Milnesium tardigradum which has extraordinary tolerance to desiccation and freezing. In this study, we present the first overview of the transcriptome of M. tardigradum and its response to desiccation and discuss potential parallels to stress responses in other organisms.The phenomenon of desiccation tolerance, also called anhydrobiosis, involves the ability of an organism to survive the loss of almost all cellular water without sustaining irreversible damage. Although there are several physiological, morphological and ecological studies on tardigrades, only limited DNA sequence information is available. Therefore, we explored the transcriptome in the active and anhydrobiotic state of the tardigrade M. tardigradum in its active and inactive, anhydrobiotic (tun) stage. Assembly of these ESTs resulted in 3283 putative unique transcripts, whereof ~50% showed significant sequence similarity to known genes. The resulting unigenes were functionally annotated using the Gene Ontology (GO) vocabulary. A GO term enrichment analysis revealed several GOs that were significantly underrepresented in the inactive stage. Furthermore we compared the putative unigenes of M. tardigradum with ESTs from two other eutardigrade species that are available from public sequence databases, namely Richtersius coronifer and Hypsibius dujardini. The processed sequences of the three tardigrade species revealed similar functional content and the M. tardigradum dataset contained additional sequences from tardigrades not present in the other two.We sequenced a total of 9984 expressed sequence tags (ESTs) from two cDNA libraries from the eutardigrade M. tardigradum, which significantly contributes to the available tardigrade sequence data and will help to establish this extraordinary tardigrade as a model for studying anhydrobiosis. Functional comparison of active and anhydrobiotic tardigrades revealed a differential distribution of Gene Ontology terms associated with chromatin structure and the translation machinery, which are underrepresented in the inactive animals. These findings imply a widespread metabolic response of the animals on dehydration. The collective tardigrade transcriptome data will serve as a reference for further studies and support the identification and characterization of genes involved in the anhydrobiotic response.This study describes novel sequence data from the tardigrade  Polypedilum vanderplanki [Echiniscus testudo Doyère 1840 [Milnesium tardigradum Doyère 1840 which shows remarkable resistance to adverse environmental conditions in all stages of life [M. tardigradum outperforms several other tardigrade species in tolerance e.g. survival of extreme temperatures above 100°C [Hypsibius dujardini Doyère 1840 [Daub et al. Unpublished data 2003] and Richtersius coronifer (Richters 1903) [H. dujardini have been focused mainly on developmental and evolutionary biology [M. tardigradum from active and inactive (anhydrobiotic/tun) stages, thereby establishing M. tardigradum as a model for anhydrobiosis research. These ESTs and the resulting unigenes were functionally annotated using Gene Ontology vocabulary. Furthermore, a cross-species comparison between M. tardigradum, H. dujardini and R. coronifer has been performed.Desiccation tolerance or anhydrobiosis is the ability of an organism to survive almost complete drying without sustaining damage. Anhydrobiosis is observed in certain micro-organisms, plants and animals such as rotifers, brine shrimp cysts, tardigrades and insect larvae, as for example those of the erplanki -3. Studyerplanki . In the erplanki -8. The hère 1840 . Anhydroère 1840 ,2,12, asère 1840 -16. We a of life  - even t of life . M. tardve 100°C  as well ve 100°C ,22. Simive 100°C ,24 suggeve 100°C . They have 100°C -30, onlyve 100°C . Howeverrs 1903) . Studies biology -38. In tM. tardigradum and subjected them to single pass Sanger sequencing. Furthermore we retrieved two EST datasets from public sequence databases  using M. tardigradum unigenes against the NCBI database showed that nearly 50% of the ESTs had no corresponding entry in GenBank. All ESTs were deposited in GenBank . FABPs have a low molecular mass and bind with high affinity to hydrophobic ligands such as saturated and unsaturated long-chain fatty acids. Various functions have been proposed for FABPs such as the uptake, transport, and delivery of fatty acids to beta-oxidation ,44. FABPdormancy . The preM. tardigradum are Kazal-type serine proteinase inhibitor and Cystatin B. Overexpression of Cystatin B (an intracellular cysteine proteinase inhibitor) in transgenic yeast and Arabidopsis showed an increase in the resistance to high salt, drought, oxidative, and cold stresses [To date, little is known about the possible mechanisms of proteolytic inhibition or suppression in anhydrobiotic organisms. Protease inhibitors are candidate genes which would offer protection against protein degradation during anhydrobiosis. Among the abundant protease inhibitors transcripts in inactive stages of stresses . Elevatestresses . The abuPlectus murrayi [Cryptococcus neoformans [Cytochrome c oxidase subunit I (COXI) is a mitochondrial gene that encodes the cytochrome c oxidase subunit I, a crucial enzyme involved in oxidative phosphorylation and thus energy production. COXI was over threefold more represented in the inactive state. Transcripts encoding COXI were also abundantly expressed during dehydration stress in the antarctic nematode  murrayi  and up roformans . The mitThe sequences, which could not be assigned any function based on homology search in NCBI, were searched for conserved domains in ProDom  and SwisM. tardigradum, R. coronifer and H. dujardini), transcripts which are potentially associated with desiccation tolerance during anhydrobiosis in other organisms were identified  or downregulated (AQP2 and AQP4) following desiccation [Many organisms adapt to desiccation stress by the activation of various water-channel proteins, called aquaporins (AQP) ,59. Datanditions . Similariccation . The uprR. coronifer, a lower level of Hsp70 protein was found in desiccated animals when compared with active ones [M. tardigradum, one isoform of the hsp70 tarnscripts showed up-regulation during the transition from active to the inactive state [hsp70 isoforms are downregulated and seem not to be directly involved in anhydrobiosis. Using the same model M. tardigradum, Reuner et al. [In the four tardigrade datasets we have identified some putative heat shock protein (HSP) encoding genes. HSPs are highly conserved throughout evolution and they function as molecular chaperones and play primary roles in protein biosynthesis and folding . In tardive ones . In M. tve state ,65, whilAphelenchus avenae under dehydration condition [P. vanderplanki by water stress imposed by either desiccation or hypersalinity [Megaphorura arctica [H. dujardini library (the less tolerant tardigrade) and also in the proteome map of M. tardigardum [Much attention was recently paid on the chaperone-like LEA (late embryogenesis abundant) proteins in anhydrobiotic animals ,67. LEA ondition -72. A sisalinity . Recentl arctica . In the digardum . These dThe translationally controlled tumor protein (TCTP) found in all tardigrada datasets is often designated as a stress-related protein because of its highly regulated expression in stress conditions and its close relation to a family of small chaperone proteins . ImportaArtemia franciscana [Aphelenchus avenae [P. vanderplanki [tps) have not been found in rotifer genomes [Adorybiotus coronifer [M. tardigardum [tps in M. tardigradum ESTs. Nevertheless, transcripts coding for trehalases have been described in M. tardigradum [M. tardigradum other strategies are probably employed.Trehalose, which accumulates in many anhydrobiotic organisms during desiccation is proposed to act as a common water replacement molecules and stabilizer of biological structures -80. The nciscana , in the s avenae  and in terplanki . Howevererplanki ,84. In a genomes . Althougoronifer , it was digardum  and we cdigradum  but we pH. dujardini, considered as one of the smallest tardigrade genomes [http://www.genomesize.com. Our dataset adds a substantial part towards the complete gene content in the tardigrada species.The datasets analysed in this study represent most of the available transcriptome data from tardigrades, and until now there is little information on tardigrade genome and transcriptome structure. The genome sizes range from very compact genomes, ~75 Mb for  genomes , up to 8M. tardigradum, H. dujardini and R. coronifer) we searched for putative orthologous sequences across all three datasets. Using a TBLASTX search with an e-value threshold of 10-5 we compared the M. tardigradum unigenes against the other two datasets. The BLAST bit-score of each top-scoring hit was extracted and M. tardigradum sequences that exhibited sequence similarity against at least one other tardigrade species are presented as a clustered heatmap in Figure M. tardigradum unigenes show similarities against both other species with some hits only present in either one of them (N = 785). A higher coverage of M. tardigradum transcripts can be seen in the H. dujardini dataset compared to R. coronifer which is likely due to the smaller R. coronifer dataset. This cross species comparison implies that the remaining 2498 unigenes contained in the M. tardigradum dataset represent further yet unknown tardigrade transcripts and expands the known tardigrade sequence data. These might be very interesting for studying the evolutionary relationships of protein families.To investigate the complementation of the three tardigrade datasets . These contained mainly abundant transcripts e.g. ribosomal proteins, ADP-ribosylation factor, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase and heat shock proteins. The resulting average transcript similarity for M. tardigradum against H. dujardini was 147.66 +/- 88.27 and M. tardigradum against R. coronifer 150.95 +/- 93.74. This is reflected in the phylogenetic distance calculated using 18S rRNA sequences  were placed in 1.5 ml Eppendorf tube and desiccated at room-temperature at 85% relative humidity (RH) for 12 to 16 hours (till they have completed the tun formation) and then at 35% RH for further 48 hours. The animals were frozen at -80°C until their experimental use.ublished . For allprotocol . BrieflySfiI restriction enzyme, products smaller than 300 bp were removed using the Chroma Spin-400 column as described in the Creator SMART™ protocol and cloned into pDNR-Lib cloning vector. This procedure was chosen because of the low amount of starting material. Plasmids were transferred via electroporation to Escherichia coli .Total RNA extraction was performed by following the instructions of QIAGEN RNeasy R Mini kit . Complete lysis of the tardigrades and especially disruption of their harsh cuticle was achieved by sonication on ice for 1 min (duty cycle 0.5s) by using a microsonicator . For cDNA synthesis 1 μg total RNA was reverse transcribed using the Creator™ SMART™ cDNA Library Construction Kit following the manufacturers recommendations . The resulting first strand cDNA was amplified by LD-PCR for 18 cycles according to the manufacturers protocol using the 5' PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3') as the forward and the CDSIII/3'PCR Primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3') as reverse primer. The amplified PCR products were then analyzed by agarose gel electrophoresis. After digestion of the amplified cDNA with the In total, 9984 cDNA clones were either picked by hand or automatically using a QPix robot  into 384 well LB-agar culture plates containing chloramphenicol. Sequencing was mostly from the 5' end using standard M13 forward sequencing primer. The sequencing of the cDNA library was sequenced on a ABI 3730XL capillary sequencer by GATC Biotech AG .M. tardigradum) and FASTA sequences (H. dujardini and R. coronifer) files are accepted as entry point to the analysis. Base calling was performed with phred [The EST analysis pipeline Figure  includesth phred ,88 usingth phred . Repetitth phred . Sequencth phred .TIGR Gene Indices clustering tools (TGICL) with standard parameters  and CAP3M. tardigradum datasets was performed using the GOSSIP statistical framework [M. tardigradum datasets.Identification of GO terms differentially enriched between the active and inactive ramework  webserviBM established and optimized the tardigrade RNA extraction protocol and constructed and managed the cDNA clone libraries, MG performed functional annotation and enrichment analysis, putative orthologue prediction and gave useful comments on sequence analysis, MF was responsible for oversight, budget, obtaining the funding for the project, and contributing advice at each step of the research. FF performed quality control, processing and assembly of ESTs and was involved in data analysis, TD contributed to the bioinformatic analysis. WW performed the phylogenetic analysis, RS provided the animals and coordinated the project and contributed comments on candidate anhydrobiotic genes, MS and DR supported the identification of anhydrobiotic genes. BM and MG wrote the main part of the manuscript. All authors read and approved the final manuscript.M. tardigradum ESTs and their GenBank accession numbersList of . This file provides a list of dbEST ID, User ID and GenBank accession numbers of all M. tardigradum ESTs.Click here for fileM. tardigradum datasetsGO-enrichment analysis statistics of . This file contains details about the GO-enrichment analysis between the active and inactive stage of M. tardigradum using GOSSIP. A list of enriched ESTs is provided.Click here for fileM. tardigradum datasetPutative anhydrobiotic transcripts identified in . This file provides a list of ESTs in active and inactive stages of M. tardigradum that are potentially associated with desiccation tolerance.Click here for fileH. dujardini datasetPutative anhydrobiotic transcripts identified in . This file provides a list of H. dujardini sequences that are potentially associated with desiccation tolerance.Click here for fileR. coronifer datasetPutative anhydrobiotic transcripts identified in . This file provides a list of R. coronifer sequences that are potentially associated with desiccation tolerance.Click here for fileM. tardigradum against H. dujardini and R. coroniferPutative orthologous sequences of . This file provides a list of the putative orthologues shared by tardigrade species investigated in this study.Click here for filePhylogenetic tree based on tardigrade 18S rRNA sequences. Displays a phylogenetic tree constructed from E. testudo, M. tardigradum, R. coronifer and H. dujardini 18S rRNA sequences.Click here for file


Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation.Phylogenetic analysis of the type 3 fimbrial genes (The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI. Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and a frequent cause of bacteremia . NosocomBiofilm formation is a trait commonly found among CAUTI isolates and results in the growth of bacteria on the inner surface of the urinary catheter. Biofilm formation promotes encrustation and protects the bacteria from the hydrodynamic forces of urine flow, host defenses and antibiotics . A perquThe expression of type 3 fimbriae has been described from many Gram-negative pathogens -28. TypemrkABCDF) arranged in the same transcriptional orientation [mrk gene cluster is similar to other fimbrial operons of the chaperone-usher class in that it contains genes encoding major (mrkA) and minor (mrkF) subunit proteins as well as chaperone- (mrkB), usher- (mrkC) and adhesin- (mrkD) encoding genes [mrkE) located upstream of mrkA has been described previously in Klebsiella pneumoniae [mrk genes have been shown to reside at multiple genomic locations, including the chromosome [mrk-containing conjugative plasmid to strains of Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes and Kluyvera species has also been demonstrated [mrk genes between Gram-negative pathogens by lateral gene transfer.Type 3 fimbriae belong to the chaperone-usher class of fimbriae and are encoded by five genes  but rare in uropathogenic E. coli and Citrobacter freundii strains  [mrk) genes from 33 CAUTI strains representing five different uropathogens . We also demonstrate functional expression of type 3 fimbriae in each of these strains and describe a common role for type 3 fimbriae in biofilm formation.Recently, we identified and characterised the role of type 3 fimbriae in biofilm formation from an th CAUTI . We alsoctively) . Despitemrk genes from 33 CAUTI strains  we amplified and sequenced an internal segment of the mrkA, mrkB, mrkC and mrkD genes from each strain. We also examined the corresponding sequence from six additional mrk gene clusters available at GenBank. A majority-rule consensus maximum likelihood (ML) tree was constructed from the 39 concatenated mrkABCD fragments. The phylogenetic analysis indicated that the sequences clustered into five major clades (referred to as clade A to E) with good bootstrap support  to 23 members , with an average inter-allelic diversity of 11.2%. Whereas the 10 C. koseri sequences clustered in a single clade (clade E), clade B (3 sequences) and clade A (23 sequences) consist of sequences from both K. pneumoniae and E. coli. Phylogenetic analysis using parsimony or distance-based methods produced tree topologies very similar to those obtained by using DNA maximum likelihood (data not shown).To investigate the phylogenetic relationship of the ort Fig. . The fivmrk consensus tree and the established phylogeny for enteric bacteria [prima facie evidence for lateral gene transfer (LGT) of mrk alleles. All K. pneumoniae chromosomal alleles cluster in Clade A, along with several plasmid-borne or chromosomal alleles from E. coli. In some cases, the K. pneumoniae and E. coli alleles are identical . Similarly, in clade B, two identical E. coli mrkABCD sequences (M184 and ECOR28) share high nucleotide sequence identity (98%) to the plasmid-borne K. pneumoniae pIA565 mrkABCD. The mrkABCD concatenated nucleotide sequences of K. pneumoniae pIA565 (clade B) and K. pneumoniae MGH78578 (clade A) share only 78.2% nucleotide sequence identity. When analysed individually, the mrkA, mrkB, mrkC and mrkD gene fragment alignments produced essentially the same tree topology as the concatenated sequence (data not shown) with little variation in within-group diversity , and at the 5' end of clusters from ECOR28 (clade B) and C. freundii M46 (clade C), indicative of recent lateral gene transfer. Downstream of mrkF, a conserved 717 bp gene was present in five of the strains, including one from each of the five defined clades. This gene  encodes a central EAL domain  suggesting that it may have a role in signalling, however, no close homologs have been functionally characterised. PCR primers designed from these sequences demonstrated that this region was also conserved in 24 other strains examined (data not shown). Notably, cko_00966 homologs were not encoded downstream of the plasmid-borne mrk clusters in E. coli pMAS2027 and pOLA52, and there is no corresponding sequence information available for this region in pIA565 [mrkE regulatory gene originally identified in pIA565 [We compared the udy Fig. . As expen pIA565 . The putn pIA565  was not mrk-positive strains examined in this study mediated mannose-resistant hemagglutination of tannic acid treated human RBC (MR/K agglutination), indicating they produced type 3 fimbriae. To specifically demonstrate a direct association between MR/K agglutination and type 3 fimbriae, the mrk locus was deleted from thirteen strains  employing λ-red mediated homologous recombination. The strains were selected on the basis of their transformation efficiency and included at least one representative from each of the mrk phylogenetic clades. Several different assays were employed to compare the thirteen sets of wild-type and mrk deletion strains. First, SDS-PAGE analysis of crude cell lysates and subsequent Western blotting was performed using a type 3 fimbriae-specific antiserum. A predominant 15 kDa band representing the MrkA major subunit was detected from all wild-type strains except C. freundii M46, which failed to react positively in this assay. In contrast, no reaction was observed for any of the mrk deletion mutants  under dynamic culture conditions. Strong biofilm growth was observed from all wild-type strains except C. freundii M46. In contrast, deletion of the mrk gene cluster caused a significant reduction in biofilm growth (p < 0.0001) in all strains except E. coli M184  genes from 33 CAUTI isolates representing C. freundii, C. koseri, E. coli, K. oxytoca and K. pneumoniae cluster into five well-supported clades on the basis of nucleotide sequence. Type 3 fimbriae were expressed by all of these strains as indicated by their positive MR/K agglutination. Type 3 fimbrial expression was also associated with biofilm growth in the majority of these strains. This is the first report describing the distinct grouping of type 3 fimbrial genes into phylogenetic clades at the species level, with strong evidence supporting inter-species lateral gene transfer. We also demonstrate the functional expression of type 3 fimbriae by strains of C. koseri and C. freundii.Type 3 fimbriae are adhesive organelles produced by a range of Gram-negative pathogens that cause CAUTI. Here we show that type 3 fimbriae  which were strongly supported by long internal branches. The majority of the sequences grouped in clade A, which is represented by the chromosomal mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. Clades A and B contained mrk gene clusters from K. pneumoniae  and E. coli (plasmid origin). Two mrk loci have been fully sequenced from E. coli; in both cases the mrk genes are located on a conjugative plasmid  and flanked by transposon-like sequences [mrk genes in the additional seven E. coli strains identified in this study remains to be determined, the data presented here and in previous studies strongly suggests inter-genera lateral gene transfer of the mrk cluster [C. koseri sequences, while clades C and D are represented by a unique sequence from C freundii and K. oxytoca, respectively. The presence of cko_00966 homologs downstream of representative mrk clusters in all 5 clades strongly suggests that the ancestral mrkABCD locus was also encoded next to a cko_00966 homolog and that the clades are largely related by linear descent. Notably, the relationship determined here is not congruent with the known evolutionary relationship of Klebsiella, Citrobacter, and E. coli [K. pneumoniae lineage, with mrk regions laterally transferred to E. coli (e.g. pMAS2027 and pOLA52) and clade E represents the C. koseri lineage. Clades B, C and D, which contain mrk sequences from K. pneumoniae, E. coli, C. freundii and K. oxytoca, are clearly under-represented and additional type 3 fimbrial gene sequences are required to confirm the groupings.Phylogenetic analysis with individual and concatenated equences ,40. Whil cluster ,28. In c E. coli , supportmrkD exhibited the highest inter-group diversity  collection [The strains and plasmids used in this study are described in Table llection . Cells wllection ,48.Taq polymerase according to the manufacturer's instructions . DNA sequencing was performed by the Australian Equine Genome Research Centre. Deletion mutants were constructed essentially as previously described using either pKD46 [C. freundii and C. koseri strains were heated at 42°C for 2 min prior to electroporation. Primers used to generate deletion mutants were as follows: 1293 and 1294 (E. coli MS2027), 1456 and 1457 (E. coli ECOR15 and K. pneumoniae strains), 1458 and 1459 (E. coli ECOR28), 1456 and 1459 (E. coli M184), 1460 and 1459 (K. oxytoca strains), 1456 and 1461 (C. koseri M546), 1462 and 1459 (C. freundii M46)  or standard PCR employing primers designed from the genome sequenced K. pneumoniae MGH78578 or C. koseri ATCC BAA895 . Restriction endonucleases were used according to the manufacturer's specifications . Chromosomal DNA was purified as previously described . PCR waser pKD46  or pKOBEer pKD46 ,51, withBacterial agglutination of tannic acid treated human erythrocytes (MR/K agglutination) was performed as previously described to detect the expression of Type 3 fimbriae . BacteriBiofilm formation on polyvinyl chloride (PVC) surfaces was monitored by using 96-well microtitre plates  essentially as previously described . BrieflyCrude cell lysates were prepared from overnight cultures and boiled in acid as previously described . ProteinmrkA (416 bp), mrkB (243 bp), mrkC (657 bp) and mrkD (778), respectively, from each of the 33 CAUTI strains and sequenced on both strands. These sequences correspond to nucleotides 112 to 530 of mrkA, 66 to 308 of mrkB, 173 to 829 of mrkC and 157 to 934 of mrkD in the reference strain K. pneumoniae MGH78578 (CP000647). Individual and concatenated gene fragments from the 33 CAUTI strains  were aligned using ClustalX [mrkD from E. coli M202 and only used the mrkABC concatenated fragments in the analysis. For comparative analysis, the complete mrk cluster (and adjacent regions) from E. coli ECOR28, C. freundii M46 and K. oxytoca M126 were amplified using an inverse PCR strategy and sequenced.PCR products were generated from an internal region of ClustalX . MaximumClustalX . A conseClustalX . We weremrk mutant strains were analysed using the ANOVA single factor test .Differences in biofilm formation between wild-type and FJ96754, FJ96756-FJ96774, and FJ96777-FJ96789 (for mrkA), FJ96793, FJ96795-FJ96811, FJ96813-FJ96814, and FJ96817-FJ96829 (for mrkC) and FJ96832, FJ96834-FJ96849, FJ96851-FJ96852, and FJ96855-FJ96867 (for mrkD). The mrkB sequences were described previously [mrk cluster (and adjacent regions) from E. coli ECOR28, C. freundii M46 and K. oxytoca M126 were deposited in GenBank under accession numbers FJ96870, FJ96871 and FJ96872, respectively.Gene fragments were deposited in GenBank under the accession numbers: eviously . The comE. coli isolates collected as part of routine methods for the diagnosis of UTI and no additional procedures on patients were involved, individual informed consent was not obtained.Approval for this study was obtained from the Princess Alexandra Hospital Human Research Ethics Committee (2005/098). Since the study used CYO carried out the majority of the experimental work under the supervision of AGM and MAS. SAB performed the bioinformatic analysis and contributed to writing the manuscript. MT performed the immunogold labelled electron microscopy and contributed to writing the manuscript. CF contributed to the construction of mutants and writing of the manuscript. AGM contributed to the design of experiments and writing of the manuscript. MAS conceived the study and wrote the manuscript. All authors read and approved the final manuscript.


A 3-year-old boy presented with iso-sexual precocious puberty for 18 months. Radiological images revealed left suprarenal mass and hormonal profile showed markedly increased testosterone and dehydroepiandrosterone-sulfate. The child underwent open adrenalectomy and histopathology revealed adrenocortical carcinoma. At 3 months follow-up, the child is doing well. Adrenocortical neoplasm has bimodal age distribution, with its peak in the first and fifth decades. Adrenocortical neoplasms in children are uncommon, comprising 6% of all adrenal neoplasms and 0.3% of all pediatric neoplasms ViriliziA 3-year-old boy presented with enlargement of the penis and pubic hair for 18 months and deepening of voice and axillary hair for 6 months. On examination, the child was otherwise healthy and normotensive Pubic hairs, axillary hairs and hairs in the beard area and upper lip were present. Stretched penile length was 9.8 cm. Bilateral testes were normally descended and normal in size. There were no skin spots and no evidence of features of Cushing syndrome. No lump was palpable on abdominal examination. Spine and digital rectal examinations were unremarkable.His hemoglobin was 14.0 g% and blood biochemistry reports were normal, except serum alkaline phosphatase which was 939 U/l. Serum electrolytes were Na: 137 meq/l and K: 4.2 meq/l. Chest X-ray was normal. Hormone profile of the patient is summarized in Ultrasonography of the abdomen revealed left adrenal solid mass with few speculated calcifications. Contrast enhanced computed tomography (CT)-whole abdomen  showed aThe child underwent left open adrenalectomy via subcostal transperitoneal approach. The postoperative period remained uneventful.Histopathology revealed a well-circumscribed, encapsulated nodular tumor (6.7 cm) in its greatest axis, weighing 125g. The cut-section showed a variegated pattern, with soft and friable intratumoral nodules with areas of hemorrhage and necrosis . MicroscAdrenocortical carcinomas with mixed virilizing and Cushing’s syndrome are typical, but pure virilizing adrenocortical carcinomas are uncommon, comprising 5–10% of the cases in most large series. ViriliziOur patient had markedly increased serum testosterone and serum DHEA-S. High concentrations of adrenal androgens, especially DHEA-S, are highly suggestive of a virilizing tumor and contrast-enhanced CT-abdomen localizes the adrenal mass.Prognosis for benign adrenal tumors is excellent. On the other hand, usual prognosis for adrenocortical carcinoma is generally poor, with an overall 5-year survival of 20–25%. Factors associated with poor prognosis, specifically for virilizing tumors, include incomplete resection, weight >80 g, volume >200 ccm, age >3.5 years at diagnosis, preoperative symptom duration >6 months and marked increase in urinary 17-ketosteroid and 17-hydroxysteroids. Pure virilizing carcinomas in general appear to have a better prognosis than other adrenal carcinomas. In our cet al. utilized the modified criteria of Weiss to analyze pediatric adrenocortical tumors.[Bugg l tumors. This claRegarding staging, in our case, the tumor was completely resected with a negative margin, weighed 125 g and there was no evidence of metastasis. The abnormal hormone levels returned to normal after surgery and thus our case was labelled as stage 1, high-grade adrenocortical carcinoma.Hence, it is concluded that a child with virilizing symptoms should be promptly investigated for biochemical profile and radiological imaging. Surgical resection is the treatment of choice. Surgery is the only definitive therapeutic modality in children, which can be curative only if adrenocortical carcinoma is diagnosed early and treated promptly.Adjuvant chemotherapy is used for metastatic or recurrent disease, but paucity of cases makes its evaluation difficult. The role


Streptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood.S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome.We have sequenced the complete genome of S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.These observations suggest that  Streptococcus agalactiae strains will increases as hundreds of genomes are sequenced [S. pyogenes belongs to the same genus, it has a smaller pan-genome and greater level of recombination in its core genome [The genomic heterogeneity within a bacterial species reflects its lifestyle, the niche it occupies, and its exposure to mobile elements, such as bacteriophages and plasmids . Even thequenced . Althouge genome . These oS. mutans [S. mutans has been classified into four serotypes  based on the chemical composition of its cell surface rhamnose-glucose polymers [S. mutans is a diverse population [c strains were widely distributed in the dendrogram, while serotype e, f, and k strains were differentiated into clonal complexes. This suggests that serotype c, the dominant serotype among S. mutans clinical isolates , is the ancestral phenotype of this organism and that serotype e and f strains have evolved strain-specific genes. Although differences in modification of cell surface polymers reflect evolutionary trends, differences in cariogenicity have not been observed, and the relationship between serotype and clinical condition remains unclear.It has long been recognized that serological, genetic, and biochemical variations exist within the species . mutans . S. mutapolymers . We prevpulation . In the S. mutans genes have revealed sequence variations, resulting in altered function of the encoded proteins [msm, bgl, cel, and gftBC loci [msm, gbl, cel, and gftBC, which they called "dispensable genes," distributed among S. mutans strains [S. mutans UA159 open reading frames (ORFs) were absent from one or more of the nine test strains, and dispensable ORF blocks (including more than one ORF) were identified by microarray analysis based on the UA159 genome [S. mutans, it seems likely that other strains of S. mutans have both unique and common genetic loci not present on the UA159 genome [S. mutans evolutionary history. However, these analyses are based on only one genome, S. mutans UA159 even though extensive genomic variation between S. mutans strains has been predicted [Studies of individual proteins -18. For proteins ,20, and proteins , serotypproteins , competeproteins ,24, and tBC loci -28. Wate strains . They al9 genome . This isredicted .Helicobacter pylori [Mycobacterium species [Chlamydia species [Escherichia coli [Salmonella species [Streptococcus species include important human and agricultural pathogens [Streptococcus sp. belonging to nine different species including S. pyogenes, S. pneumoniae, S. agalactiae, S. thermophilus, S. suis, S. sanguinis, S. gordonii, S. equi, and S. mutans are publicly available. These organisms colonize diverse habitats including tooth, oral mucosal, pharyngeal, respiratory, intestinal, and urinogenital surfaces. These species have acquired various genes for a specific niche mainly by lateral gene transfer. For example, S. pyogenes acquires or tolerates bacteriophages that are important for new virulence determinants and that induce genomic rearrangement [S. agalactiae, the main cause of neonatal infection in humans, also tolerates bacteriophages [Genome sequence data are now available for numerous species of bacteria and comparative evolutionary approaches show positive selection pressure and lateral gene transfer in the evolution of many bacterial species. These analyses have been performed for pathogenic bacteria such as r pylori ,32, Myco species , Chlamyd species , Escherihia coli , and Sal species . The patathogens ,37. Moreangement . S. agaliophages . Some ofiophages ,40. MultS. mutans serotype c strain NN2025 isolated from Japan in 2002, and we compared the genome sequence, genome structure, and gene variation with the genomes of serotype c UA159 strain isolated in 1982 from the United States, and with 95 clinical isolates from Japan and Finland, and other closely related streptococcal genera to provide useful information about the evolutionary events associated with S. mutans strains and Streptococcus sp., and to provide new insights into streptococcal species-specific survival strategies.In this context, we determined the whole genome sequence of an dnaA gene, similar to S. mutans UA159 and other genomic sequences [AP010655).The genome of NN2025 is a single, circular chromosome of 2,013,597 bp  to be common to UA159. S. mutans strains NN2025 and UA159 are classified into the same serological group c, and show the same biochemical properties , adhesive properties to glass surfaces, and cariogenic properties in rat infection models (data not shown). Waterhouse et al. reported that 80% of the S. mutans ORFs are conserved among ten strains with different serotypes by microarray hybridization based on the UA159 genome, suggesting that 80% of ORFs are conserved within S. mutans strains [S. mutans is more stable than that of other Streptococcus species, where the core genome represents only about 60% of the genome [S. mutans [S. mutans is suggested to be capable of metabolizing a wider variety of carbohydrates than many other Gram-positive organisms that have been sequenced. S. mutans resides in the oral cavity, and this varied sugar metabolism is an important survival strategy for this organism. S. mutans is predicted to possess at least five sugar ABC transport systems and at least 14 sugar PTS systems, and can use at least 16 sugars for glycolysis. Nine PTSs were confirmed to be transcribed in the presence of 13 different sugars [S. mutans including adhesins, glucan-producing and binding exoenzymes, were conserved. Adhesins of S. mutans are also important for protecting the bacterium against possible host defenses and for maintaining its ecological niche in the oral cavity. Comparative genome analysis revealed that the major S. mutans surface receptors SpaP (SMU.610 and SmuNN2025.1372)  and wall-associated antigen A (WapA) (SMU.987 and SmuNN2025.1036) are completely conserved. An uncharacterized wall-associated protein, WapE (SMU.1091 and SmuNN2025.0945), and a dextranase precursor  [S. mutans-specific genes within the sequenced Streptococcus species (data not shown), suggesting that these might be important for colonization.NN2025 contains 1,895 ORFs, of which 1,724 (90%) are predicted by reciprocal BLAST search analysis  represent the landmarks  in chromosomes . An LCB The NN2025 genome contains eight strain-specific regions  and is thought to be important for bacterial growth, but genetic variation is found within this region. Region 2 in NN2025 includes two transposase fragments, five hypothetical proteins, methyltransferase (SmuNN2025_0077), gapC (SmuNN2025_0079), transcriptional regulator gene (SmuNN2025_0082), and luxS (SmuNN2025_0080). luxS plays an important role in the production of autoinducer-2 (AI-2) in many bacterial species, and 30% of the genes of S. mutans are affected by luxS [Of the 25 regions that are unique between the NN2025 and UA159 genomes, eight regions  exist in both strains, but the contents of the ORFs are highly diverse. These regions are designated "variable regions". Region 1 contains genes of the purine nucleotide biosynthesis pathway  identifies new strain-specific content that could not be identified from only one genome. Based on their correspondence with the 'genomic islands', Region 1, 5, 8, 11, 12 and 15 appear to be horizontally transferred. The gene order of Region 15 in UA159 is different from that in NN2025, which may suggest the existence of an unknown mechanism to reorder the genomic island  genomic island, along with 29 ORFs predicted to be involved in biosynthesis and secretion of the antimicrobial peptide, bacitracin Figure . However and GS5 ,28. The  and GS5 , whose tBacillus licheniformis and B. subtilis. Its primary mode of action is to bind to undecaprenyl pyrophosphate (UPP) in the presence of a bound divalent metal cation, sequestering UPP and preventing its interaction with phosphatase [IS861 fragment (SmuNN2025_1645)  production genes  [scnM and scnT of S. pyogenes, respectively, but these two genes were not found and an apparent fragmentation of the polyketide genes has been shown in UA159 [mutIII cluster might be active in this strain, and that S. mutans has acquired peptide antibiotic synthetase genes to exclude other bacteria, perhaps for niche acquisition.The bacitracin synthesis gene cluster in TnSmu2 did not exist in most of the clinical strains, and two other bacitracin or polypeptide antibiotic synthesis gene clusters were found in NN2025 Figure . Bacitrasphatase . This insphatase ,51. Regi) Figure . Smu2025) Figure . Region ) Figure . S. mutasistance . The exiTable 1) . SmuNN20in UA159 ,30. AlthStreptococcus, three distinct CRISPR loci have been identified within the S. thermophilus chromosome; namely, CRISPR-1, CRISPR-2 and CRISPR-3 [S. thermophilus strains has revealed polymorphisms [S. thermophilus [Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25–50 bp repeats separated by unique sequence spacers of similar length ,55. It iCRISPR-3 . CRISPR-CRISPR-3 ,58. Compmophilus .S. pyogenes is an important source for new virulence factors as well as for the genomic rearrangement of the prophage regions [S. mutans have not previously described, because phage donor and acceptor strains are quite rare in S. mutans. Based on in silico and in vivo analyses, several hypotheses have been put forward proposing roles for CRISPR and cas genes, including providing immunity against foreign genetic elements via a mechanism based on RNA interference [cas and cse genes (SmuNN2025_0603-0610) in Lactobacillus casei   in NN2025. Of these 18 spacer sequences, six are identical to the sequence of the recently identified S. mutans phage M102 . The CRS. mutans. This CRISPR-2 locus consists of four cas and csn genes (SmuNN2025_0694-0696) and two hypothetical proteins . Though the CRISPR subtype (Smut1 of Sthe3 family) in NN2025 is the same as in UA159, with the gene order, the lengths of the repeat sequences are quite different not only in these two strains but also among clinical isolates, ranging from 2 to 5 kb (data not shown). To determine the spacer variability in the clinical isolates, the CRISPR-2 loci of an additional six strains were sequenced for comparison  with the lowest repeat number in any tested strains. This is the first example of a repeat length other than from 36 bp in this Sthe3 family. However, the CRISPR-2 locus gene order was completely conserved among strains. The number of the spacers is quite divergent among strains, ranging from 3 to 69  is found in both UA159 and NN2025, and has been designated as CRISPR-2 (or Smut1b ) in S. ms Figure , and theure csn2 . This is9 Figure . We next2 Figure . Of the S. thermophilus. The presence of a CRISPR spacer identical to a phage sequence adjacent to cas provides resistance against phages containing that particular sequence. Limited information is available regarding the ecological role of bacteriophages in the oral cavity, although bacteriophages have been isolated from human saliva or dental plaque [S. mutans and S. sobrinus [Streptococcus with high variation in the CRISPR repeat number), and their sequence similarity to the lytic phage, suggest that S. mutans might resist phage infection via CRISPR. This observation is in contrast with the phage-permissive S. pyogenes, because S. pyogenes possesses two to eight prophage regions within its genome (average 4.5 per genome), and the number of CRISPR-associated direct repeats and spacers is less than that of other streptococcal species , while that in the other eight S. pyogenes strains carry the same cas gene set is 3.5 . The number of prophages in an S. pyogenes genome may be affected by the CRISPR gene set present, and further examination is needed, especially considering that the many sequenced genomes of streptococci carrying prophages have rarely demonstrated natural transformation [Streptococcus, specific species may have evolved to function in a particular way to acquire foreign genes via natural transformation or bacteriophages, and the acquisition of new foreign genes via phage infection may not be favorable for the lifestyles of S. mutans in the oral cavity. Elucidation of the mechanism by which S. mutans acquires new genes will be interesting to clarify further the species-specific evolutionary strategies in the streptococci.The biological significance of CRISPR against phage infection has recently been reported in l plaque ,67. Onlysobrinus . Therefoormation . TherefoEscherichia coli [Pyrococcus horikoshii and P. abyssi [Chlamydia pneumoniae and C. trachomatis [S. pyogenes [The physical structure of the bacterial genome is highly conserved during evolution in hia coli ,70. Howe. abyssi , and betchomatis . Interespyogenes ,72. Suchori region in NN2025 are found within the 16S rRNA, tRNA-Ala, 23S rRNA, tRNA regions, and the comX1 homologue in both regions (data not shown). The genome of S. mutans NN2025 shows that the rearrangement breakpoint is located 86 bp downstream from the 23S rRNA, and this rearrangement breakpoint is conserved within the other S. mutans strains isolated in Taiwan in which Huang et al. firstly observed chromosomal inversion in 25/58 strains [recA-dependent recombination between two ribosomal operons [Salmonella typhimurium, inversion between large inverted repeats (>5 kb) separated by large intervals (>60 kb) has been shown to be recA- and recB-dependent [S. typhimurium and S. paratyphi A [The recombination sites near the  strains . These o operons . In Gramependent . In addiatyphi A . Our seqori region occurs between two replication forks and single- or double-stranded DNA breaks [rrn-comX regions were found equidistant from the ori region in the S. mutans genomes; thus, this model is in good agreement with the genomic rearrangement mechanism of S. mutans and S. pyogenes. This sequence specificity may affect site-specific recombination during homologous recombination.Although the role of homologous recombination in genomic rearrangements across the replication axis has not been elucidated, Tillier and Collins proposed an alternative model for the observed pattern of rearrangement . Gene trA breaks . In factS. mutans NN2025 and UA159 revealed extensive genomic rearrangements  [dif-like termination sequence (ter) [ori region  were almost completely conserved between NN2025 and UA159, and the other homologous genes were translocated to an inverted position on the chromosome. These chromosomal segments are symmetrical except for small gaps encoding the genes of strain-specific regions  ,78 and dce (ter)  were conce (ter) . The locS. pyogenes Manfredo and S. pyogenes SSI-1, there is a large central inversion (~1.3 Mb), which probably resulted from reciprocal recombination between rrn-comX regions that are a similar distance from the terminus of replication [S. mutans strains, therefore, could provide evidence of both intraspecies and interspecies genomic rearrangement in streptococci.For lication , and thelication . The comStreptococcus genomes from six different species were reported by Lefébure and Stanhope [S. mutans species-specific gene groups and, because limited information is available regarding overall symmetry between the genomes of various streptococcus species, on the genome-wide rearrangement within nine species (32 genomes). To identify sets of gene groups shared between two S. mutans strains or unique to each species/strain, the predicted ORFs from the 32 streptococcal strains were clustered into groups using reciprocal BLASTP analysis. The set of shared gene groups (core genome) for each species was determined to compare the numbers of species/strain-specific gene groups and those shared with S. mutans . Eighty-one percent of gene groups were shared between the two S. mutans strains. Comparison of the 1069 groups constituting the core genome of S. mutans with other bacterial species/strains showed that 49.9% to 66.3% of gene groups are conserved between S. mutans and other streptococcal genomes , the mutans group (S. mutans), the mitis group (57.4–68.3% of gene groups shared with S. mutans), the salivarius group (61.2% of gene groups shared with S. mutans), and others (51.7% of gene groups shared with S. mutans) [Extensive comparative genomic analyses of positive selection, recombination, and the genome composition of 26 Stanhope . In this mutans) . This leS. agalactiae has a larger pan-genome and less recombination than S. pyogenes as evidenced by the species having a larger habitat and gene pool diversity. This is supported by the fact that the pan-genomes of ecologically restricted organisms are smaller than those of bacteria that adopt a more generalist lifestyle [S. mutans may have a smaller pan-genome and more recombination than S. agalactiae because it is usually isolated only from dental caries, the analyses here concentrated on the S. mutans species-specific regions as discussed, comparing NN2025 and UA159 to understand the species . In addition, long-PCR analysis of the dissimilar regions in the genome reveals the genomic diversity of the species, as well as the results of MLST analysis previously reported by Nakano et al[As hypothesized by Lefébure and Stanhope , S. agalifestyle . Althougano et al  are further classified into another subgroup within the non-pyogenic streptococci [S. mutans NN2025 were identified as common to oral streptococci but not to other streptococci . The ori and ter regions of these two species are highly conserved, and there are fewer short LCBs (0–25 kb) than in other streptococcal strains. This suggests that there are fewer genome rearrangements between S. mutans and S. pyogenes than between other streptococcal species. We hypothesize that these two species occupy similar ecological niches or smaller habitats and gene pools than S. agalactiae  into one replichore may cause an unstable genome structure, and this unbalanced genome structure might induce chromosomal inversion between highly conserved regions [Salmonella genomes in culture has provided evidence that genomes with equally sized replichores  may be under positive selective pressure [Lactococcus lactis [rrn2 region of UA159. These regions are not found in NN2025. In addition, the similarity in genome structure between S. mutans and S. pyogenes suggests two possibilities regarding the chromosomal inversion in S. mutans. One option is that chromosomal rearrangement of S. mutans NN2025 and UA159 happened by chance. No characteristic differences of serotype specificity, geographical distribution, STs, or topological pattern from long-PCR analysis  was purchased from the American Type Culture Collection. Other clinical isolates and laboratory strains have been isolated at Osaka University and Helsinki University. These strains were selected from our culture collection and all of the strains were confirmed to be S. mutans based on conventional physiological tests, including rough colony morphology on mitis-salivarius agar , bacitracin resistance, and fermentation of sorbitol, mannitol, raffinose, or melibiose (1% each) in a phenol red broth base (Difco). Bacterial genomic DNA was extracted using DNAeasy kit according to the manufacturer's instruction .rrn) were amplified from chromosomal DNA using LA-Taq (Takara), sequenced, and assembled independently, as described above.The initial stage of sequencing was performed using whole genome random shotgun methods with sheared chromosomal DNA from strain NN2025. We constructed a pUC18-based library containing 1–2 kb and 4–5 kb inserts, and sequenced 48,000 clones (12.6-fold coverage) with Big-Dye terminator chemistry and ABI 3730 × l sequencer  and with ET-Dye terminator chemistry and MegaBACE 4500 sequencer . The sequence was assembled using Phred/Phrap/Consed -99. Gapssee BLAST classification of Figure ORFs >60 bp were identified and annotated separately using Metagenome gambler light , in silico Molecular Cloning Genomics Edition    and GLIMS. mutans UA159 (AE014133), S. pyogenes strains , S. pneumoniae strains , S. agalactiae strains , S. sanguinis SK36 (CP000387), S. suis strains , S. thermophilus strains , S. gordonii Challis CH1 (CP000725), and S. equi zooepidemicus MGCS10565 (CP001129) were obtained through the website of the National Center of Biological Information (NCBI) [-5 in the reciprocal BLATP analysis and the extant common cluster set (core genome) of each species was determined to compare the numbers of species-specific gene groups and those shared with S. mutans.The thirty-one genomic sequences of n (NCBI) . Alignmen (NCBI) ,114, or n (NCBI) , followin (NCBI) . SPRING n (NCBI) ,118 All S. mutans NN2025 genome, the detection of CRISPR loci was carried out using CRISPRFinder web service [Analysis of CRISPR loci was carried out based on the method of Horvath et al.  with sli service ,121 or t service  databaseS. mutans strains were examined by the LA-PCR method  using site-specific primer pairs .Click here for fileS. mutans NN2025Characteristics of CRISPR loci found in . The nomenclature, leader sequence, repeat sequence, number of repeats, and similarity were determined based on the method of Horvath et al. [see Methods for details).h et al.  with sliClick here for fileS. mutans NN2025Sequence similarities of the existing CRISPR spacers in . The spacer similarities were determined by BLASTN against viruses including bacteriophage or bacteria databases (see Methods). No description in "origin" or "BLAST E-value" indicates that no similarity was found in the database.Click here for fileStreptococcusDistribution of CRISPR-2 -associated repeat sequences in genus . Repeat sequences in the CRISPR-2 homologous region (Sthe3 family) were found in 19 of 32 streptococcal genomes .Click here for fileStreptococcusDistribution of CRISPR-2 (Smut2b)-associated repeat sequences in genus . Repeat sequences in the CRISPR-2 homologous region  found in streptococcal genomes except S. mutans NN2025 . Similarities were examined by BLASTN against each genome as a target database. The number of repeats was determined as described by Horvath et al. [see Methods).h et al.  .Click here for fileSpecific gene groups shared in oral streptococci. All the predicted ORFs from the 32 streptococcal strains were clustered into groups based on a threshold of maximum E-value = 10-5 in the reciprocal BLATP analysis to extract the ORF(s) of gene groups specific for oral streptococci . The predicted function is assigned based on the COG classification (see Methods for details).Click here for fileS. mutans (N2025 and UA159) and three strains of S. pyogenes Comparison of genomic shuffling between two strains of . A MAUVE representation of the total 64 local collinear blocks (LCBs) between chromosomal sequences of the S. mutans strains and S. pyogenes strains, at a minimum weight of 144. The S. mutans NN2025 DNA sequence given on the forward strand is the reference against which the sequence of the NN2205 was aligned and compared. LCBs placed under the vertical bars represent the reverse complement of the reference DNA sequence. LCBs placed under the vertical bars represent the reverse complement of the reference DNA sequence. The 64 connecting lines between genomes identify the locations of each orthologous LCB in the two genomes. Unmatched regions within an LCB indicate the presence of strain-specific sequence. Each sequential block represents a homologous backbone DNA sequence without rearrangements.Click here for fileS. mutans with six S. pneumoniae strains based on the chromosomal organization of the strain NN2025 or UA159Genome comparison of each . Dot plots of S. mutans NN2025 vs. six S. pneumoniae strains and of S. mutans UA159 vs the same set of S. pneumoniae strains are presented, as generated by PROmer of MUMmer software and visualized with GenomeMatcher software [see Methods).software .software  .Click here for fileS. mutans with two S. agalactiae strains, S. sanguinis SK36, S. gordonii Challis CH11, S. equi zooepidemicus MGCS10565 based on the chromosomal organization of the strain NN2025 or UA159Genome comparison of each . Dot plots of S. mutans NN2025 vs. two S. agalactiae strains, S. sanguinis SK36, S. gordonii Challis CH11, and S. equi zooepidemicus MGCS10565 and of S. mutans UA159 vs. the same set of streptococcal strains are presented, as generated by PROmer of MUMmer software and were visualized with the GenomeMatcher software (see Methods).Click here for fileStreptococcal speciesLengths of locally collinear blocks (LCBs) shared by the nine . Block lengths are taken from the S. mutans NN2025 genome. Lengths of LCBs were determined using SPRING software.Click here for file


Triatoma, includes species of high public health interest. Triatoma dimidiata, the main vector throughout Central America and up to Ecuador, presents extensive phenotypic, genotypic, and behavioral diversity in sylvatic, peridomestic and domestic habitats, and non-domiciliated populations acting as reinfestation sources. DNA sequence analyses, phylogenetic reconstruction methods, and genetic variation approaches are combined to investigate the haplotype profiling, genetic polymorphism, phylogeography, and evolutionary trends of T. dimidiata and its closest relatives within Triatoma. This is the largest interpopulational analysis performed on a triatomine species so far.Among Chagas disease triatomine vectors, the largest genus, Triatoma dimidiata populations follow different evolutionary divergences in which geographical isolation appears to have had an important influence. A southern Mexican–northern Guatemalan ancestral form gave rise to two main clades. One clade remained confined to the Yucatan peninsula and northern parts of Chiapas State, Guatemala, and Honduras, with extant descendants deserving specific status. Within the second clade, extant subspecies diversity was shaped by adaptive radiation derived from Guatemalan ancestral populations. Central American populations correspond to subspecies T. d. dimidiata. A southern spread into Panama and Colombia gave the T. d. capitata forms, and a northwestern spread rising from Guatemala into Mexico gave the T. d. maculipennis forms. Triatoma hegneri appears as a subspecific insular form.Triatomines from Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil were used. Triatoma species allows us to reach highly supported conclusions not only about T. dimidiata, but also on different, important Triatoma species groupings and their evolution. The very large intraspecific genetic variability found in T. dimidiata sensu lato has never been detected in a triatomine species before. The distinction between the five different taxa furnishes a new frame for future analyses of the different vector transmission capacities and epidemiological characteristics of Chagas disease. Results indicate that T. dimidiata will offer problems for control, although dwelling insecticide spraying might be successful against introduced populations in Ecuador.The comparison with very numerous  Triatoma includes important vector species, as T. dimidiata in Central and Meso-America. DNA sequences, phylogenetic methods and genetic variation analyses are combined in a large interpopulational approach to investigate T. dimidiata and its closest relatives within Triatoma. The phylogeography of Triatoma indicates two colonization lineages northward and southward of the Panama isthmus during ancient periods, with T. dimidiata presenting a large genetic variability related to evolutionary divergences from a Mexican-Guatemalan origin. One clade remained confined to Yucatan, Chiapas, Guatemala and Honduras, with extant descendants deserving species status: T. sp. aff. dimidiata. The second clade gave rise to four subspecies: T. d. dimidiata in Guatemala and Mexico (Chiapas) up to Honduras, Nicaragua, Providencia island, and introduced into Ecuador; T. d. capitata in Panama and Colombia; T. d. maculipennis in Mexico and Guatemala; and T. d. hegneri in Cozumel island. This taxa distinction may facilitate the understanding of the diversity of vectors formerly included under T. dimidiata, their different transmission capacities and the disease epidemiology. Triatoma dimidiata will offer more problems for control than T. infestans in Uruguay, Chile and Brazil, although populations in Ecuador are appropriate targets for insecticide-spraying.Chagas disease is a serious parasitic disease of Latin America. Human contamination in poor rural or periurban areas is mainly attributed to haematophagous triatomine insects.  Trypanosoma cruzi represent 6–8 million fewer cases than those reported in the 1980s American trypanosomiasis or Chagas disease is widespread in Latin America from Mexico to Chile and southern Argentina. Although present estimates of 10 to 12 million people infected with the haemoflagellate protozoan species Triatoma, among which two main clades appear in ribosomal DNA (rDNA) sequence phylogenies, corresponding to species of North and Central America and species of South America separated prior to the closing of the isthmus of Panama about 3 million years ago Triatoma species are distributed in three main groupings: the Rubrofasciata group (mainly North American and Old World species), the Phyllosoma group (mainly Mesoamerican and Caribbean), and the Infestans group (mainly South American), each including different complexes and subcomplexes in a classification which is progressively updated according to new genetic and morphometric data Chagas disease vectors are haematophagous reduviid (Hemiptera: Heteroptera) insects belonging to the subfamily Triatominae. Species of Triatominae are usually grouped into 17 genera forming five tribes, although other arrangements have been proposed. Of these, Alberproseniini, Bolboderini, Cavernicolini and Rhodniini are considered monophyletic, whereas Triatomini is considered polyphyletic T. cruzi. Among the species of greatest epidemiological significance as domestic vectors, three belong to the genus Triatoma: T. infestans and T. brasiliensis from South America, and T. dimidiata, distributed in Meso- and Central America from Mexico down to Colombia, Venezuela, Ecuador and northern Peru A priori, all of the over 130 species currently recognized within Triatominae seem capable of transmitting Triatoma dimidiata can be found in sylvatic, peridomestic and domestic habitats. Non-domiciliated populations may act as reinfestation sources and become involved in the transmission of the parasite to humans T. dimidiata populations studied. This study even revealed differences consistent with a specific status for populations from the Yucatan peninsula, Mexico T. dimidiata may represent a complex of cryptic species  T. dimidiata, to get a new framework able to facilitate the future understanding of the diferring peculiarities of this crucial vector species throughout its broad geographical distribution. This may also help in understanding the related differences in characteristics of Chagas disease transmission and epidemiology, as well as in responses to control initiatives in the countries concerned. After a deep analysis, it was considered that the most convenient approach would be obtained by using an appropriate marker able to furnish significant information about evolutionary trends of variation on which to construct the new baseline. This new baseline should be, whenever possible, of sufficient weight as to allow its conclusions to be reflected at systematic-taxonomic level.The aim of the present work is to analyze the intraspecific variability, haplotype profiling, phylogeography and genetic polymorphism of populations of the species For this purpose, the rDNA was preferred over mitochondrial DNA (mtDNA) because of its mendelian inheritance, evolutionary rates and overall recognized usefulness in systematics in all metazoan organism groups because of including sequences which allow to distinguish between species and between subspecies units. The better fitting of rDNA for molecular systematics has already been emphasized in large reviews on rDNA/mtDNA marker comparisons in insects Within rDNA, ITS-2 was selected as marker because of its well-known usefulness at species and subspecies levels, including the differentiation of taxa within problematic groups, as is the case of those comprising cryptic or sibling species of other insect groups T. dimidiata populations, the ITS-2 sequences of many members of the Phyllosoma, Rubrofasciata and Infestans groups were obtained and analyzed. For this purpose, a large number of rDNA ITS-2 sequences of Triatoma species from numerous geographic origins in Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil was studied. Thus, the nucleotide divergence limits between taxa within the lineage of the genus Triatoma could be established. The present study on T. dimidiata is the largest interpopulational analysis performed on a triatomine species so far.In order to be able to assess the ITS-2 evolutionary processes followed by Triatoma species of the Phyllosoma, Rubrofasciata and Infestans groups, among which 137 specimens representing T. dimidiata from 64 different geographic origins, were used for sequencing, genetic variation and phylogenetic analyses  terminology used in the present paper follows the nomenclature for composite haplotyping (CH) recently proposed Triatoma species studied together with other species and populations whose sequences are available in GenBank: T. phyllosoma (Accession Number AJ286881), T. pallidipennis (AJ286882), T. longipennis (AJ286883), T. picturata (AJ286884), and T. mazzotti (AJ286885) ; T. barberi (AJ293590)  T. rubrovaria H1 (AJ557258) T. infestans CH1A (AJ576051), and T. sordida (AJ576063) Rhodnius prolixus (Triatominae: Rhodniini) (AJ286882) Sequences were aligned using CLUSTAL-W version 1.83 http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the new ITS-2 rDNA sequences discussed in this paper are: 31 haplotypes of T. dimidiata (AM286693–AM286723), T. bassolssae AM286724, T. bolivari (AM286725), 2 haplotypes of T. hegneri , T. mexicana (AM286728), 2 haplotypes of T. pallidipennis , T. ryckmani (AM286731), T. flavida (AM286732), T. gerstaeckeri (AM286734), T. rubida (AM286735), T. nitida (AM286733), T. maculata (AJ582027), and T. arthurneivai (AM286736).The GenBank  are quite similar and potentially subject to homoplasy and recombination, alternative procedures to phylogenetic tree reconstruction revealing their relationships were tested. Therefore, a median-joining network analysis http://www.fluxus-engineering.com) with the variable positions in the multiple alignment of the different ITS-2 haplotypes from T. dimidiata populations.Phylogenies were inferred by maximum-likelihood (ML) using PAUP*4.0b10 Alternative methods of phylogenetic reconstruction allowing an evaluation of the support for each node were also applied. A distance-based phylogeny using the neighbor-joining (NJ) algorithm T. dimidiata was evaluated using DnaSP version 4 T. dimidiata haplotypes obtained. The second level corresponded to countries of sampling within each of these groups, and the third level corresponded to the different haplotypes found in each country within group. AMOVA reports components of variance at the three levels under consideration  as well as F-statistics analogs. Under the present scheme, FST is viewed as the correlation of random haplotypes within countries within groups, relative to that of random pairs of haplotypes drawn from the whole species, FCT as the correlation of random haplotypes within groups, relative to that of random pairs of haplotypes drawn from the whole species, and FSC as the correlation of the molecular diversity of random haplotypes within countries within groups, relative to that of random pairs of haplotypes drawn from the corresponding group ST's Genetic variation within and among populations of T. dimidiata studied (T.dim-H1 to T.dim-H31)  see . These fT. dimidiata haplotype sequences was 501 bp-long, of which 450 characters were constant and 24 were parsimony-informative. The interrupted microsatellite (AT)4–5 TTT (AT)5–7 was detected between positions 47 and 73 in all specimens studied. Variability in this microsatellite region and their respective sequence positions are noted in The alignment of the 31 The 51 nucleotide variable positions detected including gaps represented a 10.18% of polymorphic sites. The seven haplotypes T.dim-H25 to T.dim-H31 are responsible for this high genetic divergence . This geT. bassolsae, T. bolivari, T. hegneri, T. mexicana, T. pallidipennis, T. ryckmani, T. flavida, T. nitida, T. gerstaeckeri, and T. rubida, including haplotype length and AT content are listed in ITS-2 sequences of T. dimidiata haplotypes clearly differed from the Phyllosoma, Flavida, Protacta and Rubrofasciata complex species included in this analysis. Triatoma bassolsae differed in only one deletion in position 489 from T. pallidipennis of Morelos, Mexico (AJ286882). The T. pallidipennis sequence obtained represents a new haplotype  differing in only one deletion in position 31 from T. picturata and T. longipennis. The haplotype alignment of T. bassolsae, T. longipennis, T. mazzotti, T. picturata, T. pallidipennis and T. phyllosoma was 490 bp long showing a relatively small genetic diversity of 1.83%, with only 5 mutations (1.02%) and 4 indels (0.81%). The two T. hegneri haplotypes differ between each other in only 1 ti and, when compared with T. dimidiata H18 to H24 from Mexico and Guatemala, nucleotide differences found were only 1 ti and 2 tv.All the T. maculata and T arthurneivai, including haplotype length and AT content are listed in ITS-2 sequences of T. maculata fits very well within sequences of the Infestans complex species studied in the present work, a total of 6–19 (13.7) mutations, namely 6–11 (7.25) ti and 0–10 (6.5) tv, appearing when comparing the five Infestans complex species in question. The material of Triatoma arthurneivai here analyzed is very close to T. rubrovaria H1 (AJ557258), showing only 6 nucleotide differences (1.22%), of which only 1 ti and 5 indels.The ITS-2 of T. dimidiata haplotypes, both yielding coincident results. A maximum likelihood tree was reconstructed using the best model of evolution as determined by the lowest AIC, which was GTR+I (−Ln = 887.089), being the proportion of invariable sites (I) of 0.166. Three groups appeared with high support values indicating that their differentiation was not due to random sampling of a low variable sequence (tree not shown). The large group 1 encompassed haplotypes from all the countries, whereas groups 2  and 3  were more geographically restricted.Two different phylogenetic approaches were performed with the 31 T. dimidiata sequences using the variable sites in the multiple alignment  using the HKY+G model, according to the AIC results with a gamma distribution shape parameter = 0.58. This tree (The relevance of the ITS-2 differences among these his tree  shows thT. dimidiata haplotypes appear within a highly supported clade (95/97/100 in ML/NJ/BPP), distributed as follows: a first large subclade, also very well supported (99/97/100), comprising subgroup 1A, subgroup 1B, group 2, and group 3 of the network analysis; subgroup 1A (sequence grouping 1 = T.dim-H1 to T.dim-H10) includes populations from Central America ; interestingly, the haplotype T.dim-H10 corresponding to phenetically peculiar specimens found in cave-dwellings of Lanquin, Guatemala, appears independent although related to the rest with very high supports; subgroup 1B (sequence grouping 2 = T.dim-H11 to T.dim-H17) comprises populations from continental Colombia and Panama and appears as a monophyletic haplotype cluster; group 2 (sequence grouping 3 = T.dim-H18 to T.dim-H24) shows a well supported branch (91/92/100) and comprises populations from Mexico  and Guatemala, including the two T. hegneri haplotypes; the second large clade is also highly supported (97/96/100), corresponding to group 3 (sequence grouping 4 = T.dim-H25 to T.dim-H31) and includes populations from the Yucatan peninsula, Holbox and Cozumel islands and northern Chiapas (Mexico), northern Honduras and northern Guatemala;the 31 T. bassolsae clusters together with T. phyllosoma, T. mazzotti, T. longipennis, T. picturata and T. pallidipennis with very high support (99/91/100 in ML/NJ/BPP) in a sister clade of T. dimidiata; the separated location of the two T. pallidipennis haplotypes indicates the marked similarity of all these taxa;T. mexicana and T. gerstaeckeri cluster together in a group basal to both T. dimidiata and T. phyllosoma clades; the extremely high values (100/99/100) supporting the monophyletic clade including T. mexicana, T. gerstaeckeri, T. phyllosoma and close species, and T. dimidiata, are worth emphasizing;T. barberi, T. nitida, T. rubida, T. ryckmani and T. bolivari cluster in an unresolved branch, within which only T. ryckmani and T. bolivari appear related with a high support; the insular species T. flavida from Cuba appears as a basal lineage although with insufficient support values;T. rubrovaria, T. arthurneivai, T. sordida, T. maculata and T. infestans cluster together with the highest support.finally, the South American species Triatoma dimidiata groupings appeared well supported, with very high bootstrap proportions (BP>90%) using ML and neighbor-joining reconstruction and the highest Bayesian posterior probabilities (BPP = 100%). Similar levels were found for other well established Triatoma species, many of which showed substantially lower support values in the three statistical measurements employed. However, other species presented no ITS-2 nucleotide differences .T. dimidiata sequences using three levels as defined above  within groups, and within populations. A hierarchical analysis of molecular variance was used to test the null hypothesis of no genetic differentiation among groups considering variation at lower levels. This procedure was first applied to ed above . Most ofed above . The geoT. dimidiata ITS-2 sequences revealed the existence of three distinct groups , the first of which further subdivided into two subgroups 1A and 1B. The same AMOVA procedure was applied to ascertain whether these two subgroups could be considered as distinct populations or not. The results . Subgroup 1A includes two large populations, Honduras1 (n = 18) and Guatemala1 (n = 26), which presented a highly significant FST = 0.193, P<0.001. Although this value, under the assumption of migration-drift equilibrium, corresponds to an estimate of 2.1 migrants per generation between both populations, which would be enough to prevent their complete differentiation, such estimations shall be verified by using larger samples and markers better suited for population genetics analyses. Comparisons between each of these two populations and the smaller ones in subgroup 1A revealed that Honduras1 differed from Mexico1, Guatemala1 was different from Ecuador and Nicaragua, and none of them differed from the only two individuals from Providencia island. Similar comparisons for all pairs of populations assigned to different groups/subgroups resulted in highly significant FST values  according to a molecular clock analysis based on rDNA evolutionary rates The highest intraspecific ITS-2 variability (absolute nucleotide differences including indels) known in Triatomini members is 2.70% (13/482) in imidiata  appears ferences  and showThe divergence of 5.62% shown by the other 24 ITS-2 haplotypes  also appT. dimidiata populations are following different evolutionary divergences in which geographical isolation appears to have had an important influence  and corresponds to most of the Central American forms; (ii) T. d. maculipennis was proposed for specimens from Mexico (type specimen in Zoologisches Museum Berlin) and corresponds to forms with relatively short heads and large eyes; and (iii) T. d. capitata was proposed for large size specimens typified by longer heads and smaller eyes originally found in Colombia . However, these subspecies became later synonymized after results of a morphological re-examination which were interpreted as evidence of a clinal variation along a north-south axis Three subspecies were distinguished on the basis of morphological differences T. sp. aff. dimidiata to avoid further systematic confusion with T. dimidiata, according to taxonomic rules), and (ii) re-assign subspecific status for subgroup 1A, subgroup 1B and group 2. Results of the present study do not support the rise of the above-mentioned subspecific taxa to species level for the time being, although it is evident that in the three cases relatively long divergence processes have taken place. Similar genetic studies with other molecular markers may contribute to a more complete assessment of these evolutionary isolation and speciation processes.Present ITS-2 sequences and corresponding phylogenetic and genetic variation analyses support the appropriateness to (i) differentiate group 3 as a species of its own , and only one altitude-adapted haplotype (T.dim-H29) in its most extreme border populations in northern Honduras. The most widely spread haplotype T.dim-H28 is also present in the small island of Holbox and the large island of Cozumel, both near the Yucatan coast, suggesting that this haplotype should be considered the oldest of this species. This species is also of public health importance because of its capacity to transmit Chagas disease The taxon T. d. dimidiata corresponds to subgroup 1A and populations mainly from Guatemala and Honduras and secondarily Mexico, Nicaragua and Ecuador. The population of the Colombian island of Providence undoubtedly derives from the most widely dispersed haplotype T.dim-H1 on the nearest Caribbean coastal area of Central America and not from continental Colombia. The present populations in Ecuador may derive from introduced specimens originally from the Guatemala-Honduras-Nicaragua region, relatively recently introduced by humans T. d. dimidiata. An isolation phenomenon in caves may explain the albinic characteristics of the specimens presenting this haplotype. These cavernicole specimens from Alta Verapaz have already shown their peculiarity in morphometric and cuticular hydrocarbon studies The taxon T. d. capitata corresponds to subgroup 1B and populations from Colombia and Panama. The isthmus of Panama and the separation/joining process of South and North America towards the end of the Pliocene (3–5 Mya) T. dimidiata populations. The lack of relationship between the haplotypes of Ecuador and those of Colombia is worth mentioning, as the geographical closeness of these two countries could have given rise to the erroneous hypothesis of Colombian forms having derived from Ecuadorian populations. In a recent study of three populations of sylvatic, peridomestic and domestic T. dimidiata from Colombia, the estimated low genetic distances based on RAPD analyses did not discriminate the populations studied, indicating that they maintain the genetic identity of a single recent common ancestor The taxon T. d. maculipennis corresponds to group 2 and populations mainly from Mexico, but rarely found in Guatemala. According to the network analysis, this subspecies seems to have derived from group 1 probably by isolation in the Mexican part northward from the isthmus of Tehuantepec. Similarly as for other organisms including insects T. sp. aff. dimidiata did not represent a competition barrier, as T. sp. aff. dimidiata appears to be preferentially a low altitude species in these two countries.The taxon T. d. dimidiata, T. d. maculipennis, and T. sp. aff. dimidiata, overlap. In a morphometric analysis, populations from San Luis Potosi and Veracruz in Mexico were indistinguishable while clearly different from populations from Yucatan in Mexico and Peten in Guatemala T. d. maculipennis and the latter to T. sp. aff. dimidiata. In Guatemala, a high degree of genetic variation in T. dimidiata sensu lato was shown by RAPD-PCR Southern Mexico (including the Yucatan peninsula and Chiapas state) and almost the whole country of Guatemala (at least ten departments) constitute a crucial evolutionary area, where a high number of taxa, including T. d. maculipennis in Mexico (excluding Yucatán), T. d. dimidiata in Guatemala (excluding Petén) and probably also El Salvador; and T. d. capitata in Colombia; (ii) cytotype 2 was found in two localities  around Mérida, Yucatan, Mexico where the species T. sp. aff. dimidiata presents 5 different haplotypes ; (iii) cytotype 3 appeared in Yaxhá, Petén, Guatemala, where both T. d. maculipennis (T.dim-H18) and T. sp. aff. dimidiata  are present. Sequencing of the same specimens studied T. sp. aff. dimidiata of haplotype T.dim-H28 and T.dim-H30. Consequently, chromosomal cytotypes 2 and 3 are both found in T. sp. aff. dimidiata.Chromosome analyses and DNA genome size revealed the existence of three different cytotypes with different geographical distributions T. hegneri differ by only 3 mutations from haplotypes of T. d. maculipennis. This reduced number of nucleotide differences and the location of T. hegneri haplotypes within the clade of T. dimidiata, basal to haplotypes of group 2 . Interestingly, the most dispersed haplotypes of both T. d. maculipennis (T.dim-H18) and T. sp. aff. dimidiata (T.dim-H28) are also present on the same island, probably introduced through the intense human transport between the mainland and the island.The two haplotypes of  group 2 , does noT. d. dimidiata (subgroup 1A), T. d. capitata (subgroup 1B), T. d. maculipennis (group 2), T. sp. aff. dimidiata (group 3), and T. d. hegneri contributes giving systematic/taxonomic coherency to present knowledge about morphological and genetic concepts in these taxa. From an ancestral form close to T. sp. aff. dimidiata, it can be postulated that an original diversification focus of T. dimidiata forms took place most probably in Guatemala, with a southern spread into Panama and Colombia to give the capitata forms and a northwestern spread into Mexico to give the maculipennis forms , that there is no support to maintain them as separated species. Such a low number of nucleotide differences in the ITS is considered as pertaining to organisms able to hybridize Triatoma bassolsae is one additional taxon to be included in this situation, as has already been suggested T. pallidipennis and one in T. picturata versus the rest Genetic distances between the taxa of the Phyllosoma complex found when analyzing different mtDNA genes proved to be similar to those detected in ITS-2 at the 16S Triatoma mexicana appears to be a good species and its location in the phylogenetic tree fully supports its ascription to the Phyllosoma complex, similarly as suggested by a phylogentic analysis by means of a mtDNA CO1 fragment T. gerstaeckeri (Rubrofasciata group) clusters with T. mexicana, suggesting that it should be included in the Phyllosoma complex. All these species, i.e. T. phyllosoma , T. dimidiata , T. sp. aff. dimidiata, T. mexicana and T. gerstaeckeri constitute a well defined clade for which the generic taxon Meccus, proposed long ago Meccus might be a valid taxon.Meccus, as well as that of Nesotriatoma for species of the Flavida complex, has not been accepted because of the close relationship between T. flavida and the Phyllosoma complex T. bolivari and T. ryckmani in the Phyllosoma complex, as they appear to cluster with T. rubida of the Rubrofasciata group with relatively high support . A T. rubida - T. nitida clade previously detected with weak support under certain conditions in mitochondrial DNA marker analyses The revalidation of T. flavida as a species basal to all other North-Central American Triatoma species may be interpreted as a consequence of being a relict insular species close to the ancient first North-Central American Triatoma colonizers. Further studies with other genetic markers are needed to establish the position of T. flavida more adequately.Although not fully resolved in the tree obtained, the location of the Cuban T. arthurneivai and T. rubrovaria, a species known in southern Brazil, Uruguay and northern Argentina T. arthurneivai as independent species. Recent genetic and morphometric studies have already raised several questions about T. arthurneivai, indicating that topotypes from Minas Geraes may represent a species different from populations of São Paulo State formerly also referred to T. arthurneivai and suggesting that these São Paulo populations might probably belong to T. wygodzinskyiT. wygodzinskyi should be sequenced and compared to both true T. arthurneivai from Minas Geraes and T. rubrovaria to ascertain the status of these three taxa.The very scarce ITS-2 sequence differences between Triatoma species cluster together with maximum support (100/100/100) and well separated from that of the North and Central American species of the same genus, thus supporting results of previous analyses which indicate an early divergence of about 23–38 Mya between species of the northern (Phyllosoma complex) and southern (T. infestans) continent The South American Alternative Language Abstract S1Translation of the abstract into Spanish by S. Mas-Coma.(0.03 MB DOC)Click here for additional data file.Table S1Triatoma dimidiata populations.Summary of analysis of molecular variance for (0.06 MB DOC)Click here for additional data file.Table S2Triatoma dimidiata populations.Summary of population genetic variation parameters from ITS-2 haplotypes in the (0.08 MB DOC)Click here for additional data file.Table S3Evaluation of within groups genetic differentiation by computation of pairwise FST values for populations defined by country of origin in subgroup 1A.(0.03 MB DOC)Click here for additional data file.Table S4Triatoma dimidiata populations based on ITS-2 haplotypes.Summary of differentiation tests for (0.06 MB DOC)Click here for additional data file.


Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.The C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of  Clostridial neurotoxins (CNTs) are the most poisonous biological toxins known and molecular agents of botulism and tetanus neuroparalytic diseases . Due to C. botulinum, and also by strains of C. tetani, C. baratii, and C. butyricum , BAB87738; Pseudoalteromonas tunicata flagellin, ZP_01132756; Azoarcus BH72 flagellin, YP_934037; Desulfuromonas acetoxidans flagellin, ZP_01312630; and Burkholderia pseudomallei collagenase, ZP_01765667. The following default parameters were used in all PSI-BLAST [E-value cutoff = 0.005, with conditional compositional matrix score adjustment. The SSEARCH [C. botulinum protein database, and was obtained via the SANGER website ftp://ftp.sanger.ac.uk/pub/pathogens/cb/. SSEARCH was run with default parameters, except for the -z 11 flag, which computes the regression by reshuffling the target sequence library (removing the influence of homologous sequences present within the genome). For searching additional Clostridium species, the following protein sequence databases were retrieved from the NCBI FTP server: C. acetobutylicum ATCC 824, C. beijerinckii NCIMB 8052, C. botulinum A ATCC 3502, C. botulinum A ATCC 19397, C. botulinum A Hall, C. botulinum A3 Loch Maree, C. botulinum B1 Okra, C. botulinum F Langeland, C. difficile 630, C. kluyveri DSM 555, C. noyvi NT, C. perfringens 13, C. perfringens ATCC 131245, C. perfringens SM101, C. phytofermentans ISDg, C. tetani E88, C. thermocellum ATCC 27405.Botulinum neurotoxins A-G , and NTNH/A (YP_001253341) sequences were retrieved from NCBI. The flagellin and collagenase sequences used in the alignment of the HEXXH-containing segment . To verify that PSI-BLAST hits from CBO0798 to CNTs sequences were not due to composition, all asparagine residues were removed from CBO0798 and the top-scoring alignment detected via PSI-BLAST (C. butyricum BoNT/E), and permutation reshuffling tests were repeated using the altered sequences.To detect potential compositional bias, the composition of CNTs and CBO0798 was analyzed relative to all protein sequences in A.C.D. designed and performed the analysis and wrote the paper. B.J.M. designed the analysis and co-wrote the paper. E.M.M., M.D.J.L. and K.M.M. assisted with analysis and preparation of the manuscript.Detected sequence similarities between CBO0798 (flagellin) and CNT sequences. SSEARCH was used to screen the C. botulinum A protein database (3615 sequences) plus the target CNTs using default parameters. E-values were calculated within SSEARCH using randomly reshuffled copies of the library sequences, as described in Methods. CBO0798's rank relative to all 3615 C. botulinum proteins, associated E-value, and the alignment regions are reported for nine search cases . The flagellin aligned to two separate regions in the CNTs, suggesting an ancestral duplication.Click here for fileKey alignments between CBO0798 and CNTs produced by SSEARCH and PSI-BLAST. Smith-Waterman alignments between BoNT/A-CBO0798 and NTNH-CBO0798 were performed using SSEARCH (default parameters used with -z 11 flag). The listed E-values are based on a single pairwise alignment of both sequences rather than a database search. The alignment between CBO0798 and BoNT/E from C. butyricum is the result of a PSI-BLAST search of CBO0798 (restricted to Clostridia) using default parameters with composition-based statistics.Click here for fileMultiple alignment of clostridial flagellins. The N- and C-terminal domains are indicated, and the intermediate section represents the flagellin hypervariable region. The collagenase-like insert identified within the hypervariable region of FliA(H) is boxed in red. CBO0798 is underlined in black. Additional clostridial flagellins containing large hypervariable region inserts are grouped with CBO0798 and FliA(H) at the beginning of the alignment.Click here for file


Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne disease in ruminants, has been incriminated as the cause of Crohn disease in humans. We report the first case of human infection with MAP in a patient with HIV; infection was confirmed by obtaining isolates from several different specimen types. Mycobacterium species are among the leading AIDS indicator diseases in HIV-positive patients Mycobacterium avium complex is the most important mycobacterial species. M. avium complex includes the species M. avium and M. intracellulare, with M. avium consisting of M. avium subsp. avium, M. avium subsp. sylvaticum, and M. avium subsp. paratuberculosis (MAP). All these subspecies have identical 16S rRNA gene and 16S to 23S transcribed spacer sequences, as well as shared biochemical characteristics M. avium grows well on different solid media.Opportunistic infections caused by various MAP is the causative agent of Johne disease, a chronic granulomatous ileitis occurring mainly in ruminants 6/mL, and C-reactive protein 76 mg/L. Stained colon tissue samples, bone marrow punch, and liver biopsy showed abundant acid-fast bacilli. Endoscopic findings on colonoscopy were multiple polypoid lesions approximately 5 mm in size in the transverse and sigmoid colon.A 36-year-old HIV-positive man, who had been treated at our hospital since 1995 for HIV, hepatitis C, and hemophilia, had profuse diarrhea (6–8 episodes/day), fever as high as 39.9°C, and 10 kg of body weight loss in 5 weeks. Laboratory findings included hemoglobin 9.6 g/dL, pseudocholinesterase 2,099 U/L, HIV-DNA virus count 500 copies/mL, CD4+ lymphocyte count 29 x 10Microbiologic analyses included culture for mycobacteria  from at least 21 specimens  over a 3-year period. Of these, eight specimens  were positive for mycobacteria in liquid media after 6 to 16 weeks of incubation. Subcultures remained negative on Löwenstein-Jensen slants but after approximately 4 weeks became positive on mycobactin-supplemented Middlebrook slants with colorless dysgonic colonies. Microscopic examination of these colonies showed acid-fast bacilli .M. avium complex were performed on liquid media, all yielding strong positive results. However, repeated attempts to perform drug-susceptibility testing in the liquid BACTEC 460TB system were unsuccessful because of insufficient growth of the control. Since M. avium complex usually grows very well, the primary identification was questionable. Thus, polymerase chain reaction (PCR) for the amplification of a part of the mycobacterial gene coding for the ribosomal 16S RNA and additional sequencing was performed from two positive cultures M. avium/M. paratuberculosis, which is identical for both species and confirmed the AccuProbe results. For further differentiation between M. avium and MAP, PCR targeting the insertion sequence IS900 , which is present only in MAP strains M. avium strains remained negative  for negative .Because acid-fast bacilli were identified in biopsy specimens, treatment was started with ethambutol, ciprofloxacin, clarithromycin, and rifabutin. Initially, no clinical improvement was observed, and the patient’s weight loss and daily fever of 39C°–40°C continued. When ciprofloxacin was replaced with levofloxacin, progression of the infection appeared to stop. However, the patient died from cardiorespiratory failure.M. avium complex. Because growth was insufficient for susceptibility testing, the presence of MAP was assumed; however, the assumption was made after 2 years, because of difficulties in isolating MAP from human specimens  in media not thought to enable its growth. Finally, the demonstration of the insertion sequence IS900, an assay not routinely performed in human diagnostic laboratories like ours, confirmed this hypothesis.We describe the case of an HIV-infected patient who had a severe mycobacterial disorder thought to be caused by MAP isolated from human specimens has not yet been demonstrated by routine techniques. Several studies have reported the presence of MAP DNA in association with Crohn disease, although culture confirmation remains rare in these patients ,6.M. avium complex/M. paratuberculosis. However, because of the limited growth, we assume the presence of MAP even in those specimens not tested by IS900 PCR. These results indicate that MAP can grow to a limited extent in routine liquid media without mycobactin supplementation, at least if present in high amounts in the specimen.In the case we describe, mycobacterial growth could be detected in liquid media in 8 of 21 specimens, all confirmed as M. avium, or whether its occurrence in infections is low. However, herd prevalence of bovine paratuberculosis has been reported to range from 7% to 55% in Europe and to reach approximately 40% in stocks of >300 animals in the United States Susceptibility testing of the isolated strains could not be performed because of insufficient growth. Reports on susceptibility testing of MAP are rare, yet data obtained by a luciferase-based susceptibility assay