Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145.Whilst being closely related to the model actinomycete S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds.The high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species.This study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between  Streptomyces are of great scientific and economic importance. Streptomycetes are saprophytic, aerobic, soil dwelling bacteria which undergo complex differentiation to form mycelium, aerial hyphae and spores, and produce a diverse array of secondary metabolites, such as antibiotics and bioactive compounds ) in FASTA format. Protein-encoding gene nucleotide sequences for S. lividans TK24 were obtained from the genes.fasta file downloaded from the BROAD institute [i.e. S. coelicolor genes against the S. lividans database and vice versa). A gene was classed as present if in each of the blast results it had a sequence match of greater than 60 nt in length (at least the length of the probes on the array) with greater than 60% sequence identity and an expected value less than 0.01. If these criteria were not met then the gene was classified as absent.nstitute  on 12/12S. coelicolor protein encoding gene was calculated using the CAI tool of EMBOSS [AL645882.2) in FASTA format and the 'background' (genome frequency) codon usage table from the codon usage database [The Codon Adaptation Index (CAI) value, a measure of synonymous codon usage bias, for each f EMBOSS  The nucldatabase ,69 were S. coelicolor and/or S. lividans DNA sequence  and two independently obtained PCR products for the corresponding sequences of S. lividans 66, were separated on a 1% agarose/Tris-Acetate-EDTA gel, the relevant bands excised and the DNA extracted using a Promega Wizard™ SV Gel & PCR Clean-up system. The purified DNA was treated with PNK (NEB) and blunt end-cloned into SmaI cut dephosphorylated pUC18 (Fermentas) using T4 DNA ligase (NEB). The ligation mix was transformed into competent E. coli JM109 and insert containing clones were identified by blue/white selection. Plasmid DNA was prepared from such clones using Promega Wizard Plus™ minipreps DNA purification system and supplied to Eurofins-MWG-Operon for BigDye™ (ABI) sequencing using M13 forward (-43) and reverse (-49) universal primers.Primer pairs, each specific for a particular region of S. coelicolor M145 and S. lividans 66 and TK24 bldB were similarly obtained, and treated, to determine the sequences of the central portions of bldB in these organisms/strains.PCR products for S. coelicolor M145 and S. lividans 66 were also similarly obtained, and treated, to determine their sequences.PCR products for a region spanning SCO5082 and SCO5083 incorporating the intergenic region for S. lividans 66 and TK24 sequences obtained in the course of this study are identical to those already publicly available from the S. lividans TK24 genome sequence [All of the sequence ,66.et al [S. coelicolor and S. lividans. The reactions which require products of the genes, which according to our CGH are missing in S. lividans were removed from the network. Subsequently, the model has been refined by incorporation of additional reactions, which have been experimentally demonstrated to operate in Streptomyces, but were omitted from the initial model. The feasibility of growth of different in silico strains on different media has been simulated by the calculation of the maximal biomass synthesis rate with Flux Balance Analysis. Details of FBA methodology are described in detail elsewhere [M. tuberculosis and S. coelicolor. Models used in this work are available in SBML format from the ScoFBA website [via a web-based interface to our software.The genome scale metabolic reaction network of Borodina et al  has beenlsewhere . Linear  website . The ScoS. coelicolor GSMN model for S. lividans to accommodate the findings from the CGH analysis. RL, EL, NA, AMK and CPS wrote the paper. All authors read and approved the final manuscript.RL, NA and GB conducted the experiments. EL conducted the microarray data analysis and whole genome BLAST analysis. RL led the biological interpretation of the microarray data. RL and NA conducted other DNA sequence analysis. EL, GB, CPS, VB and MH conducted the design and validation of the 104K array. AMK conducted the flux balance analysis and, with RAL, revised the Sequences of oligonucleotide primers used in the present study.Click here for fileS. coelicolor M145 and S. lividans 66 Modules 4, 5 & 7 from CDAPSI & IIAlignments of sequences derived from . Sequences from SCO3230 (CDAPSI) and SCO3231 (CDAPSII) modules 4, 5 and 7 obtained in the course of the present study of S. coelicolor M145 ("SC") corresponding to nucleotide coordinates 3556889-3557382, 3560009-3560502 and 3567844-3568334 (Accession No. EMBL: AL645882.2) aligned with the corresponding sequences from S. lividans 66 ("SL"). Nucleotides specific to S. coelicolor M145 module 4 are in yellow, nucleotides specific to S. coelicolor M145 modules 4 & 5 are in orange, nucleotides specific to S. coelicolor M145 module 5 are in red, nucleotides specific to S. coelicolor M145 modules 5& 7 are in purple, nucleotides specific to S. coelicolor M145 module 7 are in blue and nucleotides specific to S. coelicolor M145 modules 7 & 4 are in green. Nucleotide changes where the S. lividans 66 sequence diverges from all of the S. coelicolor M145 module 4, 5 and 7 sequences are shown in pink.Click here for fileact clusterSequence diversity in the SCO5082-SCO5083 intergenic region of the . Sequences of S. coelicolor M145 (lower) and S. lividans 66 (upper) corresponding to nucleotide coordinates 5524009-5524068 present in the SCO5082-SCO5083 intergenic region of the S. coelicolor M145 actinorhodin biosynthetic cluster (Accession No. EMBL: AL645882.2).Click here for fileS. coelicolor M145 SCO6832 and SCO6833 and intergenic regionSequence of . (nucleotide coordinates 7602829-7604947) (Accession No. EMBL: AL645882.2). The coding sequences are shaded orange and the respective microarray probe positions are indicated by differently coloured text. The respective start and stop codons of the two genes are underlined.Click here for fileTable detailing probes illustrated in Additional File per GACK analysis) to S. lividans 66 and TK24 is indicated.Click here for fileS. coelicolor SCO6833 & SCO6832 and S. lividans TK24 homologues identified by BLAST searchAlignment of .Click here for fileS. lividans 66 relative to S. coelicolorGenes classified as absent/divergent from .Click here for fileS. lividans TK24 relative to S. coelicolorGenes classified as absent/divergent from .Click here for fileS. lividans TK24 onlyGenes classified as absent/divergent from .Click here for fileS. coelicolor M145 bldB regionDouble-stranded nucleotide sequence of . Double-stranded nucleotide sequence of S. coelicolor M145 bldB region with microarray probe positions marked above or below their corresponding sequences (nucleotide co-ordinates 6243830-6244327)(Accession No. EMBL: AL645882.2). bldB is highlighted in a light orange box, and the S. lividans TK24 bldB coding sequence is italicized and aligned below the S. coelicolor sequence; identical bases are indicated by asterisks. The respective start and stop codons of the two orthologous genes are underlined.Click here for fileMicroarray probes illustrated in Additional File 10per GACK analysis) to bldB regions of S. lividans 66 and TK24: binding/non-binding (as .Click here for file


Durvillaea antarctica) has a broad distribution along much of the Chilean coast. This species represents an ideal model taxon for studies of coastal marine connectivity and of palaeoclimatic effects, as it grows only on exposed rocky coasts and is absent from beaches and ice-affected shores. We expected that, along the central Chilean coast, D. antarctica would show considerable phylogeographic structure as a consequence of the isolating effects of distance and habitat discontinuities. In contrast, we hypothesised that further south - throughout the region affected by the Patagonian Ice Sheet at the Last Glacial Maximum (LGM) - D. antarctica would show relatively little genetic structure, reflecting postglacial recolonisation.South America's western coastline, extending in a near-straight line across some 35 latitudinal degrees, presents an elegant setting for assessing both contemporary and historic influences on cladogenesis in the marine environment. Southern bull-kelp (rbcL) DNA analyses of D. antarctica from 24 Chilean localities  revealed two deeply divergent  clades from the centre and south of the country, with contrasting levels and patterns of genetic structure. Among populations from central Chile (32° - 44°S), substantial phylogeographic structure was evident across small spatial scales, and a significant isolation-by-distance effect was observed. Genetic disjunctions in this region appear to correspond to the presence of long beaches. In contrast to the genetic structure found among central Chilean populations, samples from the southern Chilean Patagonian region (49° - 56°S) were genetically homogeneous and identical to a haplotype recently found throughout the subantarctic region.Mitochondrial (COI) and chloroplast  Chile has been recolonised by  Oceanic ecosystems have historically been considered relatively open, with high connectivity among populations . Recent Genetic patterns of isolation-by-distance are common in the marine realm, with neighbouring populations generally more closely related to each other than to those further away, as the chance of successful dispersal decreases with increasing distance ,6. Gene Fucus vesiculosus: [Hormosira banksii: [Macrocystis pyrifera: [Durvillaea antarctica), found evidence of recent  recolonisation of much of the subantarctic, a feat almost certainly achieved by trans-oceanic rafting of adult specimens [Although most macroalgae have only short-lived gametes or dispersive zygotes, some - particularly highly buoyant species - are hypothesised to be capable of long-distance dispersal via rafting of fertile adults . A broapecimens . Recolonpecimens -24). Butpecimens  suggestepecimens ,25. In dDurvillaea antarctica currently dominates the rocky shores of Chile from Cape Horn [D. antarctica included few samples from central Chile , and none from Chilean Patagonia, and was therefore unable to assess phylogeographic contrasts along the Chilean coast. For the current study, we analysed samples from a total of 24 localities, encompassing the full range of D. antarctica in Chile, to test the predictions that (i) D. antarctica populations in central Chile would show strong phylogeographic structure, and that (ii) D. antarctica populations in southern Chile would show little genetic structure, reflecting postglacial recolonisation.Chile presents an intriguing system for assessing the relative phylogeographic impacts of isolation-by-distance, habitat discontinuities, and historic glaciations. Central Chile has an almost straight coastline characterised by numerous extensive stretches of beach that isolate rocky-shore habitats ; this coape Horn , at arouape Horn . Fraser ape Horn  recent bD. antarctica samples from 24 localities along the coast of Chile yielded 14 distinct haplotypes for mitochondrial COI, with 34 variable sites detected across the 629 bp fragment. For a chloroplast marker, rbcL, 78 sequences from nine localities  were restricted to populations in central Chile, whereas all sampled Chilean Patagonian populations south of 44°S were fixed for a single haplotype (C-I), which was highly divergent from the others  haplotypes detected in Chile were 1.4% divergent (uncorrected distance), with one (R-I) restricted to all sampled Patagonian populations, and the other (R-II) found only in central Chile . More broadly, the Patagonian rbcL haplotype R-I has a circum-subantarctic distribution, including the Falkland Islands and South Georgia .Network analyses of Chilean COI haplotypes recovered two parsimonious networks that could not be joined at ≥90% confidence limits, with one comprising only the Patagonian haplotype C-I. All other Chilean haplotypes (CII-CXIV) formed a single network   beaches , whereas total coastal distance was not . The outlier formed by Lebu/Tirua . With or without the outlier, these results indicate that habitat discontinuity influences genetic connectivity among populations of central Chilean bull-kelp.The Mantel test revealed a significant relationship between genetic and geographic distance (ns) Fig. . DetaileD. antarctica in Chile. First, substantial phylogeographic structure was observed in central Chile (hypothesis i). Genetic isolation among populations in this region appears driven by habitat discontinuities. Second, and in direct contrast to the pattern observed for central Chile, samples from Chilean Patagonia showed complete genetic homogeneity within and among localities (hypothesis ii). These Patagonian populations were deeply divergent to those from more northern  localities, and appear to have trans-oceanic ancestry.Two broad findings were revealed by our phylogeographic research of D. antarctica , suggesllospora ,31, dispD. antarctica is in direct contrast to the broad evidence of long-distance, trans-oceanic dispersal of this species in the subantarctic [D. antarctica zygotes would suppress settlement of zygotes from such migrants. Conversely, shores swept clean by ice [D. antarctica is clearly an effective long-distance disperser, there may be differences in reproductive viability of rafts across latitudinal gradients. Numerous reports suggest that D. antarctica has an extended period of fertility in the subantarctic versus mainland New Zealand or Chile , and thences in ), perhapences in  suggest r et al. . If D. ania Fig. .D. antarctica populations could additionally be influenced by a range of processes such as seasonal variation and patchy recruitment [not show a genetic disjunction (Lebu-Tirua) is in the immediate vicinity of a large coastal island (Isla Mocha) that may effectively connect the two populations despite the beach barrier. Interestingly, Constitución, flanked by long beaches on both sides, was genetically distinct from adjacent localities but not from some more distant localities . These populations, on the northernmost edge of the range of D. antarctica in Chile, may be sensitive to any changes in water temperature. Changes to the northern range limit of an Australian congeneric, D. potatorum, have been linked to warming waters in recent years [D. antarctica in Chile  may thus be somewhat ephemeral in the long term, and could have been relatively recently colonised by kelp drifting from southern populations  in the north-flowing Humboldt Current.Local genetic structure within and among central Chilean ruitment  or harveruitment -36. Whilruitment  and Castnt years . The norD. antarctica populations indicate a biogeographic break in northern Chilean Patagonia (44° - 49°S), with both mitochondrial and chloroplast DNA phylogenies revealing a deep genetic disjunction between samples from southern Patagonia and those from central Chile  and wind (primarily eastward) through the fiords. Taitao Peninsula, however, projects across the dense system of channels that could otherwise potentially allow mixing and north-south transport of drifting D. antarctica within the fiords.Our phylogeographic analyses of Chilean ile Fig. . Althougile Fig.  has prevD. antarctica samples from southern Patagonia and those from central Chile raises the question of the taxonomic status of these two divergent lineages. A recent study of D. antarctica revealed that this taxon comprises two ecologically, morphologically and genetically distinct forms in New Zealand [D. antarctica (4.5 - 6.1% for COI) exceeds the divergence observed between the co-occurring, genetically-distinct forms in New Zealand , it is D. antarctica from all localities south of latitude 44°S  ice during the most recent ice age may also have driven kelp population extinctions. If populations of D. antarctica in southern Chile were indeed destroyed by the Patagonian Ice Sheet during the last glacial period, numerous other coastal taxa may have been similarly affected. Intriguingly, a broad-scale phylogeographic study of the rocky-shore gastropod Concholepas concholepas [Although genetic evidence of postglacial recolonisation has previously been identified from a variety of Patagonian terrestrial and freshwater taxa, including fish  is capable of long-distance dispersal via rafting of fertile adults, as evidenced by the trans-oceanic ancestry of populations throughout postglacially-recolonised Chilean Patagonia . IntrigD. antarctica were collected from 24 localities along the coast of Chile, from Pichicuy (32°27'37"S) to Cape Horn (55°59'31"S)  pieces of tissue cut from the tip of healthy fronds of attached D. antarctica, and were preserved by desiccation in either 96% ethanol or silica gel beads. DNA extraction, amplification, purification and sequencing were performed as described in Fraser et al. [c oxidase I (COI) was amplified using primers GazF1 and GazR1 [rbcL was amplified using primers KL2 and KL8 [164 samples of r et al. . A 629 bnd GazR1  for all  and KL8  for sampDurvillaea willana , Durvillaea potatorum  and Fucus (GenBank accessions: F. vesiculosus AY494079 for COI and F. gardneri AF195515 for rbcL). To place the new Chilean sequence data in a wider context, published D. antarctica DNA sequences  as selected by the AIC of Modeltest 3.06 [D. antarctica COI and rbcL sequences used in this study: FJ550093-FJ550095; FJ550097; FJ550099; FJ550119; HM103936-HM104173).Phylogenies for each DNA dataset were constructed using maximum-likelihood (ML) and Bayesian analyses, and included outgroup sequences from r et al.  with a Tr et al.  model foest 3.06 , with heest 3.06  and are D) average pairwise differences [FST values could not be used in the Mantel test: several localities were genetically monomorphic for different haplotypes, and pairwise comparisons between such fixed populations gave an FST of 1.0; hence for these locality pairs no linearised FST values (FST/(1 - FST), [Analysis of isolation-by-distance was performed on data from the 19 localities in central Chile (north of 44°S) via the Mantel  test, imferences   versus  - FST), ) could bhttp://earth.google.com/ to assess the continuity of suitable rocky shore habitat for D. antarctica among localities. As Chilean shores south of about 43°S are almost completely rocky, with few sandy beaches and, moreover, many wide oceanic channels between islands and peninsulas, measurements of beaches were only made for continental Chile and the coast of the Great Island of Chiloé (north of 43°S). The following analysis was performed only on data from sampled localities in this region . The isolating effects of beach length and total coastal distance between localities in central Chile were assessed by logistic regression. Adjacent locality pairs were first classified binomially as either genetically disjunct (D ≥1.0) or not (D < 1.0). Under this definition, locality pairs not sharing any haplotypes were considered genetically disjunct, whereas those with shared haplotypes were not considered disjunct. Logistic regression was then carried out using R [Approximate measurements of the lengths of all beaches  between pairs of adjacent rocky coastal localities in central Chile were made using the 'path ruler' tool in Google Earth  using R , using tC.I.F., J.M.W., M.T. and H.G.S. conceived the ideas; C.I.F. obtained and analysed the data; C.I.F. led the writing; and J.M.W., M.T. and H.G.S assisted with the writing. All authors read and approved the final manuscript.


Hand hygiene authorities including the WHO and U.S. CDC have recognized inherent weaknesses calling out the need for improved The European Standard EN1500 (Hygienic Handrub), ASTM Standard E1174 , and a recently approved ASTM standard, ASTM E2755-10, were critically compared based on the written protocol and empirical evidence .Staph. aureus, more representative of a hand transmitted pathogen in healthcare environments. Both EN1500 and E1174 have unrealistic hand contamination procedures which necessitate evaluation of unrealistic volumes of product at excessive contact times. E2755 employs a low volume contamination method for dry, relatively unsoiled hands, allowing ABHR evaluation at realistic volumes and contact times. Success criteria are significantly different: EN1500 uses an internal reference and ASTM methods use specific log reduction minimum endpoints.All methods use gram-negative bacteria as marker organisms and the measured endpoint is reduction on hands after test article application. ASTM E2755 includes in vivo efficacy standard would be of significant value to the infection prevention community and is a vision worth working towards.Neither method approximates actual healthcare worker ABHR usage. None has success criteria based on evidence of clinical benefit or prevention of pathogen transmission. A single, globally recognized J. Arbogast Employee of GOJO Industries, Inc., B. Okeke Employee of GOJO Industries, Inc., F. Brill Employee of Dr. Brill + Partner GmbH , T. Bradley: None declared, A. Fraise: None declared, M. Wilkinson: None declared, D. Macinga Employee of GOJO Industries, Inc.


Granulibacter bethesdensis is an emerging pathogen in patients with CGD that causes fever and necrotizing lymphadenitis. However, unlike typical CGD organisms, this organism can cause relapse after clinical quiescence. To better define whether infections were newly acquired or recrudesced, we use comparative bacterial genomic hybridization to characterize 11 isolates obtained from 5 patients with CGD from North and Central America. Genomic typing showed that 3 patients had recurrent infection months to years after apparent clinical cure. Two patients were infected with the same strain as previously isolated, and 1 was infected with a genetically distinct strain. This organism is multidrug resistant, and therapy required surgery and combination antimicrobial drugs, including long-term ceftriaxone. G. bethesdensis causes necrotizing lymphadenitis in CGD, which may recur or relapse.Chronic granulomatous disease (CGD) is characterized by frequent infections, most of which are curable.  AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test and/or complete the evaluation at http://cme.medscape.com/viewpublication/30063; (4) view/print certificate.Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians the opportunity to earn CME credit. This activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Medscape, LLC and Emerging Infectious Diseases. Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians. Medscape, LLC designates this educational activity for a maximum of 0.5 Upon completion of this activity, participants will be able to:Identify infectious organisms in cases of chronic granulomatous disease (CGD).Granulibacter bethesdensis infection in CGD.Distinguish the clinical presentation of G. bethesdensis infection in CGD effectively.Diagnose G. bethesdensis infection in CGD.Plan effective treatment for Thomas J. Gryczan, MS, Copyeditor, Emerging Infectious Diseases. Disclosure: Thomas J. Gryczan, MS, has disclosed no relevant financial relationships.Charles P. Vega, MD, Associate Professor; Residency Director, Department of Family Medicine, University of California, Irvine. Disclosure: Charles P. Vega, MD, has disclosed no relevant financial relationships.Disclosures: David E. Greenberg, MD; Adam R. Shoffner, BS; Adrian M. Zelazny, PhD; Michael E. Fenster, MD; Kol A. Zarember, PhD, BSc; Frida Stock, BS; Li Ding, MD; Kimberly R. Marshall-Batty, PhD, MS, BS; Kishore Kanakabandi; Dan E. Sturdevant, PhD; Kimmo Virtaneva; Stephen F. Porcella, PhD; Patrick R. Murray, PhD; Harry L. Malech, MD; and Steven M. Holland, MD, have disclosed no relevant financial relationships. Richard L. Wasserman, MD, has disclosed the following relevant financial relationships: served as an advisor or consultant for Baxter Healthcare, CSL Behring, Talecris Biotherapeutics; served as a speaker or a member of a speakers bureau for Baxter Healthcare, CSL Behring, Talecris Biotherapeutics; received grants for clinical research from Baxter Healthcare, CSL Behring, Talecris Biotherapeutics, Nabi Biopharmaceuticals. David F. Welch, PhD, has disclosed the following relevant financial relationships: received grants for clinical research from Prodesse (Gen-Probe); owns stock, stock options, or bonds from Cepheid, Luminex Corporation, Merck & Co., Inc., Eli Lilly and Company, Boston Scientific Corporation, Becton, Dickinson and Company. Staphylococcus aureus, Serratia marcescens, Burkholderia cepacia complex, Nocardia spp., and Aspergillus spp (–Chronic granulomatous disease (CGD) is a rare genetic disease caused by mutations in any of the 4 structural genes of the NADPH oxidase system and leads to defective production by phagocytes of superoxide and downstream oxygen metabolites .Five patients were followed up at the National Institutes of Health (NIH) Clinical Center  under protocol 93-I-0119. Patients 2, 3, and 5 had been actively followed up at NIH for at least 8 years before receiving a diagnosis of G. bethesdensis high-density microarray platform, DNA microarray hybridization, and comparative genomic hybridization analysis used for typing of the G. bethesdensis isolates have been described  and 1.5 μL of each primer at a final concentration of 0.75 μmol/L, and the PCR was performed in a thermocycler . The PCR amplification conditions were 94°C for 10 min; 40 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR fragments were visualized by electrophoresis on a 2% agarose gel and showed the expected sizes of 137 bp (16SrRNA) and 63 bp (methanol dehydrogenase subunit 1).The 16S rRNA and methanol dehydrogenase subunit 1 (GeneID YP_744165.1) genes of G. bethesdensis. Therapy with meropenem and doxycycline resulted in resolution of the lesions. However, the patient had recurrences of necrotizing cervical and axillary lymphadenitis over the next 2 years, and G. bethesdensis was isolated on 3 separate occasions. Treatment with ceftriaxone and doxycycline for 1 year resolved his lymphadenitis. He has had no further recurrence but has had persistent chronic fatigue since onset of infection.A 39-year-old man from the United States with X-linked CGD had persistent fever, chills, weight loss, and increased levels of inflammatory markers starting in April 2003, as described . ResectiA 36-year-old man from the United States with X-linked CGD had cough and fever in November 2005. He had lymphadenopathy of the supraclavicular, paratracheal, subcarinal, perihilar, internal mammary, perigastric, retroperitoneal, iliac, and inguinal lymph nodes and multiple splenic lesions . The ery3 cells/μL with 79% neutrophils. Ascitic fluid was cloudy with 3,100 leukocytes/μL  and 2,813 erythrocytes/μL. Gram stain showed no organisms. Treatment with meropenem and trimethoprim/sulfamethoxazole was begun. A CT-guided biopsy specimen of perigastric lymph nodes showed necrotizing granulomatous inflammation  agar. Routine biochemical tests did not identify the organism, but 16S rDNA sequencing confirmed that the isolate was G. bethesdensis.A 13-year-old boy from the United States with X-linked CGD was admitted for persistent fever and diffuse thoracic lymphadenopathy in January 2006. He had been followed up for al areas . A transG. bethesdensis. He responded clinically and radiographically to ceftriaxone. He was then switched to cefdinir in January 2009 after gallstones developed and continued to receive this therapy until July 2009. The patient has had no further relapses.The patient was initially treated with meropenem and voriconazole. When a repeat CT scan 2 weeks later showed enlarged mediastinal lymph nodes, meropenem was discontinued and ceftriaxone, tobramycin, and doxycycline were initiated. A CT scan 1 week later showed smaller lymph nodes. The patient stopped taking tobramycin after renal toxicity developed in March 2006, and he received ceftriaxone until May 2006. His lymphadenopathy resolved and he received doxycycline until March 2007. In December 2008, new fevers and subcarinal lymphadenopathy developed. A fine-needle aspiration specimen yielded a gram-negative rod on fungal media (IMA agar) after 15 days of incubation; 16S rDNA sequencing confirmed that the isolate was A 17-year-old boy from Panama with X-linked CGD had cervical lymphadenopathy for 2 months in June 2005. He had been treated with trimethoprim/sulfamethoxazole until the age of 15 when a chest wall infection with an unidentified basidiomycete developed. Since then, he had responded well to itraconazole and trimethoprim/sulfamethoxazole prophylaxis until lymphadenopathy developed. Cervical and mediastinal lymphadenopathy biopsy specimens , panel CG. bethesdensis. While receiving levofloxacin, adenopathy increased in the patient. Five weeks later, another cervical lymph node was removed. After 4 days, the culture grew G. bethesdensis as pinpoint colonies on sheep blood agar. The patient was treated with doxycycline and responded well clinically without further recurrences.Pinpoint colonies were seen on sheep blood agar after 6 days of incubation and were subcultured in BCYE medium. The gram-negative rod could not be identified biochemically; 16S rDNA sequencing confirmed that the isolate was S. epidermidis and α-hemolytic streptococci in liquid media. The patient was then treated with voriconazole and ceftriaxone.The 37-year-old brother of patient 2, who also had X-linked CGD, had fever, chills, and chest pain for 2 weeks in October 2008. New left lower lobe nodules and left infrahilar adenopathy prompted bronchoalveolar lavage, which showed macrophages and scattered neutrophils. Cultures were negative, but the patient was treated with levofloxacin, linezolid and posaconazole and continued receiving trimethoprim/sulfamethoxazole. Fever and pulmonary nodules resolved but adenopathy persisted. Treatment with antimicrobial drugs was stopped in December 2008. Fever and chills returned in February 2009. His ESR was 78 mm/h. Magnetic resonance imaging of the chest showed multiple enlarged anterior mediastinal and bilateral hilar lymph nodes. Bronchoscopy with transbronchial needle aspiration yielded G. bethesdensis after 7 days of incubation on BCYE and mycobacterial media. Ototoxicity developed while he was being treated with ceftriaxone, gentamicin, and vancomycin. He was then treated with ceftriaxone, doxycycline, and trimethoprim/sulfamethoxazole for 4 months and showed clinical and radiographic improvement. He was treated with cefdinir in September 2009 and has continued treatment with doxycycline and trimethoprim/sulfamethoxazole and has not had any new relapses. Patients 2 and 5 live apart but see each other a few times per year and had been together during the illness of patient 2.Despite this therapy, left supraclavicular lymphadenopathy and splenic and liver lesions developed in April 2009 . A lymphG. bethesdensis within 3 weeks on a variety of media , but growth was sparse. PCR amplification identified specific G. bethesdensis bands in excised spleen, which indicated that molecular diagnosis can be made from fresh tissue, a useful consideration for an organism that is slow growing and difficult to identify . This finding indicated that these isolates are all appropriately designated G. bethesdensis sensu stricto. The isolate reported by Lopez et al.  at multiple loci, as determined by using the Granulibacter microarray chip , and quinolones. However, ceftriaxone (MIC 8 μg/mL), aminoglycosides , doxycycline, and trimethoprim/sulfamethoxazole show activity in vitro. Our patients were treated with various combinations of these drugs; all showed initial resolution of disease. Ceftriaxone is currently our preferred treatment.Granulibacter spp., other cases of this infection in patients with CGD and those without CGD may have been overlooked. To address this issue, we now concentrate specimens, particularly from lymph nodes of patients with CGD, by centrifugation, place the specimens on BCYE and Middlebrook 7H11 agar, and incubate cultures for a minimum of 2 weeks.The reasons underlying the emergence of new pathogens are complex. The changing environments in which we and our microbial companions live make predicting when, where, and why a new pathogen will emerge difficult. Typically, we are best at recognizing clinical syndromes and working back toward the etiology. Given the difficulty of growing and identifying Clinical management of CGD has long been confounded by the relatively high rate of failure of isolating a pathogen from involved sites. This failure has been attributed to many features of CGD, including exuberant inflammation. In some cases, clinicians attribute culture-negative lesions to the inflammatory diathesis of CGD. However, the finding of a difficult-to-grow and difficult-to-identify pathogen makes this assumption unlikely, particularly in the setting of necrotizing lymphadenopathy. Identification of a fastidious, slow-growing bacterium that causes a discrete, chronic infection reminds us that new syndromes remain to be identified, even within well-described and well-characterized diseases. http://cme.medscape.com/viewpublication/30063. Credit cannot be obtained for tests completed on paper, although you may use the worksheet below to keep a record of your answers. You must be a registered user on Medscape.com. If you are not registered on Medscape.com, please click on the New Users: Free Registration link on the left hand side of the website to register. Only one answer is correct for each question. Once you successfully answer all post-test questions you will be able to view and/or print your certificate. For questions regarding the content of this activity, contact the accredited provider, CME@medscape.net. For technical assistance, contact CME@webmd.net. American Medical Association’s Physician’s Recognition Award (AMA PRA) credits are accepted in the US as evidence of participation in CME activities. For further information on this award, please refer to http://www.ama-assn.org/ama/pub/category/2922.html. The AMA has determined that physicians not licensed in the US who participate in this CME activity are eligible for AMA PRA Category 1 Credits™. Through agreements that the AMA has made with agencies in some countries, AMA PRA credit is acceptable as evidence of participation in CME activities. If you are not licensed in the US and want to obtain an AMA PRA CME credit, please complete the questions online, print the certificate and present it to your national medical association.To obtain credit, you should first read the journal article. After reading the article, you should be able to answer the following, related, multiple-choice questions. To complete the questions and earn continuing medical education (CME) credit, please go to 1. A 35-year-old man presents with a history of X-linked chronic granulomatous disease (CGD) in the hospital. He was admitted after 3 weeks of fever. Physical examination reveals obvious lymphadenopathy. Laboratory evaluation reveals an elevated erythrocyte sedimentation rate, but a Gram stain for organisms on 1 of his lymph nodes is negative.This patient is most likely infected with which of the following organisms?A. Group B Streptococcus speciesB. Mycoplasma pneumoniaeC. Aspergillus speciesD. Campylobacter jejuniGranulibacter bethesdensis in this patient is most accurate?2. Which of the following statements about a possible infection with A. This infection is unlikely, given the patient’s duration of feverB. Lymphadenopathy was present in all cases of infection with G. bethesdensisC. Infection with G. bethesdensis does not promote an elevation in the erythrocyte sedimentation rateD. G. bethesdensis was evident on bacterial staining in all casesG. bethesdensis infection is most accurate?3. The patient has a battery of tests and procedures to establish a causative organism for his infection. Which of the following statements about the diagnosis of A. G. bethesdensis cultures grew readily, but only on Middlebrook mediaB. PCR amplification testing of excised tissue could identify G. bethesdensisC. All infections with G. bethesdensis could be traced to the same strainD. There was no evidence of reactivation of latent infectionG. bethesdensis. Which of the following statements about his treatment and prognosis is most accurate?4. A bacterial culture from your patient is now growing A. Ceftriaxone is a good choice of treatmentB. Macrolide antibiotics are particularly effective for treating G. bethesdensisC. Disseminated G. bethesdensis infection was uniformly fatalD. G. bethesdensis responds to only 1 or 2 antibiotics


Background/Aim. Hepcidin, an iron regulatory hormone, is increased in response to inflammation and some infections. We investigated the relationships among serum prohepcidin, iron status, Helicobacter pylori infection status, and the presence of gastric mucosal atrophy. Methods. Seventy subjects undergoing esophagogastroduodenoscopy underwent multiple gastric biopsies, and the possibility of H. pylori infection and the degree of endoscopic and histologic gastritis were investigated. In all subjects, serum prohepcidin and iron parameters were evaluated. Results. No correlations were observed between serum prohepcidin levels and the other markers of anemia, such as hemoglobin, serum iron, ferritin, and total iron binding capacity. Serum prohepcidin levels were not significantly different between the H. pylori-positive group and the H. pylori-negative group. Serum prohepcidin levels in atrophic gastritis patients were significantly lower than those in subjects without atrophic gastritis irrespective of H. pylori infection. Conclusion. Serum prohepcidin levels were not altered by H. pylori infection. Serum prohepcidin levels decrease in patients with atrophic gastritis, irrespective of H. pylori infection. It suggests that hepcidin may decrease due to gastric atrophy, a condition that causes a loss of hepcidin-producing parietal cells. Further investigations with a larger number of patients are necessary to substantiate this point. Prohtivities . Hepciditivities . In additivities , 7. Helicobacter pylori infection with or without coexisting autoimmune gastritis has been implicated in several recent studies as an important cause of iron deficiency anemia (IDA) in patients with unexplained IDA [H. pylori [H. pylori infection seem not to respond well to oral iron therapy until the bacterium had been eradicated [H. pylori infection to respond to iron might be the production of hepcidin or hepcidin mimetics by microorganisms [H. pylori-infected patients and normalized by H. pylori eradication [ined IDA . The pos. pylori . Moreoveadicated –12. Thisadicated . It has rganisms , 15. A rdication . The stuH. pylori infection status, and the presence of gastric mucosal atrophy.Serum prohepcidin concentrations are significantly decreased in patients with hereditary hemochromatosis , increasThis was a single center, observational case-control study including 70 subjects who underwent routine endoscopic examination of gastrointestinal symptoms at the Uijeongbu St. Mary's Hospital between September 2005 and August 2006. Exclusion criteria were previous eradication therapy or the use of bisthmus compounds, proton pump inhibitors, antibiotics, or antisecretory drugs within the previous 2 months. Additional exclusion criteria were pregnancy or lactation, severe systemic illness, manifest clotting disorders or the use of anticoagulants, and a history of blood transfusion or iron supplement therapy. 13C-Urea Breath Test (UBT) was performed after an overnight fast or at least an 8 h fast. A baseline breath sample was placed into a collection tube. An aliquot of 75 mg of 13C-urea dissolved in 75 mL of citric acid solution was given orally . Another breath sample was collected after 30 min. Breath samples were subsequently analyzed to determine the 13C/12C ratio by mass spectrometry . The 13C/12C ratio of each breath sample was expressed as a milli-percentage (‰). Change in the 13C value over baseline was expressed as delta 13C. A positive result was defined as an increase of >4‰. Patients were considered to be negative for H. pylori if both histological examination and 13C-UBT results were negative. Patients were considered to be positive for H. pylori if any one of the tests was positive. During endoscopy, four biopsies  were taken. Hematoxylin and eosin (HE) staining and Giemsa staining were performed using serial sections of four specimens. The sections were independently assessed by two blinded pathologists. The Atrophic changes of the gastric mucosa on endoscopy were graded according to Kimura-Takemoto classification . Atrophiμg/L (men) or <13 μg/L (women) were considered indicative of iron deficiency.Blood samples were collected from all participants who had fasted overnight. Laboratory tests, including a complete blood count, total protein, albumin, hepatic and renal function tests, serum iron, total iron biding capacity (TIBC), and ferritin were performed using standard laboratory methods. Patients with a hemoglobin (Hb) of <14.0 g/dL (men) or <12.0 g/dL (women) were considered to be anemic, and ferritin concentrations of <30  The serum prohepcidin level was measured using the DRG Diagnostics Hepcidin Prohormone enzyme-linked immunosorbent assay (ELISA) , according to the manufacturer's instructions. The employed antibody detects both the proregion and prohepcidin (aa 25–84). The sensitivity of the assay was 3.95 ng/mL, intra-assay coefficient of variation (CV) was 4.69%, and inter-assay CV was 4.82%.t-test, whereas the categorical data were analyzed using χ2 or Fisher's exact tests. For correlation analysis, the Spearman nonparametric correlation was used. Data were processed and analyzed with SPSS, version 12.0 . A value of P < 0.05 was considered to indicate statistical significance for all tests.Values are expressed as mean ± standard deviation (SD). Continuous data were compared using an independent-samples This study protocol was approved by the Institutional Research Ethics Board of Uijeongbu St. Mary's Hospital of the Catholic University of Korea (IRB No. UCMC06BR006) and adhered to the Declaration of Helsinki. All of the study subjects completed an informed consent form before participating in the study. The informed consent was confirmed by the board.H. pylori-positive (n = 35) and H. pylori-negative (n = 35) groups, no significant differences in the hematological or biochemical parameters were observed between the two groups. However, a difference in atrophic gastritis was identified, which was significantly more frequent (P < 0.001) in the H. pylori-positive group than in H. pylori-negative group . Anemia was diagnosed in nine (12.9%) patients, three of whom were H. pylori-positive and six of whom were H. pylori-negative. Among anemic patients, only three in the H. pylori-negative group had iron deficiency. The mean serum prohepcidin level was 241.4 ng/mL  among all subjects, 238.8 ng/mL  in the H. pylori-positive group, and 244.1 ng/mL  in the H. pylori-negative group. The serum prohepcidin level correlated with the hemoglobin  and ferritin  levels in the H. pylori-positive group. No correlation was observed between the serum prohepcidin level and the other markers of anemia, such as hemoglobin, serum iron, ferritin, and TIBC, in the H. pylori-negative group.A total of 70 patients with gastrointestinal symptoms  were included in the study. When the patients were separated into H. pylori-negative subjects showed an endoscopically normal stomach with a C-0 atrophic border and histologically normal fundic mucosa, and 10 subjects showed mild atrophic change (C-1). Ten H. pylori-positive subjects showed no atrophic changes and histologically normal fundic mucosa, and 25 subjects showed various endoscopic atrophic changes . The descriptive statistics are reported in P = 0.020) irrespective of H. pylori infection (P = 0.058). No correlation was observed between the serum prohepcidin level and the other markers of anemia, such as hemoglobin, serum iron, ferritin, and TIBC, in patients with and without atrophic gastritis  in the Korean population among asymptomatic Korean adults in 2005 [H. pylori infection. This finding might be explained by the loss of hepcidin-producing cells caused by gastric atrophy. However, we did not find that serum prohepcidin levels are related to the degree of atrophy. In addition, correlation between the serum prohepcidin and the parameters for anemia was not found in patient with atrophic gastritis. This can be explained by the small number of patients enrolled and by the lack of evaluation of hepcidin expression in gastric tissues. A recent study demonstrated that gastric hepcidin expression decreased in hypergastrinemic ING-mice with chronic gastric H. pylori infection and resulted in the upregulation of the expression of various downstream iron absorption and efflux genes such as Ferroportin 1, Divalent metal transporter 1, and Transferrin receptor 1 [Schwarz et al. recently reported a new role for hepcidin in the stomach . In this in 2005 . In addi in 2005 . We founceptor 1 . These fPepsinogen, an aspartic proteinase secreted mainly by gastric cells, is classified immunologically as pepsinogen I (PG I) and pepsinogen II (PG II). Whereas PG I is secreted only from the gastric fundic mucosa, PG II is secreted from the cardiac, fundic, and antral mucosae of the stomach . The effH. pylori infection. Serum prohepcidin levels decreased in patients with atrophic gastritis, irrespective of H. pylori infection. It suggests that hepcidin may decrease due to gastric atrophy, a condition that causes a loss of hepcidin-producing parietal cells. Further studies with a larger number of atrophic gastritis patients are necessary to better investigate the relationship between hepcidin and atrophic gastritis. In conclusion, our data suggest that serum prohepcidin levels were not altered by


Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.Genes underlying important phenotypic differences between  Plasmodium parasites, the causative agents of malaria, it is now possible to use comparative genomics as a tool to look for genes that are present in some but not all Plasmodium species. Such species subset-specific genes possibly underlie important phenotypic differences between malaria parasites and could provide important clues for the development of new strategies to prevent and treat malaria in humans. In this study, we performed a comprehensive computational comparison of the published genomes of six Plasmodium species, including two human , one monkey (P. knowlesi), and three rodent malaria parasites . This comparison revealed many species subset-specific genes that are potentially linked to human pathogenicity, human-to-human transmissibility, and human virulence. These genes can now be examined further by targeted experimental analyses to test predicted phenotypic associations and to elucidate gene function.With more than 250 million infections and over a million deaths each year, malaria remains one of the most devastating infectious diseases worldwide. With the availability of complete genome sequences of both human and non-human  Plasmodium that are transmitted by mosquitoes. Four species, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, are traditionally recognized as human parasites. Other Plasmodium species are important model parasites in malaria research, including the primate malaria model Plasmodium knowlesi, which parasitizes macaque monkeys in the wild, as well as the three rodent malaria parasites Plasmodium berghei, Plasmodium yoelii, and Plasmodium chabaudi, which are natural parasites of thicket rats in central Africa.Malaria remains a serious health threat. Every year, more than 250 million people worldwide suffer from malaria and over one million people die as a consequence of the disease, mostly children in Africa under the age of five Plasmodium species that naturally infect humans, monkeys, and rodents differ in their ability to cause human disease. Firstly, laboratory experiments have shown that parasites of thicket rats are infectious to various other species of rodents but not primates P. knowlesi differs from the four human parasites in that it is not endemic in larger parts of the human population despite its known ability to infect also humans under natural conditions P. knowlesi malaria is an ancient zoonosis acquired from forest-dwelling macaque monkeys P. knowlesi malaria fails to spread in human settlements and beyond because of the known inability of P. knowlesi to develop in domestic species of AnophelesP. knowlesi the parasite might eventually become epidemic in humans P. falciparum, which accounts for up to 90% of annual infections worldwide P. vivax, the major cause of human malaria outside Africa, rarely kills, although cases of lethal P. vivax malaria have been reported P. vivax malaria in humans is commonly attributed to the inability of P. vivax-infected red blood cells to adhere to vascular endothelium and the preference of P. vivax to infect reticulocytes (immature red blood cells), which naturally limits parasitaemia because reticulocytes account for only 1–2% of erythrocytes P. vivax and P. ovale, but not P. falciparum and P. malariae, can stay dormant in the liver as hypnozoites, which can cause relapses months or even years after the primary infection in the blood has been cleared P. falciparumP. vivaxP. knowlesiP. yoeliiP. bergheiP. chabaudiPlasmodium genomes is that genes mediating parasite-host interactions are frequently restricted to a single Plasmodium species (species-specific) or restricted to a subset of Plasmodium species (species subset-specific). Perhaps the best studied and clinically most relevant example is P. falciparum erythrocyte membrane protein 1 (PfEMP1), whose different isoforms are encoded by about 60 members of the P. falciparum-specific var gene family P. falciparum malaria. PfEMP1 is therefore considered the prime virulence factor of P. falciparum malaria. Other important species- or species subset-specific gene families have been linked to host immune evasion, including the var and rif/stevor gene families in P. falciparum, vir in P. vivax, SICAvar and kir in P. knowlesi, and the cir/bir/yir family in rodent malaria parasites  were found to be highly syntenic and to contain comparably few gene differences between species. Nevertheless, important species- and subset-specific genes have been described in chromosome-internal regions as well, including members of the aforementioned var, MSP, and SERA gene families in P. falciparumP. vivaxP. vivax selectivity for young erythrocytes and/or immune evasion P. knowlesi genome is particularly rich in chromosome-internal species- and subset-specific genes, which have been identified as surface antigens of the SICAvar and kir gene families, respectively Recent genome sequencing of the two human malaria parasites Plasmodium species suggests that the search for species subset-specific genes is a promising strategy to identify new candidate genes underlying host-specific adaptations of Plasmodium species, in particular adaptations to human hosts and anthropophilic mosquito vectors. Identification and characterization of such genes may hold the key for important insights into molecular processes contributing to human disease. For example, parasite-encoded molecular factors that can explain why P. falciparum, P. vivax, and P. knowlesi but not rodent malaria parasites are infectious to humans are currently unknown. The identification of such pathogenicity factors could lead to new strategies to treat malaria in humans. Similarly, genes allowing P. falciparum and P. vivax but not P. knowlesi to complete their life cycle in anthropophilic mosquito vectors have not been identified, although an understanding of the genetic basis of this difference in human transmission success could help to prevent future host switches from monkey to human and pave the way for new transmission blocking strategies. Regarding human virulence it is likely that P. falciparum contains additional virulence genes that await functional characterization. The identification of new virulence genes would enhance our understanding of virulence mechanisms and could lead to new therapeutic interventions to treat severe malaria in humans. Finally, an understanding of the molecular mechanism underlying P. vivax hypnozoite formation is currently entirely missing but urgently needed, because hypnozoites cannot be killed by most available antimalarial drugs and complicate malaria eradication efforts The fact that parasite genes mediating parasite-host interactions are frequently restricted to a single or a subset of Plasmodium genomes harbour currently unappreciated species differences in the form of protein-coding genes that contribute to human pathogenicity, human-mosquito-human transmissibility, and human virulence. Other than subtelomeric regions, which contain mostly large and readily identifiable species-specific gene families Plasmodium genomes. Although not all subset-specific genes are expected to be functional due to stochastic processes of gene birth-and-death, a recent study in Drosophila has shown that a significant fraction of genes that differ between species do have important phenotypic effects P. falciparum, P. vivax, and P. knowlesi but absent in rodent malaria parasites, P. chabaudi, P. berghei, and P. yoelii. Second, we identified genes possibly crucial for parasite transmission success between humans by looking for genes present in P. vivax and P. falciparum but absent in the macaque monkey parasite P. knowlesi. Third, we identified genes that possibly contribute to severe human malaria by looking for P. falciparum-specific genes comparing P. falciparum with its less virulent relative P. vivax. Finally, we identified genes that potentially define unique features of P. vivax malaria by looking for genes present in P. vivax but absent in other sequenced Plasmodium genomes. Each of these comparisons resulted in the identification of several species subset-specific genes, most of which with unknown function. We propose these genes as attractive starting points for follow-up experimental analyses to test predicted phenotypic associations and to further elucidate their functions.We hypothesize that chromosome-internal regions of Plasmodium genomes sequenced to high coverage (ranging from 4 to 14.5-fold coverage) were selected for comparison, including the two clinically most important human parasites , one monkey parasite (P. knowlesi), and three rodent parasites   . Prelimie models  indicateries see . In totaed genes . Figure P. falciparum, P. yoelii, P. berghei and P. chabaudi, and the improved proteomes of P. vivax and P. knowlesi using BLASTP and genBlastG  involved in riboflavin (vitamin B2) metabolism IMP-specific 5′-nucleotidase (PFL0305c) that is involved in purine metabolism, an apicoplast phosphatidic acid phosphatase (MAL8P1.202) catalyzing the production of diacylglycerol as part of the dolichol metabolism phosphoethanolamine N-methyltransferase (MAL13P1.214) that plays a role in phospholipid metabolism Jumonji domain containing protein (MAL8P1.111) serving as one of two functionally distinct P. falciparum histone lysine demethylases nucleoside transporter (PF14_0662), a putative acyl-CoA N-acyltransferase (PFI1220w) specifically upregulated in gametocytes and sporozoites Ca++ chelating serine protease (MAL7P1.339). Taken together, the three primate parasites infectious to humans maintain a limited but conserved subset of genes that is absent in rodent malaria parasites, pointing towards new candidate pathogenicity genes required for parasitizing primate hosts, including humans.Besides the three thiamine biosynthesis enzymes, P. falciparum, P. vivax, and P. knowlesi were performed using whole-genome synteny analysis. Synteny blocks were detected with OrthoCluster imperfect synteny blocks  and non-nested synteny blocks (i.e. synteny blocks not contained within larger synteny blocks due to one-to-many orthologous relationships) Further comparisons of the genomes of the three primate parasites P. falciparum and P. vivax  is defined as the genomic region that interrupts perfect synteny within imperfect synteny blocks due to the presence of one or more consecutive non-syntenic genes (defined here either as parasite-specific genes that do not have a predicted ortholog in the compared species or as genes that do have a predicted ortholog in the compared species but not syntenic).To better characterize parasite-specific genes revealed by synteny block analysis, we define three different types of non-syntenic regions inset . A subteP. falciparum-specific genes and 173 of 388 (45%) P. vivax-specific genes in chromosome-internal regions between P. falciparum and P. vivax  P. vivax-specific genes and 222 of 364 (61%) P. knowlesi-specific genes were confirmed  than to any P. falciparum paralog (best hit PF13_0258 (TKL3); E = 0.008; PID = 48%), suggesting that the presence of this gene is of functional importance.rhoptry protein 2Deg2 chloroplast peptidases and is the sole P. falciparum member of clan PA Apicomplexa species but not non-human malaria parasites, suggesting functional gene loss in non-human malaria parasites. It should be emphasized that a syntenic gene of MAL8P1.126 annotated as DegP-like serine protease 1 precursor is present in P. knowlesi (PKH_011050) but much shorter (409 aa) with very low sequence similarity to MAL8P1.126 . Thus PKH_011050 is probably a non-functional pseudogene. The remaining two putative human malaria parasite-specific genes (PF14_0236 and PFL0360c) have no annotated function. Both contain a predicted zinc finger domain and PF14_0236 is predicted to be involved in antigenic variation Four genes in P. knowlesi could lack a functional copy of telomeric repeat binding factor 1 (TRF1). Running GeneWise with TRF1 of P. falciparum (PFI1216w) against the syntenic region in P. knowlesi reveals only residual protein sequence similarity , which is well below the degree of conservation found with P. vivax (52% PID) and probably indicative of recent gene inactivation in P. knowlesi. Both BLASTP and TBLASTN searches using PFI1216w as query against the complete P. knowlesi genome revealed the syntenic region of PFI1216w as best hit. Although almost certainly not linked to parasite transmission success, the potential absence of a fully functional copy of TRF1 in P. knowlesi is interesting, because it could offer an explanation for the presence of hundreds of variant surface antigens and telomeric repeats in chromosome-internal regions of the P. knowlesi genome  and adhesion to host , mostly because of the presence of these 26 var genes  and defense response . More generally, we find P. falciparum-specific genes enriched for GO subcellular locations membrane  and host intracellular part , indicating enrichment for proteins functional at the parasite-host interface.Looking at parasite-specific genes identified between the highly virulent parasite ar genes . The twoP. falciparum malaria, we removed known virulence genes and retained genes that contain features commonly associated with human virulence genes, including PEXEL motifs, signal peptides, or transmembrane domains. In addition, we retained P. falciparum-specific genes predicted to have virulence-associated functions based on gene co-expression or protein interaction data with known virulence genes (‘guilt-by-association’ principle) To identify novel candidate genes potentially linked to severe apyrase (PF14_0297) and a putative sugar transporter (PFE1455w) (see evasion of host defense (PF07_0107), antigenic variation (PFA0360c), biological adhesion (PF10_0350), and immune response (PF10_0044). PlasmoDB annotates another two genes with GO terms cell adhesion (PF13_0071) and immune response (MAL8P1.97), respectively. Eleven genes carry a predicted signal peptide or transmembrane domain and thus potentially function at the parasite-host interface. One of them (PF07_0107) carries an additional PEXEL motif and is thus a predicted erythrocyte surface or exported protein. Looking at RNA-seq expression data for genes with unknown function, all but two genes (MAL8P1.97 and PF10_0044) have associated expression evidence during the intraerythrocytic developmental cycle (IDC). Two genes (PF07_0107 and PF13_0194) are constitutively expressed at high levels, one gene peaks at the trophozoite stage (PF10_0350), seven genes peak at the late trophozoite/early schizont stage, and one gene (PFF0335c) peaks during schizont development. Two genes (PF10_0357 and PF10_0342) appear maximally expressed during the schizont-ring stage transition and co-localize with the MSP3 gene cluster on P. falciparum chromosome 10, suggesting a function in erythrocyte invasion.Among the resulting 15 genes  we found55w) see . The remP. vivax-specific genes compared to P. falciparum  and contains eight single-exon genes located on the same strand. The syntenic genomic region in P. falciparum maps to the MSP3 gene cluster on chromosome 10 , which harbors 13 P. falciparum-specific single-exon genes also located on the same strand. The P. falciparum gene cluster consists of several known antigens and genes involved in erythrocyte invasion, including six members of the MSP3 gene family (including MSP6), the glutamate-rich protein (GLURP) as well as the S-antigen (PF10_0343) and liver stage antigen 1 (PF10_0356). All but one of these genes (PF10_0343) have no predicted ortholog in P. vivax. It is possible that the P. vivax genes in the syntenic gene cluster on chromosome 6 have a similar function as the P. falciparum-specific genes on chromosome 10, which makes them prime candidate genes involved in erythrocyte invasion and interesting targets for further functional characterization. Three other lines of evidence support this conclusion. First, for all but one (PVX_110955) of these eight P. vivax genes, top P. falciparum BLASTP hits fall into the syntenic P. falciparum gene cluster . Second, all eight P. vivax genes carry a predicted signal peptide and are thus likely exported proteins. Third, four P. vivax genes  peak in expression during the schizont-ring stage transition, which is typical for invasion-related proteins Of the 173 identified invasion . This geP. vivax-specific genes absent in both P. falciparum and P. knowlesi that could explain unique biological features of P. vivax malaria, in particular the formation of hypnozoites. After excluding questionable open reading frames, only six candidate genes remained , suggesting recent gene inactivation in P. knowlesi. The other five genes have unknown functions. Four genes  localize to chromosome-internal RAD, MSP3 (chromosome 10), MSP3 , and MSP7 gene clusters, respectively, and are thus possibly functionally related to these gene families. The remaining gene (PVX_003710) is a 154 aa single-exon gene with EST expression evidence but of unknown function.We identified only few chromosome-internal ined see . One genPlasmodium species and proposed several chromosome-internal genes as new candidate genes underlying medically important phenotypic differences, including human pathogenicity, human-mosquito-human transmissibility, and human virulence. Previous studies have shown that important molecular processes at the parasite-host interface, including cytoadherence In this study, we compared the genomes of six P. chabaudi and P. berghei available at PlasmoDB 7.1) with genBlastG did not identify these genes in any of the three rodent malaria parasite genomes. If these genes were present in rodent malaria genomes but mis- or unannotated, then we would expect genBlastG to find them, because all 16 P. falciparum genes are well conserved in P. vivax and P. knowlesi, which have a similar phylogenetic distance to P. falciparum as the three rodent parasites. Finally, syntenic genomic regions as determined by flanking orthologs are present in latest chromosome-level assemblies of P. berghei and P. chabaudi and are assembled without gaps, making it less likely that these genes are absent in rodent parasite genomes due to incomplete genome sequences or assemblies. In some cases as shown in We identified 16 genes that are well conserved in the three primate parasites causing human disease but are not found in rodent parasites. Some of these genes could be determinants of primate (and thus human) pathogenicity . Most ofde novo and that rodent malaria parasites depend entirely on thiamine uptake from vertebrate and invertebrate hosts. Indeed, studies have shown that rodent malaria parasites have greatly impaired eryrthrocytic multiplication rates if thiamine is deliberately eliminated from the host P. falciparum thiamine enzymes, top BLASTP hits outside Plasmodium are found in bacteria, including Clostridium spp. as top hits in two of three cases (data not shown). In Clostridium ljungdahlii, the three enzymes are located next to each other on the same strand and thus form a potential operon  and subsequently lost in the common ancestor of rodent malaria parasites. Gene loss in rodent malaria parasites  as the likely cause for this species-specific difference is supported by residual sequence similarity found in syntenic genomic regions of rodent parasite genomes  biosynthesis pathway. This pathway has been proposed as attractive antimalarial drug target because of its absence in human hosts l operon , compati genomes .P. falciparum and P. vivax with the monkey parasite P. knowlesi, we identified 13 P. vivax genes that have a syntenic ortholog in P. falciparum but no predicted ortholog (neither syntenic nor non-syntenic) in P. knowlesi. The presence of such genes was unexpected because phylogenetically P. vivax is much more closely related to P. knowlesi than to P. falciparum  show only weak expression during the IDC and are specifically up-regulated in sporozoites or gametocytes. Notably, four of the identified 13 genes lack orthologs also in rodent malaria parasites and thus could mediate functions specifically required to parasitize humans. The remaining nine genes have predicted orthologs in rodent malaria parasites and thus likely represent cases of gene loss in P. knowlesi. Further experimental characterization of these 13 genes is required to confirm a potential role in human transmission success. Eventually, these studies may lead to the development of new transmission blocking strategies or to new ideas how future host switches from monkey to human can be prevented. If the ambitious goal of malaria eradication is to be taken seriously Comparing the two human parasites lciparum . Unlike P. falciparum with the less virulent human parasite P. vivax, we identified 117 chromosome-internal P. falciparum-specific genes, many of which have known virulence-associated functions  is annotated as apyrase, which is a membrane-bound enzyme converting ATP to AMP. Apyrases are involved in purine metabolism P. falciparum but not in any other Plasmodium parasite (best NCBI BLASTP hit was found in the human apicomplexan parasite Toxoplasma gondii) points towards an increased requirement of this enzymatic function in P. falciparum. Apyrase has been proposed as possible target for antimicrobial therapies P. falciparum malaria. The second gene with annotated function (PFE1455w) is a putative Na+- or H+-driven sugar symporter of the GPH family P. falciparum . NCBI BLASTP and TBLASTN searches reveal homologs of PFE1455w in T. gondii (TGME49_026020) and Neospora caninum (NCLIV_046810) but not in any other Plasmodium species. Host-derived sugars are an essential nutrient of malaria parasites for intraerythrocytic development P. falciparum.Comparing the highly virulent human parasite unctions . Subtracunctions . BecauseP. vivax from other sequenced Plasmodium species, including preference for reticulocytes and its ability to develop dormant hypnozoite forms in the liver that can cause relapses months or even years after primary infection. We hypothesized that genes present in P. vivax but absent in the other sequenced Plasmodium species are candidate genes underlying reticulocyte invasion and hypnozoite formation. We identified a currently uncharacterized chromosome-internal gene cluster on P. vivax chromosome 6 containing several P. vivax-specific genes putatively involved in erythrocyte invasion . P. chabaudi and P. berghei chromosome-level assemblies were available but unpublished. Therefore, older contig-level assemblies available at PlasmoDB (version 5.5) were used for genome-wide comparisons. The P. yoelii contig-level assembly was also downloaded from PlasmoDB 5.5. Annotated gene models (GFF3 format) were downloaded from PlasmoDB 7.0  and PlasmoDB 5.5 . If a gene had multiple isoforms, only longest isoforms ( = longest protein sequence) were kept.Published chromosome-level assemblies for P. vivax and P. knowlesi gene model annotations before genome comparisons. Using two homology-based gene predictors, including our own program genBlastG P. falciparum genes  were used as query to run both genBlastG and GeneWise against P. vivax and P. knowlesi genomes. To ensure the quality of predicted gene models, only predictions that encoded for protein sequences with high global sequence identity (PID> = 60) with the query gene were kept. If multiple predictions overlapped by more than 5% of their coding exons, only the prediction with the highest PID was kept (filtration step). We use global PID as a measure of sequence conservation because it better captures global similarity between two proteins as compared to for example the BLAST E-value, which measures local sequence similarity and is more prone to various biases, including sequence composition. In a subsequent merging step, predicted and existing gene models were merged into a hybrid gene set, retaining predictions that (a) did not overlap with existing gene models or (b) showed a PID improvement of at least 5% over overlapping existing gene models. As in the filtration step, existing and predicted gene modes were considered as overlapping if more than 5% of their coding exons overlapped. The hybrid gene set served as basis for all subsequent comparisons. A summary of improved gene models, including novel functional annotations predicted with InterProScan P. vivax) and P. knowlesi). Novel and improved gene models for both P. vivax and P. knowlesi are provided in Missing or incorrectly annotated gene models cause overestimates of genetic differences and, important for this study, false specific-specific genes. We therefore repaired the more obvious defects in P. falciparum , P. vivax , P. knowlesi , and P. yoelii  were used as query to run both NCBI BLASTP (version 2.2.21) P. berghei and P. chabaudi). Top hits of both BLASTP and genBlastG were used to compute global PIDs with the query protein using ClustalW  P. falciparum genes were considered primate parasite-specific if conserved in P. vivax and P. knowlesi but not in P. berghei, P. chabaudi, and P. yoelii. The rather conservative margin between high and low PID (25 percent points) was chosen to exclude insignificant PID differences due to fluctuating protein sequence conservation levels or imperfect gene models. Summarized results of this first initial screening are shown in P. falciparum orthologs of the 30 putative primate parasite-specific genes were inspected using the newer chromosome-level assemblies of P. chabaudi and P. berghei available at PlasmoDB 7.1. We only kept P. falciparum genes for which (a) genBlastG failed to annotate a gene with a minimum global PID of 15 in the entire genome and (b) no gene was present at the expected syntenic region as defined by the position of flanking syntenic orthologs. Sixteen out of the initial 30 genes fulfilled these two criteria and are shown in Because chromosome-level assemblies for rodent malaria parasites that became available with PlasmoDB 7.0 have not yet been published, we used older, contig-level assemblies (PlasmoDB version 5.5) and a synteny-independent, BLAST-based approach for the initial genome-wide screening for species subset-specific genes. Briefly, complete proteomes of Plasmodium genomes. Furthermore, we made no efforts to improve rodent parasite gene models based on the now obsolete PlasmoDB 5.5 genome assemblies, because greatly improved P. chabaudi and P. berghei gene models became available with PlasmoDB 7.0. It should be emphasized, however, that using the older PlasmoDB 5.5 rodent parasite genome sequences and gene models in the initial screening step did not affect our final results, because all final candidate genes in The two-step process of first screening for putative primate parasite-specific genes against PlasmoDB 5.5 versions of rodent malaria parasite genomes and gene models then verifying the absence of candidate genes in PlasmoDB 7.1 was chosen because, in agreement with pre-publication data use policies, we restricted all genome-scale comparisons to officially published http://genome.sfu.ca/cgi-bin/orthoclusterdb/download), a program recently developed in our lab, for the gene-based identification of synteny blocks. As input OrthoCluster was provided with genome coordinates of protein-coding genes as well as with gene orthology relationships predicted by Inparanoid (version 4) i.e. genes without predicted orthologs in the other genome) and ≤10% in-map mismatches . These two thresholds were chosen after observing that further increasing the percentages did not result in larger imperfect synteny blocks  We used OrthoCluster y blocks . OrthoClTo separate questionable differences from likely true genetic differences, we examined all parasite-specific genes in SGRs and SBRs. SGRs were examined in an automated manner using custom Perl scripts. The few SBRs were trickier to deal with due to ambiguous mapping locations in the other genome and were thus examined manually. Briefly, automated BLASTP and GeneWise homology searches were combined with manual visual inspections of non-syntenic regions in a genome browser. Non-random BLASTP sequence similarity (E≤1e-4 and PID≥20) between two ‘syntenic’ parasite-specific genes of which both are flanked by syntenic orthologs was interpreted as evidence of likely orthologous genes missed by Inparanoid. We refer to these genes as positional orthologs. Furthermore, non-random GeneWise alignments (bitscore ≥40) generated in syntenic regions using the putative parasite-specific gene as query are indicative of potentially split/merged genes or missing genes, depending on whether or not alignments overlap with existing gene models. Albeit due to limited sequence similarity GeneWise gene models produced in this step are not entirely reliable, we kept their protein translations for downstream proteomics analyses. In addition, we provide these tentative GeneWise gene models for further inspection  did not produce a gene model with at least 15% PID and 80% query coverage or (b) produced such a gene model but it overlapped with a P. knowlesi gene that had a different known ortholog in P. falciparum. The resulting gene list is shown in P. falciparum gene names and sequences, including PlasmoDB 7.1 Genes shared by knowlesi  were ideknowlesi  and thenother, annotated function’ (15 genes) and ‘ protein, unknown function’ (42 genes), we excluded genes representing chromosome-internal members of previously described (subtelomeric) gene families (7 genes), genes part of gene families with known members in P. vivax (6 genes), genes without expression evidence or questionable open reading frames (9 genes), and genes where visual re-examination revealed the presence of a potential positional ortholog in P. vivax that did not meet our similarity threshold for automatic detection . Of the remaining genes we only retained those with potential virulence-associated functions as predicted by (a) the presence of a PEXEL motif, a signal peptide, or a transmembrane domain, or (b) PlasmoDraft P. vivax, but was nevertheless retained in the final list because this gene is present as an extra copy in P. falciparum . As a last step, we ran genBlastG with remaining P. falciparum genes against the entire P. vivax genome and retained only those candidates that either (a) did not produce a gene model with at least 15% PID and 80% query coverage or (b) produced such a gene model but it overlapped with a P. vivax gene that had a different known ortholog in P. falciparum. The resulting gene list is shown in P. falciparum -specific genes is provided in P. falciparum gene names and sequences, including PlasmoDB 7.1, GeneDB, InterPro, and NCBI nucleotide and protein archives. Sequence-independent predictions of virulence-associated functions were obtained from.Starting with all genes in P. vivax, we overlapped the two P. vivax-specific gene sets of P. vivax-specific genes absent in both P. falciparum and P. knowlesi. We then excluded genes with named gene products , which resulted in 38 chromosome-internal P. vivax-specific genes encoding for hypothetical proteins of unknown function (To identify genes exclusively present in function . BecauseDataset S1Improved P. vivax and P. knowlesi gene models in GFF3 format.(TXT)Click here for additional data file.Dataset S2Detected imperfect synteny blocks between P. falciparum and P. vivax. OrthoCluster ‘.cluster’ output file showing both syntenic and non-syntenic genes of identified imperfect synteny blocks.(TXT)Click here for additional data file.Dataset S3Detected imperfect synteny blocks between P. vivax and P. knowlesi. OrthoCluster ‘.cluster’ output file showing both syntenic and non-syntenic genes of identified imperfect synteny blocks.(TXT)Click here for additional data file.Dataset S4Additional revised P. vivax and P. knowlesi gene models in GFF3 format. This file contains GeneWise gene models produced during syntenic examination of putative parasite-specific genes using genes from the syntenic genomic region of P. falciparum genes as query. Some predictions are based on low sequence similarity  and should therefore be considered preliminary.(TXT)Click here for additional data file.Figure S1Phylogeny (A) and selected genome features (B) of the six Plasmodium genomes compared in this study. The phylogenetic tree was computed with proml/PHYLIP v3.68 based on concatenated protein sequences from 50 randomly chosen well conserved nuclear 1-to-1 orthologs A at chromosome ends. § Only longest isoforms.logs see . Out-gro(DOC)Click here for additional data file.Figure S2Examples of improved gene models in P. vivax and P. knowlesi. Panel A shows a newly identified 60S ribosomal protein (L39) in P. knowlesi (PKH_113715), which has 86% global protein sequence identity (PID) and 98% coverage with its P. falciparum ortholog PFF0573c. Panel B shows two P. vivax split genes (PVX_090855 and PVX_090860) merged into a single gene (PVX_090860). The revised gene model is supported by EST evidence and, after improvement, recognized as CPW-WPC domain containing protein by InterProScan. Panel C shows a merged gene in P. knowlesi (PKH_132400) annotated as dynein-associated protein that was split into two genes (PKH_132400a and PKH_132400b), one of which is subsequently recognized as membrane occupation and recognition nexus (MORN)-motif containing protein. Panel D shows the replacement of a truncated hypothetical protein in P. vivax (PVX_088280) with a longer gene model, facilitating its recognition as putative acetyltransferase. Improved gene models shown in yellow. Existing PlasmoDB 7.1 gene models shown in blue (forward strand) or red (reverse strand). The complete set of improved gene models is provided in (DOC)Click here for additional data file.Figure S3Different types of non-syntenic regions.Subtelomeric regions (STR) range from the first gene on a chromosome arm to the first syntenic gene (two STRs on each chromosome). Synteny breakpoint regions (SBR) are defined as genomic regions between imperfect synteny blocks, which may or may not contain genes. Synteny gap regions (SGR) are defined as genomic regions that interrupt synteny within imperfect synteny blocks due to the presence of one or more parasite-specific genes or non-syntenic orthologs (seven SGRs shown). Shown are two real imperfect synteny blocks identified between P. falciparum (chromosome 4) and P. vivax. The syntenic architecture depicted here  is typical for all investigated Plasmodium chromosomes.(TIF)Click here for additional data file.Figure S4Influence of OrthoCluster -ip and -op parameter on the number of identified imperfect synteny blocks between P. falciparum and P. vivax. The graph shows the number of identified imperfect synteny blocks between P. falciparum and P. vivax as a function of allowed in-map mismatches (-ip parameter) and out-map mismatches (-op parameter). After allowing for 10% in-map mismatches and 40% out-map mismatches the number of identified synteny blocks reaches a plateau and does not decrease further. Similar results were observed between P. vivax and P. knowlesi (data not shown).(XLS)Click here for additional data file.Figure S5Gene organization of thiamine biosynthesis genes in Clostridium ljungdahlii suggesting an operonic gene structure. Protein sequences of the three thiamine biosynthesis genes of P. falciparum  were used as NCBI BLASTP queries to search for homologous proteins (nr database). For all three genes top hits outside Plasmodium were found in bacteria, with top hits in Clostridium ljundahlii in two of three cases. In Clostridium ljundahlii the three enzymes are located next to each other and on the same strand, suggesting that they could form an operon in this species. Screenshot adapted from the NCBI Entrez database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=retrieve&list_uids=9446921).(DOC)Click here for additional data file.Table S1List of improved gene models in P. vivax. The list includes new and replaced gene models together with characteristics of query and target genes , EST support, genomic location, method of prediction, sequence conservation levels, query coverage, and newly predicted domains after improvement.(XLS)Click here for additional data file.Table S2List of improved gene models in P. knowlesi. The list includes new and replaced gene models together with characteristics of query and target genes , genomic location, method of prediction, sequence conservation levels, query coverage, and newly predicted domains after improvement.(XLS)Click here for additional data file.Table S3Gene orthology and synteny between P. falciparum and P. vivax as well as between P. vivax and P. knowlesi. Ortholog groups and synteny blocks were identified with Inparanoid4 and OrthoCluster, respectively. Only chromosomal contigs considered for synteny analysis. Orthology prediction included genes from all contigs, chromosomal and non-chromosomal. ‡ Number of protein-coding genes after gene model improvement and excluding annotated pseudogenes and shorter isoforms. § Average percent identity of global protein sequence alignments of all one-to-one orthologs computed with ClustalW. Abbreviations: bp … base pairs; chr … chromosome totals, excluding non-chromosomal contigs.(DOC)Click here for additional data file.Table S4Complete list of identified parasite-specific genes between P. falciparum and P. vivax. The list includes both subtelomeric and chromosome-internal parasite-specific genes. Chromosome-internal parasite-specific genes are listed together with non-syntenic regions they fall into (SGR or SBR).(XLS)Click here for additional data file.Table S5Complete list of identified parasite-specific genes between P. vivax and P. knowlesi. The list includes both subtelomeric and chromosome-internal parasite-specific genes. Chromosome-internal parasite-specific genes are listed together with non-syntenic regions they fall into (SGR or SBR). Note that numbers of questionable parasite-specific genes in this table are lower than in P. falciparum were excluded for this table.(XLS)Click here for additional data file.Table S6Number of parasite-specific genes found in non-syntenic regions before (top) and after (bottom) excluding questionable differences. Questionable cases of parasite-specific genes and non-syntenic orthologs are detected and removed through a combination of both automatic and manual examination of SGRs and SBRs (see P. vivax) and 75 genes (P. knowlesi) currently located on non-chromosomal contigs.SBRs see . ‡ Inclu(DOC)Click here for additional data file.Table S7Enriched GO terms in P. falciparum-specific genes.P. falciparum-specific genes identified in comparison with P. vivax were subjected to GO term enrichment analysis using Ontologizer. GO terms statistically significantly enriched after correcting for multiple testing  are highlighted in red.(XLS)Click here for additional data file.Table S8List of species subset-specific genes as identified by BLAST and genBlastG. Genes in this table underlie the Venn diagram of (XLS)Click here for additional data file.Table S9Genes conserved in primate parasites but absent in rodent parasites. Same genes as in (PDF)Click here for additional data file.Table S10Genes shared between P. falciparum and P. vivax but absent in P. knowlesi. Same genes as in (PDF)Click here for additional data file.Table S11P. falciparum genes absent in P. vivax with possible role in human virulence. Same genes as in (PDF)Click here for additional data file.Text S1Supplementary information. Additional information about Plasmodium genome features and species phylogeny, homology-based gene model improvement, synteny block analysis, syntenic examination of chromosome-internal parasite-specific genes, compositional bias of Plasmodium genomes, and enriched GO terms of P. falciparum-specific genes.(DOC)Click here for additional data file.