The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta.We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be used for karyotyping a wide range of related and genetically uncharacterized species, significantly extending the ability to develop synteny maps for comparative and functional genomics.Comparison of the  This particularly applies to Lepidoptera (moths and butterflies), one of the most diverse and species-rich group of insects, comprising the second largest order of animals with more than 150,000 named species. A partial solution is to study widely conserved molecular mechanisms underlying basic characters and processes in model species as a guide for investigating related phenomena in less well-studied relatives. Determining orthologous genes in multiple model species will greatly enhance knowledge of evolutionary patterns and processes Great diversity, one of the most striking characteristics of insects, presents a serious challenge for genomics, and thus constructing a firm foundation for comparative genomics of insects is of critical importance Many inherited phenotypes and behaviors are restricted to a narrow range of species, and are not available or difficult to study in the sequenced models. Ultimately, positional cloning may be the only way to reveal distinctive underlying molecular mechanisms. Although a difficult task in non-sequenced species where a chromosome region responsible for the phenotype of interest is poorly defined, it can be greatly facilitated by establishing comparative maps. Providing that the location and order of genes are conserved, positional information from orthologous chromosomal regions of well-characterized model species will save the time and expense required for the analysis of species with unexplored genomes. Loci of interest can then be identified and recovered by reference to common flanking markers Bombyx mori, representing the Bombycoidea, and two nymphalid butterflies representing the Papilionoidea, Heliconius melpomeneBicyclus anynanaB. mori genome as a reference was shown in these reports to be an effective approach for initial investigation of the evolution of gene organization and arrangement, at least for Macrolepidoptera.Significant preliminary evidence indicates that genome organization at the chromosome level is well conserved between the silkworm, Manduca sexta (Sphingidae), also belongs to the superfamily Bombycoidea. For years this species has been used as a favorite experimental animal in insect biochemistry, physiology, and especially in neurobiology M. sexta genes has been increasing B. mori and M. sexta by comparatively mapping orthologous genes with the help of BAC-FISH (fluorescent in situ hybridization with BAC probes).The tobacco hornworm, M. sexta, introduced five-color BAC-FISH to facilitate mapping, and used the improved BAC-FISH protocol to show broadly conserved synteny of genes including their order between B. mori chromosome 15 and the corresponding chromosome of M. sextaLepidoptera have small, numerous, and morphologically uniform holokinetic chromosomes, which are refractory to differential cytogenetic techniques. In our previous work M. sexta BAC clones containing 159 conserved genes by means of PCR-based screening. We then mapped the BAC clones by FISH to pachytene chromosomes, constructed a complete BAC-FISH karyotype of M. sexta, and by comparison with B. mori linkage groups, examined conserved synteny of genes between the two species. We also showed the applicability of BAC-FISH to a related species by cross-hybridization of M. sexta BAC probes to selected chromosomes of the convolvulus hawk moth, Agrius convolvuli, a representative of the same subfamily, Sphinginae, but from a different tribe (Acherontiini) than M. sexta (Sphingini) In the present study, we isolated additional B. mori and the corresponding chromosome of M. sexta by mapping M. sexta orthologs of six genes using BAC-FISH B. mori and M. sexta.We previously showed that the gene order is well conserved between chromosome 15 of M. sexta were used to find single-copy orthologs in B. mori by TBLASTN search against WGS genome sequences B. mori or localized to the mapped BAC contigs in our previous report B. mori orthologs  performed in combination with two BAC probes derived from the putative Z chromosome  but inverted order of two other orthologs. This indicated the presence of a large inversion comprising about half of the Z chromosome. Nevertheless, due to conserved synteny we designated each of the 20 chromosomes of  B. mori .M. sexta, BAC-FISH mapping revealed interchromosomal rearrangements relative to the karyotype of B. mori. We found that one chromosome of M. sexta, designated as chromosome 2, was composed of chromosome 2 and chromosome 26 of B. mori; in addition, the latter part exhibited an inverted order of two orthologous genes. On the other hand, chromosome 11 of B. mori was composed of chromosomes 11 and 26 of M. sexta  or the order of orthologous genes (three events)  . RelativA. convolvuli . We used M. sexta BAC probes that mapped to three representative chromosomes, including the sex chromosome Z and two autosomes (chromosomes 5 and 15) selected as examples without and with rearrangement compared to B. mori. All twelve BAC probes derived from M. sexta cross-hybridized on the corresponding A. convolvuli chromosomes  and B. anynana (n = 28), both Nymphalidae, have been examined for genome-wide synteny to B. mori. Seventy-two loci orthologous to B. mori genes were used to examine synteny in H. melpomene after constructing foundation maps composed of AFLPs and microsatellites B. mori orthologs were compared in B. anynana, which has the most gene-rich published lepidopteran linkage maps apart from B. mori. However, localizing the orthologs first required construction of a map of 768 SNPs derived from more than 100,000 ESTs Among the molecular linkage maps constructed in Lepidoptera, most employ anonymous markers such as AFLPs and microsatellites M. sexta, we mapped 124 loci by using BAC clones as probes. This enabled us to construct a BAC-FISH karyotype and sufficiently cover all 28 chromosomes with markers for comparative studies. Besides highly reliable and efficient localization of genes, the use of BAC-FISH has several other advantages. First, it does not require polymorphism in the genes examined, whereas it is essential for linkage mapping. Second, BAC-FISH mapping is independent of genetic recombination, facilitating analysis of genomes, chromosomes or chromosomal regions with rare crossovers. Third, it does not require the use of numerous sibs from matings between genetically homogeneous strains to determine the gene order unambiguously. Selection of only 67 M. sexta BACs carrying mapped B. mori orthologs was sufficient for the identification of all 28 bivalents in a pachytene complement . M. sexta genes showing significant similarity to putative single-copy B. mori genome sequences were selected and checked for previous localization of their B. mori orthologs 2 individuals of B. mori from a single pair-mating of a strain C108 female by a strain p50 male, as reported previously Sequences of M. sexta as described previously M. sexta genes were designed to avoid including putative exon-intron junctions predicted from the alignment of cDNA sequences of M. sexta with genome sequences of B. mori .BAC clones used as FISH probes were isolated from a BAC library of  B. mori . The priBroad-complex, E75, ephrin receptor, fasciclin II and nitric oxide synthase genes were first screened by in situ hybridization using high-density replica filters Screening was performed in three steps as described previously M. sexta and A. convolvuli larvae. BAC-DNA was extracted with a Plasmid Midi kit  and labeled with a fluorochrome using a Nick Translation System , with one of four fluorochromes, which were discriminated with different filter sets. These were Green-dUTP, Orange-dUTP, and Red-dUTP , and Cy5-dUTP . For BAC-FISH, a cocktail of BAC-DNA probes was hybridized to male chromosome preparations. For genomic in situ hybridization (GISH), female genomic DNA labeled with Green-dUTP using the Nick Translation System was hybridized to the female chromosome preparation as described previously M. sexta and 60 chromosome spreads from 5 preparations of A. convolvuli.We followed essentially the procedure described previously M. sexta, we estimated the relative position of the BAC-FISH signals using Image J 1.42 software (http://rsbweb.nih.gov/ij/download.html). The length of pachytene bivalents varies considerably due to differences in condensation between nuclei and spreading procedure. Therefore, we selected 5 representative bivalents for each chromosome, except for chromosomes 14 (n = 4) and 16 (n = 3), and calculated the median distance of hybridization signals relative to the proximal end of the corresponding B. mori linkage map  of hybridization signals relative to the proximal end of the corresponding B. mori linkage map, calculated from 3–5 representative pachytene bivalents. The numbers on the right side are accession numbers of the orthologous genes used for selecting BAC clones Click here for additional data file.Figure S2Manduca sexta and Bombyx mori. Shown are M. sexta BAC probes hybridized to male pachytene bivalents of B. mori (a–c) and B. mori probes to male pachytene bivalents of M. sexta (g–i). Note the absence of hybridization signals on the bivalents; arrows indicate some conjugates of fluorochrome-labeled probes outside bivalents. Control hybridizations of the probes to bivalents of the respective species are shown in d–f (M. sexta ) and j-l (B. mori ); note specific hybridization signals on the corresponding bivalent(s). Codes of M. sexta BAC probes tested: 15J11 (cyan), 22D05 (red), 34M13 (yellow), and 18A03 (green) ; 40I18 (cyan), 39P21 (red), 49K12 (yellow), and 43G05 (green) ; 39L03 (cyan), 02I22 (red), 10O14 (yellow), and 30J18 (green) . Codes of B. mori BAC probes tested: 9B9D (cyan), 9C6H (red), 9A5H (yellow), and 5I3F (green) ; 4B2A (cyan), 6F8D (red), 1B9C (yellow), and 9A12 (green) ; 2E9E (red), 7D3E (green) (chromosome 15), 10L7B (yellow) (chromosome 20), and 8C2C (cyan) . For BAC codes, see M. sexta and Yasukochi et al. (2006) for B. mori. Bar represents 5 µm for a and 10 µm for b-l.Cross-hybridization of BAC probes between (2.07 MB TIF)Click here for additional data file.Table S1Bombyx mori orthologous genes designed for these experiments.Polymorphic STS primers of (0.11 MB DOC)Click here for additional data file.Table S2Bombyx mori orthologs of Manduca sexta genes. Genes and ESTs of M. sexta are sorted into B. mori LGs to which orthologs belong. Ref. 1. Yasukochi, Y, Ashakumary L, Baba K, Yoshido A, Sahara K. (2006) Genetics 173:1319–1328. 2. Miao XX, Xub SJ, Li MH, Li MW, Huang, JH et al. (2005) Proc Natl Acad Sci USA 102: 16303–16308.Mapping of (0.39 MB DOC)Click here for additional data file.Table S3Manduca sexta BACs. Requests for clones or the library should be sent to H.B.Z. Notes: Clones positive for multiple genes are underlined.Gene anchors in (0.16 MB DOC)Click here for additional data file.Table S4Manduca sexta BAC library.STS primers used for PCR screening of the (0.16 MB DOC)Click here for additional data file.Table S5http://kaikoblast.dna.affrc.go.jp/) a The upstream region was misintegrated to chr. 13 in Kaikobase. b The upstream region was mistakenly integrated to chr. 3 in Kaikobase.Summary of BAC-FISH analysis. * G, O, R, C5: BAC probes labeled with Green-dUTP, Orange-dUTP, Red-dUTP and Cy5-dUTP and pseudocolored for green, yellow, red and cyan if not specifically mentioned; - represents BAC not used in (0.25 MB DOC)Click here for additional data file.


Background. Panton-Valentine leukocidin (PVL-)producing Staphylococcus aureus is emerging as a serious problem worldwide. There has been an increase in the incidence of necrotizing lung infections in otherwise healthy young people with a very high mortality associated with these strains. Sporadic severe infectious complications after incision of Bartholin's abcesses have been described but involvement of S. aureus is rare.Case report. We present a 23-year-old apparently healthy female patient without any typical predisposing findings who developed severe sepsis with necrotizing pneumonia and multiple abscesses following incision of a Bartholin's abscess. Methicillin-sensitive S. aureus harbouring Panton-Valentine leucocidin genes were cultured from the abscess fluid, multiple blood cultures and a postoperative wound swab. Aggressive antibiotic therapy with flucloxacillin, rifampicin and clindamycin, drainage and intensive supportive care lead finally to recovery.Conclusions. S. aureus, in particular PVL-positive strains, should be considered when a young, immunocompetent person develops a fulminant necrotizing pneumonia. Minor infections—such as Bartholin's abscess—can precede this life-threating syndrome. Bactericidal antistaphylococcal antibiotics are recommended for treatment, and surgical procedures may become necessary. Staphylococcus aureus inducing leukocytelysis. It has been associated with diverse clinical syndromes, includingprimary and secondary skin infections such as furunculosis and abscesses anddeep seated infections such as necrotizing pneumonia. Its role and regulationare still under investigation [S. aureus has the capacityto produce a wide array of virulence factors which are responsible fordivergent clinical syndromes [S. aureus strain USA 300 contains PVL, but most infections are skin and soft tissue infections, including boils, and are not life threatening [Panton-Valentine leucocidin (PVL) isa pore-forming cytotoxin of tigation . S. aureyndromes . In theS. aureus is usually severe and often fatal, involves primarilyyoung and healthy patients, and carries a mortality rate up to 75% despiteintensive medical treatment [Necrotizing pneumonia due toPVL-positive reatment .To our knowledge, we present thefirst case of necrotizing pneumonia following incision of a Bartholin'sabscess. In October 2006, a 23-year-old woman with no systemic signs ofinfection had a surgical incision of a Bartholin's abscess (3 × 5 × 3 cm) withoutthe installation of a drainage. The patient was discharged without antimicrobialtherapy. The abscess fluid was submitted for culture.24 hours later, the woman developedfever and her state of health worsened continuously, so she was admitted to alocal hospital 4 days after surgery. Community-acquired pneumonia was diagnosedand a macrolide was administered. One day later, the patient became criticallyill and was transferred to our tertiary care hospital and immediately admittedto the intensive care unit.She presented with an influenza-likeillness with severe muscle pain in all extremities, spiking fever up to 40°C, dry cough but no sorethroat. She showed ortho- and tachypnoeawith a respiratory rate of 30 breaths/minute, oxygen saturation was 96% withthe supply of 31/minute of oxygen but no need for intubation. Her bloodpressure was 100/60 mmHg and her heart rate 140/minute. Physical examination revealedcrackles in the inferior and middle parts of both lungs, a diffuse tendernessof the abdomen, and severepain in the muscles. Auscultationof the heart was unremarkable. Vaginal examination revealed a postoperativewound without any signs of infection. She denied ever having used illicitdrugs.9/L, a very low platelet countof 24 × 109/L, elevated inflammatory markers including a C-reactiveprotein of 399 mg/L (reference range (RR) < 5 mg/L) and aprocalcitonin level of 34 ng/L (RR < 0.1 ng/L), and an elevated creatinine of1.27 mg/dl. Her hemoglobin wasonly slightly reduced with 11.5 g/dl. As the patient did not present during the influenza season and first occurence of the influenza-like symptomswas parallel with the positive blood cultures, testing was not performed. Testing for HIV wasnegative and fasting glucose was normal. As the patient had no increased rateof infections in the past, we did not test for complement of IgG deficiency. Multipleblood cultures and swabs of the postoperative wound and the vagina were obtainedand sumitted for culture.Admission laboratory data revealed anelevated leukocyte count of 13.6 × 10A computed tomography scan (CT) ofthe chest showed diffuse bilateral alveolar infiltrates and nodular opacitieswith cavity forming consistent with necrotising pneumonia .Transthoracal echocardiography on the day afteradmission showed no abnormal findings, in particular no vegetations suggestiveof infective endocarditis. Community-onset pneumonia and severesepsis with coagulopathy was diagnosed.S. aureus fully susceptible toantistaphylococcal agents with the exception of penicillin and tetracyclin.Therefore, piperacillin/tazobactam was changed to high-dose intravenous flucloxacillin4 g tid and levofloxacin was discontinued. On day 3, blood cultures and bothwound and vaginal swab taken on admission also grew S. aureus. On day 4,rifampicin 600 mg Rifampicin 600 mg (once daily) was added asblood cultures continued to be positive and the patient still suffered fromhigh fever and severe dyspnoea. CT scans of the chest and abdomen showed newlyemerged extensive bilateral pleural effusions. In addition, multiple abscesseshad evolved in the pectoralis, supraspinatus, and gluteus muscle. A reevaluationof the heart valves by transesophagalechocardiography revealed no abnormalities. On day 6, polymerase chain reaction(PCR) amplification of the lukS-lukF genes was performed as described previously [S. aureus isolates. Subsequently, clindamyin 600 mgtid was added on day 7. With this treatment, blood cultures became negative onday 8. On day 11, bilateral pleural drainage tubes had to be inserted aseffusions increased. Cultures of the pleural fluid showed no growth. All S.aureus isolates from the abscess, the postoperative wound and multiple bloodcultures had identical susceptibility profiles. Minimal inhibitoryconcentrations (MICs) were <0.25 mg/l for oxacillin, <0.25 mg/l forclindamycin, and <0.5 mg/l for rifampicin. Pulsed-field gel electrophoresis(PFGE) of SmaI digests of genomic DNAfrom all available S. aureus isolatesshowed identical patterns (data not shown).Empirical intravenous antibiotictherapy with piperacillin/tazobactam and levofloxacin was started immediately. Onday 2, the initial abscess fluid yielded eviously  and confThe patient recovered very slowlywith a 3 weeks stay at the intensive care unit with maximal supportive care.Respiratory support and vasopressors had not been necessary. Subsequently, thepatient stayed for another 8 weeks on a regular ward until recovery.Antibiotics were continued for a total treatment duration of 5 weeks forrifampicin and clindamycin and of 8 weeks for flucloxacillin.S. aureus.Swabs of persons in close contact tothe patient were taken but were negative for PVL-producing S. aureus is a major cause of respiratory,skin, bone, joint, and endovascular infections. Mostly these infections occurin persons with known risk factors such as cardiovascular disease, malignancy,or diabetes mellitus. S. aureus is responsible for at least 10% of casesof nosocomial pneumonia but only for 2% of community-acquired pneumonia [neumonia , 7.S. aureus exhibiting theputative virulence factor PVL.Our patient was a youngimmunocompetent woman with no apparent risk factors who sustained severecommunity-onset necrotizing pneumonia, multiple abscesses, and extensive pleuraleffusions. These clinical features are characteristic for invasive infectionswith S. aureus (MSSA), PVL is more often identified incommunity-acquired methicillin-resistant S.aureus (MRSA) [The true incidence of PVL-associatedpneumonia is unknown, since the number of cases published is likely to be an underestimate. Besides its occurence in methicillin-sensitive s (MRSA) .S. aureus, which was firstcharacterized in 1932. Less than 5% of all S. aureus strains harbor PVL genes. PVLis a pore-forming toxin destroying the membrane of the host defence cells anderythrocytes [S. aureus isolates to correlate toxin production with disease manifestation.They found a definite association between the occurrence of PVL genes and furunculosis andcommunity-onset pneumonia. Gillet et al. [PVL is a very virulent toxinexpressed by t et al.  comparedt et al. –14. The S. aureus strains may spread between persons in close contact to the index patient [Since PVL-positive  patient , swabs s patient , but estS. aureus nospecific guidelines have been published. Combination therapy is widely usedempirically in life-threatening infections. Bactericidal antibiotics such as β-lactamantibiotics are preferrd over bacteriostatic agents. For pneumonia, goodpenetration into the tissue has also to be considered. We added levofloxacinempirically to piperacillin/tazobactam to treat potential atypical bacteria. Assoon as cultures became positive for S.aureus the antibiotic regimen was changed from piperacillin/tazobactam tohigh-dose flucloxacillin, as this is the antibiotic of choice for β-lactamase-positive S. aureus. As blood cultures remainedcontinuously positive and the patient's condition worsened, rifampicin, anothervery potent antistaphylococcal antibiotic, was added. The efficacy ofrifampicin as an adjunctive drug in patients with life-threatening infectionsremains controversial. As soon as the S. aureus isolates turned out tobe PVL-positive, clindamycin was added. Several authors recommend the additionof clindamycin to the treatment of toxin-producing grampositive bacteria as ittargets the bacterial ribosomes thereby potentially blocking the toxinproduction [β-lactam antibiotics, but no in vivodata have supported these observations so far [S.aureus infections intravenous antibiotic therapy should be given for atleast 4 weeks after the last blood culture positive for S. aureus . Although PVL playsa key role in the pathogenesis of necrotising pneumonia and Labandeira-Rey etal. could show in a mouse model that the PVL toxin alone might be sufficient tocause pneumonia, it is not likely to be the only virulence determinantresponsible for this syndrome [For the therapy of necrotizing pneumoniacaused by PVL-positive oduction , 18. News so far , 20. Oths so far . PVL-possyndrome .The outcome of patients withnecrotizing pneumonia may be poor in many cases even if appropriate antibioticsare administered. In addition, intensive supportive care is crucial for improvingthe outcome in these severe infections .S.aureus appears to be only veryrarely involved in these cases. Clinical complications have included septicshock, toxic-like-syndrome, necrotizing fasciitis, myocarditis, and gangrenebut pneumonia has not been reported.In our patient, necrotizingpneumonia developed shortly after incision of a Bartholin's abscess. Infectionof the Bartholin's gland is the most common infectious vulvar disease anddevelops in approximately 2% of all women . SurgicaEscherichiacoli, while S. aureus is only rarely detected [Bartholin's abscesses mostly containa mixed flora of aerobic and predominantly anaerobic bacteria. The mostfrequently isolated aerobe is detected , 38.S. aureus infection should early beincluded in the differential diagnosis when young immunocompetent personsdevelop necrotizing pneumonia. Various minor infections—such asBartholin's abscess—can precede thislife threatening syndrome. Antibiotictreatment with bactericidal antistaphylococcalagents is recommended, and invasive surgical procedures may become necessary.PVL-positive


Silene latifolia is one of the most studied species. Although many sexually dimorphic traits have been described in S. latifolia, all of them are quantitative, and they usually become apparent only after the initiation of flowering.Prior to this study, no differences in gene expression between male and female dioecious plants in the vegetative state had been detected. Among dioecious plants displaying sexual dimorphism, S. latifolia, sexual dimorphism in gene expression is present long before the initiation of flowering. We describe three ESTs that show sex-specific  transcription at the rosette stage before the first flowering season.We present RT-PCR-based evidence that in To our knowledge, this study provides the first molecular evidence of early pre-flowering sexual dimorphism in angiosperms. Sexual dimorphism (the systematic difference in form or other trait(s) not present in sexual organs between individuals of different sex in the same species) is a widely studied phenomenon in animal models  and in hSilene latifolia is  the mos century , and sin century -9). Howe century . This is century . The dif century ,13.S. latifolia, odor-compounds involved in pollinator attraction differ significantly between sexes, suggesting that selection for higher attractiveness among competing males is mediated by the sensory ecology of the pollinator [et al. [S. latifolia. They statistically predicted that selection for increased flower numbers in males along with weak selection for increased flower size in females could lead to dimorphic evolution in several other traits including leaf mass [The differential expression of some genes probably results from different modes of selection operating in males and females: males are limited in their reproductive success by access to mates, whereas females are more limited by resources . In animllinator . In addillinator . This dillinator ,18. Yet,llinator . Dawson llinator  pointed llinator ). StevenS. latifolia described so far become apparent only after the initiation of flowering. Notable exceptions to this pattern include: sex-dimorphism in the long-term survival of buried seeds and burial induced dormancy in S. latifolia [S. latifolia even at the rosette stage, a long time before the initiation of flowering, and describe three ESTs with sex-specific gene expression.Almost all of the sexually dimorphic traits in atifolia , sex-dimatifolia  and in tatifolia ,21. We pS. latifolia ESTs previously described according to Northern blots or Virtual Northerns [(Men-470) expressed exclusively in male flower buds and leaves and one EST (CCLS79.1) expressed exclusively in female flower buds and leaves . Serendip2 is a new EST that was found in this study. Serendip2 has an open reading frame along the whole sequence, but it has no homology to any known gene either at the DNA or protein level. The most interesting case is probably the CCLS79.1 gene. This sequence was amplified from genomic DNA samples of both females and males, suggesting that the difference in expression is not caused by sex linkage. The results we obtained in CCLS79.1 differ from the results obtained previously in Northern blots [In this article, we present sex-specific expression patterns of iewed in ). Men-47 authors , who reprn blots  that shoS. latifolia plants differ in the expression of at least three genes long before the initiation of flowering; this situation is analogous to the pregonadal stage in mammals [(Humulus) or hemp (Cannabis)). Spatial sex segregation, which is caused by differential seed germination and seedling survival , is an iewed by . Additiol fungus . In thisl fungus . As alre studies -21. The S. latifolia in the rosette stage suggests that there may be some, as yet undetected, physiological differences between sexes. We speculate that such hidden sexual dimorphism may be present in many dioecious species, and this study should inspire other scientists to test other dioecious species for sexual dimorphism in early vegetative stages. The existence of sexually dimorphic patterns means that the S. latifolia plants "know" their sex a long time before flowering, and this situation probably enables the plants to prepare for flowering in a sex specific manner.The existence of the sex specifically expressed genes in To our knowledge, this study provides the first molecular evidence of early pre-flowering sexual dimorphism in angiosperms.A. thaliana were detected at all A. thaliana genes that grouped with a respective Silene latifolia EST with a high degree of confidence. The cut-off to detect orthology was set to 0.7. The expression patterns of the A. thaliana orthologues were searched using Genevestigator V3 [Orthology data were obtained from OrthoMCL . Files cgator V3 ,26.S. latifolia plants were grown as described by Markova et al. [a et al. . Genomica et al. . The sexa et al. . RNA froa et al. . To veriJZl conceived and designed the experiments. JZl, JZa and BJ performed the experiments. JZl analyzed the data. JZl, BJ and BV contributed reagents/materials/analysis tools. BV discussed the paper. JZl and BJ wrote the paper. All authors read and approved the final manuscript.Table S1. Complete list of the studied genes with available information concerning putative A. thaliana orthologues found in this study.Click here for fileTable S2: Comparison of the previously published results with the results obtained in this study The file compares the gene expression data obtained in this study with the previously published data. The published data on testing of Y-chromosome linkage are also summarized.Click here for fileFigures S1-11. The file contains these supplementary figures: Supplementary figure S1 - Phylogenetic analysis of the gene CCLS6. Supplementary figure S2 - Phylogenetic analysis of the gene CCLS30.2. Supplementary figure S3 - Phylogenetic analysis of the gene CCLS30.3. Supplementary figure S4 - Phylogenetic analysis of the gene CCLS57.05. Supplementary figure S5 - Phylogenetic analysis of the gene CCLS62. Supplementary figure S6 - Phylogenetic analysis of the gene CCLS120.2. Supplementary figure S7 - Phylogenetic analysis of the gene Men-194. Supplementary figure S8 - Phylogenetic analysis of the gene Men-439. Supplementary figure S9 - Phylogenetic analysis of the gene Men-484. Supplementary figure S10 - Phylogenetic analysis of the gene Men-524. Supplementary figure S11 - Phylogenetic analysis of the gene Men-604Click here for fileA. thalina orthologsGenevestigator workspace file for all the found putative . This file contains expression data available for the found putative A. thaliana orthologs of the genes: CCLS6, CCLS30.2, CCLS30.3, CCLS57.05, CCLS62, CCLS120.2, Men-194, Men-439, Men-484, Men-524 and Men-604. The file can be viewed using Genevestigator V3 at the URL: https://www.genevestigator.com/gv/user/gvLogin.jsp (Registration is recommended.)Click here for fileTable S3. List of PCR primers and conditionsClick here for fileSilene latifoliaSupplementary Table S4: Flower development in . The table compares development of male and female flowers in S. latifolia.Click here for file


Serratia marcescens is a gram-negative bacterium and often causes nosocomial infections. There have been few studies of the virulence factors of this bacterium. The only S. marcescens hemolytic and cytotoxic factor reported, thus far, is the hemolysin ShlA.S. marcescens shlAB deletion mutant was constructed and shown to have no contact hemolytic activity. However, the deletion mutant retained hemolytic activity on human blood agar plates, indicating the presence of another S. marcescens hemolytic factor. Functional cloning of S. marcescens identified a phospholipase A (PhlA) with hemolytic activity on human blood agar plates. A phlAB deletion mutant lost hemolytic activity on human blood agar plates. Purified recombinant PhlA hydrolyzed several types of phospholipids and exhibited phospholipase A1 (PLA1), but not phospholipase A2 (PLA2), activity. The cytotoxic and hemolytic activities of PhlA both required phospholipids as substrates.An S. marcescens phlA gene produces hemolysis on human blood agar plates. PhlA induces destabilization of target cell membranes in the presence of phospholipids. Our results indicated that the lysophospholipids produced by PhlA affected cell membranes resulting in hemolysis and cell death.We have shown that the  Serratia marcescens is widely distributed in natural environments and has emerged in the last two decades as an important nosocomial pathogen, mainly in immunocompromised patients  (TaKaRa). Unless otherwise specified, bacteria were grown in Luria broth (LB). Antibiotics were added as required for the following final concentrations: ampicillin, 200 μg/ml; kanamycin, 100 μg/ml; and chloramphenicol, 20 μg/ml.2, and 15 g agar (per liter), and the pH was adjusted to 7.2 , incubated in a final concentration of 1 mg lysozyme/ml for 20 min on ice, and then disrupted by sonication. Cell debris was removed by centrifugation. The supernatant was analyzed by affinity chromatography using Ni-NTA agarose (Qiagen) under native conditions without a protease inhibitor. After dialysis against PBS, the purified protein was concentrated by Amicon Ultra-15 . The protein concentration was determined using a BCA Protein Assay Kit (Pierce). We obtained approximately 1.9 mg purified recombinant protein (His-PhlA) from one liter of culture. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining.To purify the His-PhlA recombinant protein, cells were harvested, lysed by lysis buffer . These experiments showed that PhlA cleaved ester bonds within PC, CL, PI, and PE and released fatty acids in a concentration-dependent manner, but did not hydrolyze SPM in our experimental conditions  hydrolyzes the fatty acids of PLs at position ons Fig. .S. marcescens PhlA had PLA1 activity, we tried to identify the site that is hydrolyzed by PhlA using fluorescent PLs as substrates [S. marcescens PhlA and bovine pancreatic PLA2 released fluorescent fatty acids from bis-BODIPY FLC11-PC, indicating that PhlA had phospholipase A activity  is one of the products from PLs hydrolyzed by PLA1. Therefore, we investigated whether LPL could cause hemolysis of human RBC. Lysophosphatidylcholine (LPC) was found to have hemolytic activity on human RBC in the solution reaction system Fig. . Using tTo determine the range of cells affected by PhlA, we examined various kinds of RBCs. As described above, PhlA lysed human RBC, but not horse or sheep RBC, on blood agar plates. However, all three types of RBC were lysed by PhlA in a lecithin-dependent manner in the solution reaction system Fig. . An explSince PC required 6-fold more PhlA than lecithin for induction of 50% hemolysis Fig. , the eggTaken together, these results indicated that PhlA phospholipase activity hydrolyzed PL and produced LPL. Since LPL is known to be a surfactant , it may S. marcescens culture supernatants, S. marcescens was grown at 37°C for 6 h in LB containing PL. Up to 48-fold dilutions of the S. marcescens culture supernatant induced cell death of both HeLa and 5637 cells, while supernatant of S. marcescens ΔphlAB cultured under the same conditions had no effect on HeLa or 5637 cells, indicating that PhlA was an extracellular secretion product (data not shown).We examined the cytotoxicity of PhlA using HeLa and 5637 cells. PhlA had cytotoxic activity against both HeLa and 5637 cells in the presence of lecithin Fig. . To inveE. coli [H. pylori (PldA) [A wide range of pathogenic bacteria produce phospholipases, and the putative role of PLA in virulence has been studied in some of these pathogens. Outer membrane-associated PLAs (OMPLAs) were first identified in E. coli  and orthi (PldA) . The OMPi (PldA) . Anotheri (PldA) , ExoU [3i (PldA) , PlaA [1i (PldA) , and Slai (PldA) ) is secri (PldA) ,39 recomS. marcescens. Although, a ΔshlAB mutant showed hemolytic activity on blood agar plates, it did not exhibit contact-dependent hemolytic activity . Although PhlA did not produce direct cytotoxicity on cultured cells, the pathogenetic role of indirect cytotoxicity via LPL production should be investigated.Pseudomonas aeruginosa ExoU inhibited neutrophil function in the lungs of infected mice [Streptococcus (GAS) SlaA contributed to colonization of the upper respiratory tract [Y. enterocolitica YplA, has been shown to play a role in bacterial colonization of the intestinal tract and increasing the pathological changes resulting from the host inflammatory response in the mouse model [S. marcescens colonization, since S. marcescens is thought to be a commensal in the intestinal tract where PLs are supplied by the host diet. The pathogenic role of PhlA remains to be elaborated.It has been reported that ted mice  and groury tract . Furtherse model . The higS. marcescens other than the previously reported ShlA. This new factor, PhlA, had phospholipase A1 activity. It is interesting to note that the hemolytic and cytotoxic activity of PhlA depend on the presence of phospholipids in addition to those in target cell membranes and seems to be mediated by the LPL product of the PhlA enzyme. Our results contribute to understanding the pathogenic role of microbial PLA.In this report, we have identified a hemolytic and cytotoxic factor in CL: cardiolipin; EDDA: ethylenediamine-N,N'-diacetic acid; LPC: lysophosphatidylcholine; LPL: lysophospholipid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PL: phospholipid; PLA: phospholipase A; RBC: red blood cells; SPM: sphingomyelin.KS carried out most of experimental works, and drafted the manuscript. SI performed the genetic studies. NK improved some of the experimental procedures. YG provided the draft genome sequence information. MO conceived the study and co-wrote the manuscript with HW. All authors have read and approved the final manuscript.


The products were characterized by elemental analysis, IR,  and NMR spectroscopy. According to spectroscopic observations, the hybrid anions consist of a lacunary anion framework on which are graftedtwo equivalent or groups through P-O-W bridges. Some of the title compounds showedthe antigerm activity.Organothiophosphoryl polyoxotungstates R


Objective: The incidence, morbidity, and risk factors associated with Clostridium difficile-associateddiarrhea (CDAD) were studied in a group of gynecologic oncology patients.Methods: A case-control analysis of gynecologic oncology patients with CDAD was carried outfrom August 1986 through January 1989 in a university medical center.Results: One hundred twenty-three stool samples were tested for C. difficile using the CDT latexagglutination test . Thirty episodes of CDAD developed in23 patients. From August 1986 through July 1988, the incidence was stable at 1.5 episodes/100admissions. From August 1988 through January 1989, the incidence increased to 9.9 episodes/100admissions (P = 0.005). Compared with patients with nonspecific antibiotic-associated diarrhea, thestudy patients were hospitalized longer prior to the development of symptoms  and were admitted more frequently with diarrhea . Therates of surgery, chemotherapy, and radiation therapy were similar. Fever ,abdominal pain , bloody stools , and leukocytosis were more common among the study cases. The duration, indication, andnumber of antibiotics administered were similar, though once started, the mean time to symptomswas longer in the study cases . Seven relapses, 1 death, and 1 unplannedcolostomy occurred among women with CDAD.Conclusions: C. difficile is a serious cause of nosocomial morbidity in gynecologic oncologypatients. Diarrhea developing after antibiotic exposure is more likely to be associated with C. difficilein patients whose symptoms develop several days after completing antibiotics and in patients with ahistory of CDAD.


Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date.Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus and Telestes souffia. The applicability of the markers was also tested on Abramis brama, Blicca bjoerkna and Scardinius erythrophtalmus specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species.Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies. The Cyprinidae family is of special interest for conducting comparative differentiation, evolutionary and hybridization dynamics studies: (i) Cyprinidae is the most abundant and widespread freshwater fish family across the world ; and  and four endemic species . The species sampling represents 12 of the 24 European Cyprinidae genera. We also studied two sets of hybrid specimens. The first one (Table Chondrostoma hybrids specimens (i.e. hybrids between C. t. toxostoma and C. n. nasus) from the Durance River previously characterized using the mitochondrial cytochrome b gene and four nuclear intron sequences , where Ln P(D) is the estimated posterior probability of the data conditional to K. A. brama, B. bjoerkna and S. erythrophtalmus individuals were discarded from these analyses due to their too limited sample size  in the microsatellites dataset  chooize (see ). Moreov. 4.05.2 . In addiNEWHYBRID was usedA. bipunctatus and P. pictum) were discarded. Moreover, as highlighted by the results from the different populations of A. alburnus, C. n. nasus, C. t. toxosmtoma or S. cephalus, the quality of species identification, both in FCA or STRUCTURE analyses, did not depend on the population sampled  analyses for K parameter determination  is summarized in Figure C. t. toxostoma populations can hardly be differentiated on axes 1 and 2 of the FCA, whereas a differentiation between the C. n. nasus sampled in Allier River and those sampled in Rhone River is displayed on axis 2. A strong coherence was found between the genome dilution as defined by [C. t. toxostoma species whereas Hy8 were assigned toward the C. n. nasus species. F1 hybrids, which correspond to Hy4 and Hy5 depending on their mitochondrial sequence, were mainly intermediate between the two parental species. However, these F1 hybrids are not strictly homogeneous and the assignment scores fluctuate , although most of them exhibited an intermediate morphology  of polymorphic microsatellite markers applicable to European cyprinid species has never been achieved to date (but see for instance ). MoreovThe authors declare that they have no competing interests.MS, VD, JFM and JF carried out the molecular genetics laboratory work. VD, MS, EM and CC performed the statistical analyses. VD, CC and MS drafted the manuscript. AG, VD and CC conceived the study. RC participated to the financial support. All authors read, contributed to and approved the final manuscript.STRUCTURE (K = 2) and NEWHYBRID assignment scores for the 48 Chondrostoma hybrids using 41 microsatellites. Excel Table containing STRUCTURE (K = 2) and NEWHYBRID assignment scores for the 48 Chondrostoma hybrids using 41 microsatellites.Click here for fileSTRUCTURE (K = 15) assignment scores for the 15 cyprinid hybrids using 41 microsatellitesCharacterization and . Excel Table containing the characterization and STRUCTURE (K = 15) assignment scores for the 15 cyprinid hybrids using 41 microsatellites.Click here for fileMicrosatellite loci, multiplex PCR conditions, levels of variability and amplifiability of 41 microsatellite loci in 15 cyprinid species. Excel Table describing the microsatellite loci, multiplex PCR conditions, levels of variability and amplifiability of 41 microsatellite loci in 15 cyprinid species.Click here for filePairs of loci found to be at Linkage Disequilibrium in 15 cyprinid species. PDF file reporting the pairs of loci found to be at Linkage Disequilibrium in 15 cyprinid speciesClick here for fileA. bipunctatus and P. pictum) (B) using 41 microsatellitesBiplot representations of the FCA for 15 cyprinid species (A) or 13 cyprinid species (excluding . PDF file containing the biplot representations of the FCA for 15 cyprinid species (A) or 13 cyprinid species (excluding A. bipunctatus and P. pictum) (B) using 41 microsatellites.Click here for fileA. bipunctatus and P. pictum) (B) using 23 microsatellitesBiplot representations of the FCA for 15 cyprinid species (A) or 13 cyprinid species (excluding . PDF file containing the biplot representations of the FCA for 15 cyprinid species (A) or 13 cyprinid species (excluding A. bipunctatus and P. pictum) (B) using 23 microsatellites.Click here for file


Xanthomonadaceae family contains two xylem-limited plant pathogenic bacterial species, Xanthomonas albilineans and Xylella fastidiosa. X. fastidiosa was the first completely sequenced plant pathogen. It is insect-vectored, has a reduced genome and does not possess hrp genes which encode a Type III secretion system found in most plant pathogenic bacteria. X. fastidiosa was excluded from the Xanthomonas group based on phylogenetic analyses with rRNA sequences.The X. albilineans was sequenced and annotated. X. albilineans, which is not known to be insect-vectored, also has a reduced genome and does not possess hrp genes. Phylogenetic analysis using X. albilineans genomic sequences showed that X. fastidiosa belongs to the Xanthomonas group. Order of divergence of the Xanthomonadaceae revealed that X. albilineans and X. fastidiosa experienced a convergent reductive genome evolution during their descent from the progenitor of the Xanthomonas genus. Reductive genome evolutions of the two xylem-limited Xanthomonadaceae were compared in light of their genome characteristics and those of obligate animal symbionts and pathogens.The complete genome of Xanthomonadaceae, during their descent from a common ancestral parent, experienced a convergent reductive genome evolution. Adaptation to the nutrient-poor xylem elements and to the cloistered environmental niche of xylem vessels probably favoured this convergent evolution. However, genome characteristics of X. albilineans differ from those of X. fastidiosa and obligate animal symbionts and pathogens, indicating that a distinctive process was responsible for the reductive genome evolution in this pathogen. The possible role in genome reduction of the unique toxin albicidin, produced by X. albilineans, is discussed.The two xylem-limited  Xanthomonadaceae are a family of Gram negative bacteria belonging to the order Xanthomonadales in the gamma subdivision of the Proteobacteria [Xanthomonadaceae, especially species from the genera Xanthomonas and Xylella, cause plant diseases and only one, Stenotrophomonas maltophilia, is known to be an opportunistic human pathogen.The bacteria . MembersXanthomonas species and Xylella fastidiosa strains have been determined, making those bacteria attractive models for study of plant-pathogen interactions [X. fastidiosa was the first completely sequenced plant pathogen. Sequence analysis showed that this xylem-limited bacterium, which is insect-vectored to a variety of diverse hosts, had a reduced genome and did not possess hrp genes, which encode a Type III secretion system (T3SS) found in most Gram negative plant pathogenic bacteria [Xanthomonadaceae, Xanthomonas and Stenotrophomonas, form a coherent group excluding X. fastidiosa [Xanthomonas and Stenotrophomonas by genome reduction during adaptation to life within its hosts [Complete genome sequences of several ractions . X. fast system TSS found stidiosa -6. Thesets hosts .Xanthomonas albilineans is a systemic, xylem-limited pathogen that causes leaf scald, one of the major diseases of sugarcane (interspecific hybrids of Saccharum spp.) [X. albilineans produces the toxin albicidin that has phytotoxic and antibiotic properties [hrp genes in X. albilineans failed so far [ihfA and efp, which did not include S. maltophilia sequences, suggested that X. albilineans was an evolutionary intermediate between several Xanthomonas species and X. fastidiosa [um spp.) . Leaf scoperties . Albicidoperties ,9. All ad so far ,11. A phX. campestris pv. campestris or X. oryzae pv. oryzae, X. fastidiosa and X. albilineans appear to be strictly xylem-limited, living only in dead xylem cells or tracheary elements. In order to better understand the evolution of these two xylem-limited Xanthomonadaceae, we sequenced the genome of X. albilineans strain GPE PC73 from Guadeloupe [Xanthomonadaceae. This comparative analysis revealed that X. albilineans and X. fastidiosa experienced a convergent reductive genome evolution from a common ancestral parent of the Xanthomonas genus.Unlike other xylem-invading xanthomonads that interact with living plant tissues, such as adeloupe . This seX. albilineans strain GPE PC73 consists of one circular chromosome of 3,768,695 bp and three extrachromosomal plasmids of 32, 27 and 25 Kbp, respectively. The chromosome exhibits a GC skew pattern typical of prokaryotic genomes that have two major shifts, one near the origin and one near the terminus of replication, with dnaA assigned as base pair 1 of the chromosome  were assigned putative functions based on homology to other known proteins and domain analyses.The genome of X. albilineans chromosome were compared to those of the chromosomes of the following eight Xanthomonadaceae strains: X. oryzae pv. oryzae strain MAFF 311018 , X. cam pepper; ), X. axopatient; ), S. mal poplar; ), X. fasapevine; ). The chXanthomonadaceae mentioned above . On the basis of this phylogenetic tree, we conclude that X. albilineans and X. fastidiosa derived from the progenitor of the Xanthomonas genus  the albicidin biosynthesis gene cluster XALB1 which was previously sequenced from X. albilineans strain Xa23R1 [Salmonella Pathogenicity Island -1) family that is mainly found in free-living animal pathogens [X. albilineans strain GPE PC73 that are not shared with any of the other eight Xanthomonadaceae complete genome sequences compared in this study. They also identified 1256 CDSs shared by the nine Xanthomonadaceae, which represent a Xanthomonadaceae core gene set which was likely inherited from a common ancestor.In order to identify orthologs shared by the nine ns Table , we perftransfer . They aln Xa23R1  and whicathogens . The OrtX. albilineans, X. fastidiosa and the Hrp Xanthomonas group derived from a common ancestor shared with Stenotrophomonas. The chromosome sizes of X. albilineans and X. fastidiosa are smaller than those of any other xanthomonad  all annotated CDSs from the genome sequence of X. albilineans strain GPE PC73 and (ii) all available sequences from public databases excluding all sequences from the xanthomonads. On the basis of these analyses and among the 3004 CDSs, we selected the genes for which the best BLAST hit belonged to X. albilineans, X. fastidiosa or S. maltophilia. We made the hypothesis that these genes were inherited by X. axonopodis pv. vesicatoria from the ancestor of the xanthomonads. These best BLAST hit analyses confirmed that 2864 of these 3004 CDSs have the same ancestor as genes present either in X. albilineans, X. fastidiosa or S. maltophilia and were therefore inherited by X. axonopodis pv. vesicatoria from the progenitor of the Xanthomonas genus. The elimination of paralogs present in at least two copies in X. axonopodis pv. vesicatoria generated a list of 2816 CDSs representing one copy of each gene inherited by X. axonopodis pv. vesicatoria from the progenitor of the Xanthomonas genus. These 2816 CDSs are listed and individually analysed in additional file X. fastidiosa and X. albilineans , 480 CDSs are shared only with one of the two S. maltophilia strains , 112 CDSs are shared with X. fastidiosa but not with X. albilineans  and 890 CDSs are shared with X. albilineans but not with X. fastidiosa (they represent the genes lost only by X. fastidiosa). The distribution in functional COG categories of these 2816 CDSs is illustrated in Figure Further comparative analyses were performed to compare genome erosion in s Figure . OrthoMCX. axonopodis pv. vesicatoria strain 85-10 led to the identification of DNA regions constituting contiguous ancestral genes that are missing in X. albilineans or X. fastidiosa . For example, the loss in X. fastidiosa of the large DNA region constituted by ancestral genes from XCV1928 to XCV2044 seems to be due to a single event of deletion since all these ancestral genes are missing in X. fastidiosa  gene cluster contains in X. axonopodis pv. vesicatoria two ancestral genes  that are missing in both X. albilineans and X. fastidiosa. The ancestral genes XCV1912 and XCV1915 that precede and follow respectively these two lost genes are orthologs of either Xalc_1342 and Xalc_1343 or XF1110 and XF1111 that are contiguous in X. albilineans and X. fastidiosa, respectively. Using the same strategy we identified 147 and 131 ancestral genes potentially lost by pseudogenization by X. fastidiosa and X. albilineans, respectively  and a long polyserine linker (PSL) at their C termini . BLAST analyses confirmed that 11 of these 18 CDSs are unique to X. albilineans and X. fastidiosa  than the metE present in the other Xanthomonas species and in S. maltophilia (amino acid identity 89/346 = 25%). This strongly suggests that the progenitor of the two xylem-limited Xanthomonadaceae lost the ancestral metE gene  and acquired another metE by horizontal genetic transfer. The 11 genes unique to X. albilineans and X. fastidiosa also include one cysteine protease gene, one ABC transporter gene, one polysaccharide deacetylase gene, one glycosyl transferase gene, one hydrolase gene, one cell filamentation protein gene and four hypothetical protein genes  . On theiewed in ).X. albilineans encodes a T3SS that displays similarities with the Burkholderia pseudomallei bsa T3SS which belongs to the injectisome family SPI-1  and which is required for the virulence of this human pathogen. The SPI-1 injectisome family mainly includes T3SSs from human and insect bacterial pathogens or symbionts [Erwinia amylovora strain Ea273 and Erwinia tasmaniensis strain Et1/99 both contain two copies of a SPI-1 T3SS [Erwinia spp. remains unknown. E. amylovora is insect-disseminated, although the interactions between this pathogen and its insect hosts remain poorly understood. It was suggested that the presence of a SPI-1 T3SS in Erwinia spp. indicates a common ancestry and close phylogenetic relationship between Erwinia spp. and insect-related enteric bacteria, raising the possibility that an insect host might be serving as a mixing vessel for the exchange of genes between Erwinia strains and other enteric bacteria [X. albilineans could indicate an insect-associated life style of this plant pathogen.The genome of ymbionts . InteresI-1 T3SS . The rolX. fastidiosa into the Xanthomonas group. This phylogenetic tree is in accordance with the presence of the unique gum genes in both X. fastidiosa and Xanthomonas species of the Hrp Xanthomonas group. The gum genes, which are involved in the biosynthesis of extracellular polysaccharides and the formation of biofilms, play a key role in pathogenicity of these Xanthomonadaceae. These genes, which were probably acquired by the progenitor of the Xanthomonas genus, were most likely lost by X. albilineans and conserved by X. fastidiosa during their speciation. Our MLSA phylogenetic tree is also in accordance with i/the presence of 11 unique genes, including metE, in X. albilineans and X. fastidiosa, and ii/the alignment of the 5' end of the 16S RNA of Xanthomonadaceae , indicating that independent but convergent evolution events were involved in genome erosion of X. oryzae pv. oryzae and the xylem-limited Xanthomonadaceae. Some of these genes lost by three xylem-invading pathogens are orthologous of genes with assigned functions and are organized into clusters. The five following ancestral gene clusters were lost by X. albilineans, X. fastidiosa and X. oryzae pv. oryzae: i/ the ancestral genes XCV0258 to XCV265 encoding enzymes involved in the glyoxylate cycle; ii/ the ancestral genes XCV0592 to XCV0602 encoding enzymes involved in malonate metabolism; iii/ the ancestral genes XCV1316 to XCV1334 including one TonB-dependant receptor gene, a two component signal transduction system (TCSTS) and chemotaxis genes, iv/ the ancestral genes XCV2187 to XCV2196 including one TCSTS and a type I secretion system and v/ the ancestral genes XCV2796 to XCV2803 encoding enzymes involved in catabolism of polysaccharides  in which these lost functions are useless. However, only 38 of the 480 ancestral genes lost by both X. albilineans and X. fastidiosa are also absent in another xylem invading pathogen, X. campestris pv. campestris, indicating that adaptation to xylem lifestyle favoured or allowed genome erosion, but did not necessarily induce it. Alternatively, the convergent genome erosion of the two xylem-limited Xanthomonadaceae may be linked to similar insect-associated lifestyles that may have favoured genome erosion because most of the genes required for a plant-associated life style are most likely not required for an insect-associated life style.Additionally, based on this MLSA, the same 480 ancestral genes appeared to be lost by both Buchnera . In theiewed in . In X. fs Figure . Metabolbout 2%; ).X. fastidiosa during a similar stage. The accumulation of mobile elements, representing a source of chromosomal rearrangements and gene inactivation, seems to have an important role in this first stage. A similar process is likely responsible for the limited genome erosion of X. oryzae pv. oryzae, which possesses a very high number of insertion sequences (IS) covering 20% of the genome [X. fastidiosa and X. albilineans  and may be explained by a higher frequency of cytosine deaminations [recX, dinG and dinP may explain, similarly, the very high GC skew of X. fastidiosa. It may also explain the more extensive genome erosion of X. fastidiosa, compared to X. albilineans and X. oryzae pv. oryzae. Alternatively, the most important factor affecting genome erosion of X. fastidiosa may reflect the insect-associated lifestyle specific to this Xanthomonadaceae [Obligate animal symbionts and pathogens display rapid evolution and have highly biased nucleotide base compositions with elevated frequencies of adenine and thymine (A+T) (reviewed in ). X. fasae Table . Furthers Figure . A simila strain . This lainations . The losnadaceae .X. albilineans chromosome contains a lower number of distortions and has a significantly higher amplitude than the GC skew pattern of other Xanthomonas species  lower than those of most quinolones [Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis [X. albilineans has two genes conferring resistance to albicidin: an albicidin efflux pump gene that is present in the albicidin biosynthesis gene cluster XALB1 [X. albilineans [We propose a unique mechanism of genome erosion involving the unique toxin albicidin produced by inolones . DNA gyrinolones ,35. In tinolones ,37. Severculosis -40. X. aer XALB1 ,41 and ailineans . It contX. albilineans conferred a selective advantage because of the potent antibiotic activity of albicidin. The DNA damage caused by albicidin may rapidly have induced the mutation of DNA gyrase A gene and thus stopped the process of genome erosion, possibly explaining the distinctive genomic characteristics of X. albilineans. Albicidin inhibits the growth of X. axonopodis pv. vesicatoria (data not shown), suggesting that the DNA gyrase A of the ancestral Xanthomonas was sensitive to albicidin. Transfer of the albicidin biosynthesis gene cluster to X. axonopodis pv. vesicatoria led to production of functional albicidin [X. fastidiosa. Therefore, albicidin is most likely not responsible for genome erosion of X. fastidiosa. However, we cannot exclude the hypothesis that albicidin biosynthesis genes were lost during evolution of X. fastidiosa. For example, cluster XALB1 could have been lost concurrently with the flagellar biosynthesis gene cluster because these two gene clusters are close on the chromosome of X. albilineans.Acquisition of the albicidin biosynthesis genes by the ancestor of lbicidin , demonstXanthomonadaceae experienced a convergent reductive evolution. Adaptation to the nutrient-poor xylem elements and to the cloistered environmental niche of xylem vessels probably favoured this convergent evolution. Alternatively, the most important factor affecting genome erosion of X. fastidiosa and X. albilineans may reflect insect-associated lifestyles specific to these Xanthomonadaceae. X. albilineans and X. fastidiosa evolved differently: genome erosion has occurred to different extents and specific genes have been acquired independently by X. albilineans and X. fastidiosa. For example, X. albilineans has acquired a T3SS of the SPI-1 family that is mainly found in free-living animal pathogens and four NRPS gene clusters that are involved in the biosynthesis of albicidin and probably other unknown small molecules. The toxin albicidin may be responsible for the distinctive genome erosion of X. albilineans. Much progress has been recently made in understanding how X. fastidiosa spreads within the xylem vessels as well as the traits that contribute to its acquisition and transmission by sharpshooter vectors . A simiX. albilineans strain GPE PC73 was isolated from a diseased stalk of sugarcane cv. H63-1418 in Guadeloupe .. Sequen were constructed; two of them were obtained after mechanical shearing of genomic DNA and cloning of generated 3 Kbp and 10 Kbp inserts into plasmids pcdna2,1 (Invitrogen) (A) and pCNS (B) (pSU18 derived), respectively. Larger DNA fragments of about 25 Kbp  were introduced into plasmid pBeloBac11 to generate a BAC library (C). Plasmid DNAs were purified and end-sequenced  by dye-terminator chemistry with ABI3730 sequencers  leading to an approximately 17-fold coverage. The Phred/Phrap/Consed software package ([http://www.phrap.com) was used for sequence assembly and quality assessment. A total of 2151 additional sequence reactions were necessary for gap closure and sequence polishing that consisted of random sequencing of subclones (for 1625 sequence reactions) supplemented with 145 sequences of PCR-products and 381 sequences of oligonucleotide-targeted regions. Final error estimation rate as computed by phred/phrap/consed was less than 0.04 errors per 10 Kbp. The sequences reported here have been deposited in the EMBL GenBank database, and accession numbers are FP565176, FP340279, FP340278 and FP340277 for the chromosome and for plasmids plasmI, plasmII and plasmIII, respectively.The complete genome sequence of package  as descnacearum . The pronacearum  was cons process . Predict process , InterPr process . The combilineans.gyrB, groEL, recA, dnaK, efp, atpD and glnA, are typically selected housekeeping genes located at the following positions of the X. albilineans chromosome: 0.004, 0.348, 1.369, 1.983, 2.245, 3.442, and 3.655 Mb from the origin of replication, respectively. The total length of the concatenated group of full length CDSs nucleotide sequences was 10417 bp-10686 bp. The tree obtained with the concatenated data set of the seven housekeeping genes was constructed with B. pseudomallei strain NCTC 10247 as outgroup. Multiple alignments of the nucleotide sequences of the 7 housekeeping genes  for the 11 taxons were performed using ClustalW .A phylogenetic tree was constructed from MLSA, with the maximum likelihood method and GTR as substitution model (with I: 0.01 and G: 0.52). The seven loci chosen, -5; Percent Identity Cut-off = 0; Percent Match Cut-off = 80; MCL Inflation = 1.5; Maximum Weight = 316. We modified OrthoMCL analysis by inactivating the filter query sequence during the BLASTP pre-process. All CDSs of X. axonopodis pv. vesicatoria strain 80-15 listed in Additional file X. albilineans, X. fastidiosa or S. maltophilia. Best BLASTP hit analyses were performed with database UniProt by excluding all accessions from the xanthomonads using expectation value lower than 1 × 10-5.OrthoMCL clustering analyses were performed using the following parameters: P-value Cut-off = 1 × 10Salmonella pathogenicity island -1; T3SS: Type III secretion system; TCSTS: two component signal transduction system.ACURs: alternative codon usage regions; CBD: cellulose binding domain; CDSs: protein-coding sequences; Hrp: hypersensitive response and pathogenicity; IS: insertion sequences; MLSA: multilocus sequence analysing; NRPSs: nonribosomal peptide synthetases; PSL: long polyserine linker; rpf: regulation of pathogenicity factors; SPI-1: IP and MR contributed to manual annotation of the genome, analysed the data, drafted part of the manuscript and coordinated the project. VB, AC, SM, BS (Segurens) performed sequencing of the genome. SC (Carrere) and JG performed automatic annotation of the genome and OrthoMCL analysis. RK and SC (Cociancich) contributed to manual annotation of the genome and drafted part of the manuscript. CM, VV and MA conceived the study and revised the manuscript. AD, M-A J, EL, SP, BS (Szurek) contributed to manual annotation of the genome and revised the manuscript. PR conceived the study, contributed to manual annotation of the genome and drafted part of the manuscript. All authors read and approved the final manuscript.X. axonopodis pv. vesicatoria strain 85-10List and individual analysis of the 2816 ancestral genes identified in the genome of . List and individual analysis of the 2816 CDSs of X. axonopodis pv. vesicatoria strain 85-10 identified by OrthoMCL and Best hit BLAST analyses as conserved in X. albilineans strain GPE PC73, X. fastidiosa strain 9a5c, S. maltophilia strain R551-3 or S. maltophilia strain K279a.Click here for fileX. fastidiosaList of ancestral genes potentially lost by pseudogenization and short deletions in . Analysis of lost ancestral genes that are present on the chromosome of X. axonopodis pv. vesicatoria between the orthologs of two ancestral genes that are contiguous and conserved in X. fastidiosa.Click here for fileX. albilineansList of ancestral genes potentially lost by pseudogenization and short deletions in . Analysis of lost ancestral genes that are present on the chromosome of X. axonopodis pv. vesicatoria between the orthologs of two ancestral genes that are contiguous and conserved in X. albilineans.Click here for fileX. albilineans and X. fastidiosaList and description of the 11 genes unique to . Summary of BLAST analyses results of the 11 genes unique to X. albilineans and X. fastidiosa.Click here for fileXanthomonadaceaeComparison of the 5' end of 16S RNA of eight . Alignment of the 5' end of 16S RNA of the following strains: XAC = Xanthomonas axonopodis pv. citri str. 306, XOO = Xanthomonas oryzae pv. oryzae str. MAFF 311018, XCV = Xanthomonas axonopodis pv. vesicatoria str. 85-10, XCC = Xanthomonas campestris pv. campestris str. ATCC 33913, SMA = Stenotrophomonas maltophilia str. R551-3, XAL = Xanthomonas albilineans str. GPE PC73, XYL_9a5c = Xylella fastidiosa str. 9a5c and XYL_Tem = Xylella fastidiosa str. Temecula1. The yellow-highlighted region is specific to X. albilineans and X. fastidiosa.Click here for filegyrB, atpD, dnaK, efp, groEL, glnA, and recA of nine XanthomonadaceaeAlignment obtained with ClustalW of the concatenated sequences of the housekeeping genes . Alignment obtained with ClustalW of the concatenated sequences of the housekeeping genes gyrB, atpD, dnaK, efp, groEL, glnA, and recA of X. albiline , StenoK279a , StenoR551 , Vesicatori (X. axonopodis pv. vesicatoria str. 85-10), Citri (X. axonopodis pv. citri str. 306), Oryzae (X. oryzae pv. oryzae str. MAFF 311018), Campestris (X. campestris pv. campestris str. ATCC 33913), Xyl9a5C (X. fastidiosa str. 9a5c), XylTemecul (X. fastidiosa str. Temecula1), Burkholder  and Ralstonia (R. solanacearum str. GMI1000). This alignment was not modified manually.Click here for file


Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated “gene dumps” and, perhaps, simultaneously, as “gene factories”.We present the genome sequences of a new clinical isolate of the important human pathogen,  Aspergillus is an extremely diverse genus of filamentous ascomycetous fungi (molds) found ubiquitously in soil and decomposing vegetation. Being supreme opportunists, aspergilli have adapted to overcome various chemical, physical, and biological stresses found in heterogeneous environments. While most species in the genus are saprophytes, a surprising number are able to infect wounded plants and animals. Remarkably, the allergic human host also responds abnormally to the aspergilli with lung and sinus disease. The advent of immunosuppressive agents and other medical advances have created a large worldwide pool of human hosts susceptible to some Aspergillus species, including the world's most harmful mold and the causative agent of invasive aspergillosis, Aspergillus fumigatus. In this study, we have used the power of comparative genomics to gain insight into genetic mechanisms that may contribute to the metabolic versatility and pathogenicity of this important human pathogen. Comparison of the genomes of two A. fumigatus clinical isolates and two closely related, but rarely pathogenic species showed that their genomes contain several large isolate- and species-specific chromosomal islands. The metabolic capabilities encoded by these highly labile regions are likely to contribute to their rapid adaptation to heterogeneous environments such as soil or a living host. Aspergillus fumigatus is exceptional amongst the aspergilli in being both a primary and opportunistic pathogen as well as a major allergen associated with severe asthma and sinusitis A. fumigatus has been demonstrated to be a primary pathogen of the airways, sinuses, lungs, damaged skin and subcutaneous tissues. For example, it can cause post-operative infection in all human organs A. fumigatus is thought to possess particular metabolic capabilities and genetic determinants that allow it to initiate and establish an in vivo infection. This conclusion is supported by the observation that the majority of invasive aspergillosis disease is caused by A. fumigatus, even though its conidia comprise only a small percentage of the total conidia found in air-sampling studies A. fumigatus spores with the human respiratory mucosa is understood to an extent, the basic biology of the organism has until recently received little attention.A. fumigatus strain Af293 (FGSC A1100) Aspergillus nidulans and Aspergillus oryzae, has led to many unexpected discoveries, including the possibility of a hidden sexual cycle in A. fumigatus and A. oryzae, and the detection of remarkable genetic variability of this genus Recently we presented the genomic sequence of Neosartorya fischeri NRRL181 (A. fischerianus), and a more distantly related asexual species, A. clavatus NRRL1, for complete sequencing. These three species are referred to here as the Affc lineage for A. fumigatus, fischeriN. , and clavatusA.   A. oryzaeA. flavusA. fumigatus isolates Af294 and Af71, according to the array-based comparative genome hybridization (aCGH) data Consistent with previous reports The origin of 20% of Af293-specific genes can be attributed to two segmental duplication events. One of the duplicated regions (AFUA_1G16010- AFUA_1G16170) contains an arsenic detoxification cluster. The other (AFUA_1G00420-AFUA_1G00580) contains genes that may be involved in metabolism of betaine, which is often synthesized under osmotic and heavy metal stress. Interestingly the duplicated regions are also absent in Af294 and Af71 isolates, which suggests that the duplication event took place very recently.Segmental duplication events are thought to contribute to rapid adaptation of the species by increasing their expression. Since Af293 is a clinical isolate it is possible that these chromosomal aberrations were created due to selective pressures in the host.A. fumigatus isolates, we identified Af293 genes that do not hybridize with DNA extracted from the Af294 and Af71 strains in aCGH experiments A. fumigatus isolates has demonstrated that each of them harbours two or three alleles  genes involved in a programmed cell death (PCD) pathway triggered by hyphal fusion between two genetically incompatible individuals het loci have been described in A. nidulansA. fumigatus. Thus, four clinical isolates from the same multi-member incompatibility group  contained the same alleles of the polymorphic genes  polymorphism , and its close relative, nosA (AFUA_4G09710), whose orthologs encode two Zn2C6 transcriptional regulators of sexual development in A. nidulansA. clavatus ortholog, while A1163 RosA clusters with N. fischeri. This is in contrast with the NosA tree, which perfectly mirrors the species tree  is 10–15% larger than the A. clavatus and A. fumigatus genomes (A. clavatus genome (27.9 Mb) is the smallest seen to date among the sequenced aspergilli .Comparative genomic analysis has showed that the three ectively . NumbersN. fischeri, A. clavatus, A. terreus (CH476594), A. oryzaeA. nidulansA. niger CBS 513.88 A. fumigatus, fischeriN.  and clavatusA. ) constitute the Affc-core group. The genes in the Affc-core can be further divided into two groups, the Aspergillus-core (Asp-core) with orthologs in all six other aspergilli and the Affc-specific group, which is comprised of the remaining Affc-core genes. Finally, the A. fumigatus-specific (Afum-specific) group contains Af293 genes that have orthologs in neither N. fischeri nor A. clavatus.To this end, we have selected four sets of genes based on the presence of orthologs in the six other sequenced aspergilli: A. fumigatus exhibit low sequence identity to their orthologs from more distantly related fungi  than by poor annotation quality of LS genes . We made significant improvements to Af294 gene models by leveraging the comparative genomic data see . In addi average . On the  average . NonetheAnalysis of Gene Ontology (GO) terms Penicillium species and some more distantly related fungi. Similarly, only 30% of secondary metabolism Af293 genes are shared by N. fischeri and A. clavatus. The three species also vary considerably in the numbers of enzymes that control the first step in secondary metabolite biosynthesis such as nonribosomal peptide synthases (NRPS), polyketide synthases (PKS), and dimethylallyltryptophan synthases (DMATS)  and A. fumigatus (34) genomes.Although most Af293 genes involved in carbohydrate transport and catabolism are found in the Asp-core group, only 10% of secondary metabolism genes have orthologs in all sequenced aspergilli including siderophore, pigment and Pes1-related clusters. These three conserved clusters are also found in  (DMATS) . InteresLikewise, PFAM domains overrepresented among Affc- and Afum-specific genes have been shown to function in efflux or detoxification, secondary metabolite biosynthesis, resistance to antifungals, and other accessory metabolic pathways. They include MSF and ABC transporters, various oxidoreductases, cytochrome P450, glycosyl and alpha/beta fold hydrolases, polyketide synthases, glutathione transferases and methyltransferases . On the A. fumigatusS. cerevisiaePichia stipitisAspergillus species do not have large variable subtelomeric arrays arising by a series of tandem duplications found in some protozoan parasites In addition to difference in size and function, lineage specific genes display a significant subtelomeric bias. As opposed to telomere-distal Asp- and Affc-core genes, Affc- and Afum-specific genes tend to be located within 300 Kb from chromosome ends  . About 3A. fumigatus-specific blocks (2.2 and 3.1) contain gene clusters involved in biosynthesis of mycotoxin fumigaclavine and another unknown secondary metabolite Magnaporthe oryzaeA. oryzaeAlmost 50% of the Afum-specific genes can be clustered together in 13 blocks containing more than 10 Afum-specific genes separated by no more than 5 genes outside this category . TogetheAspergillus fungal species, which suggests that they were created de novo in the Affc lineage. To gain insight into the origin of the LS genes in aspergilli, we have performed phylogenetic analysis of two sets of A. fumigatus- and N. fischeri-specific genes. In Af293 and N. fischeri, Set 1 contains 790 and 1230 genes, respectively, that have an Aspergillus homolog as the best BLASTp hit; Set 2 contains 28 and 178 genes, respectively, that have a non-Aspergillus homolog as the closest relative. There is a significant difference in the numbers of trees including a non-Aspergillus species as the closest relative in N. fischeri and A. fumigatus . This is indicative of major differences in retention and/or uptake of new genetic material in these two species, consistent with differences in their reproductive modes.Most Affc- and Afum-specific genes have no orthologs in non-A. fumigatus and N. fischeri, most of the Set 1 genes exhibit topologies that do not strictly follow the Aspergillus species tree  from a non-Aspergillus species into N. fischeri or visa versa. Further analysis, however, reveals that most of the conflicts involve sparsely populated trees, long branch attraction artifacts, and other situations, where phylogenetic methods tend to mislead , fos-1 (AFUA_6G10240), pes1 (AFUA_1G10380) and pksP (AFUA_2G17600), reveal evidence of accelerated evolution in the branch leading to the two A. fumigatus isolates. This pattern can affect only a few amino acid residues (e.g. PksP) or a significant proportion of the protein (e.g. Pes1).Previous studies however have shown a high level of evolutionary conservation and phyletic retention among known  species . InteresA. fumigatus virulence. These four genes are involved in oxidative stress or nutrient availability, which is consistent with the positive selection scenario. Indeed, PabaA is involved in biosynthesis of folate, an essential co-factor for DNA synthesis. Since PABA is apparently limited in the mammalian lung, a functional pabaA gene is required for virulence A. fumigatus virulence Such a pattern can be due to either relaxation of selection or selection for rapid diversification (positive selection). In the latter case specific amino acid substitutions may decrease susceptibility to specific environmental challenges and thus enhance A. fumigatus-specific genes may contribute to its ability to survive in the human host. A recent microarray study demonstrated that the Affc-specific genes are over-represented among genes that are up-expressed in the neutropenic murine lung (Elaine Bignell submitted for publication). Many of them are found in chromosomal gene clusters associated with macromolecule catabolism and secondary metabolite biosynthesis. Similarly, clustered lineage-specific genes simultaneously induced in infected tissue have been observed in the ubiquitous maize pathogen Ustilago maydisA. fumigatus virulence may be a combinatorial process, dependent on a pool of genes, which interact in various combinations in different genetic backgrounds as suggested previously Pseudomonas aeruginosaCryptococcus neoformansThis overall lack of variability among known virulence-associated factors suggests that yet unknown A. fumigatus genome encodes 20 allergens (A. fumigatus Asp f6 (AFUA_1G14550), also known as Mn2+-dependent superoxide dismutase (MnSOD), is specifically recognized by IgE from patients with allergic bronchopulmonary aspergillosis (ABPA) and is differentially expressed during germination A. fumigatus possesses the same allergen complement as most other aspergilli and that its effect on hypersensitive individuals can be explained mostly by its ubiquity in the environment.In addition to virulence factors, the llergens  and 25 pllergens , some ofAspergillus species  selection in both sexual and asexual  species . More deA. fumigatus and the two closely related species, N. fischeri and A. clavatus, demonstrates that A. fumigatus may possess genetic determinants that allow it to establish a successful in vivo infection. LS genes that have no orthologs in the other two species comprise 8,5% of the A. fumigatus genome and often have accessory functions such as carbohydrate and amino acid metabolism, transport, detoxification, or secondary metabolite biosynthesis. Further analysis showed that these genes have distinct features  and tend to cluster in subtelomeric genomic islands, which may function as “gene dumps/factories”. The phylogenies of LS genes, their subtelomeric bias and size differences are consistent with the DDL hypothesis stating that duplication being the primary genetic mechanism responsible for the origin of species-specific genes. The presence of genomic islands indicates that A. fumigatus and may possess sophisticated genetic mechanisms that facilitate its adaptation to heterogeneous environments such as soil or a living host.Lineage-specific (LS) genes (i.e. genes with limited phylogenetic distribution of orthologs in related species) have been the focal point of many comparative genomic studies, because of the assumption that they may be responsible for phenotypic differences among species and niche adaptation. Our analyses of the genomes of A. fumigatus Af293 (FGSC A1100) was isolated from patients with invasive aspergillosis A. fumigatus A1163 (FGSC A1163) is a derivative of A. fumigatus CEA17 converted to pyrG+ via the ectopic insertion of the A. niger pyrG gene A. fumigatus clinical isolate CEA10 (CBS144.89). The type strains of A. clavatus (NRRL 1) and N. fischeri (NRRL 181) were used for sequencing and phenotypic characterization.A. clavatus, N. fischeri and A. fumigatus A1163 were deposited to the GenBank under the following accession numbers: AAKD00000000, AAKE00000000 and ABDB00000000, respectively.The genome sequences of A. clavatus and N. fischeri were sequenced using the whole genome shotgun method as previously described A. fumigatus A1163, scaffolds were compared to those of the first sequenced isolate, Af293 A1163, http://mummer.sourceforge.net/) A. flavus and A. oryzae and between the two sequenced A. niger strains.A1163 assemblies larger than 5 Kb were aligned to the Af293 chromosomes using the MUMmer package (A. clavatus and N. fischeri assemblies (supercontigs) larger than 2 Kb as described earlier The JCVI eukaryotic annotation pipeline was applied to the A1163, http://www.repeatmasker.org/), RepeatScout (http://repeatscout.bioprojects.org/), and Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.html). Putative TEs (http://transposonpsi.sourceforge.net), a program that performs tBLASTn searches using a set of position specific scoring matrices (PSSMs) specific for different TE families. TE and repeat densities were calculated as the percentage of nucleotide bases in the regions of interest  that overlap with a feature of the appropriate type (repeat or TE).Identification of repeat elements was performed using RepeatMasker (tive TEs  were idehttp://sybil.sourceforge.net/), which allows for rapid identification of discrepancies in gene structure among orthologs. The comparison with orthologous N. fischeri and A. clavatus genes resulted in significant changes to the Af293 gene catalogue. Over 1100 gene models were updated and 130 new genes were identified. Initial A. fumigatus A1163 gene models were also improved using the PASA pipeline, initially developed to align expressed sequence tag (EST) data onto genomic sequences We leveraged the comparative genomic data to significantly improve annotation quality of the Af293 genome, which was previously annotated with relatively little supporting evidence N. fischeri and A. clavatus orthologs. Previously GO terms S. cerevisiae proteins Secondary Metabolism Region Finder (SMURF) available at http://www.jcvi.org/smurf . The complete list of gene clusters can be downloaded at ftp://ftp.jcvi.org/pub/software/smurf/. Gene Ontology (GO) terms We have performed transitive functional annotation from Af293 proteins to their A1163, Aspergillus genomes were identified using a reciprocal-best-BLAST-hit (RBH) approach with a cut-off of 1e-05. In addition to the A1163, A. clavatus and N. fischeri genomes, the previously sequenced genomes of Af293 A. terreus NIH2624 (http://www.broad.mit.edu), A. oryzae RIB40 A. nidulans FGSC A4 A. niger CBS 513.55 Aspergillus Comparative database at http://www.tigr.org/sybil/asp. Orthologous, unique and divergent genes in Af293 were identified based on alignments of Af293 CDSs against A1163 assemblies using gmap as implemented in PASA After extensive computational and manual refinement, the improved protein datasets were used to generate the final set of orthologs. Orthologous groups in A. clavatus and Af293 vs. N. fischeri) were defined as areas containing a minimum of five matching (orthologous) genes with a maximum of 20 adjacent non-matching genes (having no orthologs) in the reference and target genomes. Since most syntenic regions slightly overlapped, the original blocks were merged to calculate repeat and TE density. Af293 non-syntenic blocks were defined as areas excluded from the syntenic blocks and containing at least ten Af239 non-matching genes.Syntenic blocks for each pair of genomes (Af293 vs. http://TM4.org) Genes in four lineage-specificity groups were analyzed by the EASE module A. clavatus, N. fischeri and A. terreus genomes. The rate of substitution in synonymous (dS) and in non-synonymous (dN) sites, and their ratio (dN/dS) was calculated using the PAML package dN/dS<0.1; if a gene is under weak purifying selection, 0.1<dN/dS<1; if a gene is evolving neutrally (e.g. pseudogenes), dN/dS∼ = 1; and if a gene is evolving under diversifying selection, dN/dS>1. The results are reported only for orthologous genes sets having unsaturated dS values, the same number of exons, and sequence alignment coverage >95%. For each gene, the average dN/dS ratio for five pairwise species comparisons was calculated.Selective constraints were estimated for sets of orthologous genes from the Af293, A1163, A. niger ATCC 1015, Nectria haematococca, Phanerochaete chrysosporium and Trichoderma reesei genomes projects was completed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program and the by the University of California, Lawrence Livermore National Laboratory (Contract No. W-7405-Eng-48), Lawrence Berkeley National Laboratory (contract No. DE-AC03-76SF00098) and Los Alamos National Laboratory (contract No. W-7405-ENG-36).We assembled a local database of protein sequences from the 28 publicly available fungal genome projects . All phyA. niger, A. nidulans, A. terreus, A. oryzae, A. clavatus, N. fischeri, A. fumigatus and Fusarium graminearum (teleomorph of Gibberella zeae) as an outgroup. To minimize the effect of incorrect or incongruent gene models, these proteins were chosen on the basis of having identical numbers of introns in each species and similar lengths. Sequences were aligned using MUSCLE To produce a reference tree of species phylogeny we used the protein sequences of 90 likely orthologs from N. fischeri proteins were carried out on sets of homologs identified in BLASTP searches against our fungal database. The top 20 hits with E<10−4 were retained for analysis. Sequences were aligned using ClustalW Phylogenetic analyses of individual Af293, A1163, and rosA (AFUA_6G07010) gene, several hybridizations were performed using rosA gene as the probe and genomic DNA cleaved with EcoRI, ClaI, BamHI or EcoRV. For comparison, an invariable gene for all species  was used as the hybridization probe on genomic DNA digested with HpaI.To detect polymorphisms in the 6 spores/ml suspension in PBS/Tween 80. Plates were then incubated at temperatures ranging from 25°C to 50°C and colony edges were marked using a plate microscope. Colonies were marked twice daily for 4–5 days. For each colony, two diameters perpendicular to each other were measured.Colony radial growth rate measurements were performed as described Eight replicates were measured for each isolate. The results reported here are the mean of two experiments. At least five time points during the log phase were used to calculate growth rate. The radius of the colonies was plotted against time using least-square regression analysis, and the slope of the regression line, which represents the growth rate, was calculated. Each replicate was analysed separately and the mean of the growth rate was then calculated.Text S1Allergens and sexual development genes.(0.05 MB DOC)Click here for additional data file.Table S1N. fischeri, and A. clavatus isolates at various temperatures.Growth rates of Af293, A1163, (0.02 MB XLS)Click here for additional data file.Table S2A. fumigatus clinical isolates.Resistance to antifungals among (0.02 MB XLS)Click here for additional data file.Table S3A. fumigatus Af293 genes with respect to Af294, Af71, and A1163.Divergent (0.03 MB XLS)Click here for additional data file.Table S4A. fumigatus isolates.Distribution of polymorphic alleles among (0.02 MB XLS)Click here for additional data file.Table S5A. fumigatus loci exhibiting trans-species polymorphism.Five (0.02 MB XLS)Click here for additional data file.Table S6A. fumigatus core and species-specific genes.(0.02 MB XLS)Click here for additional data file.Table S7A. fumigatus-specific genes.Features of (0.02 MB XLS)Click here for additional data file.Table S8Top biological processes overrepresented among four lineage specificity groups.(0.02 MB XLS)Click here for additional data file.Table S9Enzymes that control the first step in secondary metabolite biosynthesis.(0.02 MB XLS)Click here for additional data file.Table S10Top PFAM domains overrepresented among four lineage specificity groups.(0.02 MB XLS)Click here for additional data file.Table S11Lineage specificity and chromosomal location.(0.02 MB XLS)Click here for additional data file.Table S12Selective constraints operating on virulence-associated genes.(0.02 MB XLS)Click here for additional data file.Table S13A. fumigatus Af293 allergens.Known (0.02 MB XLS)Click here for additional data file.Table S14A. fumigatus Af293 allergens.Predicted (0.02 MB XLS)Click here for additional data file.Table S15Selective constraints operating on sex genes.(0.02 MB XLS)Click here for additional data file.Table S16Families of transposable elements identified in the Affc genomes.(0.01 MB XLS)Click here for additional data file.Table S17Fungal genomes used in phylogenetic analyses.(0.02 MB XLS)Click here for additional data file.