DAZ-like (DAZL) gene located on the short arm of autosomal chromosome 3 (3p24), an essential master gene for the premeiotic development of male and female germ cells, is the father of the Y-chromosome DAZ gene cluster and encodes for RNA-binding proteins. Reported instances of positive association of DAZL gene mutations with infertility in men have been found in a Taiwanese population but not in Caucasians. There is no study from Tamil Nadu, South India, to demonstrate the role of DAZL gene in male infertility; we, therefore, analyzed a total of 287 men, including 147 infertile and 140 normozoospermic fertile controls from rural areas of Tamil Nadu, South India, to assess the phenotypic effect of DAZL mutations in this region of the world. Interestingly, all our samples showed absence of the A386G (T54A) mutation that was found to be associated with spermatogenic failure in the Taiwanese population. Therefore, we suggest that the A386G (T54A) mutation is not associated with male infertility in Tamil Nadu, South India.The  Male infertility accounts for ~50% of the cases, with quantitative or qualitative abnormalities of sperm production leading to spermatogenic failure. Genetic abnormalities, as well as numerical and structural chromosomal abnormalities, have been identified in men with unexplained oligozoospermia  in the so-called AZFc (AZoospermia Factor c) region. Among cases with Yq microdeletions, deletion involving the DAZ gene family is the most frequent finding. The DAZ gene is believed to have been transposed to the Y chromosome during primate evolution after the divergence of the New World and Old World monkeys, i.e. 35 × 106 years ago,[DAZL) located on the short arm of autosomal chromosome 3 (3p24), arose ~30-40 million years ago. DAZL is highly homologous to the DAZ gene clustered on the Y chromosome, with 83% similarity in the coding region of the cDNA.[DAZ and DAZL are transcribed exclusively in the germ line and encode RNA-binding proteins of the highly conserved RNA-recognition motif (RRM) class. The DAZL gene has been demonstrated to be an essential master gene for the premeiotic development of male and female germ cells.[The ome Yq11.3 in the ome Yq11.3 in the ome 3 3p2, arose ~et al,[DAZL gene may be responsible for spermatogenic defects in some cases and that the genetic defect is inherited in an autosomal recessive fashion.[DAZL gene in some infertile patients in a Taiwanese population was observed by Teng et al.[DAZL gene mutations with infertility in men.Lilford et al, suggeste fashion. A mutating et al. There arDAZL plays a crucial role in spermatogenesis in humans merits investigation. There has been no study on infertile men from Tamil Nadu, South India, that has attempted to demonstrate the role of DAZL gene in male infertility. We, therefore, analyzed 147 infertile and 140 normozoospermic fertile controls from rural areas of Tamil Nadu, South India, to assess the phenotypic effect of DAZL mutations.Whether This study was conducted on subjects attending infertility clinics in Erode and Nilgiris Districts of Tamil Nadu, South India. We collected samples of blood from 45 infertile men, semen samples from 72 infertile men, and paired samples (blood and semen of the same patient) from 30 infertile men; 140 normozoospermic (> 20 million sperms / ml of semen) males (10 paired samples) of proven fertility served as controls. All the procedures followed were in accordance with the ethical standards of the Center for Cellular and Molecular Biology, Hyderabad.Semen samples were obtained by masturbation on two different occasions, separated by a 3-week interval, following a 3-day period of sexual abstinence. Semen samples were allowed to liquefy for 30 min at 37°C. Sperms in the ejaculates were analyzed for their motility, number, and morphology as per the guidelines of the WHO, 1999. Written DAZL were designed and synthesized . DNA samples were analyzed for the presence of the exon 3 of DAZL by polymerase chain reaction (PCR) in a 0.2-mL thin-wall tube under the following conditions: 50 ng of DNA, 1.2 mM MgCl2, 200 µM dNTPs, 5.0 pMol of each specific primer, and 1 IU AmpliTaq Gold in a 10 µL reaction volume. Amplification was carried out in a MJ Research Thermal Cycler  using the following cycling conditions: after an initial denaturation step at 94°C for 5 min, cycle parameters were 94°C for 1 min, 60°C for 1 min, and 72°C for 60 sec for 30 cycles, with a final extension of 72°C for 10 min. Amplified products were quantified by 2% agarose gel electrophoresis.DNA was extracted from 10 ml of peripheral blood and fromet al.[DAZL sequences of the infertile and normozoospermic fertile control men were edited and compared with the reference sequence using AutoAssembler™ software .PCR products were optimized for sequencing.et al. Purified6 spermatozoa / ml), 50 asthenozoospermic (> 60% of nonmotile sperms), 24 oligoasthenozoospermic, and 1 each of varicocele , azoospermic (no sperms in the ejaculate), teratozoospermic , and necrozoospermic (100% dead sperms) infertile men. Analysis of exon 3 of the DAZL gene in a total of 287 men, including 147 infertile and 140 normozoospermic control men of Tamil Nadu, South India, revealed the complete absence of the previously reported A386G mutation in the DAZL gene. DAZL gene showing homozygous wild-type (A/A) allele at position 386.The seminogram of 147 infertile men ranged from azoospermia to oligozoospermia. There were 69 oilgozoospermic  of exon 2, which was seen in 3.5% and 2.59% of infertile and fertile men, respectively. Another variant was A386G, with a change of threonine to alanine (T54A) in exon 3, which was seen in 7.39% of the infertile and 0.86% of the fertile men. Thangaraj et al,[DAZL gene in an Indian population comprising people of different ethnic / linguistic origins: A260G (T12A) of exon 2 in 8.1% and 7.4% of the infertile and fertile control men, respectively, and A437G polymorphism in four fertile and one infertile man. Another variant, A386G, leading to a change of threonine to alanine (T54A) in the exon 3, was not found in infertile men or fertile controls in the Indian subcontinent. Similarly, our study on 147 infertile men and 140 normozoospermic fertile control men from rural areas of Tamil Nadu, South India, belonging to Dravidian linguistic family, for exon 3 showed complete absence of A386G (T54A) mutation in both infertile and fertile normozoospermic control men.In humans some very limited studies have been carried out on the ng et al, identifiaj et al, reportedet al.[et al.[et al.[et al.[DAZL gene may not be associated with male infertility in the Indian subcontinent. Our study on infertile and fertile normozoospermic men of Tamil Nadu, South India, revealed complete absence of the T54A mutation in exon 3 of DAZL. Therefore we suggest that the A386G (T54A) mutation is not associated with male infertility in Tamil Nadu, South India.Three independent studies conducted recently also did not find the T54A mutation in Caucasian populations.–16 Similet al. The distl.[et al. As the Tl.[et al. suggestel.[et al. suggeste


Herpesviridae, little information is available about the function of the two proteins. In this study, the C-terminus of DEV UL26 protein (designated UL26c), which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb). The mAb 1C8 was generated against DEV UL26 and UL26.5 proteins and used subsequently to map the epitope in this region. Both the mAb and its defined epitope will provide potential tools for further study of DEV.The Unique Long 26 (UL26) and UL26.5 proteins of herpes simplex virus are known to function during the assembly of the viruses. However, for duck enteritis virus (DEV), which is an unassigned member of the family 520IYYPGE525, which was located at the C-terminus of the DEV UL26 and UL26.5 proteins, was identified by mAb 1C8. The result of the ELISA showed that this epitope could be recognized by DEV-positive serum from mice. The 520IYYPGE525 motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of 1C8 with several truncated peptides derived from the motif. Alignment and comparison of the 1C8-defined epitope sequence with those of other alphaherpesviruses indicated that the motif 521YYPGE525 in the epitope sequence was conserved among the alphaherpesviruses.A mAb (designated 1C8) was generated against the DEV UL26c protein, and a series of 17 partially overlapping fragments that spanned the DEV UL26c were expressed with GST tags. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using mAb 1C8 to identify the epitope. A linear motif, 520IYYPGE525. The mAb and the identified epitope may be useful for further study of the design of diagnostic reagents for DEV.A mAb, 1C8, was generated against DEV UL26c and the epitope-defined minimal sequence obtained using mAb 1C8 was  UL26.5. This protein is present in the B-capsids of the HSV-1 assembly but is absent after the completion of DNA encapsidation and is not found in the mature virion [Herpesviruses exist widely in nature. The genomes of herpesviruses consist of linear double-stranded DNA; they differ in size (from approximately 124 to 235 kb), sequence arrangement and base composition . B-capsie virion .Herpesviridae [Anseriformes. DVE is a form of hemorrhagic enteritis that occurs in captive or free-flying waterfowl [Duck enteritis virus (DEV), an unassigned member of the family sviridae , is the aterfowl  and causaterfowl . The DEVaterfowl . CurrentUL26 and UL26.5, two nested in-frame genes, encode a capsid maturation protease and the minor capsid scaffold protein of DEV [The DEV has a linear double-stranded DNA genome of approximately 180 kb with a G+C content of 64.3% . The genn of DEV .B-cell epitopes are antigenic determinants that are recognized and bound by membrane-associated receptors on the surface of B lymphocytes . They caUL26 gene  of DEV, we used prokaryotic expression of the C-terminal 360-amino acid (348-707aa) protein in E. coli BL21 (DE3) after induction by Isopropyl-β-D-thiogalactopyranoside (IPTG) and termed the recombinant protein UL26c. Western blotting using murine antibody against DEV showed that UL26c could react with DEV antibody  infected with DEV , was used as an antigen to react with mouse anti-DEV antibody in this study. The results of both western blotting and ELISA showed that this peptide defined by mAb 1C8 could react with murine anti-DEV antibody , and smaller amounts of VP24 and VP21, the products of the UL26 gene. The UL26 and UL26.5 genes are expressed as 3'-coterminal transcripts, and the promoter for the UL26.5 gene is located within the coding region of the UL26 gene in HSV-1 [UL26 gene encodes a self-cleaved protease that generates the capsid proteins VP21 and VP24 [UL26.5 gene encodes the scaffold protein VP22a [UL26.5 gene, and itself, at a site 25 amino acids from the C terminus of its product [Herpesviridae and, to date, there has been no report of the structure and function of proteins UL26 and UL26.5 in DEV. It has been reported that the genes UL26 and UL26.5 in DEV are similar to their homologues in other alphaherpesviruses [Formation of the herpesvirus capsid is the first step in viral morphogenesis. The capsid of HSV-1 is found in the mature virions and in the nuclei of infected cells from which they originate. There are three distinct types of capsid, A-, B- and C-capsids, in infected cells . The B-cin HSV-1 -29. The and VP24 . Cleavagand VP24 . The UL2in VP22a , which hin VP22a . The B-cin VP22a . The UL2 product . DEV is sviruses . It has UL26 gene. Figure UL26 and UL26.5 by the alignment of the homologous in HSV-1. The DEV UL26 gene encodes a deduced protein of 707 amino acids [UL26.5 gene is in frame with UL26; therefore, the UL26.5-encoded proteins possess the same M site as the UL26 protein. Consequently, we hypothesized that the six bands detected using western blotting in this study may indicate the six products of UL26 and UL26.5.In this study, we expressed the C-terminus of DEV UL26 and immunized mice with both recombinant protein UL26c and the DEV particles, using a prime-boost protocol, and one mAb (1C8) was generated by cell fusion. Interestingly, six bands were detected in DEV-infected CEFs in western blotting analysis using mAb 1C8 in this study Figure . These mno acids . Comparino acids , which a520IYYPGE525, because any deletion of residues from either end of 520IYYPGE525 destroyed the ability of mAb 1C8 to bind. Comparative analysis of the amino acid sequences of the identified epitope with those of another 14 alphaherpesviruses revealed that the C-terminus of the linear B-cell epitope 521YYPGE525 is conserved among the selected alphaherpesviruses, except PRV, VZV and ILTV. It has been reported previously that the motif YYPGE is conserved in the scaffold proteins of alphaherpesviruses [in vitro but had a specific effect on incorporation of the portal. This indicated that this deletion had blocked DNA packing but had not interfered with the assembly of B-capsids [A series of 17 fragments that spanned the UL26c protein were expressed with a GST tag in this study, and used to screen for the minimal epitope recognized by mAb 1C8 using western blotting and ELISA. It was demonstrated that the minimal sequence of the epitope defined by mAb 1C8 appeared to be sviruses . It was -capsids ,34. Therin vitro neutralization test showed that mAb 1C8 could not neutralize the infectivity of DEV. The absence of neutralizing activity against DEV might indicate that this region has low immunogenicity or, more probably, that this region is not exposed on the surface of the virion. Indeed, the products of the HSV-1 UL26 gene are components of viral capsids, which are located inside of the viral tegument and envelope, while the products of the UL26.5 gene are components of B-capsids [UL26.5 gene, which also contains the sequences of the epitope defined by 1C8.The result of the -capsids . The pro-capsids . The B-c-capsids  and the The mAb 1C8 and its epitope, which was defined in this study, may prove to be very useful tools for the development of immunity-based therapeutic and diagnostic techniques for DEV, although the mAb lacked neutralizing ability.In this study, we generated a mAb, 1C8, and identified a novel linear B-cell epitope on the DEV UL26 and UL26.5 proteins using this mAb. The identified epitope and mAb 1C8 may increase our understanding of the function and location of the UL26 and UL26.5 proteins in DEV. It will also be a potential tool for the design of diagnostic reagents for DEV.2 atmosphere at 37°C. Chicken embryo fibroblasts (CEF) were prepared from 9- to 11-day-old specific-pathogen-free (SPF) embryonated eggs according to standard procedures. The DEV Clone-03 strain was purified from a commercial Chinese DEV vaccine using the plaque assay, as described previously [The SP2/0 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin in a humidified 5% COeviously . The vireviously .UL26 gene, we expressed a fragment of 1083 bp at the C-terminus of the DEV UL26 gene (UL26c) by amplifying the gene fragment from DEV Clone-03. The sequences and locations of the primers used for amplification and expression of the gene in this study are shown in Table Given that it is difficult to induce prokaryotic expression of the full E. coli BL21 (DE3) . A series of fusion proteins with the expected molecular weights were induced by IPTG and stained with Coomassie blue after SDS-PAGE as described previously [After construction, each recombinant expression construct was transformed into eviously . For preThree 8-week-old BALB/c mice were primed subcutaneously with DEV virus particles mixed with an equal volume of Freund's complete adjuvant , followed by two boosts of immunization with the recombinant protein UL26c. The protocols used for the preparation of mAbs and ascitic fluid have been described previously ,38. All The specificity and reactivity of the mAb 1C8 were also determined by western blotting using recombinant UL26c protein and CEF infected by DEV, respectively. Purified recombinant UL26c protein, truncated proteins and DEV in CEF were separated by denaturing SDS-PAGE. For western blotting, all the proteins and the virus were transferred onto nitrocellulose membranes, and detected as described previously . PurifieThe reactivity of the mAb with different truncated recombinant UL26 proteins was determined further by ELISA as described previously . BrieflyThe mAb 1C8 was tested for the presence of DEV-neutralizing antibodies. The CEF lysates containing DEV were mixed with ascites fluid containing mAb 1C8 and incubated at 37°C for 1 h; unrelated ascites fluid and PBS, used as negative controls, were treated in the same way. The mixture was added to the prepared CEF and incubated at 37°C. After 2 h of incubation, the mixture was removed and the cells were overlaid with 1% low-melting-point agarose containing 8% FBS. At 72 h post-incubation, the cells were overlaid again with 1% low-melting-point agarose containing 0.1% Ponceau. After further incubation at 37°C for 24 h, the plaques were counted and compared.To investigate whether the peptides could be recognized by anti-DEV antibody, the epitope peptide F12 was purified and used as antigen to coat ELISA plates (10 μg/well) to react with mouse anti-DEV antibody and mouse sera, respectively. Purified rGST was used as the negative control for F12. In addition, the purified F12 and rGST were also used to detect the reactivity of the epitope peptide by western blotting.Alphaherpesvirinae using the MEGALIGN program in Lasergene (DNAStar) with CLUSTAL W multiple alignments, as described previously [The mAb 1C8-defined epitope sequences and flanking sequences of DEV were compared with those of 14 other selected herpesviruses of the eviously . The 14 The authors declare that they have no competing interests.XL, SL and XK designed research; XL, ZH, YS and DY performed research; XL, SL, XK, HL and YW analyzed data; and XL, SL and XK wrote the paper. All authors read and approved the final manuscript.


Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb3 oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host.Three closely related bacterial species within the genus  N. meningitidis, N. gonorrhoeae and N. lactamica exclusively colonise mucosal surfaces in humans. While N. gonorrhoeae leads to gonorrhea, the other two species persist mainly in their host in the absence of disease. N. meningitidis does occasionally cause severe, life threatening illness, however. Little is known about the factors and elements that dictate the unique human interactions exhibited by each species. Moreover, the evolutionary relationships between these species are poorly characterized. Here, we describe two successive alterations in a single gene that can be linked first to all species within the genus Neisseria and then the species N. meningitidis. We also show these signature alterations have phenotypic consequences by affecting core respiratory metabolic processes. These findings have significant implications for the evolution of related bacterial species within a single host and provide a novel perspective on the episodic and reversible nature of innovative adaptation.The closely related bacterial species  Neisseria comprises Gram-negative, oxidase-positive diplococci that are frequently isolated from the mucosal surfaces of humans and two closely related species are important pathogens of man Neisseria gonorrhoeae is the etiologic agent of gonorrhea that remains one of the most common sexually transmitted diseases contributing to worldwide morbidity, mortality and infertility. Although treatable with antibiotics, no vaccine is currently available against the gonococcus. Neisseria meningitidis is primarily a commensal of the human oropharynx that, under incompletely understood circumstances, causes invasive disease and meningitis. Most cases of meningococcal disease are caused by clonal complexes of related sequence types (STs), the so-called hyper-invasive lineages The genus N. gonorrhoeae and N. meningitidis display remarkable conservation and uniformity at the levels of coding sequences, gene content and synteny. Nonetheless, comparative genome analyses have identified genes and gene clusters unique to either N. gonorrhoeae or N. meningitidis but few if any of the corresponding products can be specifically connected to the differential host interactions observed. A prime example of this situation would be the genes required for biosynthesis of polysaccharide capsule, which is essential to systemic meningococcal disease, and that are absent in N. gonorrhoeae. However, only a limited subset of capsular serogroups is associated with disease and 16–20% of meningococcal carriage isolates do not possess these genes N. meningitidis but how this element might relate to speciation or specifically to meningococcal biology remains unclear N. meningitidis and N. gonorrhoeae are further complicated by the existence of the closely related species Neisseria lactamica, a harmless commensal found predominantly in the upper respiratory tracts of infants and children N. meningitidis in which carriage is low during infancy and rises to high levels in adolescents and young adults, carriage of N. lactamica is high in young children but declines with age N. gonorrhoeae and N. meningitidis were identified iga1 and pptA genes encoding an extracellular endopeptidase and a protein targeting phosphoethanolamine transferase Despite their differing host interactions, mechanisms of transmission and ecology, N. gonorrhoeae and N. meningitidis are most consistent with allopatric divergence from a single common ancestor. Such a model was first proposed by Vazquez and colleagues based on the relatively reduced diversity of N. gonorrhoeae strains measured by multilocus enzyme electrophoresis (vs N. meningitidis) of house keeping genes and the fact that the primary niche for all human Neisseria species other than N. gonorrhoeae is the oropharynx N. gonorrhoeae arose as a clone of N. meningitidis that could colonize the urogenital tract. Prolonged physical isolation, niche specialization and genetic isolation would thus have driven speciation. This model was further supported by the analyses of the porA gene (encoding the class 1 outer membrane porin protein PorA) that is found in all strains of N. meningitidis and N. gonorrhoeae but absent in N. lactamica and other commensal Neisseria species N. gonorrhoeae examined to date carry an identical frameshift mutation that disrupts the integrity of the porA ORF Neisseria, ggt and adhC (encoding gamma-glutamyl transpeptidase and S-nitrosoglutathione oxidoreductase respectively), are also intact in N. meningitidis but inactivated due to frameshift mutations in N. gonorrhoeaeN. gonorrhoeae denotes descent from an organism carrying active forms of the genes, it remains unknown how extant isolates of N. meningitidis and N. gonorrhoeae might relate to such an ancestral population. Despite the potentially confounding contributions of shared ancestry and genetic exchange, a recent study utilizing multilocus sequence typing (MLST) demonstrated the ability to readily categorize isolates of N. meningitidis, N. gonorrhoeae and N. lactamica into three distinct species The highly conserved genetic structure and human host restriction observed for c oxidases have been implicated in major evolutionary transitions in anthropoid primates and carnivorous plants Neisseria genomes is of the c-family or cytochrome cbb3 oxidase type cbb3 oxidases consist of four subunits encoded by tandemly arranged genes (B) where dioxygen is reduced c-type cytochromes believed to channel electrons to the dinuclear center cbb3 oxidase in some organisms has a high affinity for oxygen and is often associated with growth under conditions of oxygen-restriction N. gonorrhoeae and N. meningitidis inhabit niches associated with low oxygen tension cbb3 oxidases couple oxygen reduction to translocating protons across the inner membrane such that metabolic energy is conserved for subsequent ATP synthesis Oxygen reductase members of the heme-copper superfamily act as terminal oxidases in all domains of life and play a central role in aerobic energy generation and conservation ed genes  [23]. CcN. gonorrhoeae and N. meningitidis also possess a truncated denitrification pathway in which nitrite (NO2−) is first reduced to nitric oxide (NO) by NirK (AniA) that is then reduced to nitrous oxide (N2O) by NorB nirK and norB genes are differentially controlled by a number of transcriptional regulators that are responsive to changes in the levels of oxygen, NO2− and NO N. gonorrhoeae and N. meningitidisN. gonorrhoeae and N. meningitidis is their highly recombinogenic nature that results from inter- and intraspecies genetic exchange Neisseria species, this largely restricts imports to donors from within the genus. In the particular case of N. meningitidis, interspecies recombination at numerous loci encoding surface antigens has been widely documented N. gonorrhoeae, N. meningitidis and N. lactamica species represent cohesive, differentiated entities Neisseria species remains poorly understood. Moreover, it remains unclear what genetic elements dictate the unique human interactions exhibited by each species.A cardinal feature of N. meningitidis as it is found in all strains examined and absent from all isolates of N. gonorrhoeae and N. lactamica and of other commensals tested. Although this mutation results in the truncation of an essential component of the cytochrome cbb3 oxidase, the sole neisserial respiratory oxidase, it conditionally affects nitrite consumption by the nitrite reductase that functions in the denitrification pathway. These findings provide evidence that an alteration in the circuitry of respiratory electron-transfer networks is associated with N. meningitidis speciation.Rare genomic changes, signature mutations occurring in the genomes of particular clades, have frequently been employed to resolve phylogenetic uncertainties O-linked protein glycosylation system in N. gonorrhoeae strain MS11 c-type cytochrome domain architecture as opposed to the di-heme form found in all other CcoP proteins (as annotated by possessing the IPR004678 domain of the InterPro database)  to that in N. gonorrhoeae save for the presence of transitional substitution in codon 366 (CAG to TAG resulting in a premature stop codon). The N. meningitidis forms would then be predicted to be truncated at the end of the AlaSerPro-rich, low complexity region (LCR) that separates the second and third c-type heme domains and encompasses the glycan attachment site ccoP from 78 additional N. meningitidis isolates used in this study  . When this study  further  species . Thus, tccoP genes using the majority rule consensus tree constructed with Clonalframe revealed five distinct branches corresponding to each of the Neisseria species: N. meningitidis, N. gonorrhoeae, N. lactamica and other Neisseria including N. cinerea, N. polysaccharea, N. sicca, N. subflava and N. flavescens further differentiating these isolates from one another. Within each clade, ccoP genes were well conserved  although ccoP genes belonging to commensal isolates other than N. lactamica were much more diverse (p-distance = 0.165)  . This wa= 0.165)  where synalFrame .N. meningitidis, we sought to determine what phenotypic consequences might ensue from such an alteration. We therefore took a comparative approach to addressing this point by constructing strains of N. meningitidis MC58 expressing the tri-heme CcoP form found in N. gonorrhoeae and strains of N. gonorrhoeae VD300 expressing the truncated di-heme form from N. meningitidis . Like CcoP, c5 is predicted to be membrane-associated and also possesses an AlaSerPro-rich LCR that encompasses the attachment sites of its O-linked glycan between its two c-type heme domains N. meningitidis, a tetracycline resistance gene cassette insertion mutation in cycB that disrupts the integrity of the ORF distal to the first heme domain was shown to abolish nitrite reduction and nitrite-dependent growth under microaerobic conditions N. gonorrhoeae, a cycB null mutation was reported to result in increased sensitivity to growth inhibition by excess oxygen and small decreases in respiratory capacity c5 is a prime candidate to act as an electron carrier in pathways ultimately targeting both cytochrome cbb3 oxidase and NirK.In another approach, BLAST searches were performed using the third heme encompassing domain immediately C-terminal of the major AlaSerPro-rich LCR in c5 and the tri-heme CcoP form, we examined what genetic interactions might exist between cycB and the different ccoP alleles with regard to the truncated dentrification pathway. To this end, a cycB null mutation was generated in which the entire open reading frame was deleted. Derivatives of N. gonorrhoeae carrying this allele were distinctive in that they exhibited a severe growth defect manifested as poor plating efficiency and slow growth that was not seen in equivalent strains carrying the previously characterized cycB insertion mutation (data not shown). While profiling of c-type heme proteins confirmed the absence of intact c5 in both backgrounds, strains carrying the cycB insertion mutation expressed a c-type heme protein whose migration corresponded to that predicted for a truncated, mono-heme c5 form resulting from disruption of the open reading frame at residue 171  backgrounds (data not shown). Taken together, these results document clear differences in the requirements for nitrite reduction associated with di- and tri-heme encoding ccoP alleles. Accordingly, the findings strongly suggest that strains of N. meningitidis are fundamentally distinct from those of other neisserial species with regard to respiratory denitrification.Reciprocal experiments were carried out in nditions . Both N.sumption . These rc growth . Measurec growth . These pc growth . To ensuN. meningitidis, transposon insertion mutations were generated in the cloned ccoP gene that were then introduced into a wildtype background. However, transformants were only recovered for those insertions that mapped 3′ to the ORF encompassing the second c-type heme domain (i.e. within the LCR and third c-type heme domain). To ensure that this selectivity was not due to some peculiarity of the constructs themselves, a strain carrying a second active copy of ccoP at an ectopic site was created. Transformants for all insertion mutations were recovered using this merodiploid strain . Here as a control, a strain carrying a tandem duplication of ccoP (by Campbell-type plasmid integration) was used in which the distal gene copy was non-functional due to a deletion of the promoter and translational initiation sites. Transformants for all insertion mutations were recovered using this strain and importantly, all those that could not be recovered in the wildtype background mapped exclusively in the non-expressed ccoP copy  were likely identical in both species.To examine the role of CcoP in d strain . A similcoP copy . As mutaccoP, a transcriptional fusion construct was made in which expression was linked to tac-UV5 control sequences and introduced at an ectopic site in the neisserial genome. As this construct does not include an associated QlacI gene, repression requires a background expressing LacIQ. For practical purposes, we therefore carried out these experiments in N. gonorrhoeae which enabled us to first introduce the ectopic de-repressible ccoP allele in a background expressing di-heme CcoP from the endogenous locus  and the third heme domain of N. gonorrhoeae CcoP was constructed. This was done in a manner such that the relative spacing between the two heme domains in the hybrid was maintained as seen for wildtype c5 and was facilitated by using a conserved stretch of residues in the AlaSerPro-rich linker domains  and a di-heme expressing ccoP allele. The c5-CcoP chimaera was detected as a novel cytochrome c species that migrated with a mobility equivalent to that seen for endogenous c5  share 74% sequence identity with the corresponding C-terminal segment of c5 and 47% with the first heme domain of c5. Such a scenario would account for the presence of the AlaSerPro-rich region between the second and third CcoP heme domains. Although it is difficult to establish amino acid identity within these AlaSerPro rich regions (due to their underlying low complexity), both the lengths of the AlaSerPro stretches (34 residues in c5 versus 42 in CcoP) and overall alanine richness (47.1% Ala residues in c5 versus 47.6% in CcoP) are similar between the two. Moreover, these stretches each bear serine occupancy sites for the glycans associated with the general O-linked glycosylation system in N. gonorrhoeaec5 and the tri-heme CcoP provide strong evidence for modular-based evolution underlying CcoP neofunctionalization.Given its unusual activity and unique distribution within oP forms . This hyNeisseria represent an intriguing model system in which to investigate the evolution trajectories of related pathogenic and commensal bacteria in a single host. Here, we report the identification of a SNP unique in its association with the species N. meningitidis that leads to truncation of the c-type heme protein CcoP, an essential component of its sole, terminal respiratory cytochrome oxidase. The dichotomy in neisserial phylogeny revealed through this altered molecular character raises the obvious question as to why this change appears to be so adaptive on the one hand and yet at the same time, so restricted in its distribution. From the sole viewpoint of aerobic respiration, CcoP functions by supporting cytochrome cbb3 oxidase activity. However, we failed to discern any phenotypic alterations in either the N. gonorrhoeae or N. meningitidis strains varying solely in expression of the di- versus tri-heme forms under standard lab conditions. Therefore, a key factor here may be the altered circuitry of electron transfer favoring microaerobic (oxygen reduction by cytochrome cbb3 oxidase) versus combined microaerobic denitrifying (oxygen reduction by cytochrome cbb3 oxidase supplemented by nitrite reduction by NirK) respiration. For example, the ability of di-heme CcoP to promote solely cytochrome cbb3 oxidase activity (at the expense of reduced electron carriage to NirK) could be adaptive under more aerobic conditions or in situations where nitrite levels might be diminished. Moreover, nitrite reduction could come with significant metabolic cost as it generates nitric oxide that can be toxic and growth inhibitory N. meningitidis strains express the NorB nitric oxide reductase (reducing nitric oxide to innocuous nitrous oxide), toxic NO can accumulate so rapidly that growth inhibition occurs before sufficient NorB activity has been expressed  N. meningitidis as NirK-dependent, nitrite sensitivity occurring under a number of growth conditions N. meningitidis strains are under relaxed selective constraints with regard to NirK-mediated nitrite reduction. Specifically, nirK was not detected in 7 of 26 N. meningitidis strains examined by microarray-based genome hybridization technology nirK found frameshift and inactivating missense mutations in 5 of 23 and 8 of 31 strains respectively N. meningitidis, this is not the case in N. gonorrhoeae and other Neisseria species. In the latter strains, alleles encoding intact NirK are found in all available genomic and individual sequences  is a molecular marker of species within the genus Neisseria. The ability of tri-heme CcoP to act as a redox partner in a pathway that ultimately can transfer electrons to NirK would dramatically reshape the organization of electron transport chain, providing broader connectivity to electrons emanating from bc1 through c4 as well as c5  and N. lactamica isolates used here that were derived from carriage studies within the United Kingdom as well to other commensal species for which the data set is small. Nonetheless, the ccoP tri-heme allele is present in all Neisseria species for which data are available and it is conspicuously absent in any other species including members of the most closely related genera in the family Neisseriaceae such as Chromobacterium, Eikenella, Kingella, and Laribacter and more distant relatives within Betaproteobacteria.Another extraordinary aspect of the l as c5  [51], [5ccoP allele suggests that N. meningitidis as a clade has either undergone a relatively recent, dramatic reduction in population size (with a ccoP SNP -bearing strain first to pass the bottleneck) or that there has been a selective sweep of the SNP allele through the population via natural genetic transformation. Both scenarios are plausible given the relatively short time period over which sequential bottlenecks associated with epidemic spread can lead to clonal descent N. meningitidis, N. lactamica and other commensal Neisseria species inhabit the same apparent oropharyngeal niche and bi-directional, interspecific genetic exchange of some loci occurs frequently N. meningitidis expressing tri-heme CcoP and of N. lactamica and other commensal species expressing di-heme CcoP arise but are purged due to reduced fitness. Consistent with this idea, the low p-distances observed by ClonalFrame for ccoP genes belonging to N. meningitidis, N. lactamica and N. gonorrhoeae are indicative of the conserved nature within species such that diversity may result in reduced fitness of the organism. The lack of recombination observed by RDP3 analysis  further support this hypothesis. Therefore, other as yet unidentified differences in gene repertoire or expression are likely epistatic to ccoP.The SNP-associated analysis  among N.in vitro studies have identified both azurins (members of the cupredoxin family) and c-type heme cytochromes as electron donors to other members of the blue, copper - containing nitrite reductase (CuNIR) family in vivo. Although both N. meningitidis and N. gonorrhoeae express a lipoprotein form of azurin termed Laz, it has been reported that Laz is nonessential for growth under either aerobic or anerobic conditions (in the presence of nitrite) c550 protein was recently shown to disrupt NirK dependent growth and nitrite utilization in Bradyrhizobium japonicumc-type heme domains translationally fused to the carboxy-termini of the nitrite reductase domain have been identified in other proteobacterial species by genome mining c-type cytochrome provided conclusive evidence for the direct transfer of electrons between these partners cycB and ccoP here along with related observations c5 and CcoP (presumably in the context of cbb3) come in close contact with NirK. However, the NirK lipoprotein in its active trimer form is proposed to be linked to the outer membrane cbb3 oxidases must be integrated into the inner membrane in order to fulfil their role in proton pumping essential to ATP synthesis. While the carboxy terminal extensions  found in CcoP and c5 might function to bridge a potential periplasmic gap, more detailed studies of the spacial distribution and organization of the structures involved are needed to address this matter. Regardless of the molecular nature of electron transfer between CcoP and NirK, our finding here that a component of the oxygen reducing respiratory complex can be essential to the activity of an alternative reductase is unprecedented.An important aspect of this topic relates to which redox partners carry out direct electron transfer to neisserial NirK. Although Neisseria. They also demonstrate that microaerobic denitrification is a metabolic pathway of major influence in these bacteria and support the position that N. gonorrhoeae, N. lactamica and N. meningitidis fully deserve their designation as a distinct species, because they are clearly evolving independently. These findings also provide a dramatic example of how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host.In summary, these findings provide a novel perspective on the evolution of species within the genus N. meningitidis isolates initially employed in the evaluation of the (MultiLocus Sequence Typing) MLST typing method were used for ccoP sequence analysis th century, included several isolates from each of seven recognised hyper-invasive clonal complexes: (Sequence Type) ST-1, ST-5, ST-4, ST-11, ST-32, ST-8, and ST-41/44 in addition, to isolates belonging to clonal complexes ST-22, ST-23 and ST-13 (cc269) as well as isolates with unique sequence types. A total of 11 unencapsulated N. meningitidis isolates containing the capsule null locus were also investigated and these included isolates from clonal complexes ST-53, ST-198 and ST-334 (Neisseria and N. gonorrhoeae isolates used for sequence studies are listed in 2 atmosphere. DNA was extracted using an IsoQuick Nucleic Acid Extraction kit (Orca Research Inc.) according to manufacturer's instructions. The neisserial strains used for functional studies are described in 2 as described previously 3, in 25 ml Sterilin McCartney bottle shaken at 190 r.p.m. 2. Growth was monitored by measuring the optical density at 600 nm (OD600) in a WPA biowave CO 8000 Cell Density Meter. E. coli DH5α and HB101 were used for plasmid propagation and cloning experiments and were grown on Luria-Bertani media (LB). Antibiotics were used at the following concentrations for N. gonorrhoeae: chloramphenicol, 10µg/ml; erythromycin, 8µg/ml; tetracycline, 4µg/ml; for N. meningitidis: erythromycin, 8µg/ml; tetracycline, 20µg/ml; for E. coli: chloramphenicol, 30 µg/ml; erythoromycin 300µg/ml; kanamycin 50µg/ml; ampicillin 100µg/ml; tetracycline, 15µg/ml; streptomycin, 100µg/ml. pUP6 is a derivative of pHSS6 that carries two gonococcal DNA uptake sequences lac promoter/operator at an intergenic chromosomal site located between the gonococcal genes lctP and aspC. Cloning in front of the lac promoter thus allows for chromosomal integration of the insert between lctP and aspC in N. gonorrhoeae and subsequent IPTG inducible expression A total of 71 d ST-334 . The nonccoP genes from strains described in Amplification and sequencing of Nucleotide sequence data for forward and reverse strands were assembled with the STADEN software package ccoP locus of N. meningitidis with the tri-heme ccoP allele of N. gonorrhoeae and vice versa, the endogenous ccoP locus of N. gonorrhoeae with the di-heme ccoP allele of N. meningitidis, the strains MC58 and VD300 were transformed with the plasmids pUP6NGOccoP::mTnerm#22 and pUP6NMEccoP::mTnerm#17 respectively, and selected on GC agar plates containing erythromycin  reactions into an otheriwse wildtype allele. Each pair of PCR fragments containing the mutation was created using primers FE1141 (5′-CGGAATTCGAGCTCTCTTTATCTGTTTCCTGTTAGTAC-3′) in combination with FE1156 (GTAGACCTATTTGCCATCCGCTTTGGCGGC-3′5′-AACGGTTTC), and FE1155 (GTCTACTAGGAAACCGTTTGTGCCGCCTGCC-3′5′-CGGATGGCAAA) in combination with Av2045 (5′-TTGGACGACGGACGAAGTCTC-3′) was used to make the second overlapping PCR fragment. Altered base pairs including the novel stop codon and an AccI restriction site (underlined) are shown in bold. The overlapping PCR fragments were spliced together using primers ccoP seq5′ (5′- TCGGTTATCTGGTTATGTATCC -3′) and Av2044 (5′-GAATACGCTCTCCCTCTTTACC-3′). Purified PCR products were used to genetically transform N. gonorrhoeae strains AccI digestion of PCR fragments. Direct DNA sequencing of PCR fragments derived from the transformants was done, using appropriate primer sets, to verify the introduction of the stop codon and the absence of any other alterations.The premature stop codon was introduced into N. gonorrhoeae ccoP gene along with 133 bp upstream of the gene and 151 bp downstream of the gene was amplified using primers FE1140 (5′-TACTCTATATCGTCTTCAACAGG-3′) and FE1144 (5′-CAAAAATATCAGTCGGTCTGACTGC-3′). The resulting PCR fragments were digested with unique, flanking EcoRI/SacI sites and cloned into the polylinker of pUP6, yielding plasmid pUP6NGOccoP. Similarly, the N. meningitidis ccoP gene along with 133 bp upstream of the gene and 350 bp downstream of the gene was amplified using primers FE1140 and FE1181 (5′-GCGGGATCCGAGCTCTTACAACAAATAGGCAGTCTGCG-3′). The resulting PCR fragments was digested with unique, flanking EcoRI/BamHI and cloned into the polylinker of pUP6, yielding plasmid pUP6NMEccoP. Mini-transposon (mTn) mutagenesis was performed on both pUP6NGOccoP and PUP6NMEccoP as previously described cm) or erythromycin (mTnerm), were isolated, mapped by PCR and sequencing using appropriate primer sets, depending on the location of the transposon insertion. Primer sequences and the detailed location of each transposon insertion site are available upon request.The ccoP gene the coding region of ccoP and 20 bp of upstream DNA, to include the RBS, was amplified by PCR from N. gonorrhoeae strain VD300 by using the forward primer av2226 (TTAATTAATGTGATAACGGAGCAAAACAATG - 3′5′ - CGAAAACC), PacI site underlined, and the reverse primer FE1144 (5′ - CAAAAATATCAGTCGGTCTGACTGC - 3′). The PCR reaction was performed by Advantage HD polymerase (AH diagnostics) to create blunt ends. The resulting PCR product was cut with PacI and cloned into pGCC6, digested with PmeI and PacI, at an intergenic chromosomal site located between the gonococcal genes lctP and aspC and linked to the lac promoter/operator. The resulting plasmid pGCC6NGOccoP was then used to transform strain KS351 (ccoP2x) and transformants were selected for growth on GC agar plates containing chloramphenicol. To inactivate the ccoP allele from the endogenous locus, the N. gonorrhoeae strain carrying the ectopic de-repressible ccoP allele (KS345) was transformed with pUP6NGOccoP::mTnerm#26  integration of pUP6NGOccoP on the chromosome. The resulting N. gonorrhoeae strain carrying a tandem duplication of ccoP (KS344) was transformed with a series of characterized pUP6NGOccoP::mTnerm and pUP6NGOccoP::mTncm plasmids and transformants were selected on GC plates containing erythromycin and chloramphenicol respectively. The location of each transposon insertion, to either the expressed or non-expressed copy of ccoP, was determined by PCR using appropriate primer sets (available upon request).To create an N. meningitidis strain MC58 was transformed with the plasmid pGCC6NGOccoP to create a strain carrying an ectopic copy of N. gonorrhoeae ccoP. Transformants were selected on GC agar plates containing chloramphenicol and confirmed correct by appropriate PCR primer sets and immunoblotting followed by heme-staining to visualize expression of the tri-heme form of CcoP. The resulting N. meningitidis strain (KS350) carrying the ectopic N. gonorrhoeae ccoP and the wildtype strain MC58 were transformed in parallel with a series of characterized pUP6NMEccoP::mTnerm plasmids and transformants were selected on GC plates containing erythromycin.For this purpose, the wildtype cycB insertion mutants, cycB::tet was introduced into MC58 and into VD300 by transformation and transformants were selected for growth on GC agar plates containing tetracycline. To obtain a complete deletion of cycB in N. gonorrhoeae the primers FE2223 (5′ - TCCGCAAAGCGGTGGAAATG- 3′) and FE2220 (5′ – GTTTAAACTGTCGCGGAGTTGTTTCATTTG - 3′) were used to amplify an 800 bp fragment of genomic DNA upstream of the cycB gene and the primers FE2221 (5′-TGAAACAACTCCGCGACAGTTTAAACACTATATGGCAAACCAATCCGGTGC -3′) and FE2225 (5′ –TATTTTGACAAACCACCGGAG - 3′) were used to amplify a 900 bp fragment of genomic DNA downstream of the cycB gene. The PCR products contained regions of homology at the 3′ end of the upstream fragment and at the 5′ end of the downstream fragment such that they could be spliced together by PCR-based splicing-by-overlap extension leaving out the entire cycB gene. This was done by using the primers FE2224 (5′–TTCATCCGGACAAACGCGTTG -3′) and FE2222 (5′–AACCTGTCGCTCTACGGCGAAC- 3′). The purified PCR product was used to genetically transform N. gonorrhoeae by a non-selective transformation technique c5 expression, including the truncated c5 form seen in the cycB::tet mutants, was verified by heme-stained blots of whole cell extracts.To generate the cycB and the cycB-ccoP hybrid allele was performed by cloning PCR amplified products into a unique SacI restriction site in plasmid p2/16/1 iga locus of the gonococcal chromosome. The resulting plasmids were then used to transform the mutant KS337  and transformants were selected for growth on GC agar plates containing erythromycin.Ectopic expression of cycB allele the coding region including about 250 bp of upstream DNA was amplified by PCR from N. gonorrhoeae strain VD300 by using the forward primer c555F_SacI (GAGCTCAATTGGCAAAGGTTATCTTGCG - 3′5′ – TGCA) in combination with the reverse primer FE2200 (GAGCTCACACCCATTTGATGTCATTTCC - 3′5′ – ATTC), SacI sites underlined.For cloning of the wildtype cycB-ccoP hybrid allele was constructed by PCR-based splicing-by-overlap extension such that the region encoding the amino-terminus of cycB encompassing the first c-type heme domain and about 250 bp of upstream DNA was fused to the region encoding the third heme domain of ccoP. The relative spacing between the two heme domains in the hybrid protein was maintained as seen for wildtype cycB by facilitating a conserved stretch of four residues (Ala-Ala-Pro-Ala) in the C-terminal end of the AlaSerPro-rich linker domains. The cycB part of the hybrid allele, including about 250 bp of upstream DNA, was amplified by PCR using the flanking primer c555F_SacI in combination with the primer FE2216 (5′ – TCCGCTTTGGCCGCAGGGGCTGCCGCACCCTTGTC – 3′). The region encoding the third heme domain of ccoP was amplified by PCR using the flanking primer FE2218 (GAGCTCATTCGATATGAATCCGGATTTCTG – 3′5′ – CCGG), SacI site underlined, in combination with the primer FE2217 (5′ – ACAAGGGTGCGGCCGCACCTGCCGCCAAAGCGGATG– 3′). The two overlapping PCR fragments were spliced together by using the flanking primers c555F_SacI and FE2218.The Nitrite concentrations in culture media were measured by a colourimetric assay as previously detailed c-type cytochromes were prepared by harvesting bacteria from plates into 10mM Hepes buffer pH 7.0, subjecting the suspension to 5 cycles of freezing and thawing, and finally suspending the bacteria in 1% w/v n-dodecyl β-D-maltoside (Sigma-Aldrich). After 10 min incubation at 50°C samples were separated on 10% or 12% Criterion XT Precast Gels  and blotted onto PVDF membranes. To visualize heme-dependent, peroxidase activity of the c-type cytochromes, the membranes were first incubated with SuperSignal West Pico Chemiluminiscent Substrate, according to manufacturers instructions , and then exposed to X-ray film  Whole-cell extracts for detection of Figure S1ccoP in the 33 alleles among Neisseria species. The scale above the graph is in amino acids. CcoP functional domains are represented as distinct blocks with green blocks representing heme groups and the red bar depicting the AlaSerPro-rich region. Black vertical bars above this represent synonymous nucleotide polymorphisms with non-synonymous polymorphisms depicted below the diagram. Red vertical bars both above and below represent synonymous and non-synonymous polymorphisms respectively detected among N. gonorrhoeae, N. lactamica and N. meningitidis isolates only. The asterisk (*) indicates the position of the stop codon found among N. meningitidis isolates. The vertical bars found beside to the left of the alignment beside allele numbers indicate the species to which the ccoP alleles belong; black: N. gonorrhoeae; red: N. lactamica; blue: N. meningitidis; green: other Neisseria species including N. cinerea, N. polysaccharea, N. subflava, N. mucosa, N. flavescens and N. sicca.CcoP polymorphisms. Distribution of polymorphisms along the 1, 336 nt of gene (7.54 MB TIF)Click here for additional data file.Figure S2N. meningitidis. Cultures of wild-type (MC58) (open squares); NgoccoP (KS348) (open circles); cycB (c5-) (filled squares) and NgoccoP, cycB (KS349) (filled circles) growing under aerobic conditions (A) and under microaerobic conditions without nitrite (B). The results shown are representative of three independent experiments.Effects of CcoP domain alterations on aerobic and microaerobic growth in (0.10 MB TIF)Click here for additional data file.Figure S3N. gonorrhoeae. Cultures of wild-type (VD300) (open squares); and mutants ccoP2x (KS335) (open circles); cycB (KS336) (filled squares); ccoP2x, cycB (KS337) (filled circles); NmeccoP (KS340) (open triangles); NmeccoP, cycB (KS341) (filled triangles) growing under aerobic conditions (A) and under microaerobic conditions without nitrite (B). The results shown are representative of three independent experiments.Effects of CcoP domain alterations on aerobic and microaerobic growth in (0.09 MB TIF)Click here for additional data file.Figure S4cycB insertion mutant expresses a truncated c5 protein. The cycB gene, encoding the c5 protein, was disrupted by insertion of a tetracycline resistance (TetR) gene c5 ORF that terminates at residue 171 followed by eleven residues derived from sequences within the tetracycline resistance gene insertion before a stop codon (indicated by an asterisk). This results in a 182 residue c5 protein retaining the membrane-proximal heme domain (in gray).The (0.05 MB TIF)Click here for additional data file.Figure S5N. gonorrhoeae strains: 1, wild-type (VD300) and mutants 2, cycB (KS336); 3, ccoP2x (KS335); 4, ccoP2x, cycB (KS337); 5, NmeccoP(KS340); 6, NmeccoP, cycB (KS341); were grown under microaerobic conditions plus 5 mM nitrite. Samples were taken after one (1) and six (6) hours of growth, and whole cell lysates were analyzed by immunoblotting with anti-NirK antibodies.NirK expression during growth under microaerobic conditions. (0.15 MB TIF)Click here for additional data file.Figure S6c5-CcoP translational fusion. A translational fusion consisting of the amino-terminus of c5, encompassing the first c-type heme domain, and the third c-type heme domain of CcoP was made by exploiting a conserved stretch of residues  in the AlaSerPro-rich linker domains. Also underlined are the two cysteine and single histidine residues found in a CXXCH motif required for disfulfide bonding to the vinyl groups of heme. The total number of amino acids (and thus the relative spacing) between the two c-type heme domains was maintained as seen for wildtype c5. The hybrid-encoding gene was then expressed from an ectopic site.Strategy for construction of a (15.81 MB TIF)Click here for additional data file.Table S1(0.15 MB DOC)Click here for additional data file.Table S2(0.09 MB DOC)Click here for additional data file.Table S3(0.04 MB DOC)Click here for additional data file.Table S4(0.03 MB DOC)Click here for additional data file.


We present a tool for tracking coronary vessels in MRIscans of the human heart to aid in the screening of heartdiseases. The vessels are identified through a single click insideeach vessel present in a standard orthogonal view. The vesselidentification results from a series of computational stepsincluding eigenvalue analysis of the Hessian of the MRI imagefollowed by a level set-based extraction of the vessel centerline.All identified vessels are highlighted using a virtual contrastagent and displayed simultaneously in a spherical curvedreformation view. In cases of over segmentation, the vessel tracescan be shortened by a click on each vessel end point. Intermediateanalysis results of the vessel computation steps can be displayedas well. We successfully validated the tool on 40 MRI scansdemonstrating accuracy and significant time savings over manualvessel tracing.


Phytophthora are among the most destructive plant pathogens and therefore have attracted considerable attention during the past two decades. Although it has been realized that a close phylogenetic relationship exists, so far sharp distinction has been made between the obligate biotrophic downy mildews and the hemibiotrophic Phytophthora. In the study presented here, it is shown that a continuum of character states from hemibiotrophic Phytophthora species to obligate biotrophic downy mildews is present. Intermediate character states between downy mildews and Phytophthora species exist in several rare parasites of grasses, which are not embedded within the major clades of the downy mildews but are placed sister to these, with unresolved affinities to both these clades and to Phytophthora. They still have retained traits hitherto thought to be exclusive for Phytophthora. A careful review of previous research is presented and it is highlighted that uniquely for downy mildews, Poakatesthia may form an intracellular mycelium, growing through several host cells. In addition, scanning electron microscopy reveals that sporangiophore growth is not determinate in Viennotia and that outgrowth from sporangiophores is very similar to Phytophthora infestans. It is concluded that the sharp morphological distinction between downy mildews and Phytophthora species (that are often placed in separate families and even different orders), is rather artificial, since all features thought to be exclusive to Phytophthora or the downy mildews are united in the rare grass-parasitizing down mildew genera Viennotia and Poakatesthia and the enigmatic genus Sclerophthora. Therefore, several paradigms regarding the distinction between Phytophthora and the downy mildews need to be reconsidered.Downy mildews and root and foliar rots caused by  Phytophthora species are among the most destructive rot-causing pathogens of plants, responsible for several catastrophic events, like the sudden oak death in North America, caused by Ph. ramorumPh. infestansPhytophthora species often have a much wider host range. In addition to these characteristics, there are three main morphological characteristics that are thought to distinguish Phytophthora species from downy mildews. First, downy mildews do not form intracellular mycelium, but invade host cells only by haustoria Phytophthora is able to grow through both living and dead cells Phytophthora the sporangiophore may grow further after sporangia have formed. Third, it is generally believed that in downy mildews all sporangia ripen simultaneously, while in Phytophthora sequential maturation takes place. These differences were thought to be of major importance and have been used as an argument to postulate a deep divide between downy mildews and Phytophthora, placing the downy mildews in a family of its own, the Peronosporaceae, while Phytophthora – on the basis of its thallus growth and general morphological characteristics – has usually been placed in the family Pythiaceae of the order Pythiales Phytophthora. Only in one study Phytophthora was found to be monophyletic, often with maximum support Phytophthora infected plants usually rot and die, whilst those infected with downy mildews may remain almost or completely asymptomatic for long periods and may even recover, particularly in secondary infections of natural populations. However, the effectors secreted by these pathogens are similar Phytophthora which is initiated by necrosis inducing proteins Bremia, only seldom do plants die from infections with this pathogen. Bremia is not culturable on artificial media and therefore it is unlikely that necrotic parts of infected plants can serve as a source of nutrition for the hyphae. In general, the downy mildews have evolved into well adapted obligate biotrophic pathogens, which may be transmitted with the seeds of their host plants and many hardly cause observable symptoms. This is especially the case in Basidiophora and some species of Hyaloperonospora, which systemically infect their hosts and in effect develop endosymbiotically Phytophthora and the obligate biotrophic downy mildews, the question then whether some missing links between these two groups still exist. Recent molecular phylogenetic studies Hyaloperonospora, Perofascia; downy mildews with coloured conidia – Peronospora, Pseudoperonospora; downy mildews with pyriform haustoria – Basidiophora, Benua, Bremia, Novotelnova, Paraperonospora, Plasmopara, Plasmoverna, Protobremia). However, in none of the analyses to date did these apparent sister relationships receive high bootstrap support and therefore this molecular evidence must be treated cautiously. If the before mentioned artefact in phylogenetic reconstruction Phytophthora remains unclear. Göker et al. Hyaloperonospora and Perofascia had started from hosts in the Poaceae and Thines et al. Phytophthora and the rather basal position of the graminicolous downy mildews that was revealed by recent molecular phylogenetic investigations.Because of the close phylogenetic relationship revealed by molecular studies and the similarity in the effector genes between the hemibiotrophic Phytophthora, can also be observed in Viennotia , the apophyses, which are typical for iennotia . In scanapparent . In bothapparent . These aPhytophthora-like outgrowth in the genus Viennotia is unique for downy mildews. Outgrowth in Viennotia and Phytophthora takes place trough the middle of the scar left after sporangium dispersal and not as previously depicted by de Bary Plasmopara halstedii, abnormal sporulation with outgrowth from a sporangiophore bearing sporangia has been observed on sunflower roots by Novotelnova Phytophthora has been considered the most important characteristic in delimitating downy mildews. Therefore the finding that in Viennotia sporangiophore growth is indeterminate provides important evidence that sharp distinction made between Phytophthora and downy mildews based on morphogenetic criteria is rather artificial.The possibility for Phytophthora mycelium may grow through both living and dying cells. Intracellular mycelium is not uncommon in Phytophthora species, , although many species of this genus also produces digit-like haustoria. In Poakatesthia penniseti, a rare downy mildew parasite of Pennisetum glaucum, it has been observed that intracellular, callose-covered, hyphae form from haustoria, which may grow through several host cells before they enter the apoplast again PoakatesthiaSclerophthora, some basal Pseudoperonospora and Peronospora species, as well Peronosclerospora, which general mycelium morphology is much alike PoakatesthiaPoakatesthia penniseti raises questions regarding the obligate biotrophy of this downy mildew. Unfortunately, as only the type collection is available as a herbarium specimen, this conclusion can not be supported by cultivation experiments.It is generally assumed that in downy mildews hyphal growth only takes place extracellularly and only the determinate haustoria intrude into host cells, whilst in Sclerophthora is able to grow on artificial media is not unexpected, as there is a long-standing and ongoing debate Phytophthora. In a recent phylogenetic reconstructions, the placement of this genus could not be unambiguously resolved Sclerophthora is controversial becomes already apparent from its name: “Sclero” is referring to the thick-walled oospores, which are similar to those of the graminicolous downy mildew Sclerosporaphthora” refers to the Phytophthora-like vegetative mycelium including sporangiophores. The main reasons for the inclusion of Sclerophthora in the downy mildews have been the morphology of the oospores and the determinate sporangiophore growth. However, like in Phytophthora, sporangia do not form synchronously in this genus Sclerophthora in Phytophthora has been the similarity in the shape of sporangia und the supporting sporangiophores Phytophthora on grounds of oospore morphology might still be justified. Also the apparently fast and rather long independent evolutionary history, which can be deduced from the long branch for Sclerophthora in Thines et al. Phytophthora.The finding of Tokura SclerosporaSclerospora amongst the downy mildews Sclerophthora nor Sclerospora has been reported again and so it is possible that the observations made by Tiwari & Arya Sclerophthora the ripening of sporangia is asynchronous Viennotia, sporangiogenesis is synchronous.The second downy mildew genus which has been reported to be culturable on artificial media is Graminivora, Poakatesthia, Viennotia), Sclerophthora and Phytophthora. Taking into account the comparison given in Phytophthora is always present in downy mildews and that most characteristics thought to be exclusive to Phytophthora can also be observed in lineages of the graminicolous downy mildews.Therefore, in addition to the LM and SEM observations reported here, a careful examination of the literature also reveals that several characters states are in common for some genera of the graminicolous downy mildews with lasting sporangiophores  need to be rethought, as there are downy mildews species which show traits that are at variance with these basic assumptions.Phytophthora. Phytophthora and downy mildews are both phylogenetically and morphologically connected by “bridging taxa” and share an intimate relationship. The downy mildews are therefore to be considered a more specialised sister-taxon to some advanced Phytophthora lineages, as they have evolved from a Phytophthora-like ancestor at a time, when some other basal lineages of Phytophthora s.l. had already parted from the downy mildew-Phytophthora s.str. lineage . Whether the “bridging taxa” of the graminicolous downy mildews take an intermediate phylogenetic position between Phytophthora and all other downy mildew clades or whether the plesiomorphic, Phytophthora-like, character states that can be observed in some downy mildews have been lost several times independently can so far not be resolved.The arguments listed above necessitate a critical revision of the paradigm that downy mildews are much different from Sclerophthora and in the rare downy mildew species Poakatesthia penniseti and Viennotia oplismeni, several pathogenicity effector genes and developmental genes intermediate to the model organisms in Phytophthora and the downy mildews could be found. Therefore, the investigation of these organisms might shed light on the shift from hemibiotrophy to obligate biotrophy and thereby help to decipher the most basal chapter in downy mildew evolution.It seems likely that in the enigmatic genus Of the extremely rare parasites Poakatesthia penniseti and Viennotia oplismeni only the type collections are available , which were investigated in cause of this study. The Phytophthora specimen depicted in this study is deposited in the herbarium of the University of Hohenheim (HOH) as HUH 992.For light microscopy (LM) of sporangia and sporangiophores, small pieces of the samples were transferred to 5% chloral hydrate to restore turgidity. Preparation for scanning electron microscopy was done as previously described


There is no validated gold-standard diagnostic support tool for LSS, and therefore an accurate diagnosis depends on clinical assessment. Assessment of the diagnostic value of the history of the patient requires an evaluation of the differences and overlap of symptoms of the radicular and cauda equina types; however, no tool is available for evaluation of the LSS category. We attempted to develop a self-administered, self-reported history questionnaire as a diagnostic support tool for LSS using a clinical epidemiological approach. The aim of the present study was to use this tool to assess the diagnostic value of the history of the patient for categorization of LSS.The initial derivation study included 137 patients with LSS and 97 with lumbar disc herniation who successfully recovered following surgical treatment. The LSS patients were categorized into radicular and cauda equina types based on history, physical examinations, and MRI. Predictive factors for overlapping symptoms between the two types and for cauda equina symptoms in LSS were derived by univariate analysis. A self-administered, self-reported history questionnaire (SSHQ) was developed based on these findings. A prospective derivation study was then performed in a series of 115 patients with LSS who completed the SSHQ before surgery. All these patients recovered following surgical treatment. The sensitivity of the SSHQ was calculated and clinical prediction rules for LSS were developed. A validation study was subsequently performed on 250 outpatients who complained of lower back pain with or without leg symptoms. The sensitivity and specificity of the SSHQ were calculated, and the test-retest reliability over two weeks was investigated in 217 patients whose symptoms remained unchanged.The key predictive factors for overlapping symptoms between the two categories of LSS were age > 50, lower-extremity pain or numbness, increased pain when walking, increased pain when standing, and relief of symptoms on bending forward . The key predictive factors for cauda equina type symptoms were numbness around the buttocks, walking almost causes urination, a burning sensation around the buttocks, numbness in the soles of both feet, numbness in both legs, and numbness without pain . The sensitivity and specificity of the SSHQ were 84% and 78%, respectively, in the validation data set. The area under the receiver operating characteristic curve was 0.797 in the derivation set and 0.782 in the validation data set. In the test-retest analysis, the intraclass correlation coefficient for the first and second tests was 85%.A new self-administered, self-reported history questionnaire was developed successfully as a diagnostic support tool for LSS. Lumbar spinal stenosis (LSS) is a well-recognized spinal disorder and a term used to describe a complex set of symptoms, physical findings, and radiological abnormalities caused by a narrowed spinal canal. The presence of a narrow canal in radiographic imaging does not in itself define the syndrome, and a diagnosis of LSS is defined by symptoms and clinical findings that must be supported by radiographic evidence. Computed tomography and magnetic resonance imaging are often non-specific and there may be discrepancies between clinical symptoms and imaging findings in cases of LSS -3.There is no validated gold-standard diagnostic support tool for LSS, and therefore an accurate diagnosis depends on clinical assessment. However, there are few scientific evaluations of the sensitivity and specificity of diagnoses based on clinical history and physical examinations, or appropriate correlations of these data with imaging and operative findings. Katz et al. used the opinion of two expert orthopedic surgeons to define the presence or absence of LSS , and fouThere are two categories of leg symptoms caused by LSS . One typFull-blown cauda equina syndrome only occurs in rare instances, but the above symptoms can occur as a part of cauda equina syndrome ,7. ThereAssessment of the diagnostic value of the history of the patient requires an evaluation of the differences and overlap of symptoms of the radicular and cauda equina types; however, no tool is available for evaluation of the LSS category. Therefore, we attempted to develop a self-administered, self-reported history questionnaire as a diagnostic support tool for LSS using a clinical epidemiological approach. The aim of the present study was to use this tool to assess the diagnostic value of the history of the patient for categorization of LSS.A series of 137 patients with LSS and 97 with lumbar disc herniation who successfully recovered following surgical treatment in our department during 2000 and 2003 were included in this study  was developed as a diagnostic support tool for LSS.This study was performed in six university hospitals, ten medical centers, and thirty one hospitals and clinics affiliated with university hospitals or medical centers during January and March in 2004. A series of 115 patients with LSS gave informed consent to participate in the study and answered the SSHQ before surgery. All these patients recovered following surgery. Patients with cervical myelopathy, diabetic neuropathy, previous surgery, inflammatory disorders, and degenerative scoliosis were excluded. All patients were evaluated by study investigators using the same protocol as that in derivation study 1. Operative and follow-up visit notes were reviewed to determine if stenosis was confirmed intraoperatively and if symptoms improved following surgery. Nerve root compression resulting exclusively from a herniated nucleus pulposus was not considered as a part of LSS syndrome. All LSS patients were categorized into radicular or cauda equina types based on history, physical examination, and MRI findings using the same criteria as those in derivation study 1. There were 55 patients with radicular type LSS and 60 patients with the cauda equina type  and inflammatory disorders were also excluded. This study included 250 patients who complained of leg symptoms, including cases of LSS (n = 165), lumbar disc herniation (n = 61), diabetic neuropathy n = 13), and peripheral vascular disease (n = 11)  curve were estimated. History and physical examination variables were dichotomized at clinically sensible cut-off values. Pinprick, strength, and Achilles reflexes were each classified as always normal or with at least 1 abnormal finding. Univariate analyses were performed to derive predictors of LSS using logistic regression analysis. Two-by-two contingency tables were prepared to calculate the sensitivity, specificity, and likelihood ratio of the SSHQ. The area under the ROC curve for the derivation data set was estimated to investigate the internal validity of the clinical prediction rule, and the area under the ROC curve for the validation data set was estimated to examine the external validity. Reliability was investigated based on the reproducibility in the test-retest method. Test-retest analysis was performed in 217 patients with a 14-day period between the first and second tests. Test-retest data were examined graphically by plotting the difference between tests against the mean of the 2 tests . The intKey factors for predicting overlapping symptoms between the two types of LSS are shown in Table Based on the results of univariate analysis for predictors of LSS, we developed the SSHQ as a diagnostic support tool for LSS  declare that they have no competing interests.SKonno, SKikuchi, SKokuban, YT, YS, KY, MO, TY and HT conceived the study and participated in the study design. SK performed the statistical analysis. SK drafted the initial manuscript for journal submission and participated in revisions. SKonno, SKikuchi, SKokuban, YT, YS, KY, MO, TY and HT coordinated data collection at each site. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Japanese version of the SSHQ. A copy of the Japanese version of the SSHQ for Japanese readersClick here for fileEnglish version of the SSHQ. A copy of the English version of the SSHQClick here for file