Lactobacillus strains isolated from the intestinal content of rats fed with a diet supplemented with sorbitol.Comparative genomic hybridization (CGH) constitutes a powerful tool for identification and characterization of bacterial strains. In this study we have applied this technique for the characterization of a number of recA, pheS, pyrG and tuf sequences identified five bacterial strains isolated from the intestinal content of rats as belonging to the recently described Lactobacillus taiwanensis species. DNA-DNA hybridization experiments confirmed that these five strains are distinct but closely related to Lactobacillus johnsonii and Lactobacillus gasseri. A whole genome DNA microarray designed for the probiotic L. johnsonii strain NCC533 was used for CGH analysis of L. johnsonii ATCC 33200T, L. johnsonii BL261, L. gasseri ATCC 33323T and L. taiwanensis BL263. In these experiments, the fluorescence ratio distributions obtained with L. taiwanensis and L. gasseri showed characteristic inter-species profiles. The percentage of conserved L. johnsonii NCC533 genes was about 83% in the L. johnsonii strains comparisons and decreased to 51% and 47% for L. taiwanensis and L. gasseri, respectively. These results confirmed the separate status of L. taiwanensis from L. johnsonii at the level of species, and also that L. taiwanensis is closer to L. johnsonii than L. gasseri is to L. johnsonii.Phylogenetic analysis based on 16S rRNA gene, L. taiwanensis. The microarray-based CGH technology has been shown as a remarkable tool for the identification and fine discrimination between phylogenetically close species, and additionally provided insight into the adaptation of the strain L. taiwanensis BL263 to its ecological niche.Conventional taxonomic analyses and microarray-based CGH analysis have been used for the identification and characterization of the newly species  Lactobacillus are indigenous to the gastrointestinal tract of humans and animals [Lactobacillus acidophilus complex. The first major study on this group using DNA-DNA hybridization defined two main homology groups, A and B, with six subgroups [L. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus and Lactobacillus gallinarum, and strains of the group B became Lactobacillus gasseri and Lactobacillus johnsonii [L. acidophilus and L. johnsonii are commercialized as probiotics and they have been subjected to extensive studies aimed to infer the mechanisms governing their beneficial health effects. L. acidophilus strain NCFM increased the immunogenicity of an oral human rotavirus vaccine in animals [L. acidophilus strain Bar13 interfered with the adhesion of enteropathogenic bacteria to enterocytes and inhibited IL-8 production by HT-29 cells, suggesting a potential to protect intestinal cells from acute inflammatory response [L. johnsonii strain La1 (NCC533) has been shown to suppress gene expression of proinflammatory cytokines, supposedly involved in atopic dermatitis [L. johnsonii strain NCC533 has been sequenced [in vivo and those whose expression is specifically necessary for long persistence in the gut [A number of species belonging to the genus  animals -3. Due tubgroups , that laubgroups . Strainsohnsonii . Some st animals . L. acidresponse . L. johnrmatitis -12. The  the gut ,15.ad hoc committee for the re-evaluation of the species definition in bacteriology considered sequencing of housekeeping genes, DNA profiling and DNA arrays as very valuable methods for species delineation and phylogenetic positioning [The bacterial species concept has been widely discussed ,17, but itioning .L. acidophilus group constitutes a paradigm of a compact cluster showing closely related species that offers special difficulties for the correct taxonomic allocation of new strains. Several techniques have been applied to identify members of the L. acidophilus group, such as 23S-rRNA-targeted oligonucleotide probe hybridization [L. acidophilus complex [L. johnsonii, L. gasseri and L. acidophilus constitute three clearly independent and consistent genomic groups, although L. gasseri was more closely related to L. johnsonii than to L. acidophilus. The bacterial genomic variability between strains belonging to the same species or between closely related species comes from mobile DNA elements and from variable regions. The latest may be required for environmental adaptation and could be acquired by lateral gene transfer. The L. johnsonii NCC533 chromosome harbors three regions that contain two prophages, Lj928 and Lj965, and a 6-kb potentially autonomous unit [L. johnsonii NCC533 also carries four further integrases and several IS elements flanking or disrupting diversity regions [The dization , random dization ,23, sequdization  and autodization . Recentl complex . They shous unit ,28. Thesous unit -28. L. j regions -28.L. johnsonii, Lactobacillus intestinalis, Lactobacillus murinus, Lactobacillus reuteri and Lactobacillus sp. BL263 (previously named as AD102) [L. reuteri and Lactobacillus sp. BL263 cell numbers [Lactobacillus sp. BL263 could be assigned to the L. acidophilus group, its species allocation was still uncertain. In the present study, standard taxonomic methods and microarray-based CGH analysis have been implemented for the identification and characterization of Lactobacillus sp. BL263 and four additional rat intestinal isolates  displaying identical PCR-DGGE pattern.A previous work studying the influence of sorbitol intake on the population of rat intestinal lactobacilli showed the presence of five species: s AD102) . Sorbito numbers . AlthougLactobacillus strains used in this work are listed in Table The Lactobacillus species [recA), phenylalanyl-tRNA synthase (pheS), CTP synthetase (pyrG) and translational elongation factor Tu (tuf). PCR reactions were performed with the Expand High Fidelity PCR System (Roche), using chromosomal DNA and the primers listed in Table recA gene, the PCR fragments were cloned in Escherichia coli using the pMOS Blue vector contained in the Blunt- ended cloning kit  following the manufacturer instructions. Plasmid DNA was isolated from each clone containing a portion of the recA gene from the Lactobacillus strains studied here. The partial recA genes were sequenced using the M13 universal and reverse primers.The housekeeping genes selected for multilocus sequence analysis based on the results of previous studies on  species ,31 were The sequences were either obtained in this work, retrieved from the GenBank database or from the Ribosomal Database Project II (RDP) . MultiplCarbohydrate fermentation patterns were determined by using the API 50 CH  according to the supplier instructions. Results were recorded after 48 h at 37°C. Growth at 15°C, 45°C, pH 4.5 or in the presence of NaCl (4.5% and 7.0%) was determined in MRS broth (Difco). All the growth analyses were done at least in duplicate. Data were analysed using Pearson's correlation coefficient (XLSTAT programme). A tree was created by cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA).550 of 1.0. Cells were collected by centrifugation, washed with Tris-HCl 100 mM, pH 7.5, resuspended in 1 ml of the same buffer plus sucrose 0.5 M and lysozyme 2.5 mg/ml. After incubation for 1 h at 37°C, cells were washed, collected by centrifugation and resuspended in 50 μl of SDS-PAGE loading buffer. Cells were lysed at 100°C 5 min and 20 μl of total extract were separated on a 12% SDS-PAGE using standard protocols [Cultures were grown in 10 ml MRS at 37°C to an ODrotocols . The gelLactobacillus strains were spotted on duplicated membranes. Six digoxigenin-labelled probes were prepared by random priming using the DIG DNA Labelling kit (Roche) and genomic DNA isolated from the type strains of L. intestinalis, L. johnsonii, L. crispatus, L. gasseri, L. acidophilus or L. taiwanensis. Those probes were hybridized against six identical macroarrays with 26 Lactobacillus strains. Hybridization, washing and staining was carried out as recommended by the manufacturer using the CDP-Star chemiluminescent substrate (Roche). Chemiluminescence was detected in a Fujifilm LAS 1000 imaging system (Fuji Photo Film Co. Ltd.) and it was measured using the Image Gauge Version 4.0 programme. The DNA-DNA hybridization experiments were done in triplicate.DNA macro-arrays on nylon membranes  were performed by using a dot blot assay according to Hänninen et al.  with somL. johnsonii NCC533 [http://www.python.org. Probes were eliminated from the analysis if their signal strength was lower than twice the standard deviation of the local background when hybridized with L. johnsonii NCC533 genomic DNA. The signal ratio (Cy3-labeled unknown strain DNA versus Cy5-labeled L. johnsonii NCC533 DNA) of each spot was calculated without background subtraction and normalized based on our previous experience of CGH [T/NCC533 (intra-species comparison), and BL263/NCC533 (inter-species comparison with the novel Lactobacillus).Based on the genome sequence of i NCC533 , Agilenti NCC533 . Hybridie of CGH . Having recA, pheS, tuf and pyrG sequences generated in this study were deposited in the GenBank under accession numbers FJ556999 to FJ557013, FJ557014 to FJ557023, GU121623 to GU121628 and HM777006, GU121629 to GU121634 and HM777008, and GU121635 to 121640 and HM777007, respectively. The microarrays data have been deposited in NCBI's Gene Expression Omnibus [The 16S rRNA gene,  Omnibus  and are Lactobacillus sp. BL263, BL301, BL302, BL303 and BL304 were determined. Sequences of BL263, BL301, BL302 and BL303 were identical (data not shown) and shared a 99.9% sequence identity with BL304 strain. A preliminary analysis showed that these sequences were very similar to sequences of L. gasseri and L. johnsonii. Subsequently, a phylogenetic tree was obtained by using the tools available in RDP [L. gasseri and L. johnsonii were selected for a detailed phylogenetic reconstruction by maximum likelihood. During the preparation of this manuscript, the novel L. taiwanensis species isolated from silage was described [L. taiwanensis species and that they are closely related to L. johnsonii and L. gasseri  of the rat isolates e in RDP . On the escribed . The phyus group .recA, pheS, pyrG and tuf gene sequences from strains BL263, BL301, BL302, BL303, BL304 and L. taiwanensis DSM 21401T also led to phylogenetic trees that clearly separated a cluster encompassing L. taiwanensis and the rat intestinal isolates from all the L. johnsonii and L. gasseri strains as indicated by the bootstrap values  at different temperatures and salt concentrations were used to construct a dendrogram . Results obtained with L. johnsonii, L. gasseri and L. taiwanensis were less clear-cut. L. johnsonii and L. gasseri intra-species hybridization values were greater than 71%, but L. johnsonii BL261 and L. gasseri BL223 only showed values of 57% and 62% with their respective type strains. Nevertheless, those values still fall within relaxed species delineation boundaries (50-70% DNA reassociation) [L. taiwanensis showed intra-specific hybridization values in the range of 73% to 95%. Inter-species hybridizations between L. johnsonii and L. gasseri strains rendered values up to 46%, and always higher than with the other species included in this assay, supporting that they are closely related but distinct species. L. taiwanensis strains also showed high reassociation rates with L. johnsonii and L. gasseri, their inter-species hybridization values ranging from 21-40% and from 10-26%, respectively. Therefore, these results also demonstrated the separate status of L. taiwanensis strains at the species level, and undoubtedly placed them within the L. acidophilus complex. Inside this group they showed closer DNA homology to L. johnsonii and L. gasseri than to L. crispatus and L. acidophilus in agreement with the previous phylogenetic analyses.DNA similarity values were determined by DNA-DNA hybridization with 26 be Table . Relativciation) . L. taiwL. johnsonii strain NCC533 was tested in a self-hybridization experiment to validate the design of the microarrays. This step was necessary since the microarrays previously used for inter-species CGH in the L. acidophilus group [L. johnsonii NCC533 strain was used as both the reference and the test strain, and as expected the distribution of the log2 ratios showed a normal distribution around zero (data not shown). The performance of the oligonucleotide microarray was further evaluated by hybridization with DNA from L. johnsonii ATCC 33200 type strain and L. johnsonii BL261. In the DNA-DNA hybridization assays, this latter strain fell into the relaxed definition of species. The distribution of the log2 ratios of the CGH results are shown in Fig. 2 ratio of -3.5 or greater with these two strains, suggesting a majority of very similar DNA sequences. The rest of ORFs showed log2 values under -3.5, reflecting high divergence or absent genes in the test strains. The two L. johnsonii strains profiles were typical of intra-species comparisons. In contrast, L. taiwanensis and L. gasseri strains showed a significant deviation from zero for nearly all the genes, suggesting global sequence divergence between L. johnsonii and both tested strains  with ratios of -3.5 or greater than L. gasseri (47%), showing slightly better gene conservation. This is in agreement with the results of DNA-DNA hybridization experiments. These results confirmed the separate status of L. taiwanensis from L. johnsonii at the level of species, and they showed that L. taiwanensis is closer to L. johnsonii than L. gasseri is to L. johnsonii.Firstly, the DNA microarray constructed with oligonucleotides representing all the coding regions from us group  were ampins Fig. . These sL. johnsonii NCC533 genome  for a complete cluster, between L. johnsonii and L. taiwanensis form a clear-cut cluster containing an ABC transporter and hypothetical proteins, which is flanked by transposases (LJ1292-LJ1298). This cluster is absent in all other strains of L. johnsonii and L. gasseri analyzed to date. Taken together, these observations strongly argue for an horizontal gene transfer. Considering the marked conservation of this cluster compared to the rest of the L. taiwanensis genome, the hypothesis of its loss in the rest of the analysed strains is very unlikely.CGH results were mapped on the ome Fig. . The genroarrays , furtherL. taiwanensis BL263, L. gasseri ATCC 33323T and L. johnsonii ATCC 33200T versus L. johnsonii NCC533, their CGH results were combined in Fig. L. taiwanensis and L. gasseri were not always shared by the two species. These "species-related" variable genes counted for almost one quarter of the L. johnsonii NCC533 genome. Since they were in minority related to mobile elements, this observation strongly supports three evolutionary branches. The number of genes conserved in L. taiwanensis and absent/divergent in L. gasseri was 257, whereas those conserved in L. gasseri and absent/divergent in L. taiwanensis were only 186. These results clearly showed that L. taiwanensis is closer to L. johnsonii than L. gasseri is to L. johnsonii. The colour code of the Fig. L. johnsonii ATCC 33200T absent/variable genes are also absent in the two other tested species. However, 63 genes absent in the L. johnsonii type strain are present in L. taiwanensis .In order to compare the genome conservation of Lactobacillus strains from the intestinal content that could not be assigned conclusively to known species [recA, pheS, pyrG and tuf phylogenetic reconstructions and DNA-DNA hybridization experiments revealed close relatedness between them at the genomic level (> 72%). They were therefore regarded to belong to the same species. During the preparation of this manuscript a new L. taiwanensis species isolated from silage was described [L. taiwanensis species. This species is placed within the L. acidophilus group, but they constituted a different cluster from their closest relatives, L. johnsonii and L. gasseri. The five L. taiwanensis strains have been isolated from the same environment and showed very high homology in the phylogenetic analyses; however, the carbohydrate utilization and growth profiles clearly distinguish L. taiwanensis BL301 from the groups made by L. taiwanensis strains BL304 and BL302 and by L. taiwanensis strains BL303 and BL263 , and the general trend towards an increase in genome sequencing projects will offer the opportunity of applying the DNA microarray-based CGH as an alternative to the classical whole genome DNA macroarray hybridizations for bacterial species determination. At a more affordable price than full genome sequencing, the CGH analysis renders many more information parameters than whole DNA re-association. Although the CGH microarrays presented the limitation of be unable to detect novel genes, it offers the re-association ratio for each ORF, the plot of the signal to ratio frequency, and the mapping of the conserved genes on the reference genome, thus providing a different ground for intra and inter-species comparisons. This work has shown that the inter-species profile comparison of the novel species L. taiwanensis clearly demonstrated its separate status respect to L. johnsonii.The microarray-based CGH has been used to characterize bacterial intra-species genetic diversity at the whole-genome level -46. It h complex . Here, mL. taiwanensis BL263 and L. johnsonii NCC533 using DNA-DNA hybridization with macro or microarrays may look dissimilar: whole DNA re-association values were 25% for the macroarray method, but in microarray CGH analysis 51% of the L. johnsonii NCC533 genes were conserved in L. taiwanensis. In this latter experiment, the hybridization ratios distribution of the conserved genes in L. taiwanensis is centred on -1  and several of them, including the ones annotated for fructose, glucose and cellobiose transport, are specifically induced in the gastrointestinal tract (GIT) [L. taiwanensis BL263. These predictions are supported by the API 50CH results showed for L. taiwanensis BL263 . These enzymes have been almost exclusively found in bacterial species associated with the GIT and its role has been largely discussed [see review [L. taiwanensis BL263 exhibited taurocholic and taurodeoxycholic acid deconjugation activity . This is in agreement with the CGH analysis showing that a putative operon encoding a BSH and two bile salt transporters (LJ0056 to LJ0058) on L. johnsonii NCC533 genome is conserved in L. taiwanensis BL263, although this strain has only one of the two transporter encoding genes. Homologues of these genes in L. johnsonii 100-100 were shown to be gene duplicates and to have a function in taurocholic acid uptake [L. johnsonii NCC533 to interact with mucins and epithelial cells has been shown to likely rely on cell-surface associated elongation factor Tu and heat shock protein GroEL [L. taiwanensis BL263, offering a putative role of the encoded proteins in the interaction of this strain with the host. This interaction may also be influenced by the presence of another conserved gene encoding a putative adhesin (LJ0391) that showed similarities with Fap1 fimbrial protein from Streptococcus parasanguinis [A number of features on 3 genome  that arect (GIT) . CGH resd uptake ,52. The in GroEL . As expeanguinis .in silico comparative genomic analysis showed a wide sequence similarity and an accurate genome synteny between L. johnsonii and L. gasseri species [Lactobacillus species described here is placed in a third branching of similar closeness, which raised once more the question about the limits of species delineation. Additionally, the CGH analysis showed that the new species is genetically slightly closer to L. johnsonii than L. gasseri is. This result is also supported by our DNA-DNA hybridization data and our phylogenetic analysis of recA, pheS, pyrG and tuf sequences in these three species.Previous studies using DNA microarray-based CGH and  species ,26. In sLactobacillus species within the L. acidophilus group, and with microarray-based CGH analysis we confirmed the status of L. taiwanensis BL263 as species showing gene by gene differences and similarities with its closest relative L. johnsonii strain NCC533.Since the genome is the final target of all the molecular taxonomic indicators of species determination, the DNA microarray-based CGH analysis is the most powerful technology for strain typing and species assignment, just behind the costly genome sequencing. In this work we have characterized with conventional taxonomic analyses a novel The authors declare that they have no competing interests.L. taiwanensis species and performed all the experiments related to conventional taxonomic analyses. BB designed and analyzed the microarray-based CGH experiments. DM performed the microarray-based CGH experiments. MZ designed and performed the phylogenetic analyses. GPM and MJY designed the study and supervised it. MJY drafted the manuscript. LSAR, BB, MZ and GPM helped improving the draft. All authors read and approved the final manuscript.LASR has isolated the five strains of the L. johnsonii NCC533 that are conserved on L. taiwanensis BL263 but not in L. johnsonii ATCC 33200T or L. johnsonii BL261Genes on . This table contains 63 and 34 genes on L. johnsonii NCC533 that are conserved on L. taiwanensis BL263 and absence on L. johnsonii type strain or L. johnsonii BL261, respectively.Click here for file


An 18-year-old Ethiopian woman presented with debilitating back pain and high fever. X-ray examinations showed diffuse pulmonary tuberculosis and a psoas abscess. After starting rifampicin, isoniazid, ethambutol and pyrazinamide, acute interstitial nephritis developed that spontaneously recovered. According to Harrison's Online rifampicin should have been causative, but the spontaneous recovery excluded that possibility. The clinical course fit the diagnosis of a paradoxical response, for which recently risk factors have been described. Thus, a paradoxical response should be added to the list of causes of interstitial nephritis in tuberculosis patients and in such cases rifampicin could be continued. Rifampicin, mostly in combination with isoniazid, ethambutol and pyrazinamide, is the first-line therapy for tuberculosis. Rifampicin's most feared side effect is hepatotoxicity. However, nephrotoxicity such as acute tubular necrosis and interstitial nephritis (IN) have also been reported. In case of acute tubular necrosis, rifampicin-dependent antibodies have been found, suggesting a causal relationship between rifampicin and renal failure . In acut9/l, creatinine 93 micromol/l, blood urea nitrogen 3.8 mmol/l and serum albumin 20 g/l. Urinalysis showed no protein, cells or casts. HIV serology was negative. Acid-fast bacilli were found in the pus of the psoas abscess, so the diagnosis of hematogenously disseminated tuberculosis was established. Treatment with rifampicin, isoniazid, ethambutol and pyrazinamide was started. After 6 weeks on this four-drug regimen laboratory tests were repeated. Serum creatinine had risen to 272 micromol/l with a simultaneous rise of the lymphocyte count to 1.5 × 109/l. Urinalysis showed 2+ protein, but no cells or casts. Protein excretion was 2.1 g/day. All four drugs were continued, albeit that the dosage of ethambutol was reduced with 50%. In the ninth week of treatment a renal biopsy was performed. Light microscopy revealed normal glomeruli but the interstitium showed a dense mononuclear infiltrate, existing of lymphocytes, some plasma cells and a few eosinophils. There were no signs of acute tubular necrosis. A Ziehl-Neelsen stain was negative for mycobacteria . Furthermore, during PR a significant rise of the lymphocyte count  is seen [PR is defined as a transient worsening of pre-existing, often symptomless, lesions during anti-tuberculosis treatment. The etiology is unknown, although an interaction between the host's immune response and mycobacterial products has been suggested. PR occurs more frequently in HIV-positive patients. Risk factors for developing PR in HIV-negative patients are: anemia Hb<6.8 mmol/l), low serum albumin (< 30 g/l) and a low lymphocyte count (< 0.8 × 10 mmol/l, Transient IN during rifampicin treatment in HIV-negative tuberculosis patients has been described before, but not recognized as PR. One patient recovered after withdrawal of pyrazinamide while rifampicin was continued. The authors postulated pyrazinamide as the cause of the nephritis . AnotherHIV-negative tuberculosis patients with well-defined risk factors may develop IN during anti-tuberculosis therapy. This nephritis should be considered a PR, thus anti-tuberculosis drugs could be continued and corticosteroids eventually added.IN: Interstitial nephritis; PR: paradoxical response; CT: Computed tomography; CRP: C-reactive protein.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.JvdM reviewed the case and relevant literature and prepared the manuscript. GJ reviewed and edited the manuscript. PW performed the histological examination. All authors read and approved the final manuscript.


Caenorhabditis elegans, an MSP (major sperm protein) dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known.MAP (mitogen-activated protein) kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode We investigated two key elements of oocyte-to-embryo transition, MSP expression and MAP kinase signaling, in two parthenogenetic nematodes and their close hermaphroditic relatives. While activated MAP kinase is present in all analysed nematodes irrespective of the reproductive mode, MSP expression differs. In contrast to hermaphroditic or bisexual species, we do not find MSP expression at the protein level in parthenogenetic nematodes. However, genomic sequence analysis indicates that functional MSP genes are present in several parthenogenetic species.C. elegans, thus requiring intact MSP genes. However, MSP expression has been shifted to non-sperm cells and is reduced below the detection limits, but is still sufficient to trigger MAP kinase activation and embryogenesis.We present three alternative interpretations to explain our findings. (1) MSP has lost its function as a trigger of MAP kinase activation and is not expressed in parthenogenetic nematodes. Activation of the MAP kinase pathway is achieved by another, unknown mechanism. Functional MSP genes are required for occasionally emerging males found in some parthenogenetic species. (2) Because of long-term disadvantages, parthenogenesis is of recent origin. MSP genes remained intact during this short intervall although they are useless. As in the first scenario, an unknown mechanism is responsible for MAP kinase activation. (3) The molecular machinery regulating oocyte-to-embryo transition in parthenogenetic nematodes is conserved with respect to  Throughout the animal kingdom, female gametes interrupt their development during oogenesis at various stages of meiosis. In response to external stimuli, this arrest is released, and oocyte maturation can take place. Then oocytes resume meiotic divisions, ovulate and get competent for fertilisation.An important step during oocyte maturation of all animals is MAP kinase activation (reviewed in -3). MAP C. elegans. The Erk1/2 ortholog MPK-1 [There are MAP kinase orthologs from several subfamilies present in og MPK-1 ,12 is thog MPK-1 ,14).C. elegans sperm representing 15% of its total protein content [C. elegans, MSP comprises a large multigene family of about 50 highly conserved members [Ascaris suum to 1-13 in other mammalian intestinal parasites, 1-4 in filarial nematodes or 5-12 in plant and insect parasitic species [C. elegans are expressed at the same time and only during the terminal stages of spermatogenesis [Oesophagostomum dentatum, Brugia malayi, Dictyocaulus viviparus and Ascaris suum [MSP is a small basic protein of ~15 kDa , as well as the MAP kinase gene of a second parthenogenetic nematode, Diploscapter coronatus, a close relative of C. elegans  and C. elegans/D. coronatus are slightly more similar to each other than sequence pairs derived from nematodes with identical reproduction , but also in the three Acrobeloides species. MAP kinase activation sometimes extended over three or four proximal oocytes (-1 to -4), occasionally interrupted by a non-activated cell, or could be restricted to the -2 or -3 oocyte without a signal in -1. We noticed such staining patterns in 4/11 (A. nanus) and 11/22 cases (Acrobeloides (herma)). As an example, an unusual staining of the second and fourth, but not the first, oocyte from the Acrobeloides (bisex) gonad is shown . While C. elegans oocytes mature at a fast rate of one every 23 min [D. coronatus (data not shown) giving a likely explanation for the less frequent staining of oocytes in this species.Furthermore, the percentage of stained oocytes was considerably lower in y 23 min , this inAcrobeloides species a more pronounced MAP kinase signal in the nuclei of late-stage oocytes than in C. elegans . Stainii Figure . The sams Figure  indicatii Figure , the cor elegans  and suggus paper  indicatis Figure . LikewisA. nanus and D. coronatus  and Acrobeloides (herma). This demonstrates cross-reactivity of the antibody to MSPs from other species as expected from the conservation of MSP C-termini in a wide range of nematodes  lane  lane).Additional smaller bands in the e Figure  likely r elegans . In some elegans , this siIn conclusion, our results show that MSP is not detectable and thus appears to be absent at the protein level in the parthenogenetic species we analysed.A. nanus, from its hermaphroditic sister species Acrobeloides (herma) and from the parthenogenetic D. coronatus. We isolated and characterised several MSP genes from all three nematodes.Parthenogenetic nematodes do not undergo spermatogenesis during which MSP is normally expressed. Indeed, our experiments gave no evidence for its expression. We therefore expected that they do not require MSP, allowing the accumulation of mutations in MSP genes over time. A decay of genes specific to sex and recombination is predicted as an effect of the loss of sex on the eukaryotic genome . To inveD. coronatus and Acrobeloides (herma) MSP. As in many other MSPs   Figure , excludiA. nanus where several stops in the coding region indicate a pseudogene (not shown). Pseudogenes containing frameshifts or short deletions were also present in some MSP clones from Acrobeloides (herma) (not shown), in agreement with a number of pseudogenes in the large C. elegans MSP gene family [Besides intact genes with no signs of degeneration we also detected an exception in e family ,53.A. nanus and D. coronatus, a single intron of 60-70 bp is situated at amino acid position 33 [D. coronatus is the only nematode so far where a second MSP intron is present . As whole genome sequences do not exist for either nematode, we looked for MSP coding regions in parthenogenetic species with a sequenced genome [Meloidogyne incognita and its facultatively parthenogenetic [Meloidogyne hapla. We compared this MSP set to our above obtained sequences and to published MSP sequences from other, non-parthenogenetic nematodes. The deduced protein sequences of M. incognita and M. hapla are almost identical with each other  and number (one intron) are identical in all Meloidogyne MSPs investigated here and agree with our data from the parthenogenetic nematodes A. nanus and D. coronatus as well as with results from other species [Meloidogyne MSPs (60-70 bp) and comparable to MSP sequences from other nematodes . In  species .Meloidogyne MSPs, we investigated the regions flanking the AUG initiator codon. According to Kozak's rules, a consensus sequence surrounding the AUG codon is critical for a favourable context of translation initiation [Meloidogyne MSP loci analysed, we find purines (A) at the -3 position and the nucleotide G in position +4 (not shown) as it was previously reported for MSP transcripts isolated from G. rostochiensis and D. viviparus [To assess the translation potential of itiation -67. In aiviparus ,55. ThisThus, a remarkable conservation in genomic organisation and deduced protein sequence exists in MSPs from several parthenogenetic nematodes positioned in different clades.C. elegans, the hormonal trigger for oocyte maturation and MAP kinase activation is MSP, a cytoskeletal protein secreted from sperm [C. elegans. As different modes of reproduction exist in the phylum Nematoda, including many examples of parthenogenesis , we C. elegans and parthenogenetic nematodes  and Acrobeloides (bisex) which are closely related to the parthenogenetic A. nanus . We chose Acrobeloides (herma), the hermaphroditic sister species to A. nanus, because cloning of A. nanus MSP was not successful. The C-terminal MSP sequences we isolated were identical to the respective C. elegans epitope at the amino acid level , punctata . However elegans . Close ia Figure .A. maximus and Z. punctata. Although we have used a different batch of the same antibody, our inability to reproduce this result can not be caused by a lower sensitivity. Expression levels of MSP in the parthenogenetic species as presented by [C. elegans and still suggest abundant expression. Using serial dilutions we showed that it is possible for us to detect at least 1% of MSP input . To test this assumption, we analysed cell extracts from A. maximus and Z. punctata, but MSP was undetectable in these nematodes either  Although we could never find males in blished; ). The hiescribed ,72. Malencognita ,74 whichncognita . Althoug(ii) The preservation of MSP genes in parthenogenetic nematodes may be explained by a recent transition to parthenogenesis. Evolution did not yet modify MSP genes, and although they are obsolete, we find them still intact. A model where parthenogenesis constantly emerges and disappears in different branches of nematodes could account for these observations. A feature of this model, supported by theoretical considerations ,76, is tAlthough this is an attractive hypothesis, presently no data exist for its direct proof which would e. g. require the inactivation of intact MSPs in a parthenogenetic background. Nevertheless, a detailed comparison of MSP sequences in non-parthenogenetic vs. parthenogenetic root knot nematodes proposed a recent origin of parthenogenesis based on the absence of an increased substitution rate, changed substitution pattern or pseudogene formation . These rC. elegans. Albeit this view is not supported by our data, we cannot exclude the presence of MSP at levels too low for detection via Western blotting and immunofluorescence. MSP is deprived of structural and motility functions in sperm-less parthenogenotes. Thus, a low expression level could be sufficient for acting like a hormone in MAP kinase signaling and oocyte maturation. This model would implicate a shift in MSP expression from sperm to non-sperm cells which could be achieved by mutations in regulatory sites. As C. elegans MSP is exported from sperm by a non-classical vesicle budding mechanism [(iii) As a third possibility, MSP genes might be conserved because MSP is needed to trigger oocyte maturation in parthenogenetic species like in echanism , one wouWith the presently available data it is not possible to distinguish between the presented alternatives. Unlike the first two scenarios, the last hypothesis provides a number of reasonable predictions and may serve as a starting point for further analysis.C. elegans oocyte-to-embryo transition is governed by MSP, a cytoskeletal protein released from sperm, and is therefore coupled to the presence of sperm. Within the Nematoda, reproduction is highly versatile, and parthenogenesis, reproduction without the contribution of sperm, has evolved many times independently. To elucidate oocyte-to-embryo transition in nematodes that lack sperm, we have investigated two of its hallmarks, MAP kinase activation and MSP signaling, in two representative parthenogenetic species. Activation of MAP kinase in parthenogenetic nematodes at the time of oocyte maturation implies that this signal does not constitute a simple switch whose presence or absence determines the reproductive mode. Instead, it might regulate meiotic cell cycle progression of maturing oocytes independent of the reproductive mode.The MSP, the major sperm protein, has no apparent function in parthenogenetic nematodes that lack sperm. Indeed, we demonstrate its absence at the protein level. However, the unexpected discovery of intact MSP genes suggests either their strong maintenance by natural selection or a very recent origin of parthenogenesis in the nematode branches we examined.E. coli OP50 as a food source. To reduce contamination with other bacteria, we used minimal medium plates [C. elegans N2 ), Acrobeloides sp. 0/5061 ), Acrobeloides nanus ES501 (parthenogenetic [A. nanus), Diploscapter coronatus PDL0010 .All nematodes were cultivated under standard conditions , feedingm plates . Studieshroditic ), Acrobeogenetic ; called C. elegans MSP [® 488 coupled goat anti-mouse antibody at a 1:1.000 dilution (Invitrogen). Microscopy was carried out with a Zeiss Axioskop 2 fluorescence microscope. Pictures were taken with an AxioCam MRc camera (Zeiss) and arranged with the Keynote software (Apple).For visualisation of MAP kinase and MSP, we prepared and immunostained nematode gonads as described ,80. Briegans MSP . Seconda2O, freeze-cracked twice in liquid nitrogen/ice, and boiled for 10 min in SDS loading buffer. After electrophoresis on a 12% SDS gel, proteins were electroblotted onto PVDF membranes under semidry conditions and subjected to Western detection. Primary antibody was the monoclonal anti-MSP antibody directed against the C-terminus of MSP [α-tubulin antibody (Sigma # T9026). Secondary antibody was a HRP-coupled goat anti-mouse antibody at 1:2.500 . Antibodies were diluted in TBST/2% milk powder. Autoradiographic films (Fuji) were developed according to standard protocols, digitally scanned and processed with Keynote.For detection of MSP in nematode lysates, 40 mg animals (mixed stage population) of the respective species were washed in Hs of MSP  at 1:400E. coli XL1-Blue bacteria (Stratagene) and sequenced with the BigDye® Terminator V3.1 sequencing kit (Applied Biosystems). Sequence assembly and editing were carried out using the Phred/Phrap/Consed package [http://www.biology.utah.edu/jorgensen/wayned/ape/. The mentioned MAP kinase and MSP sequences were deposited at the EMBL Nucleotide Sequence Database .To clone MAP kinase sequences, RNA was extracted from various nematodes and cDNA was synthesised as described earlier . Initial package ,85 and tM. incognita and M. hapla [Whole genome sequences from M. hapla ,61 were M. hapla . After cM. hapla , BLASTP M. hapla  and the http://www.ncbi.nlm.nih.gov/protein: C. elegans MPK-1 and MPK-2 (Accession numbers P39745.2 and AAN60533.1), human and zebrafish Erk1 (P27361.4 and AAY57804.1), human Erk2 (NP_620407.1), human and zebrafish Erk5 (Q13164.2 and ABC94477.1), human, zebrafish and C. elegans p38 , human and zebrafish Jnk (NP_620707.1 and NP_571796.1), zebrafish Erk3 (ABC94479.1) and human Erk4 (NP_002738.2). A protein multiple sequence alignment was computed from the retrieved and our isolated MAP kinase sequences using Muscle [For alignment and phylogeny of MAP kinase proteins, following MAP kinase sequences were retrieved from NCBI g Muscle . The alig Muscle . Detailsg Muscle . Phylogeg Muscle  according Muscle . The iniPH performed most experiments and wrote the paper. MK and NN cloned the MAP kinase genes. NN and PH cloned the MSP genes. MK, ES, and PH designed the study and discussed the results. ES critically revised the manuscript. All authors read and approved the final manuscript.


Lactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains.in silico approach to refine the distinction of subfamilies with different enzymatic activities. Comparison of protein 3D structures of proline peptidases PepI/PepR/PepL and esterase A allowed identification of a conserved core structure, which was then used to improve phylogenetic analysis and functional annotation within this protein superfamily.We performed a genome-wide comparative genomics analysis of proteolytic system components, including cell-wall bound proteinase, peptide transporters and peptidases, in 22 sequenced LAB strains. The peptidase families PepP/PepQ/PepM, PepD and PepI/PepR/PepL are described as examples of our Lactococcus lactis strains was explored using pangenome comparative genome hybridization analysis. Variations were observed in the proteinase PrtP and its maturation protein PrtM, in one of the Opp transport systems and in several peptidases between strains from different Lactococcus subspecies or from different origin.The diversity of proteolytic system components in 39 The improved functional annotation of the proteolytic system components provides an excellent framework for future experimental validations of predicted enzymatic activities. The genome sequence data can be coupled to other "omics" data e.g. transcriptomics and metabolomics for prediction of proteolytic and flavor-forming potential of LAB strains. Such an integrated approach can be used to tune the strain selection process in food fermentations. Lactic acid bacteria (LAB) have been used for centuries as starter or adjunct cultures in dairy fermentations. The breakdown of milk proteins (proteolysis) by LAB plays an important role in generating peptides and amino acids for bacterial growth and in the formation of metabolites that contribute to flavor formation of fermented products. The proteolytic system of LAB comprises three major components: (i) cell-wall bound proteinase that initiates the degradation of extracellular casein (milk protein) into oligopeptides, (ii) peptide transporters that take up the peptides into the cell, and (iii) various intracellular peptidases that degrade the peptides into shorter peptides and amino acids. In particular, as caseins are rich in proline, LAB have numerous proline peptidases for degrading proline-rich peptides -3. AminoLactococcus lactis subsp. cremoris MG1363 [Lactobacillus helveticus strain [Lactobacillus rhamnosus GG [L. lactis strains [Several reviews have described the proteolytic system of LAB with respect to their biochemical and genetic aspects ,5-8. In s MG1363 , a Lactos strain  which is strains  providesLactococcus lactis strains by pangenome CGH analysis [In this study, we systematically explored the diversity of the cell-wall bound proteinase, the peptidases and the peptide transporters in twenty-two completely sequenced LAB strains. The distinctions between subgroups in large peptidase families such as the PepP/PepQ/PepM family, the PepD family and the PepI/PepR/PepL family are described in detail as examples. The PepI/PepR/PepL family was compared with the EstA family of esterases, the key enzyme for synthesizing various ester flavors ,15, sincanalysis .http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi. The genomes include: Lactobacillus acidophilus NCFM , Lactobacillus johnsonii NCC 533 , Lactobacillus gasseri ATCC 33323 , Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 , Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA365 , Lactobacillus plantarum WCFS1 , Lactobacillus brevis ATCC 367 , Lactobacillus sakei 23 K , Lactobacillus salivarius UCC118 , Oenococcus oeni PSU1 , Pediococcus pentosaceus ATCC 25745 , Leuconostoc mesenteroides ATCC 8293 , Lactobacillus casei ATCC 334 , Lactococcus lactis subsp. lactis IL1403 , Lactococcus lactis subsp. cremoris MG1363 , Lactococcus lactis subsp. cremoris SK11 , Streptococcus thermophilus CNRZ1066 , Streptococcus thermophilus LMG18311 , Streptococcus thermophilus LMD9 , Lactobacillus reuteri F275 , Lactobacillus helveticus DPC 4571  and Lactobacillus rhamnosus GG . Incomplete genome sequences of Lactococcus lactis subsp. lactis strains KF147 and KF282 [L. lactis strain diversity by pangenome CGH analysis [Complete genome sequences of LAB were obtained from the NCBI microbial genome database http://www.uniprot.org/[http://hmmer.janelia.org/. The homologous sequences of each proteinase, peptidase and peptide transporter were collected on basis of the BLAST and HMM search results and redundancies were removed. Orthologous groups (subfamilies) were identified by an in-house developed method [Protein sequences of experimentally verified proteolytic system members, i.e. cell-wall bound proteinase, various peptidases and peptide transporters, were derived from the non-redundant protein database Uniprot prot.org/. These sprot.org/ search aprot.org/ and utild method ,20. Multd method . Bootstrd method . The tred method  and orthd method  to improhttp://www.rcsb.org/pdb/[http://ekhidna.biocenter.helsinki.fi/dali_server/ using the protein structures of the BLAST hits as input.Peptidases PepI/PepR/PepL and esterase EstA belong to the same protein superfamily, but they possess different functionalities. In order to identify substrate specificity of each protein subfamily, a comparison of known protein 3D structures was carried out. As described above, protein sequences of experimentally characterized peptidases PepI, PepR, and PepL, together with EstA esterases were used to search against all the sequenced LAB genomes and other prokaryote genomes in the NCBI database by BLASTP . Moreove.org/pdb/. The proThermoplasma acidophilum (PDB ID: 1MTZ), proline iminopeptidases from Xanthomonas campestris pv. citri (PDB ID: 1AZW) and Serratia marcescens (PDB ID: 1WM1) as members of PepI/R/L subfamilies, and the esterase (PDB ID: 2UZ0) from Streptococcus pneumoniae which belongs to the EstA subfamily. These 3D structures were superimposed and visualized by the YASARA program . Conserved superimposable regions (core regions) of the catalytic domain were identified based on the 3D-structure alignment, and these consisted of 4 discontinuous sequence segments that are connected by loops of variable structure.The retrieved 3D structures of the proteins used as templates in this study are: the tricorn interacting factor F1 with proline iminopeptidase (PIP) activity from The amino acid sequences of the four core region segments were aligned with MUSCLE or ClustalW as described . The aliL. lactis strains was acquired from pangenome arrays [L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris SK11, and incomplete genome sequences of L. lactis strains KF147 and KF282, as described by Bayjanov et al. [Gene Expression Omnibus) database http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE12638.Comparative genome hybridization (CGH) data of 39 e arrays . The panL. lactis strains were used to query the database containing pangenome CGH data. We obtained a statistical score of the hybridization signal for each gene from the reference strains against 39 L. lactis strains. A cut-off value 5.5 was used to assign presence or absence of every gene from the proteolytic system in query strains, as described by Bayjanov et al. [The genes encoding predicted proteolytic system components of the three sequenced v et al. . In mostv et al. .An overview of the distribution of components of the proteolytic system identified in 22 completely sequenced LAB is given in Figure L. acidophilus group [L. acidophilus, L. johnsonii, L. gasseri, L. bulgaricus, and L. helveticus strains, encode a relatively higher number and variety of proteolytic system components. Some enzymes are only found in a few LAB strains, such as the cell-wall bound proteinase (PrtP). PrtP was only found on the chromosome of L. acidophilus, L. johnsonii, L. bulgaricus, L. casei, L. rhamnosus and S. thermophilus strain LMD9, as well as on the plasmid of L. lactis subsp. cremoris SK11 [L. johnsonii. Other essential peptidases  such as endopeptidase PepO and dipeptidase PepV are encoded by multiple paralogous genes.The LAB genomes in the us group , includiris SK11 . MembersL. acidophilus, L. brevis, L. casei, L. rhamnosus and L. lactis strains possess all three known LAB peptide transport systems, i.e. the di/tripeptide Dpp and DtpT systems and the oligopeptide Opp system [L. reuteri strain only has one functional peptide transport system, the DtpT system. Several peptide transporters or peptidases fall into larger protein superfamilies. Examples are (i) the oligopeptide-binding protein OppA and di/tripeptide-binding proteins DppA/DppP in the same peptide-binding protein family, (ii) aminopeptidase PepC together with endopeptidases PepE and PepG belonging to MEROPS peptidase family C1-B, (iii) proline peptidases PepI, PepR and PepL belonging to MEROPS family S33, and (iv) aminopeptidase PepM together with proline peptidases PepP and PepQ belonging to MEROPS family M24  will be discussed in detail in the following sections.PepP, PepQ and PepM belong to the MEROPS peptidase family M24 which requires metal ions for catalytic activity. PepM is a methionyl aminopeptidase cleaving N-terminal methionine from proteins. PepP is a member of the proline peptidases which cleave off any N-terminal amino acid linked to proline in an oligopeptide. PepQ is also a proline peptidase, however specific for Xaa-Pro dipeptides, where Xaa represents any amino acid Figure Our phylogenetic analysis shows that PepP, PepQ and PepM are separated into three distinct subgroups in accordance with the known different substrate specificities of each peptidase Figure . PepP anL. sakei and Pediococcus pentosaceus. The absence of the pepP genes in both genomes is very likely due to a gene loss event. The family tree also includes an experimentally verified pepP gene from L. lactis whose protein product has been purified and characterized [pepP genes are always flanked on the chromosome by a gene encoding an elongation factor for protein translation. The conserved gene context of pepP among LAB genomes is consistent with the putative important physiological role of PepP in protein maturation, as suggested by Matos et al. [In the PepP subgroup, one gene is found in each LAB genome except in cterized . Moreoves et al. .L. delbrueckii bulgaricus strains have two pepQ paralogs. One paralog is clustered with the other orthologs of LAB, whereas the second paralog is located in a separate cluster (LBU_116514595 and LDB_104774485). This might be the result of an ancient duplication  are added and highlighted in the tree, supporting the annotation of the subgroups.Genes from the PepQ cluster are distributed equally in all LAB genomes, generally as one copy per genome. However, the n Figure  or horiztic gene . Experimlgaricus  and L. hL. brevis has an extra paralogous gene, which clusters together with the L. plantarum pepM gene. Gene context analysis suggests that pepM genes in all Lactobacillus strains share the same neighbor genes, except the pepM gene from L. plantarum and both the paralogs from L. brevis. One of the L. brevis pepM genes (LBE_116334483) is located in the same operon as a transposase. Based on the protein family tree, we hypothesize that an extra pepM gene was acquired first in the ancestor of L. brevis and L. plantarum, after which one gene was lost from L. plantarum. The L. plantarum pepM gene (LPL_28377183) is flanked by a methionine metabolism related operon (cysK_cblB/cglB_cysE). Therefore, the pepM gene in L. plantarum may have a broader function, probably utilizing proteins and peptides as methionine pool, in addition to the classic PepM function for N-terminal maturation of proteins.In the aminopeptidase PepM subgroup, Leuconostoc mesenteroides (LME_116618966) is located as an intermediate between the PepP/PepQ and PepM subfamilies. It shares higher sequence homology with a putative pepP gene from Clostridium botulinum  [Xanthomonas campestris pv. citri (PDB ID: 1AZW) [Serratia marcescens (PDB ID: 1WM1) [2UZ0) from Streptococcus pneumoniae [The proline iminopeptidase PepI possesses aminopeptidase activity toward N-terminal proline peptides, preferably tri-peptides, while prolinase PepR has a broad specificity for dipeptides including Pro-Xaa dipeptides . The onlresidues . InteresD: 1MTZ) , XanthomD: 1AZW) , and SerD: 1WM1)  as membeeumoniae  as a memS. pneumoniae. The third cluster PepIb contains proline iminopeptidases from Proteobacteria and Actinobacteria, and PepI from Firmicutes (including the ones from LAB), as well as the known structure 1MTZ from Thermoplasma acidophilum. The last cluster PepR/L consists of putative PepL proteins from LAB and the subgroup of prolinase PepR. Experimentally verified proteins PepR from L. helveticus CNRZ32 [L. delbrueckii subsp. bulgaricus CNRZ 397 [L. delbrueckii subsp. lactis DSM7290 [L. lactis [L. casei BL23 [L. helveticus strain 53/7 has also been experimentally characterized [A MSA of the concatenated sequences of the four conserved structural regions of the PepI/PepR/PepL and EstA superfamily members from various microorganisms was constructed and manually curated [See Additional File s CNRZ32 ,39, PepICNRZ 397 ,41, PepL DSM7290  and EstA. lactis  and L. csei BL23  also supcterized .L. salivarius strains lack the genes encoding proline peptidases PepI, PepR or PepL. This agrees with the observation from gene deletion experiments in strains harboring those peptidase genes that the physiological role of PepI, PepR or PepL is not essential for cell growth [L. helveticus, the growth rate in milk was slower for a PepI-deletion mutant as compared to the wild type [L. helveticus and L. rhamnosus toward several proline dipeptides was significantly reduced in a PepR-deletion mutant [Lactobacillus strains.Sequenced lactococcal, streptococcal, leuconostoc and l growth ,45,46. Hn mutant ,46. ThosL. lactis strains was studied by comparative genome hybridization (CGH) analysis. PanGenome arrays were made based on ORFs found in four sequenced L. lactis strains, and subsequently used to determine the presence or absence of orthologs in 39 L. lactis strains [L. lactis strains. All other components described in Figure L. lactis strains. Those genes are excluded from the table, as well as all genes of strains P7304 and P7266 (see explanation in Table legend). Some plant-derived L. lactis strains such as KF24, NIZOB2244W, LMG9446 and KW10 have the largest set of proteolytic system genes.The distribution of proteolytic system components in various  strains . Table 1prtP, prtM and pcp genes are located on one large plasmid, while the pepO2, pepF2 and oppABCDF2 are co-localized on a different plasmid. The co-presence or co-absence of these genes in other L. lactis strains .Variations are found for proteinase PrtP and its maturation protein PrtM, for peptidases Pcp, PepO2, PepF2 and PepX2, and for genes from peptide transport systems Opp and Dpp Table . Most ofns Table , is largL. lactis subsp. lactis IL1403. It is mainly found in L. lactis subsp. lactis strains from dairy origin. This putative pepX2 gene was originally annotated as a hypothetical protein named YmgC. It contains both a C-terminal domain of X-prolyl dipeptidyl aminopeptidase and a Peptidase_S15 catalytic domain which are usually found in PepX, whereas the PepX N-terminal domain is missing in PepX2. No experimental evidence for the enzyme activity of PepX2 is known. The family tree of PepX shows that this putative pepX2 gene is not clustered in the same orthologous group as its paralogous gene from L. lactis subsp. lactis IL1403 [Additional File L. lactis subsp. lactis IL1403 and Pediococcus. Their best BLAST hits against the non-redundant protein database are from Listeria monocytogenes, suggesting a HGT event [See Additional File PepX2 is a PepX homolog of Lactobacillus, Lactococcus, Streptococcus, Pediococcus, Oenococcus, and Leuconostoc strains. The comparative genomics analysis was shown to distinguish various subgroups within a protein superfamily, allowing a highly improved annotation of genes and clarification caused by inconsistent annotation.In this study, we performed a systematic genome-wide analysis of all the proteins involved in proteolysis, including cell-wall bound proteinase, peptide transporters, and peptidases, from twenty-two fully sequenced LAB genomes, including L. bulgaricus and L. helveticus have a very extensive set of proteolytic enzymes, which is consistent with previous knowledge that L. bulgaricus serves as the proteolytic organism in yoghurt rather than S. thermophilus [L. helveticus is a proteolytic cheese adjunct culture that has been used to degrade bitter peptides in cheese [L. bulgaricus encodes the Dpp system with preference for uptake of hydrophobic di/tripeptides, complementing S. thermophilus which encodes the general di/tripeptide transporter DtpT in its genome, suggesting that more peptides can be utilized by both bacteria when grown together. LAB species of plant origin, such as L. plantarum, O. oeni, and Leuconostoc mesenteroides, encode less proteolytic enzymes in their genomes, which agrees with their ecological niche that is fiber-rich but contains less proteins.This information on the distribution of the proteolytic system genes can be used to predict the proteolytic potential of various LAB strains. For instance, mophilus . L. helvn cheese . InteresSeveral examples have been provided for the division of large superfamilies into subfamilies. Clear separation of major subgroups can be observed from the family trees. By including the experimentally characterized genes, different substrate specificities can be assigned to various subfamilies. The PepP/PepQ/PepM and PepI/PepR/PepL superfamilies include subfamilies with distinct substrate specificities. The general dipeptidase superfamily PepD consists of several distinct orthologous groups of which the substrate specificities are still unknown. In most cases, the prediction of orthologous groups and the evolutionary events leading to the variation of substrate specificities are straight-forward using the phylogenetic analysis. However, some orphan genes are present as intermediate groups between the subfamilies with unknown functions and some of them may originate from HGT events.Torpedo californica and a lipase of Geotrichum candidum [L. plantarum and L. casei (LPL_28379307 and LCA_116494294) which are not grouped into the PepR subfamily  neighbor-joining tree for PepF members in LABClick here for fileComparison of important residues of the conserved core regions and the cap region. The file contains a table describing the four structurally conserved regions and the cap regions. The identified residues within those regions, which are functionally important and/or conserved in PepI/R/L or EstA families, are highlightedClick here for filePepX superfamily tree. The file contains a bootstrapped (n = 1000) NJ tree for PepX family homologs of LABClick here for file


Orientia tsutsugamushi.Scrub typhus is an acute febrile illness caused by We encountered a patient with sensorineural hearing loss complicating scrub typhus, and three patients with scrub typhus who complained of otalgia, which was sudden onset, severe, paroxysmal, intermittent yet persistent pain lasting for several seconds, appeared within 1 week after the onset of fever and rash. The acute sensorineural hearing loss and otalgia were resolved after antibiotic administration.When patients in endemic areas present with fever and rash and have sensorineural hearing loss or otalgia without otoscopic abnormalities, clinicians should suspect scrub typhus and consider empirical antibiotic therapy. Orientia tsutsugamushi, an obligate intracellular bacterium; it is transmitted via the bites of Leptotrombidium chigger mites [O. tsutsugamushi spreads throughout the body via the blood and lymphatic vessels, so that patients infected with this microorganism manifest a variety of clinical symptoms and signs such as myalgia and diffuse lymphadenopathy [Rickettsia rickettsii (R. rickettsii), R. typhi or R. coronii [Scrub typhus is an acute febrile illness caused by er mites . O. tsutenopathy . Complicenopathy -5. A sen coronii -8. Heari coronii ,4. HowevWe report four confirmed cases of scrub typhus with otological complaints. One patient presented with sensory-neural hearing loss and other three patients presented with severe otalgia.3; platelets, 120,000/mm3). Biochemical tests were normal except for increased serum levels of aminotransferases , and her urinalysis was normal. Serum C-reactive protein was 5.5 mg/L, erythrocyte sedimentation rate was 51 mm, blood urea nitrogen was 20.7 mg/dL and serum creatinine was 0.93 mg/dL. Peripheral blood smears for malaria and parasites, and blood and urine cultures, were all negative. Chest X-rays were normal. Indirect immunofluorescence assays revealed an IgM titer of 1:160 against O. tsutsugamushi on admission, and an IgM titer of 1:320 against O. tsutsugamushi at 2-week follow-up. The initial IgG titer of 1:1,024 rose to 1:4,096 two weeks later . Hematologic tests revealed normal leukocyte count and hemoglobin, and low platelet count , R. typhi or R. coronii [Our patients presented with scrub typhus and associated sensory neural hearing loss and severe otalgia. A sensory neural type of hearing loss has been reported in patients infected with  coronii -8. Altho coronii ,11,12. I coronii . In this coronii . SimilarWhen patients in endemic areas present with fever and rash and have sensorineural hearing loss or severe otalgia without otoscopic abnormalities, clinicians should suspect scrub typhus and consider empirical antibiotic therapy.Written informed consent was obtained from the patient or their relative for publication of this study. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.JIK took care of the patients and drew up the first draft of the report, JHL, consultant otorhinolaryngologist, made a substantial contribution to draft the manuscript, and revised the draft all over the course of submission, DMK conceived of the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/173/prepub


Acute kidney injury in the setting of adult minimal change disease is associated with proteinuria, hypertension and hyperlipidemia but anemia is usually absent. Renal biopsies exhibit foot process effacement as well as tubular interstitial inflammation, acute tubular necrosis or intratubular obstruction. We recently managed a patient with unique clinical and pathological features of minimal change disease, who presented with severe anemia and acute kidney injury, an association not previously reported in the literature.A 60-year-old Indian-American woman with a history of hypertension and diabetes mellitus for 10 years presented with progressive oliguria over 2 days. Laboratory data revealed severe hyperkalemia, azotemia, heavy proteinuria and progressively worsening anemia. Urine eosinophils were not seen. Emergent hemodialysis, erythropoietin and blood transfusion were initiated. Serologic tests for hepatitis B, hepatitis C, anti-nuclear antibodies, anti-glomerular basement membrane antibodies and anti-neutrophil cytoplasmic antibodies were negative. Complement levels  were normal. Renal biopsy unexpectedly displayed 100% foot process effacement. A 24-hour urine collection detected 6.38 g of protein. Proteinuria and anemia resolved during six weeks of steroid therapy. Renal function recovered completely. No signs of relapse were observed at 8-month follow-up.Adult minimal change disease should be considered when a patient presents with proteinuria and severe acute kidney injury even when accompanied by severe anemia. This report adds to a growing body of literature suggesting that in addition to steroid therapy, prompt initiation of erythropoietin therapy may facilitate full recovery of renal function in acute kidney injury. Acute kidney injury (ARF) in the setting of adult minimal change disease (MCD) has been well documented in the literature -6, but iA 60-year-old Indian-American woman with a history of hypertension and diabetes mellitus for 10 years presented to the emergency room with progressively worsening shortness of breath, abdominal pain, nausea, vomiting and a decreased urine output for the past 2 days. One month earlier, the patient had undergone an elective total knee replacement for osteoarthritis, and 1 week earlier the patient had been treated for urinary tract infection (UTI) with Bactrim™ (trimethoprim/sulfamethoxazole).Hypertension and diabetes mellitus had been well controlled. The patient took diltiazem, lisinopril, metformin, glipizide and acetaminophen for knee pain. Her baseline renal function had been normal, with a serum blood urea nitrogen (BUN) level of 8 mg/dl and creatinine of 0.8 mg/dl 1 month earlier. She did not use non-steroidal anti-inflammatory drugs, whether prescribed or over the counter. The patient did not use tobacco or alcohol, nor did she have any risk factors for immunodeficiency virus (HIV) infection.At initial presentation, her blood pressure was 148/76 mmHg, pulse 61 per minute and respiration 20 per minute. The patient was awake and oriented. There was mild bilateral ankle edema. The rest of the physical examination was unremarkable.3 . Hematocrit was 31.9 mg/dl. The patient's baseline hematocrit was 39.0 mg/dl. Glycosylated hemoglobin was 6.8%. Urine analysis showed 4+ proteinuria and microscopic hematuria but no crystals or casts. Lipid profile was normal: cholesterol 177 mg/dl and low-density lipoprotein (LDL) 95 mg/dl.The serum sodium was 128 mmol/l, potassium 8.3 mmol/l, bicarbonate 19 mmol/l, BUN 52 mg/dl, and creatinine 7.0 mg/dl. The plasma albumin concentration was 1.5 g/dl. Liver function tests were normal. White blood cell count was 7200/mmThe patient was admitted to the intensive care unit. Emergent hemodialysis was initiated Figure . ErythroThe patient's hematocrit level continued to drop during the first week of hospitalization, reaching a nadir of 23.6 mg/dl and requiring packed red blood cell transfusion on two occasions Figure . There wA renal biopsy was performed. Examination by light microscopy showed 14 glomeruli, all of which appeared normal. Cytoplasmic swelling was noted in the proximal tubules Figure . ElectroPrednisone 60 mg/day was initiated Figure . Within Oliguric acute kidney injury occurs infrequently in adult MCD ,2. The uThaysen et al.  describeTo date, anemia has not been reported in ARF associated with adult MCD, although this may be due to the paucity of cases of severe ARF from MCD. Our patient presented with severe ARF and moderately reduced hemoglobin, which continued to drop dramatically during the first several days of hospitalization. Although our patient had microscopic hematuria during the oliguric phase, a finding seen in about one-third of patients with MCD , it is nIn vitro and animal studies have suggested that EPO might promote renal recovery and decrease mortality in ARF [One intriguing recent observation is the possible tissue-protective effect of EPO. y in ARF ,9. The Ey in ARF ,9. Therey in ARF , decreasy in ARF . This may in ARF . In thisy in ARF . Renal fIn combination with the experimental data, our findings then support the notion that EPO, administered early in the course of ARF, may have salutary effects beyond its effects on hematopoiesis, a hypothesis that should be further explored.Adult MCD may present with severe ARF, significant anemia and a normal lipid profile. Severe anemia associated with EPO deficiency may occur abruptly in this setting. Erythropoietin may attenuate renal injury and facilitate renal recovery in addition to its function on hematopoiesis if treatment is initiated early. Regardless of the age at onset or severity of ARF, renal and hemopoietic function can recover completelyARF: acute kidney injury; MCD: minimal change disease; UTI: urinary tract infection; BUN: blood urea nitrogen; HIV: immunodeficiency virus; LDL: low-density lipoprotein; EPO: erythropoietin; CT: computer tomography; GBM: glomerular basement membraneThe authors declare that they have no competing interests.YQ reviewed the literature and drafted the paper. SM interpreted patient data, identified the unusual nature of the case, advised on content of the paper and critically reviewed the content of the paper. NG provided clinical information and critically reviewed the content of the paper. EF interpreted patient data and the literature and critically reviewed the content of the paper. All authors read and approved the final manuscript.


A genomic analysis of the annual fish Nothobranchius furzeri, a vertebrate with the shortest known life span in captivity and which may provide a new model organism for aging research. Nothobranchius furzeri is the vertebrate with the shortest known life span in captivity. Fish of the GRZ strain live only three to four months under optimal laboratory conditions, show explosive growth, early sexual maturation and age-dependent physiological and behavioral decline, and express aging related biomarkers. Treatment with resveratrol and low temperature significantly extends the maximum life span. These features make N. furzeri a promising new vertebrate model for age research.The annual fish N. furzeri as a new model organism, we provide a first insight into its genome and a comparison to medaka, stickleback, tetraodon and zebrafish. The N. furzeri genome contains 19 chromosomes (2n = 38). Its genome of between 1.6 and 1.9 Gb is the largest among the analyzed fish species and has, at 45%, the highest repeat content. Remarkably, tandem repeats comprise 21%, which is 4-12 times more than in the other four fish species. In addition, G+C-rich tandem repeats preferentially localize to centromeric regions. Phylogenetic analysis based on coding sequences identifies medaka as the closest relative. Genotyping of an initial set of 27 markers and multi-locus fingerprinting of one microsatellite provides the first molecular evidence that the GRZ strain is highly inbred.To contribute to establishing N. furzeri.Our work presents a first basis for systematic genomic and genetic analyses aimed at understanding the mechanisms of life span determination in  Studies in invertebrate model organisms such as yeast and worm have identified a number of genes and pathways that regulate life span and aging , and somNothobranchius furzeri, might represent an alternative vertebrate model for the study of aging n. Correspondingly, in FISH experiments using the 5'-TTAGGG-3' motif as probe, specifically the terminal ends of chromosomes were labeled . Thus, characteristic vertebrate telomeric structures are present in N. furzeri and will be described elsewhere . The version described in this paper is the first version ABLO01000000. The 111.6 Mb of N. furzeri GRZ genomic sequence generated by Roche/454 technology have been deposited at GenBank under project id (PID) 29535. The 5.4 Mb of N. furzeri MZM-0403 genomic DNA sequence generated by the Sanger technique have been deposited under the project accession [GenBank:ACCZO00000000]. The version described in this paper is the first version [GenBank:ACCZ01000000]. The 5.4 Mb of N. kunthae genomic sequence generated by the Sanger technique have been deposited under the project accession [GenBank:ACDA00000000]. The version described in this paper is the first version [GenBank:ACDA01000000]. N. furzeri GRZ major rRNA gene cluster [GenBank:EU780557]; Cdkn2b [GenBank: EU271680]; Cdkn2d [GenBank: EU400615]; Msra [GenBank EU400617]; Sirt1 [GenBank:EU271679]; Tp53 [GenBank:EU271681].The 5.4 Mb of in situ hybridization; FITC: fluorescein isothiocyanate; GRZ: Gona Re Zhou; NCBI: National Center for Biotechnology Information; PI: propidium iodide.DAPI: 4',6-diamidino-2-phenylindole; FISH: fluorescence N. furzeri GRZ. AC and MaS provided biological specimens. KR, CL, and MP analyzed the sequences. JK constructed the genomic library of N. kunthae and performed genotyping. NH cloned aging related genes. IN, SS, MiS and MaS performed cytogenetic studies and genome size estimation by flow cytometry. GG helped with the genome size estimation based on sequences. UG and KS performed phylogenetic analyses. KR, UG, KS, MaS, AC, CE and MP contributed to writing the manuscript. All authors read and approved the final manuscript.CE and MP initiated the project. KR coordinated the project, and generated and assembled most sequences. ST and MBS helped with sequencing N. furzeri based on sequence data. Additional data file N. furzeri GRZ gene fragments identified by BLASTX searches in Swiss-Prot/TrEMBL. Additional data file N. furzeri genome size. Additional data file N. furzeri strain MZM-0403 and the closely related species N. kunthae. Additional data file N. furzeri GRZ. Additional data file N. furzeri GRZ gene fragments lacking homologs in medaka, stickleback, tetraodon, and zebrafish. Additional data file N. furzeri GRZ major rRNA cluster in tetraodon and human. Additional data file N. furzeri strains GRZ and MZM-0403 and the closely related species N. kunthae. Additional data file N. furzeri strains GRZ and MZM-0403 and N. kunthae. Additional data file N. furzeri GRZ. Additional data file The following additional data are available with the online version of this paper. Additional data file N. furzeri genome size are given.Coding sequences identified in random genomic sequence samples of medaka, stickleback, tetraodon and zebrafish, their genome sizes, and inferred Click here for fileN. furzeri GRZ gene fragments identified by BLASTX searches in Swiss-Prot/TrEMBL.Click here for fileN. furzeri genome size.Flow cytometry measurements to estimate the Click here for fileN. furzeri strain MZM-0403 and the closely related species N. kunthae.G+C histograms of Click here for fileN. furzeri GRZ.Microsatellite frequencies of Click here for fileN. furzeri GRZ gene fragments lacking homologs in medaka, stickleback, tetraodon, and zebrafish.The subgroup of Click here for fileChromosomal locations and accession numbers of sequences used for analyses of aging related genes.Click here for fileN. furzeri GRZ major rRNA cluster in tetraodon and human.Conservation of the Click here for fileN. furzeri strains GRZ and MZM-0403 and the closely related species N. kunthae.Genotypes of microsatellites analyzed in Click here for fileN. furzeri strains GRZ and MZM-0403 and N. kunthae.Analysis of 19 gene-associated markers in Click here for fileN. furzeri GRZ.Primers used to clone and sequence five aging related genes in Click here for fileNucleotide and amino acid sequences used for phylogenetic analyses.Click here for file


Puccinia striiformis f. sp. tritici is an obligate biotrophic pathogen that causes leaf stripe rust on wheat. Although it is critical to understand molecular mechanisms of pathogenesis in the wheat stripe rust fungus for developing novel disease management strategies, little is known about its genome and gene functions due to difficulties in molecular studies with this important pathogen. To identify genes expressed during early infection stages, in this study we constructed a cDNA library with RNA isolated from urediniospores of P. striiformis f. sp. tritici germinated for 10 h.PMK1, GAS1, and GAS2 of the rice blast fungus. We selected six ESTs  for assaying their expression patterns during urediniospore germination and wheat infection by quantitative real-time PCR. All of them had the highest transcript level in germinated urediniospores and a much less transcript level in un-germinated urediniospores and infected wheat tissues (1–7 dpi). The transcript level of Ps159 increased at later infection stages (6–7 dpi). Our data indicated that these genes were highly expressed in germinated urediniospores and may play important roles in fungal-plant interactions during early infection stages in the wheat stripe rust fungus.A total of 4798 ESTs were sequenced from the germinated urediniospore library and assembled into 315 contigs and 803 singletons. About 23.9% and 13.3% of the resulting 1118 unisequences were homologous to functionally characterized proteins and hypothetical proteins, respectively. The rest 62.8% unisequences had no significant homologs in GenBank. Several of these ESTs shared significant homology with known fungal pathogenicity or virulence factors, such as HESP767 of the flax rust and P. striiformis f. sp. tritici were identified by EST analysis. Six of them were confirmed by quantitative real-time PCR assays to be highly expressed in germinated urediniospores.Genes expressed in germinated urediniospores of  Puccinia striiformis Westend f. sp. tritici Eriks is the causal agent of wheat stripe rust. It is an obligate biotrophic basidiomycete with an incomplete life cycle. Because of the high variability in the pathogen population, race-specific resistance in many newly developed cultivars often fails within a few years of cultivation and results in severe yield losses. In the past century, most of the P. striiformis f. sp. tritici studies had been focused on the identification of physiological races, virulence variation, and ultrastructural and histological examinations [Wheat stripe rust is one of the most important diseases of wheat throughout the world. It is a major constraint in wheat production and is a serious threat to food security worldwide ,2. Pucciinations ,3,4.P. striiformis f. sp. tritici has not been identified. Urediniospore is the most common spore form that has been observed in the wheat stripe rust fungus, which is strictly dependent on living host cells for growth and reproduction. To date, no stable transformation system has been established for P. striiformis f. sp. tritici and other Puccinia species. These biological characteristics make molecular and genetic studies of this important fungus relatively more difficult. Although much progresses have been achieved in researches on its genetic diversity, population structure, and evolution [Unlike the wheat stem rust fungus, the sexual stage of volution -8, thereVarious genomic approaches, such as expressed sequence tag (EST) , serial Uromyces fabae, Hahn and his colleagues sequenced ESTs from purified haustoria. A major shift in gene expression was observed between urediniospore germination and the biotrophic growth stage [Puccinia triticina, including resting and germinated urediniospores, appressorium and haustorium formation stages during infection of a susceptible wheat cultivar, and infected leaves of a resistant wheat cultivar. While 38% unigenes matched sequences in various databases and collections, the annotation rates were low for ESTs from germinated urediniospores (4%) and appressoria (2%). Gene sets obtained from these different libraries appeared to be remarkably different, suggesting drastic reprogramming of the transcriptome during these major differentiation processes [P. striiformis f. sp. tritici. A total of 4798 ESTs were sequenced to generate 1118 unisequences (uniseqs). The majority of these ESTs (over 60%) had no significant homologs in GenBank, indicating that many of them may represent genes unique to P. striiformis f. sp. tritici. Several of these ESTs share significant homology with known fungal pathogenicity or virulence factors, such as HESP767 of the flax rust and PMK1 of the rice blast fungus. The high transcript level of six selected genes in germinated urediniospores was confirmed by quantitative real-time PCR (qRT-PCR) assays. Some of these genes highly expressed in germinated urediniospores may be important for early infection processes in P. striiformis f. sp. tritici.EST analyses have been conducted in a few rust fungi. To examine gene expression during infection of broad bean by th stage ,16. A torocesses . In thisP. striiformis f. sp tritici (race CY32) in sterile distilled water in plastic plates. After incubating for 10 h at 9°C, about 60% of urediniospores attached to the plastic surface and produced long, un-branched germ tubes . On average, about 95% of the cDNA clones had inserts longer than 0.2 kb. The insert size varied from 200–3500 bp, with an average of 750 bp. A total of 5500 random cDNA clones were sequenced from the 5'-end to obtain 4810 quality reads or ESTs.To identify genes related to early events of urediniospore germination and differentiation, we incubated freshly harvested urediniospores of ES321929 – ES323046. Most of the uniseqs had the insert size of 200–900 bp. Only 36 uniseqs were longer than 1000 bp. The G+C content of these ESTs ranged from 40.74% to 53.73%, with an average of 45.08%, which was similar to the G+C content of ESTs in P. graminis [Before clustering analysis, ESTs containing no insert sequence or insert shorter than 100 bp were removed. The remaining 4798 ESTs were aligned and assembled into 315 contigs (containing two or more ESTs) and 803 singletons. A total of 1118 unisequences (uniseqs) were submitted to dbEST at NCBI [graminis .Saccharomyces cerevisiae gene encoding a hypothetical protein. Five of these most abundant contigs [see Additional file Ixodes scapularis. Contig Ps28 consisting of 128 ESTs was homologous to a differentiation-related protein Inf24 from Uromyces appendiculatus. Inf24 was identified as infection structure protein that was highly expressed during urediniospore germination [Kluyveromyces lactis.The number of individual ESTs belonging to each contig ranged from 2 to 608 [see Additional file mination . For conP. striiformis f. sp. tritici ESTs is similar to that has been observed in other fungal EST projects [All uniseqs were subjected to similarity searches against sequences in the non-redundant protein (nr) database at GenBank using the BLASTX algorithm. InterProScan was used to analyze uniseqs with no BLASTX matches for known protein domains. The vast majority (703) of the uniseqs had no significant homolog in GenBank by BLASTX search. Among 415 uniseqs displayed similarity  to entries in the nr database, about 56% of them were similar to genes coding for proteins with unknown functions (not classified in Gene Ontology). The frequency of orphan sequences in projects .Among the 415 uniseqs that exhibited similarity to entries in the non-redundant protein database at GenBank, about 40.72% shared homology to proteins from filamentous fungi while the rest were homologous to proteins from a wide variety of organisms, including yeast, bacteria, nematodes, plants, insects, and animals [see Additional file P. graminis [P. striiformis f. sp. tritici.A total of 267 uniseqs were identified to be homologous to proteins with known cellular functions. Figure graminis . Many ofMagnaporthe grisea, Ustilago maydis, and several other fungi [M. grisea and Gpa1 of Cryptococcus neoformans [M. grisea, which is important for regulating plant infection processes in a number of phytopathogenic fungi [To date, many genes important for fungal pathogenesis have been identified in plant and human pathogens -24. Homoer fungi -28. Ps25oformans ,30. The ic fungi -33.U. appendiculatus and appeared to be members of a multigene family. Inf24 is highly expressed during urediniospore germination. Microinjection of antisense oligonucleotides of Inf24 inhibits appressorium development in U. appendiculatus [P. striiformis f. sp. tritici.Ps8 and Ps28 may be related to the infection structure differentiation  transporter genes. In M. grisea and other plant pathogens, several ABC transporter genes have been implicated in fungal-plant interactions, possibly by controlling the efflux of phytotoxic fungal metabolites or plant defensive compounds [Contigs Ps6 and Ps159 encoded proteins homologous to gEgh16 of graminis , which ietration . Both GAompounds .Candida albicans, superoxide dismutase is required for tolerance to oxidative stress and full virulence [C. albicans [Common in several Fungal Extracellular Membrane proteins) domain that is unique to filamentous ascomycetes [M. grisea, the CFEM domain-containing protein Pth11 is known to play an important role in pathogenesis as a receptor [in vitro but is involved in host surface recognition. The protein encoded by contig Ps85 may play a similar role in P. striiformis f. sp. tritici, and is involved in appressorium differentiation.We also identified an EST, Ps238 , Ps85 (a CFEM domain-containing protein), Ps159 (gEgh16 homolog), Ps259 , Ps261 (Pmk1 homolog), and Ps87 (HESP767 homolog), were selected for assaying their transcript levels during different developmental and infection stages by quantitative real-time PCR (qRT-PCR). All these six genes were expressed in urediniospores, germinated urediniospores, and infected wheat tissues harvested at 1 through 7 days post inoculation (dpi). However, their transcription levels were much higher in germinated urediniospores than in un-germinated urediniospores or infected wheat tissues Fig. ; Fig. 4.P. striiformis f. sp. tritici.Although the transcript level of these genes in infected wheat tissues was relatively low, they had different expression patterns during plant infection. In comparison with the others, Ps159 had a relatively high transcript level in infected wheat plants. Its expression was up-regulated from 2 to 6 dpi and then slightly decreased at 7 dpi. The transcript level of Ps85 and Ps28 increased at early infection stages but decreased after 5 dpi. The transcript level of Ps259 and Ps261 in wheat tissues was lower than that of the other ESTs examined. Those two genes may be specifically and highly expressed during urediniospore germination. These data suggest that a considerable reprogramming of gene expression occurs during urediniospore germination, early stages of plant infection, and the biotrophic growth in During incompatible interactions, Ps87 also displayed an expression pattern that decreased at early stages but slightly increased after 3 dpi Fig. . The higM. grisea [Phakopsora pachyrhizi [F. oxysporum [P. triticina [P. graminis [P. striiformis f. sp. tritici [For plant pathogens with little or no history of genetic research, single-pass sequencing of random cDNA clones as in EST projects represents a relatively inexpensive and rapid procedure for finding novel genes and information about their expression. To date, extensive EST databases have been established for various plant pathogenic fungi, including . grisea , Phakopschyrhizi , and F. xysporum . For wheriticina  and P. ggraminis . Recentl tritici . Among 1 tritici . DiffereP. striiformis f. sp. tritici with host plants. As an obligate biotroph, germ tubes of the wheat stripe rust fungus fail to progress beyond this stage of development in vitro [P. striiformis f. sp. tritici library. Among the 4798 ESTs generated in this study, 3995 of them could be assembled into contigs. A few contigs consisting of over 100 EST clones may represent genes that are highly expressed during urediniospore germination. When the uniseqs were queried against the nr protein database, about 76.2% of them had no significant homology with proteins of known functions, which may be related to the difficulty of functional characterization of genes in rust fungi. Among these contigs encoding proteins of known functions, 18 of them are ribosomal proteins. ESTs corresponding to genes encoding ribosomal proteins also are abundant in other fungal ESTs, such as those of B. graminis [M. grisea [Urediniospore germination represents an early stage of the interaction of in vitro ,48. In tgraminis  and M. g. grisea .U. appendiculatus, which is specifically expressed during urediniospore germination [P. graminis f. sp. tritici. Ps55 and Ps1010 were homologous to a deacetylase and a cell wall organization and biogenesis-related protein from C. neoformans, respectively [M. grisea and other plant pathogens [The 1118 uniseqs identified in this study contained a wide range of genes involved in different cellular functions. The most abundant genes during urediniospore germination were those involved in metabolic activities as well as those responsible for protein biosynthesis, which accounted for 10.29% and 5.28% of the uniseqs, respectively. A few genes are involved in RNA synthesis, cell signal and communication, cell structure and growth, and cell/organism defense, indicating that active metabolism and protein synthesis is important for urediniospore germination and germ tube growth. Several ESTs, including Ps8, Ps28, and Ps228, shared similarity to differentiation-related protein Inf24 from mination . Ps88 haectively . These tathogens ,54. In tU. fabae [Expression patterns of six selected uniseqs, Ps28, Ps85, Ps87, Ps159, Ps261, and Ps295, were examined by qRT-PCR analysis. The transcript level of these genes in germinated urediniospores was several-folds higher than in un-germinated urediniospores or infected wheat tissues, indicating that gene expression in the wheat stripe rust fungus changes dramatically during urediniospore germination and parasitic growth in wheat plants Fig. ; Fig. 4.U. fabae .P. triticina ESTs, including the Pmk1 homolog PtMAPK1 [PtMAPK1 gene has increased transcript levels during urediniospore germination and plant infection. When expressed in U. maydis, it can complement the defects of the kpp2 mutant in mating and plant infection [A number of genes that are important for plant infection have been identified in various phytopathogenic fungi . Several PtMAPK1 . The PtMnfection .U. appendiculatus. In U. appendiculatus, microinjection of Inf24 antisense fragment into germinated urediniospores blocked its transcription and appressorium formation [Other putative fungal virulence or pathogenicity factors identified in this EST analysis included contigs Ps8, Ps28, Ps6, Ps159, and Ps87. Contigs Ps8 and Ps28 shared similarity to differentiation-related protein Inf24 from ormation . InjectiGAS1 and GAS2 of M. grisea [GAS1 and GAS2 are specifically expressed during appressorium formation and important for appressorial penetration in M. grisea. They are homologous to gEgh16 of B. graminis and members of a small protein family unique to filamentous fungi. In several fungal pathogens, gEgh16 homologs are expressed at early plant infection stages [P. striiformis f. sp. tritici  was used for cDNA synthesis and library construction. The MaxPlaxTM Lambda Packaging Extract  was used for in vitro packaging and transfection of Escherichia coli strain XL1-Blue. The resulting directional library consisting of 6.05 × 105 primary clones was amplified and stored in 15% glycerol at -80°C. Randomly selected cDNA clones were sequenced with primer seq1 (5' CGACTCTAGACTCGAGCAAG 3') from the 5'-end with an ABI 3130-XL DNA sequencer.Total RNA was isolated from 100 mg of germinated urediniospores with the RNeasy Plant Mini RNA purification kit  following the instruction provided by the manufacturer. The SMARTSequence reads longer than 100 base pairs were processed by cross_match  that allFor functional classification, the resulting unisequences were searched against the NCBI non-redundant protein database using the BLASTX program ,62. The st-strand cDNA products synthesized with RNA from urediniospores, germinated urediniospores, and infected wheat leaves were used as the templates for qRT-PCR assays.Standard protocols  were use2O, 1 μl 10 μM forward primer, 1 μl 10 μM reverse primer, 1 μl TaqMan probe, and 2 μl the cDNA template. The Smart CyclerSystem  was used for PCR reactions that consisted of 10 s at 95°C (1 cycle) and 40 cycles of 95°C for 5 s and 60°C for 20 s. The transcript level of selected ESTs was calculated by the 2-ΔΔCT method [P. striiformis f. sp. tritici as the endogenous reference for normalization. Relative quantification of these ESTs was computed with their transcript levels in different stages in comparison to that in urediniospores.Four sets of primers and fluorescently labeled TaqMan probes [see Additional file T method  with theYZ constructed the cDNA library, participated in EST sequencing and sequence analysis, conducted qRT-PCR assays, and prepared the manuscript; ZQ, LX and XW contributed to EST sequencing and Blast searches. WZ participated in various experiments; BL and XX assisted in qRT-PCR assays; LH, JZ and QH coordinated the project; ZK conceived of this study, contributed to experimental designs, provided supervisions, and revised the manuscript; All authors read and approved the final manuscript.P. striiformis f. sp tritici ESTs derived from the germinated urediniospore cDNA library. The number of contigs consisting of 1, 2, 3–5, 6–10, 11–35, and more than 36 ESTs was presented by the columns.The redundancy of Click here for fileP. striiformis f. sp. tritici germinated urediniospore library. These data provided represent the nine most abundant expressed ESTs.The most abundant ESTs identified in the Click here for fileUniseqs with significant homology to genes from different organisms. These data provided display the number of uniseqs have homology to genes from diversity organisms.Click here for fileUniseqs with similarity  to proteins in public databases were grouped into functional categories according to Gene Ontology. These data provided represent the original EST number and best hit.Click here for fileSequences of primers and probes used for qRT-PCR. The primer pairs, probes and reference sequences used for qRT-PCR were listed.Click here for file


We compared the characteristics of a cultured human "Helicobacter heilmannii" isolate with those of other helicobacters found in animals. Phenotypic, protein profile, 16S rDNA sequence, and DNA-DNA hybridization analyses identified the human strain as H. bizzozeronii, a species frequently found in dogs. Thus, H. bizzozeronii may have zoonotic potential.