Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid.Lipoproteins are virulence factors of Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed.We exploit the vaccine strain M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in  Proteins posttranslationally modified by covalent lipid attachment are present in eukaryal and bacterial organisms. In bacteria, 1–3% of the genome encode for lipoproteins. Bacterial lipoproteins are anchored in the membrane surface where they fulfill various cellular functions, ranging from cell wall integrity, secretion, nutrient uptake, environmental signaling to virulence + signals at m/z values which we calculated for the unmodified N-terminal peptides were not found. Instead, we found MS signals at m/z values which indicate that the N-terminal peptides are modified in a lipoprotein-specific manner with different combinations of saturated and unsaturated C16, C18 and C19 fatty acids. The calculated m/z values are summarized and compared with the experimentally determined m/z values in Table To characterize the modifications of lipoproteins at the molecular level, the four recombinant lipoproteins LprF, LpqH, LpqL and LppX were expressed in proteins -44. Therproteins ,46. Someyristic C4:0, palm+ signals in the MS spectrum were confirmed by MS/MS fragmentation and thereby information about the linkage of the modification was obtained. The structures of the di- or triacylated N-terminal tryptic or AspN-digested peptides from LprF, LpqH, LpqL and LppX were investigated by MS/MS. All eliminations found in MS/MS of lipoproteins isolated from the parental strain are summarized in Table The modifications we estimated from the [M+H]N-acylation was shown to be a common motif in lipoproteins of high GC-rich Gram-positive M. smegmatis[, we proposed Lnt modification also taking place in slow-growing mycobacteria. This proposal was based on the observation that M. tuberculosis apolipoprotein N-acyltransferase Ppm1 could complement a M. smegmatis lnt mutant [Since smegmatis,13, we pt mutant .M. bovis BCG, differences in molecular mass of about 831.36 Da for LprF, LpqH, LpqL and LppX, 993.60 Da for LppX, 1035.69 Da for LprF and 1155.84 Da for LppX between the experimentally determined peptide and unmodified N-terminal peptide were found  with default settings to compare both M. bovis ORFs to E. coli lnt, M. tuberculosis lnt Rv2051c, as well as M. tuberculosis Rv2262c/2261c sequences. Pairwise sequence alignment revealed the highest sequence identity (100%) between BCG_2070c and Rv2051c from M. tuberculosis. Interestingly, pairwise sequence alignment of BCG_2279c and Rv2262c/2261c reveals that both sequences differ by a 2 bp insertion in Rv2262c   and ClustalW2  with default settings to analyze the conservation of essential residues  among the 23 residues of E. coli Lnt. However, different residues among the 23 were conserved  mutant also formed an acyl-enzyme intermediate as the wildtype Lnt C387, but did not have any detectable Lnt activity [M. tuberculosis. Beside the three essential residues of the catalytic triad, four other essential residues W237, E343, Y388 and E389 are absolutely required for Lnt function. Among these seven essential residues, five residues are conserved in M. tuberculosis Rv2051c, Rv2262c/2261c and M. bovis BCG BCG_2070c, BCG_2279c Lnt homologues.Using bacteria . A furthactivity ,47. MoreN-acyltransferases. BCG_2070c is identical to an ORF with proven N-acyltransferase activity since M. tuberculosis Lnt complemented the M. smegmatis lnt deletion mutant and all three residues of the catalytic triad essential for Lnt function in E. coli are conserved. Lnt activity of BCG_2279c may be buried by the Lnt activity of BCG_2070c. Therefore we generated a BCG_2070c lnt deletion mutant and characterized lipoprotein modifications in the mutant. The lnt deletion mutant was constructed by transformation of M. bovis BCG with the suicide plasmid pMCS5-rpsL-hyg-ΔlntBCG applying rpsL counter-selection strategy, a powerful tool to generate deletion mutants in mycobacteria [M. bovis BCG Δlnt. Deletion of lnt was verified by Southern blot analysis using a 5’lnt DNA probe  or 280.49 Da (1035.69 Da - 162.24 Da - 592.96 Da) between the C16/C19/C16 or C16/C19/C19 triacylated modification found in the parental strain and the corresponding estimated C16/C19 modification in the Δlnt mutant indicate a lack of N-acylation with a C16 or C19 fatty acid in the Δlnt mutant. In MS/MS analysis, this indication of missing N-acylation in the mutant was confirmed by identification of the estimated modifications and information about its linkage  is uniquely found in lipoproteins of M. smegmatis[Lipoproteins are present in all bacterial species, but their biogenesis and lipid moieties differ, especially between Gram-negative and Gram-positive bacteria. The three enzymes involved in lipoprotein biosynthesis, namely Lgt, LspA and Lnt first were identified in escribed . Mycobacsmegmatis,13.M. tuberculosis, lgt, the first enzyme of the lipoprotein biosynthesis pathway is essential. A targeted deletion of lgt was not possible [lspA deletion mutant was viable, but the mutant strain showed a reduced number of CFU in an animal model and induced hardly any lung pathology. This confirmed a role of the lipoprotein biosynthesis pathway in pathogenesis of M. tuberculosis[All three enzymes of the lipoprotein biosynthesis pathway, Lgt, LspA and Lnt are essential in Gram-negative, but not in Gram-positive bacteria. However, in possible . In conterculosis,24.M. tuberculosis lipoproteins in particular have been shown to suppress innate immune responses by TLR2 agonist activity [M. tuberculosis mutants deficient in individual lipoproteins are attenuated in virulence as shown for LppX [M. tuberculosis deletion mutant, defective in lipoprotein LpqS showed attenuation in macrophages [M. tuberculosis lipoproteins in immunogenicity and pathogenicity and all the achievements in knowledge about the lipoprotein modification in apathogenic M. smegmatis, still little is known about the molecular structure of lipoproteins in pathogenic mycobacteria. The elucidation of lipoprotein structure can build the fundamental knowledge for future development of lipoprotein based subunit vaccines and antitubercular drugs targeting enzymes of the lipoprotein synthesis pathway [M. bovis BCG belong to this sub-group.Lipoproteins itself are well known virulence factors in pathogenic bacteria. activity . Newest activity . Severalfor LppX , LprG [5for LppX  and LpqHfor LppX . Recentlrophages . Despite pathway . TherefoSn1 or Sn2 position could not be determined by mass spectrometry and therefore currently remains elusive. In LprF, a novel triacylation with C16/C19 diacylglycerol and C19 N-acyl was identified. This differs from previous lipoprotein analyses in M. smegmatis, where C16 fatty acid was the single substrate for Lnt [N-acylation. Lipoprotein modifications with acyl residues of different length, stiffness and bulkiness may influence membrane fluidity and localization of lipoproteins. In Francisella novicida, an environmentally regulated membrane remodelling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme is reported. By incorporation of shorter or longer N-acyl fatty acid chains to the outer membrane lipid A, the bacterium regulates the maintenance of membrane fluidity and integrity [N-acyl lipoprotein modification for mycobacteria in terms of adaptations to environmental alterations or specific bacterial conditions. Adaptation of lipoprotein modification to environmental conditions has been shown for S. aureus. A combination of conditions including acidic pH and post-logarithmic growth phase induced the accumulation of diacylated lipoproteins [In the present study, we investigated the lipid moieties of four mycobacterial lipoproteins representing lipoproteins with different functions. By MALDI-TOF/TOF analyses of a Trypsin digest of purified LpqH, LpqL and LppX and an AspN digest of purified LprF, we unambiguously identified modifications at the universally conserved cysteine in the parental strain. All four proteins were found to be triacylated carrying a thioether-linked diacylglyceryl residue with C16 and C19 fatty acid (C16/C19) to the sulfhydryl group of the lipobox cysteine and an amide-linked C16 fatty acid. Whether the fatty acids of the diacylglyceryl residue are in the  for Lnt ,13. Likentegrity . Therefoproteins .E. coli Lnt. E. coli Lnt utilizes all three major phospholipids of E. coli phosphatidylethanolamine, phosphatidylglycerol and cardiolipin as its fatty acid source in vivo [N-acyltransferase activity in vitro [E. coli Lnt incorporates palmitic (C16) fatty acids from the Sn1 position of phospholipids to diacylated lipoproteins [Sn1 position is esterified principally with octadecanoic or tuberculostearic acid (C18 related fatty acids), whereas palmitic acid (C16) is mainly located at the Sn2 position [N-acylation of lipoproteins in M. smegmatis[M. smegmatis Lnt uses fatty acids from the Sn2 position as substrates and therefore has a different specificity than E. coli Lnt [M. bovis BCG. Our results provide strong evidence, that not only palmitic acid from the Sn2 position, but also tuberculostearic acid (C19), a fatty acid from the Sn1 position of phospholipids is transferred by Lnt [By the usage of C19 fatty acid, mycobacterial Lnt strongly differs in substrate specificity from  in vivo . Subsequin vitro . E. coliproteins . In mycoposition . Based osmegmatis,13, Nakacoli Lnt . This spd by Lnt .S-diacylglyceryl residue are recognized by TLR2/6 heterodimers, triacylated lipoproteins carrying the additional N-acyl are recognized by TLR1/2 heterodimers. The two ester-bound fatty acids are inserted into a pocket in TLR2 while the amide-bound fatty acid is inserted into a hydrophobic channel in TLR1. Therefore the N-acyl of the lipoprotein is indispensable for the heterodimerization of TLR2 and TLR1 and thus the initiation of TLR2/1 signaling [M. tuberculosis[N-acylation with C19 fatty acid is only characteristic for LprF or also for other lipoproteins and whether it is a feature of M. bovis BCG Lnt remains to be investigated.Lipoproteins are recognized by TLR2 in association with TLR1 or TLR6. While diacylated lipoproteins carrying the ignaling ,59. Receerculosis,61. It mBeside the triacylated forms, also diacylated forms of the N-terminal peptide were found in proteins from the parental BCG strain. A modification with C16/C19 diacylglycerol was found in LpqL and a C16/C16 diacylglycerol was found in LppX. These molecules probably indicate N-terminal peptides from unmature proteins which have not been converted to mature lipoproteins by Lnt yet.M. bovis BCG were identified to be triacylated at their N-terminus which suggests that N-acylation by an Lnt enzyme also exists in slow-growing mycobacteria. We identified the open reading frame, encoding the Lnt enzyme responsible for the N-acylation. M. bovis BCG Pasteur genome analysis revealed two open reading frames BCG_2070c and BCG_2279c homologous to E. coli Lnt. Our biochemical analyses of four lipoproteins expressed in a BCG_2070c Δlnt mutant demonstrated that BCG_2070c is the major if not the only functional mycobacterial Lnt in M. bovis BCG. When we subjected lipoproteins LprF, LpqH, LpqL and LppX expressed in the Δlnt mutant to MALDI-TOF/TOF analyses, none of the proteins was found to be N-acylated. All four proteins were found to be only diacylated in contrast to the triacylated proteins in the parental strain. Diacylglyceryl residues composed of C16/C19 fatty acid, C16/C16 fatty acid or C16/C18 were found. Hereby the usage of oleic acid as a substrate for lipoprotein modification in mycobacteria, to our knowledge is shown for the first time.Lipoproteins from N-acylation and that BCG_2279c is not able to compensate Lnt function. BCG_2279c has a C to S amino acid substitution in C387, a residue essential for Lnt function in E. coli. In E. coli, a C387 alteration absolutely abolishes Lnt function, because this residue is part of the catalytic triad of Lnt [We showed that the lack of BCG_2070c results in a failure of lipoprotein d of Lnt . AlteratStreptomyces spp., bacteria closely related to mycobacteria, also encode two Lnt homologues. Deleting Streptomyces scabies lnt1 and lnt2 genes individually or in combination revealed that Lnt1 is a functional Lnt sufficient and required for N-acylation. Lnt2 could not compensate for the Lnt1 deletion. However, both Lnts seem to be required for efficient lipoprotein N-acylation as the lack of Lnt2 alone resulted in a marginal N-acylation activity. This implies a subsidiary but inessential role for Lnt2, not directly involved in N-acylation of lipoproteins [M. bovis BCG. But, in contrast to the Lnts in S. scabies, BCG_2279c is missing one of the three essential residues required for Lnt activity in E. coli. This, in our opinion diminishes the possibility for BCG_2279c to be an Lnt with N-acylation activity and favours a contributive role for it. In vitro biochemical assays [proteins . Likewisl assays  with purO-glycosylation occurs at Ser and Thr residues respectively. Although glycosylations of the tryptic or AspN-digested N-terminal peptides of LprF and LppX were identified, the exact glycosylation site within the peptide could not be determined. No glycosylations were found for N-terminal fragments of LpqH and LpqL. This possibly is due to the use of proteases which have cleavage sites close to the N-terminus and therefore the peptide fragment may be too short to include O-glycosylation sites. The information about the exact molecular nature and function of the glycosylation is scarce, but its influence on subcellular lipoprotein localization and its protection from proteolytic degradation are proposed [B. subtilis lipoprotein glycosylation is discussed to control a lipoprotein “shaving” mechanism and thus their release into the culture medium [lnt mutant, demonstrating that N-acylation is not a prerequisite for glycosylation. Lnt independent glycosylation was also demonstrated in C. glutamicum[C. glutamicum Cg-Ppm1 is responsible for glycosylation. Cg-ppm1 (Ppm synthase) and Cg-ppm2 (Lnt) are similar organized as MSMEG_3859 (Ppm synthase) and MSMEG_3860 (Lnt) in M. smegmatis  transport system and there is good evidence that n E. coli,65. Lnt membrane . Mycobacmembrane -68 and amembrane . NeverthM. bovis BCG, the live vaccine for tuberculosis are triacylated and we identified the lipid modifications at the molecular level. BCG_2070c is a functional homologue of E. coli Lnt, but differs in substrate specificity. The identification of N-linked tuberculostearic acid shows for the first time, to our knowledge, that mycobacteria-specific fatty acids are used by mycobacterial Lnts.In the present study we show that lipoproteins from M. tuberculosis pathogenesis and immunogenicity have been shown. Our results about lipoprotein structure therefore may contribute to provide the knowledge which is required to develop novel vaccines and antituberculosis drugs to eliminate this worldwide epidemic.The antituberculosis drug pipeline is not sufficiently filled and the vaccines used at present do not provide effective protection against tuberculosis in adults. For lipoproteins and their biosynthesis pathway potential implications in M. bovis BCG wildtype and BCG_2070c lnt deletion mutant were analyzed at the molecular level. N-acylation of lipoproteins was only found in the wildtype strain, but not in the mutant strain, which confirmed BCG_2070c as functional lnt in M. bovis BCG. Moreover, we identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.Lipoproteins are triacylated in slow-growing mycobacteria. By MALDI-TOF/TOF analyses lipoprotein modifications in The authors declare that they have no competing interests.M. bovis BCG mutant strain and participated in the MS/MS data analyses. PS conceived of the study, participated in its coordination and helped to draft the manuscript. All authors read and approved the final manuscript.JKB designed the study, performed experimental work and drafted the manuscript. AT carried out the genetic engineering of M. bovis BCG wildtype and Δlnt mutant strain.Western blot analysis of purified lipoproteins of Click here for fileM. tuberculosis Rv2262c/Rv2261c and M. bovis BCG_2070c using EMBOSS Needle.Sequence alignment of Click here for fileMultiple sequence alignment of Lnt homologues using Clustal W2.Click here for fileConservation of essential residues in Lnt homologues.Click here for fileMycobacterium bovis BCG lnt (BCG_2070c).Disruption of Click here for fileMALDI-TOF analysis of the N-terminal peptides of LprF.Click here for fileMALDI-TOF analysis of the N-terminal peptides of LppX.Click here for file


Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain’s mechanisms to dominate the cocoa bean fermentation process.A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition.The genome sequence of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.Genome analysis of  Acetobacteraceae of the Alpha-proteobacteria  indicated a bidirectional replication mechanism of the chromosome Figure A, which s Figure B. This eomal map . The seqif sites . The difwith oriC. The occA. pasteurianus 386B and A. pasteurianus IFO 3283 , which are involved in the truncation of 32 genes [A. pasteurianus 386B . For instance, pAPA01-011 and pAPA01-020, the largest plasmids of A. pasteurianus IFO 3283, shared only 44 and 16 CDS with APA386B_1P, the largest plasmid of A. pasteurianus 386B, respectively  pathway, except for the phosphofructokinase-coding gene, indicating incomplete glycolysis. The absence of this gene has been reported before in A. pasteurianus IFO 3283, G. diazotrophicus Pal5, and G. oxydans 621H [A. pasteurianus IFO 3283 and A. aceti NBRC 14818 [glpK; Apa396B_92; Figure A. pasteurianus 386B genome contained genes coding for a glycerol uptake facilitator protein (APA386B_93), suggesting that this strain is able to take up glycerol from the environment to use it as an energy source, which might be present as a substrate during the cocoa bean fermentation process owing to yeast metabolism. DHA-phosphate may be channeled into the lower part of the EMP pathway. The A. pasteurianus 386B genome sequence provided further evidence that acetate is formed out of ethanol by soluble, NAD(P)+-dependent ADH  and glutamine synthetase (APA386B_2129), could be taken up by a specific ammonia transporter (APA386B_239). Furthermore, the genome of A. pasteurianus 386B contained genes to synthesize and use trehalose, which can protect the cell from high osmolarity and/or can be used as an energy source in bacteria and yeast [otsA; APA386B_1724), trehalose-6-phosphate phosphatase , and trehalase . In addition, genes coding for the mechanosensitive channels MscL (APA386B_2572) and MscS (APA386B_1440) were present in the genome sequence of A. pasteurianus 386B, which generally play an important role in osmotolerance [Genome analysis showed that nd yeast . The patolerance .Acetobacter pasteurianus 386B possessed several membrane-bound dehydrogenases that channel electrons into the respiratory chain  was separated from the other two genes . This gene organization in A. pasteurianus has been suggested before [A. pasteurianus 386B contained two uncharacterized, membrane-bound, PQQ-dependent oxidoreductases with five transmembrane helices . In contrast to other AAB, the genome of A. pasteurianus 386B did not possess the major polyol dehydrogenase (SldAB), an enzyme of industrial importance [n Figure A. A firsd before . Furtherportance . Indeed,portance .A. pasteurianus 386B genome, which points to the ability of this strain to produce fructose from sorbitol. However, it has been shown previously that this dehydrogenase is also responsible for the conversion of mannitol, an important intermediate of the cocoa bean fermentation process, into fructose [A. pasteurianus 386B . In addition, A. pasteurianus 386B possessed six membrane-bound oxidoreductases with unknown function  and a cytochrome bd-type ubiquinol oxidase , whereby the encoding genes of the latter were present twice in the genome sequence of A. pasteurianus 386B . Both terminal oxidases reduce oxygen to water when reoxidizing ubiquinol into ubiquinone -dependent sorbitol dehydrogenase (APA386B_1096 - APA386B_1098) were present in the fructose . As the n Figure . These oA. pasteurianus 386B  as well as cytochrome c  were identified in the genome of A. pasteurianus 386B. Furthermore, genes for the transport of heme c and the maturation of cytochrome c were present  [c, as cytochrome c oxidase (complex IV) is probably inactive, because only subunit I (APA386B_609) was present in the genome.The genes coding for a proton-translocating nicotinamide nucleotide transhydrogenase were present in the genome of ent ALDH . Indeed,h yields . Further6B_2400) . HoweverA. pasteurianus 386B to tolerate high levels of acetic acid may consist of a cytosolic acetate-assimilating detoxification pathway. This involves a conversion of acetate to acetyl-CoA, which is performed either by acetyl-CoA synthetase , an efflux pump in the cytoplasmic membrane capable of exporting acetic acid [A. pasteurianus 386B contained the gene cluster involved in pellicle polysaccharide formation , preventing the diffusion of acetic acid into the cytoplasm [ureDABCEFG; APA386B_1179 - APA386B_1184), an urea transporter , an allophanate hydrolase (APA386B_936 - APA386B_937), and an urea carboxylase (APA386B_218) were present, indicating the ability to transport urea and convert it into ammonia, which may contribute to the survival of A. pasteurianus 386B in acidic environments, such as the cocoa pulp-bean mass (pH 3.5 – 4.5). The human pathogenic Gr. bethesdensis CGDNIH1 contains this mechanism too, although it may not be widespread among AAB strains, as it is absent in G. oxydans 621H and Ga. diazotrophicus Pal5 [A. pasteurianus 386B revealed the presence of genes coding for cytoplasmic components that are adapted to intracellular acidification. This is the case for N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase , a protein involved in purine biosynthesis. Indeed, N5-CAIR mutase of A. pasteurianus 386B is 99% identical to its orthologue in A. aceti 1023, the latter strain being adapted to an acid cytosol [alr; APA386B_1310), a protein involved in peptidoglycan biosynthesis, is 92% identical to the A. aceti 1023 orthologue, a protein known to function at low pH [N5-CAIR mutase and alanine racemase between A. pasteurianus 386B and A. aceti 1023 was higher than between A. pasteurianus 386B and any other sequenced AAB strain (data not shown), indicating that the presence of acid-stable proteins is not widespread.A first strategy of es place . This suCA cycle . Indeed,tic acid . Thirdlyytoplasm ,61,62. Fcus Pal5 . Lastly,A. pasteurianus 386B revealed that this strain is most related to the thermotolerant strain A. pasteurianus NBRC 101655. In addition, adaptive mutation resulted in 14 validated mutations, involved in improved thermotolerance of this strain [A. pasteurianus 386B and not in A. pasteurianus NBRC 101655 , contained three synonymous mutations and one nonsynonymous mutation. Furthermore, the genes necessary for growth at high temperatures (42°C) of the thermotolerant strain A. tropicalis NBRC 101654 have been identified recently [A. pasteurianus 386B, all genes of A. tropicalis NBRC 101654 necessary for growth at high temperatures were found in the genome sequence of A. pasteurianus 386B as well  and stresses (such as acidic and heat stress) during the cocoa bean fermentation process.The complete genome sequence of A. pasteurianus 386B contained a low number of transposases, resulting in the absence of truncated genes, which might be important for expression under cocoa bean fermentation conditions.Comparative genome analysis provided information regarding niche adaptations of this strain. For example, the presence of a gene coding for an endopolygalacturonase was discovered. This enzyme is involved in the breakdown of pectin, a compound responsible for the viscosity of the cocoa pulp-bean mass. Although depectinization is mainly important in the beginning (anaerobic yeast activity phase) of the cocoa bean fermentation process, the activity of the pectinolytic enzymes allows air to enter the cocoa pulp-bean mass, which promotes the growth of obligate aerobic AAB. Therefore, the presence of this gene could be an important prerequisite for survival and performance of AAB during cocoa bean fermentations. Furthermore, the comparative genome analysis revealed that the genome of A. pasteurianus 386B to tolerate stress conditions occurring in a cocoa bean fermentation ecosystem. As also active prophages were absent in the genome sequence, these findings indicate that A. pasteurianus 386B is genetically more stable compared with other fully characterized AAB, contributing to the prerequisites of a starter culture strain.Genome analysis unraveled various mechanisms of A. pasteurianus 386B, which will provide insight into its metabolic activity. Finally, the characteristics of A. pasteurianus 386B revealed in this study are essential to generate further insights into the functional role of AAB in general, and A. pasteurianus in particular, during the cocoa bean fermentation process, which is of great importance to select an appropriate starter culture for homogeneous, fast, and successfully controlled fermentation processes.All these findings support that this strain is a suitable functional starter culture for controlled cocoa bean fermentation processes. In addition, the results presented in this study will enable analysis of the transcriptome of Acetobacter pasteurianus 386B was originally isolated from a spontaneous cocoa bean heap fermentation carried out in Ghana [A. pasteurianus 386B was propagated in MYP medium twice (aerobic incubation at 30°C overnight), followed by centrifugation  of 2-ml cultures.in Ghana . The strTotal genomic DNA was extracted from cell pellets using the High Pure PCR Template Preparation Kit , followed by RNase treatment and purification using the High Pure PCR Product Purification Kit (Roche Applied Science), always according to the manufacturer’s instructions. To confirm the identity of the bacterial strain grown, a 16S rRNA gene-specific region was amplified based on the genomic DNA extracted, as described previously . AmplicoFor genome sequencing, a total amount of 5 μg of genomic DNA was used for the construction of an 8-kb paired-end library with the GS FLX Titanium Library Paired-End Adaptors Kit and the GS FLX Titanium Rapid Library Prep Kit (Roche Applied Science) according to the manufacturer’s instructions. The optimal DNA copy per bead ratio was determined by an emulsion PCR titration using a GS FLX Titanium SV emPCR kit (Lib-L) (Roche Applied Science). The final emulsion PCR was performed using the GS FLX Titanium LV emPCR kit . Pyrosequencing was performed on a Genome Sequencer GS FLX instrument using Titanium chemistry (Roche Applied Science) with the sample occupying one region of a four-region gasket. Library preparation and pyrosequencing were performed by the VIB Nucleomics Core Facility . Reads were assembled using the GS De Novo Assembler version 2.5.3 with default parameters.Pfu DNA Polymerase (Fermentas), and sterile ultrapure water in a final volume of 50 μL. Following amplification, PCR product sizes were verified using a 1.0-% (w/v) agarose gel and the remaining reaction mixture was purified using the Wizard SV Gel and PCR Clean up system (Promega). Amplicons were sequenced in a commercial facility using Sanger sequencing technology . All DNA sequences obtained were uploaded into the Consed program, manually inspected, and integrated into the genome assembly to generate the complete genome sequence of A. pasteurianus 386B. To facilitate gap closure and assembly validation, contigs were mapped to the A. pasteurianus IFO 3283 genome by means of the r2cat tool [To close remaining gaps in the assembled genome sequence, PCR primers were designed based on contig ends using the Consed program  and syntcat tool ,69.A. pasteurianus 386B was generated with the DNAPlotter tool [A. pasteurianus 386B was predicted with the Ori-Finder tool [Automatic gene prediction and annotation of the assembled genome sequence were carried out using a local installation of the bacterial genome annotation system GenDB v2.2 . A combiter tool . The orider tool . CRISPRsAcetobacteraceae. Therefore, the annotated genome sequences of the finished genomes of A. pasteurianus IFO 3283 (including plasmids), Acidiphilium multivorum AIU301, Acidiphilium cryptum JF-5, Ga. diazotrophicus Pal5, Ga. medellinensis NBRC 3288 (formerly Gluconacetobacter xylinus NBRC 3288), and G. oxydans 621H were used [A. pasteurianus subsp. pasteurianus LMG 1262T, A. pasteurianus NBRC 101655, A. pomorum DM001, A. tropicalis NBRC 101654, and A. aceti NBRC 14818 were included [A. pasteurianus 3P3 [A. pasteurianus 386B, together with the plasmids identified, was incorporated as well. Comparative analysis of these genome sequences, including synteny analyses, identification and classification of orthologous genes, and phylogenetic analysis was accomplished by the EDGAR software framework using default parameters [A. pasteurianus 386B and A. pasteurianus IFO 3283 [A phylogenetic analysis was performed using complete and draft genome sequences of members of the family ere used -27,63,87included ,22,23. AIFO 3283 , using tIFO 3283 .A. pasteurianus chromosome and plasmids were deposited in the DDBJ/EMBL/GenBank database (Sequencing Project PRJEB1172). The accession numbers are listed in Table The annotated sequences of the N5-carboxyaminoimidazole ribonucleotide; PPP: Pentose-phosphate pathway; PQQ: Pyrroloquinoline quinone; SCACT: Succinyl-CoA:acetate CoA transferase; TCA: Tricarboxylic acid.AAB: Acetic acid bacteria; ACT: Artemis comparison tool; ADH: Alcohol dehydrogenase; AIMS: Architecture imparting sequences; ALDH: Aldehyde dehydrogenase; CDS: Protein-coding sequences; COG: Cluster of orthologous groups; CPSM: Cocoa pulp simulation medium; CRISPR: Clustered regularly interspaced short palindromic repeat; DHA: Dihydroxyacetone; EMP: Embden-Meyerhof-Parnas; FAD: Flavine adenine dinucleotide; GO: Gene ontology; LAB: Lactic acid bacteria; MYP: Mannitol-yeast extract-peptone; N5-CAIR: The authors declare that they have no competing interests.KI carried out DNA extraction, conducted genome assembly and finishing, performed sequence annotation, bioinformatics analyses, and comparative genome analyses. SW and LDV designed and coordinated the study and participated in the analysis of the results. KI, SW, and LDV wrote the manuscript. All authors read and approved the final manuscript.Prophage- and transposon-related genes of Acetobacter pasteurianus 386B. Homologues of the genes involved are shown for A. pasteurianus IFO 3283, Ga. diazotrophicus Pal5, and G. oxydans 621H.Click here for fileOverview of genes in Acetobacter pasteurianus 386B truncated by transposons in A. pasteurianus IFO 3283. Overview of genomic positions of the homologues of truncated gene fragments of A. pasteurianus IFO 3283 in A. pasteurianus 386B.Click here for fileStrain-specific genes of Acetobacter pasteurianus 386B. Unique genes were identified by comparative analysis of five available genome sequences of A. pasteurianus strains using EDGAR.Click here for filePCR assay for endopolygalacturonase gene. Assays were performed on selected A. pasteurianus strains, originating from spontaneous cocoa bean fermentations.Click here for fileUncharacterized oxidoreductases of Acetobacter pasteurianus 386B. Features of the genes that could be annotated are listed. The oxidoreductases are classified according to their protein family.Click here for fileGenes of Acetobacter pasteurianus 386B coding for membrane-bound oxidoreductases with unknown specific function. Homologues of the genes involved are shown for A. pasteurianus IFO 3283, Ga. diazotrophicus Pal5, and G. oxydans 621H.Click here for fileGenes of Acetobacter pasteurianus 386B involved in thermotolerance. List of homologous genes between A. pasteurianus 386B and A. tropicalis NBRC 101654, and between A. pasteurianus 386B and A. pasteurianus NBRC 101655.Click here for file


We studied whether the change in antibiotic susceptibility testing (AST) guidelines from CLSI to EUCAST influenced cumulative antibiograms in a tertiary care hospital in Switzerland.Antibiotic susceptibilities of non-duplicate isolates collected within a one-year period before (period A) and after (period B) changing AST interpretation from CLSI 2009 to EUCAST 1.3 (2011) guidelines were analysed. In addition, period B isolates were reinterpreted according to the CLSI 2009, CLSI 2013 and EUCAST 3.1 (2013) guidelines.Escherichia coli / cefepime, 95.8% (CLSI 2009) vs. 93.1% (EUCAST 1.3), P=0.005; Enterobacter cloacae / cefepime, 97.0 (CLSI 2009) vs. 90.5% (EUCAST 1.3), P=0.012; Pseudomonas aeruginosa / meropenem, 88.1% (CLSI 2009) vs. 78.3% (EUCAST 1.3), P=0.002. These differences were still evident when comparing susceptibility rates according to the CLSI 2013 guideline with EUCAST 3.1 guideline. For P. aeruginosa and imipenem, a trend towards a lower antibiotic susceptibility rate in ICUs compared to general wards turned into a significant difference after the change to EUCAST: 87.9% vs. 79.8%, P=0.08 (CLSI 2009) and 86.3% vs. 76.8%, P=0.048 (EUCAST 1.3).The majority of species/drug combinations showed no differences in susceptibility rates comparing periods A and B. However, in some gram-negative bacilli, decreased susceptibility rates were observed when comparing CLSI 2009 with EUCAST 1.3 within period B: The change of AST guidelines from CLSI to EUCAST led to a clinically relevant decrease of susceptibility rates in cumulative antibiograms for defined species/drug combinations, particularly in those with considerable differences in clinical susceptibility breakpoints between the two guidelines. The European Committee for Antimicrobial Susceptibility Testing (EUCAST) was initiated to harmonize minimum inhibitory concentration (MIC) breakpoints across Europe . In linePseudomonas aeruginosa [Escherichia coli causing bacteremia [E. coli and Klebsiella pneumonia [E. coli [In general, EUCAST recommends lower resistance MIC breakpoints than CLSI, in particular for Gram-negative bacteria, and, in part, abandoned the intermediate susceptibility zone. These changes have been shown to result in different susceptibility rates, e.g. higher cefepime and meropenem resistance rates in ruginosa , higher cteremia , higher neumonia , and hig[E. coli . However[E. coli ,9, and c[E. coli . This study was designed to determine whether and to which extent susceptibility rates in cumulative antibiograms of the five most prevalent bacterial species in our tertiary-care hospital would differ between two consecutive years before and after changing from CLSI 2009 to EUCAST 1.3 (2011) AST guidelines. Furthermore, we determined whether differences in cumulative antibiograms represented true changes in antimicrobial susceptibility, or if they were merely an effect of guideline changes. In addition, as resistance rates of cumulative antibiograms from general ward specimens reportedly differ from those found on intensive care units (ICUs), we aimed to determine whether guideline dependent changes differed between ICUs and general wards ,12.The University Hospital Zurich, Zurich, Switzerland, is an 871 bed tertiary-care teaching hospital covering all medical specialties except paediatrics and orthopaedics. Six intensive care units  with a total of 65 beds are assigned to different departments. Hematopoietic stem cell transplantations are performed in a specialized unit. All microbiologic samples are tested in the clinical microbiology laboratory of the Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.P. aeruginosa. Data were collected in two consecutive one-year periods just before (period A) and after (period B) the change of AST interpretation from CLSI 2009 to EUCAST 1.3 on 1 July 2011. All bacterial isolates from samples collected on general wards and intensive care units were taken into account. In addition, samples of the outpatients’ clinic for respiratory medicine were included in a subgroup analysis for E. coli, K. pneumoniae, Enterobacter cloacae, P. aeruginosa, and Staphylococcus aureus. According to the guidelines for analysis and presentation of cumulative antibiograms, we excluded all repeated isolates, i.e. only the first isolate of a certain bacterium per patient and year was analysed, regardless of the material, the donor site, or the resistance profile [The species analysed comprised  profile . In addition, isolates collected on the general wards and on the ICUs were analysed separately. The “ICU section” consisted of the six ICUs and the hematopoietic stem cell transplantation unit. The “ward-section” consisted of all general wards.For susceptibility testing, the disc diffusion method according to Kirby-Bauer was used . AntibioFor certain drugs, e.g. ceftazidime, cefotaxime, and piperacillin/tazobactam, EUCAST guidelines contain other antibiotic disc loads than CLSI guidelines, preventing a direct comparison of disk diffusion AST results ,3. Thus,The first one-year period (period A) was interpreted according to CLSI 2009 guidelines only. Period B was originally interpreted according to EUCAST 1.3 guidelines. In order to analyze whether or not changes in susceptibility rates over time were due to the guideline change alone, results of period B were reinterpreted according to CLSI 2009 guidelines. Additionally, period B was reinterpreted according to CLSI 2013 and EUCAST 3.1.Isolates of intermediate susceptibility were classified together with resistant isolates to the “non-susceptible”-group. P-values <0.05 were considered statistically significant.Differences in group proportions were assessed using chi-square or Fisher’s exact tests, as appropriate. We used Stata  for statistical analyses. E. coli, 277 K. pneumoniae, 186 E. cloacae, 271 P. aeruginosa, 721 S. aureus) were included. Between July 2011 and June 2012 (period B), 2688 isolates  were included.Between July 2010 and June 2011 (period A), 2540 isolates .For CAST 1.3 . MoreoveThis observational study was designed to analyse cumulative hospital antibiograms in two adjacent one-year periods before and after the clinical laboratory changed antibiotic susceptibility test interpretation from CLSI 2009 to EUCAST 1.3 (2011) guidelines, and to analyse whether possible differences are due to true epidemiologic changes or result only from CLSI / EUCAST guideline differences. Differences resulting from guideline changes alone may misdirect physicians in the interpretation of antibiotic susceptibility trends in that truly increasing resistance rates may be missed or unchanging resistance rates may be reported as increasing or decreasing, resulting in a change of antibiotic use patterns and thus patient management and quality of care. Clinical breakpoint (CBP) setting is a multi-step process comprising the determination of epidemiological cut off (ECOFF) values from MIC distributions, correlating these ECOFFs to zone diameters distributions, comparing putative CBPs to available PK/PD data and, finally, clinical validation of putative CBPs in clinical outcome studies . CLSI and EUCAST use different methods for the determination of disc diffusion CBPs: CLSI uses a variant of the error-rate-bounded method, sometimes incorporating an intermediate zone , and EUCSuch different CBP determination methods inevitably lead to different CBPs in CLSI and EUCAST AST guidelines for many species/drug combinations. EUCAST 1.3  disk diffusion CBPs are frequently higher as compared to CLSI 2009, and in many cases even higher as compared to revised CLSI guideline versions 2010 to 2013 . SeveralE. coli / cefepime and E. cloacae / cefepime; P. aeruginosa / meropenem; E. coli/amoxicillin and clavulanic acid; E. coli / ceftriaxone; Instead of a uniform trend towards lower reported susceptibility rates for all species/drug combinations after implementation of EUCAST guidelines, this study showed four distinct patterns of effects of guidelines changes on susceptibility rates: i) a clearly artificial change of susceptibility rates due to changes in AST guidelines  showed a statistically significant, yet artificial, decrease in susceptibility rates when period B was analysed according to EUCAST 1.3 (2011) instead of CLSI 2009 guidelines. For these three species/drug combinations, susceptible clinical inhibition zone diameter breakpoints differ substantially between EUCAST 1.3 and CLSI 2009 guidelines, i.e., 6 mm difference for cefepime susceptible CBP and Enterobacteriaceae , and 8 mm difference in meropenem CBP for P. aeruginosa . Three species/drug combinations  or less effective antibiotic regimens. Empirical antibiotic treatment is not only guided by individual patient characteristics, but also by epidemiological data such as local susceptibility rates . UsuallyE. coli, and clindamycin susceptibility rates of S. aureus when applying both CLSI and EUCAST guidelines. However, for imipenem susceptibility rates of P. aeruginosa, a significant difference between ICU’s and general wards was found only if applying EUCAST 2011, but not with CLSI 2009 (P.aeruginosa isolates collected in the outpatient clinic for respiratory medicine decreased significantly when EUCAST 2011 guidelines were applied. The most likely reason is a different inhibition zone diameter distribution (i.e. a shift towards lower inhibition zone diameters) of isolates originating from this patient population, resulting in a comparably stronger effect of guideline changes on susceptibility rates. These two examples show that guideline changes can affect different hospital wards to a different extent. The results of this study are in concordance with those of other authors demonstrating that susceptibility rates differ between ICU’s and general wards ,27. CorrLSI 2009 . MoreoveThus, effects of AST guidelines are not easily predictable and are dependent on the epidemiological situation. Both clinicians and microbiologists should, therefore, know their local epidemiology to be able to foresee effects of AST guideline changes on clinical practice. In addition, switching guidelines should be accompanied by thorough analyses during one time period where both guidelines are applied in order to detect bacteria/drug combinations belonging to one of the patterns described here. This might be particularly important in settings with higher resistance rates.Despite a broad general set of data, the smaller sample size for the general ward/ICU distinction may have obscured significant differences in this study. In addition, this study is limited to the epidemiological situation in one single institution in north-eastern Switzerland, which is a low prevalence region for antibiotic resistance, hampering generalizability to other geographic settings. Effects of CBP changes as shown in this work may, thus, differ from the situation in high-prevalence regions. In conclusion, this study demonstrates that changes in AST guidelines, e.g. from CLSI 2009 to EUCAST 1.3 (2011) guidelines, can influence a hospital’s cumulative antibiogram in various ways. These changes cannot be easily predicted and may differ between hospital units. Even cautious interpretation can only serve as an approximation of the real epidemiological changes. Nevertheless, these issues have to be taken into account when interpreting cumulative antibiograms in a period after significant AST guideline changes. Further studies are needed to assess the effect of the guideline changes on different local epidemiological situations.


Daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod Parhyale hawaiensis. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models.Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod P. hawaiensis. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid Acyrthosiphon pisum. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against nr . These included over 400 predicted transcripts with significant similarity to D. pulex sequences but not to sequences of any other animal. Annotation of several hundred genes revealed P. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways.To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of P. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as D. pulex and P. hawaiensis, as well as the need for development of further substantial crustacean genomic resources.The amphipod  The water flea Daphnia pulex was recently added to this list, and is the only crustacean to date with a publicly accessible sequenced genome . The current state of understanding of crustacean-hexapod phylogenetic relationships (see Conclusions) therefore does not allow straightforward predictions of which pair of these three transcriptomes should be most similar to each other.The assembly yielded isotigs as long as 7,936 bp, with average length 1,128 bp Figure . Howeverh Figure . These rP. hawaiensis will likely focus on elucidating the function of highly conserved metazoan genes in this amphipod. We therefore annotated the transcriptome for the presence of genes belonging to major conserved animal signaling pathways , our data suggest that even these unassembled reads can be a rich source of gene discovery. The transcriptome sequences for these genes ranged in length from 276 bp  to 4,882 bp , and the majority are at least 500 bp long, making them immediately useful for in situ hybridization, RNAi-mediated gene knockdown, and RACE  using BLASTX. Unless otherwise specified, all BLAST searches were conducted using BLAST v2.2.24+ [The sequence data from this study have been submitted to GenBank under study accession number VZ performed experiments, helped design data analysis and analyzed data. KEV performed experiments and analyzed data. BEC helped design research, performed experiments, collected and analyzed data. FA performed ovary dissections and embryo collections. WEB helped design the research, performed experiments, analyzed data and obtained funding for the research. CGE proposed the idea for the research, helped design the research and analyze the data, wrote the manuscript with input from all authors, and obtained funding for the research. All authors read and approved the final manuscript.P. hawaiensis transcriptomeEmbryonic stages pooled for creation of the . Staging as per [g as per .Click here for filede novo assembly of the P. hawaiensis transcriptomeComparison of read lengths from Newbler v2.5 . (A) Distribution of read lengths after assembly with Newbler v2.5 (red). (B) Distribution of read lengths of the shortest assembled reads and raw reads. The assembly yielded assembled reads of over ~4000 bp.Click here for filede novo assembly of the P. hawaiensis transcriptomeDistribution of average coverage (reads/bp) within contigs produced by Newbler v2.5 . The coverage within contigs is calculated by dividing the total number of base pairs contained in the reads used to construct a contig by the length of that contig.Click here for filede novo transcriptome assemblyAnalysis of the effect of trans-splicing transcripts on . Assembly of all trimmed sequences compared to assembly of sequences lacking the trans-splicing leader sequences [nr database with an E-value cut-off value of 1e-10.equences . Number Click here for fileP. hawaiensis transcriptomePhylogenetic distribution of species of top unique BLAST hit for Newbler v2.5 assembly of the . Of the unique BLAST hits to all non-redundant assembly products (isotigs + singletons), 90% were from species belonging to the clades shown. Over 50% of these top BLAST hits are from arthropod species. The large number (12.2%) of top BLAST hits in the complete assembly to sequences from Branchiostoma floridae is due to the high similarity of C2H2 zinc finger domain-containing sequences with a particular linker sequence (TGEKP) that is also highly represented in the genome of the aphid Acyrthosiphon pisum [D. mojavensis, D. willistoni, D. ananassae, D. grimshawi, D. pseudoobscura pseudoobscura, Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus (Flies & Mosquitoes), Bombyx mori (Moth), Tribolium castaneum (Beetle), Harpegnathos saltator, Camponotus floridanus, Apis mellifera, Nasonia vitripennnis , Acyrthosiphon pisum (Aphid), Pediculus humanus corporis (Louse), Ixodes scapularis (Tick), Penaeus monodon, Lepeophtheirus salmonis, Litopenaeus vannamei (Crustaceans), Caenorhabditis remanei (Nematode), Gallus gallus, Taeniopygia guttata (Birds), Rattus norvegicus, Mus musculus, Monodelphis domestica , Xenopus laevis, X. tropicalis (Amphibian), Danio rerio, Tetraodon nigroviridis (Fish), Branchiostoma floridae (Amphioxus), Ciona intestinalis (Sea Squirt), Saccoglossus kowalevskii (Acorn Worm), Strongylocentrotus purpuratus (Sea Urchin), Trichoplax adherens (Trichoplax), Hydra magnipapillata, Nematostella vectensis (Cnidarians), Perkinsus marinus (Dinoflagellate).on pisum . Red: vaon pisum ,74,75. HClick here for fileDaphnia pulex gene sequences identified in the de novo P. hawaiensis transcriptomeSequences with strong similarity to . Because the D. pulex genome and nr are databases of inevitably different sizes, E-values shown here are for information only and are not strictly comparable. See text for additional details.Click here for fileP. hawaiensis GenBank accessions in the de novo transcriptomePresence of existing . Sequences of P. hawaiensis developmental genes from GenBank were used as a query to BLAST the de novo transcriptome. Most genes with hits had several matches in the transcriptome, among both assembled reads and singletons.Click here for fileP. hawaiensis transcriptome adds sequence data to GenBank accession number HM191476, the P. hawaiensis prospero homologueThe . Extended contig for Ph-prospero, comprising the complete mRNA GenBank accession , one isotigs and one contig from the Newbler assembly of the transcriptome (dark grey). The isotig provides an additional 445 bp of 3' UTR sequence and 116 bp of 5' UTR sequence (black) to the GenBank sequence. Comparison with the GenBank sequence shows that isotig24415 and singleton GAP9EXG06HFGHB belong to the same contig.Click here for fileP. hawaiensis transcriptomeSelected signaling pathway genes identified in the . Hit ID indicates if gene hits were found assembled reads (A) or singletons (S). Sequence length (range) indicates the shortest and longest A or S hit sequences for each gene. These results are shown graphically in Figure Dm = D. melanogaster; Dr = Danio rerio; Xt = Xenopus tropicalis. Query sequence details: 1. Kinase domain was masked. 2. FERM domain used as query. 3. Amino acids 500-833 (Dl/Ser domain) used as query. 4. Amino acids 1-250 (groucho/TLE domain) used as query. 5. Kinase domain masked; amino acids 420-1390 used as query. 6. Kinase domain masked; amino acids 175-372 used as query. 7. Kinase domain masked; amino acids 150-516 used as query. 8. Kinase domain masked; amino acids 1-100 used as query. 9. Kinase domain masked; amino acids 1-890 used as query. Asterisks indicate genes that appear elsewhere in the same table (in a different pathway).Click here for fileP. hawaiensis transcriptomeSelected developmental process genes identified in the . Hit ID indicates if gene hits were found assembled reads (A) or singletons (S). Sequence length (range) indicates the shortest and longest A or S hit sequences for each gene. Groups of hits of a given colour indicate transcriptome sequences that mapped to the same overlapping region of the BLAST target; hits of different colours indicate transcriptome sequences that map to different, non-overlapping regions of the BLAST target. Query organism was D. melanogaster for all cases. Boldface indicates genes also present in other tables  or singletons (S). Sequence length (range) indicates the shortest and longest A or S hit sequences for each gene. Groups of hits of a given colour indicate transcriptome sequences that mapped to the same overlapping region of the BLAST target; hits of different colours indicate transcriptome sequences that map to different, non-overlapping regions of the BLAST target. Query organism was D. melanogaster for all cases. Query sequence details: 1. S/T kinase domain was masked. 2. Dead box/Zn finger domains were masked. 3. HLH domain was masked 4. Peptidase C14 domain was masked. 5. Kinase domain masked; amino acids 175-372 used as query. 6. BTB domain used as query. 7. Kinase domain masked; amino acids 1-890 used as query. Boldface indicates genes also present in other tables (Additional Files Click here for fileRepresentative of consecutively numbered isotigs with highly similar lengths. An example of two isotigs which both have Cyclin D as their top BLAST hit (see Additional File Click here for file


Glycyrrhiza glabra L.) is an important medicinal plant. In this study, the antimicrobial activities of ethanolic and aqueous extracts from licorice leaves were studied compared to root extracts activities. Bacillus subtilis, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli, and Candida albicans were used as test organisms. Antimicrobial activity was tested by paper disc agar diffusion and serial dilution methods in order to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The root and leave extracts showed activity against Candida albicans, and tested gram-positive bacteria in a dose dependent manner. The ethanolic extract of the leaves was the most active extract against gram-positive bacteria. Its effectiveness against strains provides hope that it can serve as an alternative therapeutic agent. Licorice ( Because of the side effects and resistance that pathogenic micro-organisms build against the antibiotics, much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine. Medicinal plants may offer a natural and new source of antibacterial agents for use.Glycyrrhiza glabra L. (Fabaceae) is a native of south-east Europe and south-west Asia, including Iran. Glycyrrhiza glabra L. (licorice), is a very sweet, moist, soothing herb with anti-inflammatory and expectorant properties, controls cough, and has hormonal effects. Moreover, it detoxifies and protects the liver. Medicinally, it is used internally for Addison’s disease, asthma, bronchitis, coughs, peptic ulcer, and arthritis  from Abadeh in Iran. This plant was identified and stored in the Herbarium of Biosciences Faculty, Shahid Beheshti University (voucher number 8600100).Roots and leaves of Preparation of plant extractsGlycyrrhiza glabra L., leaves and roots were shade dried at room temperature and powdered by an electric blender. 50 g of each of the powdered materials were macerated with 500 mL of water or ethanol 80% for 24 h. All the extracts were concentrated by rotary vacuum evaporator. The condensed products were weighed and kept at 4 °C prior to test.In order to prepare various extracts from different parts of Test microorganismsCandida albicans (ATCC10231) was used as the fungal tested organism and Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 465), Enterococcus faecalis (ATCC 29737), Escherichia coli (ATCC 25922), Pseudomonas aerouginosa (ATCC 85327), and Klebsiella pneumoniae (ATCC 10031) were used as the bacterial tested organisms. For the suspension, 1 × 108 cells/mL was prepared and compared to that of the 0.5 Mc Farland standard tube. Antimicrobial assayAntimicrobial activity of the above mentioned extracts was determined using the paper disc agar diffusion method. Petri dishes containing the Muller Hinton agar were prepared and each plate was separately inoculated with different cultures of organisms by swabbing aseptically on the whole surface of the agar with cotton wool. The filter paper discs (6 mm diameter) were impregnated with 20 μL of each extract . The discs were dried and placed aseptically at the plates. Disks injected with 20 μL of solvent were served as negative controls, and Chloramphenicol discs (30 μg) were used as positive control. The petri dishes were incubated at 37 ± 0.1°C for 20-24 h. At the end of period, the inhibition zones formed on the media were measured. The positive antimicrobial activity was read based on growth inhibition zone. Evaluation of the inhibitory properties was carried out in triplicates.The Minimum Inhibitory Concentration (MIC) value of extracts was determined by serial dilution method . The extIn order to determine Minimum Bactericidal Concentration (MBC) values, 100 μL of the content of the tubes with no turbidity, were cultured on the Muller Hinton agar medium and incubated at 37 ± 0.1°C for 24 h. This test was repeated in duplicates.Extraction of dried powder gave the following yields: leave ethanolic (5.4%), leave aqueous (4%), root ethanolic (10%) and root aqueous (9.6 %) extracts.Glycyrrhiza glabra L. exhibited a strong activity against C. albicans and tested gram-positive bacteria , except aqueous extracts from root that showed no inhibition zone against E. faecailis. Results obtained from the controls indicated that solvents had no effect on the microorganisms. The relative antibacterial activity of ethanolic extract was higher than aqueous extract. As shown in G.glabra showed the highest activity, whereas the value of aqueous root extracts against C. albicans was 625 μg/mL. This matter may be due to the materials extracted from various solvents.The MIC and MBC values are shown in Helicobacter pylori  of the alcohol, acetone, and chloroform extracts of Glycyrrhiza glabra roots against the microorganisms tested; the alcohol extracts inhibit B. cereus, K. pneumoniae, S. aureus; the acetone extracts inhibit B. cereus, B. subtilis, K. pneumoniae, S. aureus; the chloroform extracts showed inhibition effect against B. cereus, B. subtilis, E. faecalis, K. pneumoniae, S. aureus (Ates The majority of antimicrobial effects from licorice is due to isoflavonoid components particularly hispaglabridin and B,4’-O-methylglabridin, glabridin, glabriol and 3-hydroxyglabrol .S. aureus is a major clinical pathogen. During the past decade, this bacterium has developed resistance to many commonly used antibiotics (Glycyrrhiza glabra L. showed activity against S. aureus and can be used as raw materials for phytotherapy.ibiotics . In thisG. glabra.The present study provides further evidence about leaves antimicrobial activity of


Diaphorina citri is a notorious agricultural pest that transmits the phloem-inhabiting alphaproteobacterial ‘Candidatus Liberibacter asiaticus’ and allied plant pathogens, which cause the devastating citrus disease called Huanglongbing or greening disease. D. citri harbors two distinct bacterial mutualists in the symbiotic organ called bacteriome: the betaproteobacterium ‘Candidatus Profftella armatura’ in the syncytial cytoplasm at the center of the bacteriome, and the gammaproteobacterium ‘Candidatus Carsonella ruddii’ in uninucleate bacteriocytes. Here we report that a putative amino acid transporter LysE of Profftella forms a highly supported clade with proteins of L. asiaticus, L. americanus, and L. solanacearum. L. crescens, the most basal Liberibacter lineage currently known, lacked the corresponding gene. The Profftella-Liberibacter subclade of LysE formed a clade with proteins from betaproteobacteria of the order Burkholderiales, to which Profftella belongs. This phylogenetic pattern favors the hypothesis that the Liberibacter lineage acquired the gene from the Profftella lineage via horizontal gene transfer (HGT) after L. crescens diverged from other Liberibacter lineages. KA/KS analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. These findings highlight the possible evolutionary importance of HGT between plant pathogens and their insect vector’s symbionts that are confined in the symbiotic organ and seemingly sequestered from external microbial populations.he Asian citrus psyllid  Diaphorina citri (Hemiptera: Psyllidae) is an important agricultural pest that transmits a serious citrus disease, Huanglongbing (HLB) or greening disease. This insect is widely distributed in Asia, and is spreading into other citrus growing regions worldwide Candidatus Liberibacter: L. asiaticus, L. americanus, and L. africanusD. citri vectors L. asiaticus and L. americanus in Asia and the Americas, and the African citrus psyllid Trioza erytreae (Hemiptera: Triozidae) vectors L. africanus in Africa L. solanacearum  Liberibacter species are very fastidious, but L. crescens, the species recovered from mountain papaya, has recently been reported to be readily culturable L. asiaticusL. solanacearumL. crescensL. americanusThe Asian citrus psyllid D. citri possesses a large yellow symbiotic organ called the bacteriome, where two distinct symbionts are harbored Candidatus Profftella armatura’ is located in the syncytial cytoplasm at the center of the bacteriome, whilst the gammaproteobacterium ‘Candidatus Carsonella ruddii’ is found in uninucleate bacteriocytes on the surface of the bacteriome. Our previous study revealed that Profftella is a toxin-producing defensive symbiont that potentially protects D. citri from natural enemies, while Carsonella_DC is a nutritional symbiont that provides the host with essential amino acids, which are scarce in the psyllid’s diet of phloem sap In its abdomen, Liberibacter lineage horizontally acquired a putative transporter gene from a bacterium closely related to the extant Profftella.Here we report that the Profftella genome were extracted by BLASTP searches Profftella genome as queries. Amino acid sequences were aligned using MAFFT 6.847 HGT candidates in the KS and KA values were calculated as described previously KA/KS value was tested against a bootstrap distribution of KA/KS values, which was generated by 10,000 bootstrap resamplings of codons from the original alignment. When KS values calculated from resampled alignments were close to saturation values (larger than 2.0 per site), the KS values was set as 2.0 for the estimation of KA/KS value.L. asiaticus str. psy62 [accession no. NC_012985], L. asiaticus str. gxpsy [NC_020549], L. solanacearum CLso-ZC1 [NC_014774], L. americanus PW_SP [AOFG01000001-22], and L. crescens BT-1 [NC_019907] were obtained from GenBank.To analyze the structural organization, the genomic sequences of Profftella is significantly similar to its counterparts of Liberibacter spp. The top BLAST hit was the “putative homoserine/homoserine lactone efflux protein” of L. asiaticus str. psy62 and str. gxpsy . Subordinate hits were “putative homoserine/homoserine lactone efflux protein” of L. americanus  and L. solanacearum , followed by LysE superfamily proteins, such as “lysine transporter LysE”, “threonine transporter RhtB”, and “homoserine/homoserine lactone efflux protein”, of various betaproteobacterial species belonging to the order Burkholderiales. Putative orthologs of the Profftella lysE were observed in L. asiaticus, L. americanus, and L. solanacearum, but not in L. crescens, the most basal Liberibacter lineage currently known Profftella was 40–43% identical to its orthologs of Liberibacter spp (Profftella and Liberibacter spp.BLASTP searches against the NCBI nr database demonstrated that the putative LysE protein (accession no: YP_008343788) of cter spp . No otheProfftella forms a highly supported clade with the proteins of Liberibacter spp.  after L. crescens diverged from other Liberibacter lineages. The structural organizations of the lysE flanking regions were partially conserved among genomes of L. asiaticus, L. americanus and L. solanacearum . Whereas the KS values both between L. asiaticus and L. americanus and between L. solanacearum and L. americanus were saturated (> 3.00), the KA values were still as low as 0.42 and 0.39, respectively. These results support the hypothesis that the lysE genes of Liberibacter spp. are under purifying selection and thus are functional.The Liberibacter lineage horizontally acquired a lysE-type transporter gene from the Profftella lineage, an endosymbiont of their vector insect. KA/KS analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. Although their true functions are yet to be identified, LysE superfamily proteins of various bacteria are generally involved in exporting substrates, playing important roles in resistance to toxic substances, in maintenance of optimum intracellular concentration of metabolites, and in excretion of regulatory molecules Liberibacter have acquired novel functions through this HGT. Whereas HGTs are rampant among bacteria Profftella, the putative donor lineage of the lysE gene, is this type of endosymbiont. In this context, infection style of Liberibacter, the putative accepter of the gene, would be noticeable. As Liberibacter spp. are transmitted by psyllids in a persistent manner, exhibiting near systemic infection of various organs and tissues The present study demonstrated that the


Physicians depend on reliable information on the local epidemiology of infection and antibiotic resistance rates to guide empiric treatment in critically ill patients. As these data are scarce for Central Africa, we performed a retrospective analysis of microbiological findings from a secondary care hospital in Gabon.Microbiological reports from 2009 to 2012 were used to assess the non-susceptibility rates of the three most common isolates from six major types of infections .Staphylococcus aureus was predominant in the majority of infections. Overall, the three most prevalent pathogens in children were S. aureus (33.7%), Streptococcus pyogenes (8.1%) and Escherichia coli (4.5%) and in adults S. aureus (23.5%), E. coli (15.1%) and Klebsiella pneumoniae (7.4%). In total, 5.8% (n = 19) of all S. aureus isolates were methicillin resistant. The proportion of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae was 15.4% (n = 78), 49.4% of all K. pneumoniae were ESBL-producer (n = 42).A high diversity of pathogens was found, but The high diversity of potential pathogens and high resistance rates in Gram-negative bacteria challenge a rational empiric use of antibiotics. Countrywide continuous sentinel surveillance is therefore urgently needed. Salmonella enterica Typhi reported from DR Congo . (18.8%) ,20. Howe (18.8%) . This obOur report on bacterial spectra of major infections and antimicrobial non-susceptibility patterns enables a more rational use of antibiotics. This is of outmost importance, not only for patients in Gabon but also for travelers returning from the tropics which have acquired infections in the visited country . Our stuAlthough the data we present are very valuable in a setting where information on antimicrobial resistance is severely limited, some limitations need to be addressed. First, based on the reports of microbiological findings we were not able to distinguish community from hospital acquired infections. The source of infection is therefore blurred. Second, some samples have also been taken after starting an antibiotic treatment, which may lead to selection and over-representation of resistant isolates. Third, we were unable to provide further epidemiological data . Fourth, the collection of samples for microbiological culture was not systematic but based on the physician’s judgment which might be a sampling bias.S. aureus is the predominant pathogen and the high proportion of ESBL-producing K. pneumoniae is alarming. The high diversity of pathogens challenges an empirical use of antibiotics. A countrywide continuous sentinel surveillance is therefore urgently needed.Our analysis provides important data of bacterial spectra and antimicrobial resistance in a semi-urban setting in Africa. Staphylococcus aureus.CLS: Contract laboratory services; CLSI: Clinical laboratory standards institute; EARSS: European antimicrobial resistance surveillance system; EQA: External quality assurance; ESBL: Extended-spectrum beta-lactamase; HAS: Albert Schweitzer Hospital; MRSA: Methicillin resistant The authors declare that they have no competing interests.AA, LF, HK and FS carried out microbiological studies, AA, KK, MAH, BCK, MPG, GP, SI, PGK and FS were involved in the study conception and design and data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/455/prepub


Dirofilaria immitis and Angiostrongylus vasorum are both important potentially fatal canine nematodes with overlapping endemic areas, especially in Europe. The preadult and adult stages of both species are living in the Arteria pulmonalis and the right heart, and diagnostically detectable circulating parasite antigens have been demonstrated for both species. For the detection of D. immitis infections, a variety of commercial tests have been developed, however, they have not been evaluated for cross-reactions against circulating antigens of A. vasorum.A. vasorum and which had circulating antigens as confirmed by a species-specific ELISA, were evaluated for the detection of A. vasorum antigen in six commercially available D. immitis test kits.In this study, potential cross-reactions of sera from 16 dogs, which were experimentally infected with A. vasorum-infected dog (6.2%) tested positive.In three fast tests , all sera were negative. One fast membrane ELISA (SNAP® HTWM RT Test) was positive with four sera (25%), and one serum delivered a non-valid result twice. In the PetChek® HTWM PF Test, depending on the interpretation protocol, 5 or 8 dogs (31.2 – 50%) were positive. With the DiroCHEK®-ELISA, a single A. vasorum in commercially available test kits for the detection of D. immitis antigen, the simultaneous use of highly specific diagnostic methods for the differentiation of these two canine heart worms is recommended.Due to potential cross-reactions with  Dirofilaria immitis, a filarial nematode, and Angiostrongylus vasorum, a metastrongylid nematode, are both localized in the Arteria pulmonalis and the right heart of their definitive hosts. Dogs, foxes and some other carnivores are the definitive hosts of both parasites, while Culicidae and Gastropoda are the intermediate hosts of D. immitis and A. vasorum, respectively.The adult stages of D. immitis is present in coastal Mediterranean areas with expansion northwards, while in North America the parasite has expanded from the south-eastern coastal areas northwards and westwards [A. vasorum was diagnosed for the first time in France in 1913 [A. vasorum in North America, with a potential for expansion to further regions [A. vasorum and D. immitis have therefore to be accounted for. Furthermore, in non-endemic areas of D. immitis, this agent has to be considered based on anamnestic information (travelling with pet dogs or imports) and differentiated from A. vasorum infections.In Europe, stwards  up to Castwards . A. vasoin 1913 ,5. Its pin 1913 ). Reportin 1913 -10 or biin 1913 ,12 may hregions ,13,14. OD. immitis infection are usually prevented by the monthly use of macrocyclic lactones in known endemic areas [Wolbachia with doxycycline supported by administration of macrocyclic lactones [A. vasorum, instead, are treated using macrocyclic lactones such as moxidectin [Fatal clinical consequences of c areas -17, and c areas  and/or, actones . Dogs inidectin  or milbeidectin , or applidectin . Prophylidectin .D. immitis is based on the detection of microfilariae or circulating antigens released by mature adult female worms into the blood circulation, both being detectable starting from 6 months after infection [Dipetalonema reconditum[Dirofilaria repens, indicating that modern test kits may overcome cross-reactions detected in previously developed test kits for these parasites [Ancylostoma caninum and Trichuris spp. [A. vasorum infections in dogs is the isolation of first stage larvae (L1) from faecal samples, which are produced by the parasites approximately 6–7 weeks after infection. Larval migration techniques such as the Baermann-Wetzel method [A. vasorum have been described [A. vasorum have been developed. These latter ones have been evaluated for cross-reactions against Crenosoma vulpis[Toxocara canis, Ancylostoma caninum) and, importantly, against D. immitis[The currently used diagnostic laboratory methods for the detection of these parasites are divergent. The diagnosis of fection . A variefection -24. Testfection , while tfection ,26,29,30econditum,31 or Dirasites ,33, and,is spp. ,34. The  method  are commma vulpis,10 and a. immitis, showingA. vasorum and D. immitis may share epitopes responsible for potential cross-reactions in antigen detection tests. This hypothesis has been confirmed during the development of the ELISA for the detection of circulating antigen for A. vasorum[Due to their common localization within the definitive hosts, their considerable size and particularly the well documented production of circulating antigens ,36, it w. vasorum.A. vasorum in six different commercially available tests for the detection of D. immitis antigen.The aim of the study was to evaluate potential cross-reactions of sera from dogs experimentally infected with A. vasorum were obtained during previously performed studies [A. vasorum antigen [Dirofilaria immitis infection was excluded based on the fact that the dogs were living in a non-endemic area under controlled experimental conditions, and at necropsy.A total of 16 sera from dogs experimentally inoculated with 200 third stage larvae (L3) of studies . Infectistudies , by the antigen  and by tAll tests were performed blinded with an identity code from 1 to 16, by veterinarians (fast tests) or by experienced laboratory technicians from the IPZ (ELISAs).All sera were non-haemolytic, stored at −20°C and tested within 11–40 months after collection. The following test kits were used, adopting the manufacturer’s instruction and within the indicated expiry dates:1) Witness® Dirofilaria, lateral flow 2) SensPERT® Canine Heartworm, lateral flow 3) SNAP® HTWM RT, membrane ELISA 4) SNAP® 4Dx® Plus, membrane ELISA 5) Petchek® HTWM PF Antigen Test , ELISA 6) DiroCHEK®, ELISA Single test results are shown in Table A. vasorum antigen was highly positive for all experimentally infected dogs, with absorbance values  varying between 0.263 and 2.007 , with aA. vasorum infected dogs were positive for D. immitis antigen, and one serum delivered a non-valid result twice. In the PetChek®-ELISA two methods for interpretation were adopted: following the instruction for veterinary practitioners based on eye detection, 5 dogs resulted positive for D. immitis infection, while following the instructions under laboratory conditions with OD measurements, a total of 8 dogs were seropositive. With the DiroCHEK®-ELISA, a single A. vasorum infected dog was D. immitis seropositive. With one exception (76 dpi), all cross-reactions were observed in dogs infected with A. vasorum for more than 90 days, with worm burdens varying from 10–170.In three fast tests  all sera resulted negative, while in one fast membrane ELISA (SNAP® HTWM RT) four D. immitis antigen detection kits with sera of dogs infected with A. vasorum. The two ELISAs for D. immitis detection (PetChek® and DiroCHEK®) and the membrane ELISA SNAP® HTWM RT showed single cross-reactions against A. vasorum, which had not been considered so far. In contrast, the adopted ELISA for the detection of circulating A. vasorum antigen has been developed evaluating different monoclonal antibodies, which had been selectively chosen based on their absence of cross-reactivity against D. immitis circulating antigens, resulting in an overall high specificity [This study provides evidence of false positive reactions in ificity .D. immitis antigen tests are considered to be more sensitive than microfilariae concentration methods or other procedures [D. immitis – positive dogs have been discussed in previously performed studies evaluating different D. immitis test kits. Low worm burden and low number of female worms have been shown to reduce sensitivity of the tests [A. vasorum. An unknown number of dogs with travel anamnesis and testing positive for circulating heartworm antigen may have falsely been diagnosed positive due to A. vasorum cross-reactions, and erroneously treated with melarsomine and/or macrocyclic lactones. Therefore, serological results for D. immitis should be confirmed or excluded by additional diagnostic tests  or diagnostic imaging frequently delivering pathognomonic findings for heart dirofilariosis [Generally, cedures . In partcedures  for the e tests ,26,39. Hariosis ,41 or anariosis ,43.A. vasorum cross-react in commercially available test kits for the detection of circulating D. immitis antigen. The simultaneous use of highly specific diagnostic tools is recommended for epidemiological studies where both heart worm species occur or for individual dogs with a suspected heart worm infection.In this study we confirmed that sera of dogs infected with The authors declare that they have no competing interests.MS participated in the design of the study, collected the samples, carried out the diagnostic assays and drafted the manuscript. PD conceived the study and implemented the draft of the manuscript. Both authors have read and approved the final manuscript.


Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent  evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea.Cultivated peanut  and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago.More than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.The first high-density, gene-based linkage map for  Arachis hypogaea L.) is a major crop in most tropical and subtropical areas of the world and provides a significant source of oil and protein to large segments of the population in Asia, Africa and the Americas. In the U. S., peanut is a high-value cash crop of regional importance, with major production areas concentrated in the Southeast. Plant breeding efforts to pyramid genes for disease and insect resistances, quality, and yield is hampered by the polyploid genetics of the crop species, the multigenic nature of many traits , and the difficulty of selecting for many traits in the field . Thus, secondary selection methods that are environmentally neutral would greatly facilitate crop improvement efforts. Molecular markers fit this criterion, but only recently have markers been developed that reveal sufficient polymorphisms in A. hypogaea and related species to have wide-spread application in peanut breeding. Preliminary steps for utilizing molecular markers for crop improvement are developing collections of polymorphic markers and utilizing them to construct dense and high-resolution genetic maps.Cultivated peanut (n = 4x = 40) A. hypogaea throughout the 1990s and early 2000s with the marker systems available at that time [n = 2x = 20) peanut species. This polymorphism led researchers to create molecular maps for Arachis. The first molecular map in peanut was constructed between the diploids A. stenosperma Krapov. and W.C. Gregoryx and A. cardenasii Krapov. and W.C. Gregory by Halward et al. [A. batizocoi Krapov. and W.C. Gregory (A. cardenasii × A. diogoi Hoehne)]. Another interspecific tetraploid linkage map of 298 loci and 21 linkage groups was derived from a backcross population between A. hypogaea and a synthetic amphidiploid  are closely allied to the cultivated peanut [The domesticated peanut is thought to have arisen from a single hybridization event between two diploid wild species followed by whole genome duplication approximately 3,500 years ago . This shd peanut , mappingd peanut ,18, alfad peanut ,20, oat d peanut , and othA. ipaensis and 67 accessions of A. duranensis have been collected in South America. The largest concentration of A. duranensis is in southern Bolivia and northern Argentina, with a few populations being reported in Paraguay and one in central Brazil [A. duranensis was produced using large numbers of molecular markers derived from transcribed sequences.One accession of l Brazil ,23. The l Brazil . Due to A. duranensis accessions using 556 markers indicated that the species is highly polymorphic at the molecular level and individual accessions could be separated based on a cluster analysis  progenitor species, clustered with the A-genome (AA) species A. stenosperma and not with the B-genome species A. batizocoi. Recent molecular cytogenetic analysis of A- and non-A-  genome species suggests that karyotype diversity among non-A-genome species is extensive enough to support separation into additional genome classes where A. ipaensis remains in B sensu stricto while A. batizocoi is placed into a separate group [A. batizocoi is less typical of B-genome species.A preliminary study of SSR marker variation among 37 s Figure . Intereste group . TherefoA. duranensis accessions ranged from 160 to 375 out of 556, which is 29 to 67% of the total number of SSR markers screened. This is a significant amount of variation, which indicates the high genetic diversity within the species. Based on cluster analysis, success of crosses, and fertility of F1s, accessions PI 475887 and Grif 15036 were selected for subsequent mapping studies using 94 F2 progenies. Screening of the parental accessions with 2,138 SSR markers derived from A. hypogaea EST sequences resulted in 1,768 (82.7%) that were scorable (detected by ABI3730XL genotyping systems) and 896 (41.9%) that were polymorphic . The same markers were used to create a map between two A. batizocoi accessions and to determine syntenic relationships between the A and B genome species .The number of polymorphic SSR markers between paired A. duranensis map  to around 6,000 . Those mp Figure , 24 wereenosperma. These menosperma except fi et al.  was not i et al. .A. duranensis were developed to produce Single Nucleotide Polymorphism (SNP) markers for mapping . Although this program has been reported to be accurate for large-scale mapping projects [The projects , few indprojects . To confA. batizocoi was syntenic with chromosomes 4A and 9A of A. duranensis implicating inversion and reciprocal translocation events as the underlying chromosomal rearrangements in this B-genome species. Recombination frequencies were generally low in the central, presumably centromeric chromosomal regions of A. duranensis and increased toward the telomeres, a pattern typical of many plant species [Significant segregation distortion (p = 0.05) was observed for 513 29.8%) markers  and included 172.4 markers (119–266) .The map produced in the current study is the first high-density map available in peanut, and because it was generated from a progenitor species of y et al.  to more Homology search of the 1,724 mapped loci resulted in significant hits for 1,463 loci in at least one of the three databases: Medicago, Uniprot and GenBank NR database, and 580 of the mapped loci gave significant similarities in either of the two gene ontology databases: Medicago Gene Atlas and TAIR . While a majority of the dots in the synteny plots appear to be random , one accession of A. ipaensis, and eight accessions of A. batizocoi (B genome) were obtained from the USDA or NCSU germplasm collections. Plants were then grown in greenhouses at the University of Georgia at Athens. The accessions evaluated are shown in Figure A. duranensis accessions, including PI 468200 × PI 468198, PI 468319 × PI 475885, and PI 475887 × Grif 15036. The hybrid combination PI 475887 × Grif 15036 was one of the most polymorphic as revealed by using a panel of SSR markers as described below and thus was selected for subsequent mapping. PI 475887 was originally collected by Krapovickas, Schinini, and Simpson near Salta, Argentina during 1980, and Grif 15036 was originally collected by Williams, Simpson, and Vargas near Boqueron, Paraguay during 2002 [2 population was developed by self-pollinating multiple F1 individuals. The intraspecific F2 population (n = 94) from a hybrid between two A. duranensis accessions was then used for mapping studies.Thirty-seven accessions of ing 2002 . CrossesA. duranensis, A. ipaensis, A. stenosperma, and A. batizocoi accessions using a modified CTAB method [DNA was isolated from leaf samples of B method ,46. The B method ,47-53 anB method  was usedB method  for the A. hypogaea genome were mined for 2,138 EST-SSR markers (GM710-GM2847) . Unigenes in the transcript assembly were screened for perfect repeat motifs using SSR-IT http://www.gramene.org/db/markers/ssrtool) and for imperfect motifs using FastPCR . The repeat count (n) threshold for each motif type was set for n ≥ 5. SSR markers were genotyped on an ABI3730XL Capillary DNA Sequencer  using forward primers labelled with FAM, HEX, or TAMRA fluorophores. PCR was performed in a 12 μL reaction mixture containing 1.0 × PCR buffer, 2.5 mM Mg++, 0.2 mM each of dNTPs, 5.0 pmol of each primer, 0.5 unit of Taq polymerase, and 10 ng of genomic DNA. Touchdown PCR was used to reduce spurious amplification. The SSR markers were screened for length polymorphisms using GeneMapper 3.0 software . Of the 2,138 EST-SSR primer pairs tested, markers derived from 598 could be mapped. A set of 34 SSR markers from genomic sequences of Arachis previously screened for polymorphism between parents of the A. duranensis mapping population  were also mapped  and 63,216 singletons) from tetraploid peanut ESTs  representing ca. 37 Mb of the Arachis unigenes showing similarity to R-gene homologs identified by mining a peanut transcript assembly [Arachis hypogaea[SSCP markers were developed from genomic DNA templates for previously described NBS sequences isolated by targeting conserved sequence motifs in NBS-LRR encoding genes ,57 and fassembly . SSCP frassembly -60. A to hypogaea,61 was aA. duranensis PI 475887 and Grif 15036. These were assembled into 81,116 unique transcripts (unigenes) (GenBank Accn. No. HP000001-HP081116). Assemblies were searched for single nucleotide polymorphisms (SNPs) that fulfilled the following two criteria: (a) the SNP position is covered at least by two reads from each genotype, and (b) at least 80% of the reads call the SNP in the particular genotype. Using these criteria, we identified 8,478 SNPs in 3,922 unigenes. To facilitate the selection of candidate SNPs for designing and building Illumina GoldenGate SNP genotyping arrays, putative intron positions were predicted by aligning Arachis contigs with Arabidopsis and Medicago genomic DNA sequences identified by BLAST analyses. SNPs within 60 bp of a putative intron were eliminated, thereby reducing the collection of candidate SNPs to 6,789 in 3,264 unigenes from which 1,236 high-quality SNPs, each representing separate unigenes, were selected for genotyping. SNPs were also detected by allele re-sequencing in a subset of 768 conserved legume orthologs identified by coauthors , and 300 of these SNPs were added to the GoldenGate array. SNP genotyping on the GoldenGate array was conducted at the Emory Biomarker Service Center, Emory University. The BeadStudio (Illumina) genotyping module was used for calling genotypes. Markers with GC quality scores lower than 0.25 were excluded from subsequent analysis.Total RNA was isolated from roots of young seedlings (up to four trifoliate) and from developing seeds  of the two parental genotypes, PI 475887 and Grif 15036 . cDNA libraries were developed using the Mint cDNA synthesis kit (Evrogen) and normalized using the Trimmer cDNA normalization kit (Evrogen). cDNA sequences were generated by Sanger and 454 GS-FLX sequencing methods and assembled using the tool Mira . Altoget-12 for clustering markers into linkage groups. The recombinant inbred line2 (RIL2) algorithm and Kosambi function were used to calculate genetic distances. The program Joinmap 3.0 [The program, MSTMap  was usednmap 3.0  was usednmap 3.0 .http://www.medicago.org), Uniprot (http://www.uniprot.org) and GenBank NR (http://www.ncbi.nlm.nih.gov/genbank) databases using various blast algorithms. Gene ontology annotations were also added by searching Medicago Gene Atlas (http://mtgea.noble.org) and The Arabidopsis Information Resource  databases. A significance threshold of E =1e-5 was applied in all inquiries.The cDNA sequences included in the genetic map have been used to search for homologous genes in the Medicago  not anchored to the chromosomal assembly in the target genomes were discarded. Plotting the data and processing of blast results were performed with Visual Basic programs written for this study.The EST sequences used for marker-development were compared to the whole genome sequences of The authors declare that they have no competing interests.EDN developed the cDNA libraries, organized sequencing and SNP genotyping, and constructed the initial genetic maps. YG conducted parental genotyping and constructed final genetic maps. EDN and ST developed the mapping population. EDN and RAO genotyped the SSR, RGC, and RS markers. JEB performed the synteny analysis. ST, YG, SK, AFH, NK developed the SSR markers. SK performed the genetic diversity study. CAT and DZ assembled the ESTs and nominated SNPs for the genic sequences. ADF, NCG, RVP and DC nominated the conserved legume ortholog SNPs. EDN and JEB participated in manuscript writing. DC, HTS and NN contributed to project design. POA was Co-PI of the project and finalized the manuscript. SJK was the PI of the project, designed experiments, coordinated the study, and participated in manuscript writing. All authors read and approved the final manuscript.SNP markers on Illumina GoldenGate array. Marker ID along with sequence information for OPAs and target ESTs are provided.Click hereb for fileComparative genetic mapping ofArachis duranensisusing two different software programs on the same dataset. Genetic maps were constructed by MSTMap (left) using 1673 co-dominant markers and Joinmap 3.0 (right) using 1724 markers. Linkage group assignments, marker orders and genetic distances were highly consistent, except for the order among a few closely linked loci. Marker positions determined by Joinmap 3.0 are provided in Additional file Click here for fileMapped markers with segregation distortion (p = 0.05) and their position on the map. Column A lists the marker positions on each chromosome, column B indicates marker name, column C is the chromosome number, columns D to H list the number of individuals in each genotype class, and columns J and K provide χ2 and P values, respectively.Click here for fileAnnotated loci mapped inArachis duranensis. Columns B to J include homologs identified in the Medicago (B-D), GenBank-NR (E-G) and Uniprot-Sprot (H- J) databases. Columns L to S include gene ontology (GO) terms identified in the three major GO categories: molecular function , biological process  and cellular component .Click here for fileSequences associated with SSR and SSCP markers for synteny analysis. Column A is the marker name and column B is the Genebank ID or sequence used as query for synteny analysis.Click here for fileMarker positions for the linkage map.Click here for file