complex proteins encoded by xptA1A1C1 (MM1780 through MM1782), the organisation of flagellar genes is similar to that of P. mirabilis.Fifty three genes that encode proteins required for flagellar structure  and eight chemotaxis-related genes , umoB (MM3381), umoC (MM1924), umoD (MM2830), rssBA (MM2256 and MM2257), and the rcsBCD phosphorelay regulatory system (MM1705 through MM1707), were also identified.The genes that flank the locus that encodes flagellar structural proteins encode a methyl-accepting chemotaxis protein (MM0264) and an aerotaxis protein (MM1607). Genes that encode regulators of flagellar motility, such as M. morganii genome encodes the hmuSTUV haeme uptake system (MM0575 through MM0578), the afuABC system for ferric and ferrous iron transport (MM1068 through MM1070), feoAB (MM2728 and MM2729), the ferric siderophore receptor locus ireA (MM3404), the B12 transporter locus btuCD (MM0560 and MM0561), btuB (MM2756), and the siderophore iron uptake systems yfeDCBA (MM0546 through MM0549) to circumvent host iron-sequestering mechanisms. A possible haeme-binding system is encoded by MM1650 and MM1651. A gene that encodes a putative iron receptor (MM1952) is located adjacent to a fecR homologue (MM1953). There are two other iron-related ABC transporters  and 12 additional TonB-dependent receptors .The znuACB high-affinity zinc transporter system was recently shown to be a fitness factor for E. coli and P. mirabilis during experimental urinary tract infection [M. morganii gene products MM2146 through MM2148.The nfection ,47. The P. mirabilis [P. mirabilis  was also found. The IpaC and IpaD proteins have low similarity  to those of P. mirabilis, whereas the IpaB protein has the highest homology with ipaB of P. mirabilis but low similarity (20-30%) with that of Shigella spp. and Salmonella enterica subsp.A T3SS, which comprises gene products MM0224 through MM0247 and has a low GC-content (43.7%), resides in a 20.8-kb pathogenicity island. This island of 24 genes, which shares homologous syntenic blocks with irabilis , containqseBC , yedWV , and BarA/UhpA of the LuxR family . Nineteen orthologues that encode TCS components were identified in P. mirabilis HI4320 [P. alcalifaciens NCTC 10286; r04, P. rettgeri DSM# 1131, P. rustigianii DSM# 4541, Enterobacter aerogenes KCTC 2190, Photorhabdus luminescens laumondii TTO1, and Y. enterocolitica 8081.Nineteen potential TCS were identified. These include the quorum sensors M. morganii genome identified seven sigma factor subunits of RNA polymerase. These are the major sigma factor rpoD  encoded by MM1372, and six alternative sigma factors: rpoH  encoded by MM1254, rpoN  encoded by MM1306 to control promoters for nitrogen assimilation, rpoS  encoded by MM1208 to activate stationary-phase promoters, rpoE  encoded by MM1906 to regulate extra-cytoplasmic stresses, rpoF  encoded by MM1736 to regulate flagellum-related functions, and a FecR family sigma factor encoded by MM1954.Analysis of the ryhB found in E. coli and P. mirabilis [The small RNA regulatory gene irabilis , which rzapA gene encodes a zinc metalloprotease that cleaves a broad range of host proteins, including serum and secretory IgA1, IgA2 and IgG, complement proteins, and antimicrobial peptides [M. morganii KT, P. mirabilis HI4320, and P. luminescens laumondii TTO1 genomes all encode zapABCD , the P. mirabilis HI4320 genome encodes zapEEEABCD [The peptides . WhereasM. morganii have been investigated [rcsB (MM1706), rcsC (MM1707), rcsD (MM1705), and rcsF (MM2111), are implicated in the regulation of capsule synthesis.Lipopolysaccharide (LPS), the main structural component of the outer membranes of Gram-negative bacteria, consists of a lipid A molecule and a variable O-antigen. Lipid A released during bacterial lysis induces endotoxic shock. Several O-specific polysaccharides of stigated ,51, and stigated . The genstigated  are listM. morganii genome with that of the other 14 members of the Enterobacteriaceae analysed revealed orthologues in the pathogens P. stuartii ATCC# 25827, E. coli UTI89, and E. coli APEC O1, all of which have been implicated in causing urinary tract infections [Clusters of regularly interspaced short palindromic repeats consist of multiple short nucleotide repeats, which are separated by unique spacer sequences flanked by characteristic sets of CRISPR-associated genes ,55. The fections .hmpA, encoding a secreted haemolysin, was originally identified in uropathogenic isolates [hmpBA, encoding secreted proteins that are highly conserved in P. mirabilis [M. morganii KT. Functional similarity of the E. coli and M. morganii homologues of HmpBA was reported previously [The gene isolates ,58. The irabilis , are enceviously .Proteeae organisms [M. morganii, which revealed a high degree of amino acid conservation to P. mirabilis urease, has been purified and characterised [M. morganii and P. mirabilis urease gene cluster are required for virulence [ureABCEFGD were the orthologues from Yersinia pseudotuberculosis and Y. enterocolitica, with amino acid identities that ranged from 69% to 94%. Unlike the urease gene cluster of P. mirabilis, the M. morganii genome has a gene that encodes the transporter MM1968, in addition to MM1961 to MM1967. On the other hand, the P. mirabilis ureR gene, which encodes a transcriptional activator, was not found in the genome.Rapid urea hydrolysis is a prominent phenotype of rganisms . The urerganisms . The urecterised . Ureasesirulence . The anaM. morganii KT contains genes for two pathways involved in putrescine production. The first of these pathways involves ornithine decarboxylase, which is encoded by speF (MM3013), and the putrescine transporter, which is encoded by potE (MM3012). The enzymes in the second pathway, which are encoded by carAB (MM2009 and MM2010), argI (MM0127), argG (MM1552), argH (MM1551), speA (MM2553), and speB (MM2554), also participate in urea production. All of the urease cluster genes that encode enzymes from both pathways have orthologues in P. mirabilis HI4320 [The genome of XaxAB genes (MM0454 and MM0455) that encode apoptotic toxins, a putative intimin gene for host-cell invasion (MM0208), a HlyD-family toxin scretion protein (MM2481), and a transporter gene (MM2482). Among the nine insecticidal toxin-related genes  are orthologous to the insecticidal toxin genes of Pseudomonas spp. The three other insecticidal toxin genes xptA1A1C1 (MM1780 through MM1782) were identified in a continuous locus between the flagellum-related genes and chemotaxis genes.Several CDSs that encode potential toxins were found. These include a gene that encodes the cytotoxin RtxA MM0676), the two 6, the tweut operon it were found to vary substantially between species [eutSPQTDMNEJGHABCLK-pduST-eutR (MM1148 through MM1130). This region carries two extra genes compared to the 17 eut genes found in other Enterobacteriaceae genomes [pduST located between eutK and eutR. As shown in Table The ethanolamine utilisation system, which is encoded by the  species , is comp genomes . In addiKlebsiella pneumonia, E. coli, and Salmonella enterica serovars. The pdu operon, a paralogous operon required for use of propanediol that is found in these three species, was not found in the M. morganii KT genome.The comparison revealed that the 17-gene operon is present in eut operon, MM1168 through MM1187 encode the enzymes of the cob-cbi operon, which is required for cobalamin (vitamin B12) biosynthesis [Located upstream of the ynthesis -67. UndekatA, soxS, sodC, sodB, oxyR, and sodA.Pathogenic bacteria produce superoxide dismutase to protect them from being killed by the reactive oxygen species generated by their hosts . As showpspABC operon (MM0627 through MM0629) encodes a phage-shock-protein (psp), which ensures the survival of E. coli during late-stationary-phase stresses [The stresses -71.M. morganii KT and the 14 members of the Enterobacteriaceae family, including 5 Proteeae species , 4 E. coli strains , K. pneumonia NTUH K2044, Enterobacter aerogenes KCTC 2190, Photorhabdus luminescens laumondii TTO1, S. enterica serovars Typhimurium LT2, and Y. enterocolitica 8081, revealed that 459 CDSs found in M. morganii are not found in the other Proteeae species studied, and 295 CDSs found in M. morganii are not found in any of the 14 Enterobacteriaceae genomes studied , motility, iron acquisition, ethanolamine use, and urease production, as well as components of an immunity-like system (CRISPR) and fimbrial adhesins, toxins, and haemolysin.Among the orthologous genes, 2,411 CDSs were found in the genomes of the 5 M. morganii is frequently isolated from clinical specimens collected from patients with nosocomial bacterial infections [M. morganii that confirm the chromosomal origin of the plasmid-located cephalosporinases [M. morganii that encode cefotaxime-hydrolysing β-lactamases have also been reported with increasing frequency [M. morganii [M. morganii. In the event of an outbreak, this situation poses a potential threat owing to the absence of a proper antibiotic therapy.Epidemiological studies have revealed that fections ,11,72-74orinases  are now orinases . Genes frequency . Plasmidmorganii ,27. GeneProteeae, which have GC contents that range from 39% to 43%, the GC content of M. morganii is 51%. which is genetically different from other species [Morganella [Morganella [Compared with other members of the genus  species ,77 and trganella ,15,61,76rganella ,79. Howerganella ,81.P. mirabilis genome has seven rRNA operons (six 16S-23S-5S operons and one 16S-23S-5S-5S operon) [M. morganii KT genome sequence revealed eight duplications of the 16S and 23S rRNA genes while 5S rRNA were also 8. The prophage genes that were found in the other Enterobacteriaceae species appeared to comprise 7% of the M. morganii genome.Information on the 16S rRNA gene and paralogs in genome is important for evolution and bacterial population studies . The P.  operon) . AnalysiPm1 and ICE/R391 genes, which are present in many P. mirabilis isolates [Proteeae genomes, which implies that different transposition events occurred during the evolution of M. morganii and of these species.Interestingly, the ICEisolates , are notP. mirabilis, M. morganii KT has LysR family transcriptional regulatory genes (MM1739 and MM1765), genes MM1743 through MM1749 that encode the camphor resistance gene CrcB and related proteins, a short-chain dehydrogenase/reductase SDR gene (MM1764), and the insecticidal toxin complex genes xptA1A1C1 (MM1780 through MM1782).Unlike the flagellar genes of M. morganii KT and P. mirabilis have duplicated MR/P fimbrial operons, albeit with different numbers of genes [M. morganii KT, only one copy is in the P. mirabilis genome. In M. morganii KT, the mrp' operon is oriented opposite to mrpI, whereas mrpI is not present in the P. mirabilis mrp' operon.Both of genes . As showM. morganii and P. mirabilis. However, genes that encode components of T3SSs have low sequence similarities.More than half of the pathogenicity island genes are conserved and found to show collinear synteny between Proteus spp. and E. coli clinical strains but are not produced by Providencia spp. and Morganella spp. [zapABCD genes found in M. morganii KT differ from those in P. mirabilis HI4320 zapEEEABCD [zapE gene downstream of zapABCD. Although the M. morganii urease shares a high degree of sequence similarity to P. mirabilis ureases, the presence of the unique transporter gene (MM1968) and the absence of ureR in M. morganii suggest that the two species use different systems to regulate urease transport.EDTA-sensitive protease Zap gene clusters have been found in many lla spp. . The zapP. mirabilis, some were found in Xenorhabdus [Pseudomonas [Yersinia, and Photorhabdus [Photorhabdus, the related toxin complex is released upon invasion of the nematode host [M. morganii clinical isolate harbours eight insecticidal toxin genes remains to be investigated.Although none of the insecticidal toxin genes were found in orhabdus ,83, Pseuudomonas , Yersiniorhabdus . In Photode host . Why theM. morganii [P. mirabilis HI4320 [M. morganii has fewer types of fimbriae (three vs. five) and fewer gene clusters (8 vs. 17). This implies that M. morganii may be less virulent than P. mirabilis in the context of urinary tract infections.The urinary and hepatobiliary tracts are two major portals of entry for morganii . Compareeut operon likely plays a critical role in enabling M. morganii, E. coli, Klebsiella spp., and Yersinia spp. to use ethanolamine as a source of carbon and/or nitrogen to colonise the intestine. However, the other Proteeae genomes lack the eut operon and pduTS, which together encode proteins that help to establish a microcompartment [cob-cbi operon, which encodes the enzymes needed for cobalamin biosynthesis.Given that the intestine is thought to provide a rich source of ethanolamine , the eutpartment , and theeut operon, which includes pduST, and the cob-cbi operon likely provide the fitness factors required for colonisation by intestinal bacteria. This may explain why M. morganii is more frequently associated with nosocomial bacterial infections than other members of Proteeae.The Margonella may be considered as the new drug targets.Typically antibiotics target the essential cellular function, like cell-wall synthesis, ribosomal function, or DNA replication . The whoM. morganii genome encode drug resistance determinants and factors that influence virulence, such as fimbrial adhesins, flagellar structural proteins, components of the iron acquisition system, T3SS, and TCS, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins. Comparative analysis with 14 other Enterobacteriaceae genomes revealed several systems that vary between species. These include transposes of the IS4 family, insecticidal toxins, T3SS components, and proteins required for ethanolamine utilisation and cobalamin biosynthesis. It is interesting to note that neither the eut operon (which includes pduST) nor the cob-cbi operon is found in other Proteeae genomes. Nevertheless, the eut operon is also found in several other non-Proteeae enterobacteria genomes, albeit with different gene organisation.The pathogenicity-related genes identified in the M. morganii genome sequence. Comparative genome analysis revealed several pathogenicity-related genes and genes not found in other Proteeae members. The presence of the eut operon (which includes pduST) and the cob-cbi operon in M. morganii but not in the other Proteeae genomes studied may explain why M. morganii is more frequently associated with nosocomial bacterial infections. Moreover, the evidence that M. morganii shares features with other non-Proteeae enterobacteria suggests that horizontal gene transfer has occurred between M. morganii and other intestinal bacteria.In summary, this is the first report of an M. morganii strain KT (year 2009) from blood of a 57-year-old man during postoperative care. The patient had a medical history of type 2 diabetes mellitus, hepatocellular carcinoma, rectal cancer and gallbladder stone. He was admitted for rectal cancer surgery, and received chemotherapy and radiotherapy. Following surgery, bacteraemia caused sepsis.With approval by the institutional biosafety committee, we isolated M. morganii as the agent responsible for the infection. A presumptive diagnosis of infection with M. morganii was confirmed using API-20 kit reagents  and the BACTEC NR-860 apparatus .Colony morphology, Gram staining, oxidase testing , catalase testing, and routine biochemical reactions identified M. morganii in trypticase soy broth or on trypticase soy agar. Bacteriological media were purchased from Biostar Inc. (Taiwan).We cultured M. morganii strain KT was isolated using reagents from QIAGEN DNeasy kits .Genome DNA from the Genome sequencing was performed using the whole genome shotgun strategy . GenomicShort sequencing reads were assembled into contigs using ABySS version 1.2.7 software . ContigsM. morganii was analysed using our own integrated annotation pipeline composed of prediction and database search tools. Glimmer (version 3.02) was used to predict assembled contig sequences for prokaryotic CDS regions [The draft genome of  regions . PotentiWe used BLAST to query non-redundant protein databases with all predicted CDS regions  and therdnaA gene and DnaA boxes of the genome, using Ori-Finder [We used tRNAscan-SE to predict prokaryotic transfer RNA (tRNA) genes . Ribosomi-Finder . HorizonEnterobacteriaceae genomes were downloaded from the National Center for Biotechnology Information and draft genome and protein sequences (four Providencia spp.) of Enterobacteriaceae genomes were downloaded from the Genome Institute at Washington University.The previously published genome and protein sequences of Enterobacteriaceae organisms as databases for comparison, validate orthologous protein with E value (E < 1e-4), identity (> 30%) and threshold to length percentage of alignment.We used BLAST program and formatted proteins of other eutK-pduS-pduT-eutR (MM1133-MM1130) . PCRs were carried out in 25-μl reactions containing 30 ng of genomic DNA, 1 × buffer, 250μM deoxynucleoside triphosphate, 0.75 μl of Taq DNA polymerase, and 0.4 μM concentrations of forward and reverse primers. Reactions were amplified in a thermocycler at 95°C for 2 min, followed by 30 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 60 s. Purified PCR products were sequenced (ABI model 3730).PCR was conducted using primer flanking ORFs: The plasmid DNA was isolated by the procedure of alkaline denature method  and thenM. morganii KT has been deposited in the DDBJ/EMBL/GenBank under the accession number ALJX00000000. The version described in this paper is the first version, ALJX01000000.The draft genome sequence of Information regarding bacteria responsible for clinically relevant infections was collected over a 6-year period for all patients diagnosed with infection by the Infectious Control Committee of Changhua Christian Hospital, a 1,600-bed medical center in central Taiwan. The reports identified bacteria isolated from samples and their sensitivities to the antibiotics amikacin, ampicillin, amoxicillin-clavulanate, cefepime, cefazolin, cefuroxime, cefmetazole/flomoxef, cefotaxime, ertapenem, gentamicin and meropenem.The authors declare that they have no competing interests.THK, YTC, CYT, WCS and HLP helped design the research project. YTC carried out computational work, conducted sequence analysis and interpreted the results, and drafted the manuscript. THK wrote parts of the manuscript related to clinical analyses. WCS, FRH and MFH assisted with assembly work. HLP and FRH assisted with experiments related to biochemistry and molecular biology. CHC, YMT, CEL and HCC isolated, identified, and cultured bacteria. THK, HLP and CYT refined the manuscript. All authors read and approved the final manuscript.Supplementary Figure 1. The origin of replication was assigned based on the GC deviation of the genome using Ori-Finder (*.pdf)Click here for fileSupplementary table 1. Resequencing analysis on assembled contigs (*.xls)Click here for fileSupplementary table 2M. morganii genes involved in multidrug efflux genes (*.pdf). Click here for fileSupplementary table 3M. morganii (*.pdf). Flagellum-related genes and chemotaxis genes located in 58.8-kb locus of Click here for fileSupplementary table 4M. morganii (*.pdf). Protein similarity search of Type III secretion system (T3SS) of Click here for fileSupplementary table 5M. morganii genes involved in lipopolysaccharide or enterobacterial common antigen biosynthesis (*.pdf). Click here for fileSupplementary table 6M. morganii (*.pdf). Protein similarity search of insecticidal toxin of Click here for fileM. morganii genes involved in superoxide stress (*.pdf)Supplementary table 7. Click here for fileSupplementary Table 8. 2 prophages and 12 degenerate prophages (*.xls)Click here for fileSupplementary Table 9. Specific CDSs in M. morganii compared to other Enterobacteriaceae (n = 14) and/or Proteeae (n = 5). (*.xls)Click here for fileSupplementary Table 10. Comparison of CDSs in M. morganii compared to other Enterobacteriaceae (n = 14) and Proteeae (n = 5). (*.xls)Click here for file