Mycobacterium intracellulare isolates showed that 143 (86%) strains could be assigned to Mycobacterium chimaera sp. nov. Of 97 patients from whom M. chimaera sp. nov. was isolated, only 3.3% exhibited mycobacterial lung disease, whereas all M. intracellulare isolates caused severe pulmonary infections.Retrospective molecular genetic analysis of 166  Mycobacterium avium complex (MAC) play an important role among infections caused by nontuberculous mycobacteria (NTM). MAC consists of the 2 well-established species, M. avium (which has 4 subspecies) and M. intracellulare, as well as several other closely related mycobacteria  by 20 nt mismatches  region, this species genetically corresponds to sequevar MAC-A and differs from M. chimaera sp. nov. are sparse, we performed a retrospective study to determine the frequency of its occurrence within the group of MAC-positive clinical specimens and its possible role in causing human disease in comparison to M. intracellulare. We reanalyzed mycobacterial isolates from 97 in-house patients of the Charité University Hospital that have been processed in our laboratory from 2002 through 2006. An additional 69 isolates were provided by the National Reference Center (NRC) for Mycobacteria in Borstel, Germany. All strains had previously been classified as M. intracellulare by 16S rDNA–based methods. In addition to the partial 16S rRNA gene, we sequenced the 16S–23S ITS region to allow for unambiguous identification. Amplification of the partial 16S rRNA gene was performed according to a standard procedure  for the identification of the ITS PCR product to distinguish M. intracellulare, sequevar Min-A (DSMZ 43223), and M. chimaera sp. nov., sequevar MAC-A (DSMZ 44623), showed reproducible peak profiles. M. avium spp. avium, sequevar Mav-A, DSMZ 44156  and M. intracellulare, sequevar Min-C  served as negative run controls providing a different peak location. All clinical M. intracellulare, sequevar Min-A strains, and M. chimaera sp. nov. strains (sequevar MAC-A) could be allocated unequivocally to the 2 reference strains by their congruent peak patterns  suggests relatively low pathogenicity. As most other studies assessing the pathogenic potential of clinical NTM isolates referred either to members of the whole MAC group (inclusively M. avium) or to the complete NTM spectrum, their results cannot be compared (M. chimaera sp. nov. was highly virulent), possibly because of the low number of cases analyzed (12 patients) (M. intracellulare, sequevar Min-A, isolates were unequivocally associated with severe mycobacterial lung disease. Despite the low case numbers, these findings suggest that this species is more virulent and justify further epidemiologic investigations to verify this observation.DHPLC was a rapid and reliable method for distinguishing ,In agreement with other authors, our observations indicate that a precise differentiation of MAC isolates may provide clinically relevant data (If one assumes substantial differences in pathogenicity, the allocation of MAC isolates to defined species may facilitate diagnosis of mycobacterial lung disease. However, implementation of a staged identification procedure in routine microbiologic laboratories requires the availability of commercial, easy-to-use test kits. Because diagnosis of NTM infections remains a challenge and often results in indecisive situations that prolong the administration of adequate therapy, the rapid identification of MAC-related species highly predictive for mycobacterial disease would be very useful.Comparison of characteristics of Mycobacterium chimaera sp. nov. and M. intracellulare, sequevar Min-A-positivepatients


To the Editor: Tuberculosis is a chronic wasting disease in domestic birds  and free-ranging birds worldwide (Mycobacterium avium subsp. avium (mainly domestic birds) or by M. genavense . Nontuberculous  mycobacteria  occasionally have been isolated from necropsied pet birds  ≈6 months of age that was brought to a clinic because of inappetence over a 3-day period and polydipsia and yellow coloration of urine. Clinical examination showed slight emaciation, heavy biliverdinuria, ascites, and melena. By coprologic examination, 3 eggs of Ascaridia sp. worms were found in 1 field of view using 40× magnification. A Gram stain of fecal material showed sporadic gram-positive rods. On the basis of these signs, chlamydiosis was suspected. Differential diagnosis suggested liver cirrhosis, neoplasia, and Pacheco disease. Enrofloxacin  was administered subcutaneously (0.15 mL injected subcutaneously) and albendazole  were administered orally (0.2 mL injected subcutaneously). The bird died 1 day later.We report a case of Ascaridia sp.) were observed in the intestinal lumen.Necropsy showed ascites (clear yellowish fluid), hepatomegaly , mild splenomegaly, and hemorrhagic enteritis with thickening of the intestinal wall; the finding of hemorrhagic enteritis was unclear because the intestinal mucosa was hyperemic and covered with a thick layer of viscous mucus that contained blood. Twenty worms  were free of histopathologic lesions. Hypertrophic cirrhosis (chronic active hepatitis) was diagnosed. Neither granulomatous nor other lesions were observed.M. avium spp., and a subsequent PCR assay for M. avium differentiation indicated M. avium subsp. hominissuis (IS1245+ and IS901–); both PCR assays were performed as described  analysis according to Van Soolingen et al.  were microscopically detected in the liver and intestine. Cultivation according to Matlova et al. (position .M. avium subsp. hominissuis is not considered an avian pathogen and rarely has been isolated from tuberculous lesions (M. avium subsp. hominissuis from multiple organs of 1 exotic bird that had developmental anomaly and liver fibrosis (fibrosis . In addiM. avium subsp. hominissuis may not lead to tuberculous lesions in birds, particularly when the infection occurs without complications. Susceptibility to mycobacterial infection, including M. avium subsp. hominissuis , depends on the host’s immune and nutritional status, environmental conditions unfavorable for the host, and genetic factors (The etiology of mycobacteriosis, especially in pet birds, is rarely identified. This may be because intravitam and postmortem findings are nonspecific. Infection with 1245 RFLP analysis showed isolates with 2 profiles that differ in the presence of only 1 band. The additional band in the rest of the isolates probably represents the transpositional event. The variability in 1 or 2 bands of 1 strain was also observed previously (M. avium subsp. hominissuis . Unfortunately, the source of infection for this bird was not identified.IS1245 RFLP profile was described in a M. avium isolate from a parrot (M. avium subsp. hominissuis .A multibanded IS


Erwinia pyrifoliae is a newly described necrotrophic pathogen, which causes fire blight on Asian (Nashi) pear and is geographically restricted to Eastern Asia. Relatively little is known about its genetics compared to the closely related main fire blight pathogen E. amylovora.E. pyrifoliae strain DSM 12163T, was sequenced using both 454 and Solexa pyrosequencing and annotated. The genome contains a circular chromosome of 4.026 Mb and four small plasmids. Based on their respective role in virulence in E. amylovora or related organisms, we identified several putative virulence factors, including type III and type VI secretion systems and their effectors, flagellar genes, sorbitol metabolism, iron uptake determinants, and quorum-sensing components. A deletion in the rpoS gene covering the most conserved region of the protein was identified which may contribute to the difference in virulence/host-range compared to E. amylovora. Comparative genomics with the pome fruit epiphyte Erwinia tasmaniensis Et1/99 showed that both species are overall highly similar, although specific differences were identified, for example the presence of some phage gene-containing regions and a high number of putative genomic islands containing transposases in the E. pyrifoliae DSM 12163T genome.The genome of the type strain of E. pyrifoliae genome is an important addition to the published genome of E. tasmaniensis and the unfinished genome of E. amylovora providing a foundation for re-sequencing additional strains that may shed light on the evolution of the host-range and virulence/pathogenicity of this important group of plant-associated bacteria.The  Erwinia amylovora, a quarantine pathogen in Europe, is the most important global threat to pome fruit production  and to a wide-variety of Rosaceae , including amenity and forest species [E. amylovora is native to the North Eastern USA, and it was the first phytopathogenic bacterium described . This ibed  and is considered to have a restricted geographic distribution in East Asia (Korea and Japan) [E. pyrifoliae is rarely conducted, although a quantitative PCR method based on differential 16S rRNA and 16S-23S ITS sequences [E. pyrifoliae causes fire blight disease symptoms essentially indistinguishable from those of E. amylovora infection. Limited host germplasm screening with E. pyrifoliae strains indicates a slightly wider host-range than originally thought, with virulence observed on selected commercial pear (Pyrus communis) and apple  varieties [E. amylovora, suggesting that important genetic differences remain to be elucidated.d Japan) -8. Monitequences ,10 has rarieties . The levE. amylovora [E. pyrifoliae [E. amylovora, E. pyrifoliae intra-species genotypic diversity appears low, with strain differences primarily observed in plasmid content [E. pyrifoliae and E. amylovora [In contrast to the considerable genetic information available for mylovora ,12, relarifoliae . As for  content . Thus famylovora ,15.E. pyrifoliae, strain DSM 12163T, and then compare it to the recently sequenced genome of the non-pathogenic pome fruit epiphyte, E. tasmaniensis Et1/99 [The objectives of our work were to sequence the complete genome of the type strain of E. pyrifoliae, strain DSM 12163T . We sequenced and annotated two additional small plasmids of sizes 4,960 bp (pEP5) and 3,070 bp (pEP3).Earlier reports identified four plasmids in a large group of M 12163T ,19. Receublished . These tE. pyrifoliae DSM 12163T .From the coverage of the individual plasmids, it is possible to estimate the copy number of each plasmid in oriV region. The rest of the plasmid contains a large region that is similar (78% identity) to a part of plasmid pEP5 , when compared to the E. amylovora OT1 or E. tasmaniensis Et1/99 rpoS gene sequence. This 140 bp deletion results in a frameshift and a coding interruption after the RpoS subregion 1.2 [rpoS gene resumes in a different frame, starting from subregion 2.4 in RpoS [During genome assembly, it was observed that rpoS region, we confirmed that this deletion is present in E. pyrifoliae DSM 12163T  are widely distributed among proteobacterial pathogens of plants, animals, and humans, and constitute a fundamental virulence determinant . In othet plants ,30.E. pyrifoliae DSM 12163T contains two distinct T3SSs. One T3SS is closely related to the hrp/dsp cluster of E. amylovora, while the second T3SS is more similar to the inv/spa cluster of Salmonella typhimurium [hypersensitive response and pathogenicity related region (hrp) of E. pyrifoliae DSM 12163T is composed of 25 genes (EPYR_03319-EPYR_03343) organized in four operons encoding the T3SS machinery. The number and the arrangement of these genes are similar to the known hrp pathogenicity island found in E. amylovora [hrpA and hrpS genes in E. amylovora. Both ORFU1 and ORFU2 are also absent in the genome of E. tasmaniensis Et1/99 [The genome of himurium . The hypmylovora , except hrp region in E. pyrifoliae DSM 12163T is identical to the T3SS arrangement found in E. pyrifoliae strain WT3 [E. pyrifoliae DSM 12163T the hrp region is bordered by the hrp effector and elicitors region  and the Hrp-associated enzymes  region. Both regions are organized identically as in E. amylovora [E. tasmaniensis Et1/99 [hrpN and hrpW, dspA/E encoding a secreted effector essential for E. amylovora virulence [orfA, orfB, orfC, and dspB/F. The HAE region includes hrp-associated systemic virulence genes (hsvABC), the gene hrpK encoding for a putative type III translocator [The genomic arrangement of the rain WT3 . In E. pslocator , and twoinv/spa system are organized in a single gene cluster (EPYR_02139-EPYR_02160) in the genome of E. pyrifoliae DSM 12163T  and Vgr , are secreted by a T6SS [Pectobacterium atrosepticum SCRI1043 (syn. Erwinia carotovora subsp. atroseptica), vgr and hcp genes are up-regulated when this pathogen is grown in the presence of potato extract, suggesting a potential role in virulence [P. atrosepticum in potato tuber and stem virulence bioassays [Rhizobium leguminosarum, a locus linked to the T6SS was discovered that negatively affects pea nodulation [T6SS gene clusters have recently been found to be widespread among pathogenic and non-pathogenic Gram-negative bacteria . Bacteriy a T6SS . In the irulence . Two mutioassays . In the dulation .E. pyrifoliae DSM 12163T is composed of 30 genes  and the VgrG2 of E. pyrifoliae DSM 12163T is more closely related to VgrG1 of E. tasmaniensis Et1/99. These genes (EPYR_00656-EPYR_00659) have a lower G+C content than the backbone of the chromosome, indicating an acquired foreign origin of these genes. The genes downstream of vgrG2 in E. pyrifoliae DSM 12163T are homologous to genes downstream of vgrG1 in E. tasmaniensis Et1/99 .The T6SS gene cluster of E. pyrifoliae DSM 12163T is 91.9% identical to its ortholog in E. tasmaniensis Et1/99. Apart from this hcp gene, E. pyrifoliae DSM 12163T possesses two additional hcp genes on the plasmids pEP3 and pEP5  than to the Hcp proteins of the large chromosomal T6SS clusters. The related enterobacterium Serratia proteamaculans 568 has a similar gene organization for its T6SS , and only one vgrG gene. In comparison, Hcp1 is more closely related to the Hcp genes of E. pyrifoliae DSM 12163T and E. tasmaniensis Et1/99 than to Hcp2. The P. atrosepticum SCRI1043 T6SS gene cluster is completely different from that in E. pyrifoliae DSM 12163T regarding its organization [The Hcp encoded in the T6SS cluster of 6SS Fig. , but hasnization ,43.E. pyrifoliae DSM 12163T (EPYR_02109-EPYR_02113) and E. tasmaniensis Et1/99 (ETA_18657-ETA_18659)  and one conserved gene were found (EPYR_02112), while in E. tasmaniensis Et1/99 one of these core genes was absent. Genes in this second set show low homology to each other and to the large T6SS clusters. A frameshift in COG3523 (EPYR_02110/02111) within this T6SS cluster of E. pyrifoliae DSM 12163T was identified which leads to an early stop-codon in the gene. It can be deduced that this gene cluster is inactivated, and prone to mutations and deletions.A second set of designated T6SS-related genes was found in the genomes of 59) Fig.  which arE. pyrifoliae DSM 12163T. One set is tightly clustered (EPYR_00976-EPYR_01028) and the encoded proteins show higher identity with the corresponding proteins of Salmonella and Escherichia spp. than with those of E. tasmaniensis Et1/99 [E. pyrifoliae DSM 12163T, as discernible by a reduced G+C-content bordered by an IS2-type integrase (EPYR_01040) flanking smpB (EPYR_01041), a gene also found the E. tasmaniensis Et1/99 genome (ETA_09720). The left border of the genomic island does not display distinctive insertion features. The first upstream-gene with high homology to E. tasmaniensis Et1/99 is tsx (EPYR_00974), which matches a gene that encodes a nucleoside-specific channel-forming protein in E. tasmaniensis Et1/99 (ETA_09440). Notably, in E. tasmaniensis Et1/99 the region included between ETA_09440 and ETA_09720 contains quorum-sensing genes expRI which are absent in E. pyrifoliae DSM 12163T.Two sets of genes encoding for flagellar assembly and chemotaxis related proteins were found in the genome of E. pyrifoliae DSM 12163T . This second set closely resembles the flagellar genes found in E. tasmaniensis Et1/99 and appears to be ancestral in E. pyrifoliae DSM 12163T. Compared to the acquired flagellar assembly apparatus, the native gene cluster contains two extra genes (EPYR_01615 and EPYR_02267) encoding FliZ, a putative alternative sigma factor regulatory protein and the putative CheV chemotaxis signal transduction protein, respectively.A second complete set of flagellar genes is present but split among different clusters in the genome of E. amylovora [E. pyrifoliae DSM 12163T contains a complete sorbitol gene cluster (EPYR_00602-EPYR_00606), and the organism is able to utilize sorbitol as a sole carbon source [E. tasmaniensis Et1/99 lacks a sorbitol gene cluster in its genome [E. pyrifoliae and E. amylovora which carry the respective metabolic genes.Metabolism of sorbitol has been described as a contributing virulence factor in mylovora  on pome mylovora . The genn source . The nons genome . Associas genome  suggest s genome , and botE. pyrifoliae produces an exopolysaccharide (EPS) that is similar to amylovoran produced by E. amylovora [E. amylovora, mutation of one of the exopolysaccharide genes, wceB, impaired pathogen EPS biosynthesis and reduced virulence on apple [E. pyrifoliae DSM 12163T (EPYR_01479-EPYR_01490) is more related to the gene cluster of E. amylovora Ea7/74 [E. tasmaniensis Et1/99 [P. stewartii subsp. stewarii DC283 [E. pyrifoliae DSM 12163 T are 87.7 - 96.3% identical to the corresponding proteins in E. amylovora Ea7/74, while the identities to E. tasmaniensis Et1/99 range between 68.2 and 92.8%, with no match for WceN and WceM  refers to the ability of bacteria to regulate gene expression in accordance with the presence of extracellular signalling molecules, or autoinducers (AI), that are produced in a cell density-dependent manner . Two priN-acyl homoserine lactones (AHLs), produced by the LuxI family of proteins, as signal molecules. This system was first described in the marine bacterium Vibrio fisheri [lux box). Several AHLs have since been described in a wide-variety of Gram-negative pathogenic and non-pathogenic bacteria, differing principally in the N-acyl side chain moiety of the AHLs, with each unique AHL signal controlled by its own pair of luxRI gene orthologs [The AI-1 system utilizes  fisheri , where irthologs .E. pyrifoliae, proteins encoded by one gene set (EPYR_02385-EPYR_02386) have low sequence identity with the autoinducer biosynthetic and receptor proteins PhzI/PhzR of Pseudomonas chlororaphis (22 and 26% respectively) and ExpI/ExpR of P. atrosepticum SCRI 1043 (17% and 22%). However, EPYR_02386 also shows 45% identity with the DNA-binding transcriptional activator SdiA, which regulates cell division in Escherichia coli and S. typhimurium [E. coli or in S. typhimurium [E. pyrifoliae and other Erwinia species.In himurium , and whihimurium . Thus, SE. pyrifoliae DSM 12163T to induce a positive reaction in AHL-biosensors Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL/pZLR4 (data not shown), it is hypothesized that the gene set composed of EPYR_02385 and EPYR_02386 contributes to production and detection of a yet unknown extracellular signal not obviously related to known QS signals.Given the very low identity of EPYR_02385 to known AHL synthesis protein and the failure of luxS, but also on presence and functionality of the genes coding for the AI-2 receptors [E. pyrifoliae DSM 12163T has a functional luxS gene (EPYR_03027) but it lacks orthologs for both of the two known AI-2 receptors: the LuxPQ-receptor of Vibrio harveyi [Salmonella typhimurium [E. amylovora Ea273 [luxS but lacks AI-2 receptors, this gene probably has a metabolic rather than QS role in E. pyrifoliae.A second QS system, based on production of an AI-2 signal molecule and controlled by the LuxS protein, is also widespread among Gram-negative and Gram-positive bacteria , and puteceptors . E. pyri harveyi  and the ra Ea273 , which aE. pyrifoliae DSM 12163T contains four TonB-dependent receptor genes, one of which is also a putative copper receptor .Iron is an essential nutritional factor for plant pathogenic bacteria, which have high-affinity iron acquisition systems in order to supply this need . Iron reE. amylovora ferrioxamine-receptor FoxR [E. amylovora, foxR mutants retain ability to synthesize desferrioxamine E, but lose their ability to utilize this as an iron substrate, are impaired in growth under iron-limited conditions such as found on flower surfaces, have reduced virulence, and are less resistant to plant defence responses [foxR, E. pyrifoliae also has a biosynthetic gene cluster dfoJAC (EPYR_03489-EPYR_03491) containing an ortholog of dfoA, one of the biosynthetic genes for desferrioxamine in E. amylovora CFBP1430 [E. pyrifoliae DSM 12163T synthesizes desferrioxamine E and has an iron-acquisition system similar to E. amylovora [E. pyrifoliae DSM 12163T, a ferrihydroxamate TonB-dependent receptor and ABC transport system fhuACDB; EPYR_00903-EPYR_00906) is also present, which may contribute to uptake of ferrihydroxamate siderophores .Another TonB-dependent receptor gene (EPYR_03492) encodes an ortholog of the tor FoxR . The genesponses ,68. AdjaCFBP1430 , indicatmylovora . In E. pE. pyrifoliae DSM 12163T is predicted to belong to the Transport Classification DataBase  group TC 1.B.14.2.-, which consists primarily of heme and porphyrin uptake systems. An inactivated homolog of the TonB-dependent receptor yncD (EPYR_02731-EPYR_02732) is also present in the E. pyrifoliae DSM 12163T genome, but is inactivated by frameshifts. The ent-fep-fec gene cluster encoding enterobactin biosynthesis and transport in many Enterobacteriaceae, is absent in the genome of E. pyrifoliae DSM 12163T, as has been observed for E. tasmaniensis Et1/99 [A fourth TonB-dependent receptor (EPYR_01969) found in E. tasmaniensis Et1/99 is 3,883,467 bp [E. pyrifoliae DSM 12163T. Genomic comparison of the complete genomes of the two related bacteria was performed using a Mauve progressive alignment [The chromosome of 3,467 bp , comparelignment , and larE. pyrifoliae DSM 12163T and E. tasmaniensis Et1/99 is largely due to the insertion of mobile genetic elements in E. pyrifoliae DSM 12163T. At least three regions contain phage genes, indicating insertion of prophages or remnants thereof. Additionally, a large number of genomic islands have inserted into the genome of E. pyrifoliae DSM 12163T, as indicated by the large number of possibly active or inactivated transposases identified. Of the remaining areas, two regions encode restriction systems and one cluster carries a complete additional set of flagellar genes .The difference in genome size between E. tasmaniensis Et1/99 contains a large number of fimbrial gene clusters spread across the chromosome that are lacking in E. pyrifoliae DSM 12163T. Additionally, the nas/nir cluster is present in E. tasmaniensis but absent in E. pyrifoliae DSM 12163T, which explains the inability of E. pyrifoliae to generate nitrite from nitrate [E. tasmaniensis Et1/99 encodes part of the central region of E. pyrifoliae plasmid pEP36 carrying the thiOSGF and the betB genes, but not the entire plasmid. E. tasmaniensis Et1/99 also carries genes for the ferric citrate uptake system fecRABCDE and for achromobactin uptake, which are all lacking in E. pyrifoliae DSM 12163T. From the alignment . Whole-genome sequencing was performed by GATC  using both a 454 GS-FLX and a Solexa sequencer.From a single run with the 454 GS-FLX, a total of 533,966 high-quality filtered sequence reads were generated with an average read length of 232 bp . The assembled contigs were aligned to the sequence using the SeqMan program of the LASERGENE package .A total of 4,835,468 high-quality filtered sequence reads with an average read length of 36 bp were generated by Solexa sequencing Table . Averageam EDENA  with manE. pyrifoliae DSM12163T genome sequence was done in two steps. For the chromosome, GenBank accession FP236842 was used as reference. The sequences for plasmids pEP36 and pEP2.6  were published [Assembly of the The reference sequence was also used as a template for the previously assembled contigs using SeqMan. Most contigs aligned to the template, and the non-aligned contigs were mainly due to false assembly in an earlier phase and could be resolved by realignment. The leftover contigs were checked by BlastN versus the template, and two contigs that were not covered by any part of the template were extended by several rounds of assembly versus the 454 reads until they were closed. This assembly was then confirmed by assembly using the Solexa reads, and by shifting the zero-point in the newly assembled plasmid sequences (pEP5 and pPE3).Genes were predicted using a combined strategy  based onRoutine sequence manipulations were done using several subroutines of the LASERGENE package. Whole-genome comparisons were done using the progressive alignment option of the Mauve comparison software (Version 2.0 ).E. pyrifoliae DSM 12163T genome sequence, the Transport Classification DataBase (TCDB) http://www.tcdb.org/ was used for improving the automated annotation of transporters. For comparative classification of the T3SS effectors, the Pseudomonas syringae Hop database http://pseudomonas-syringae.org/Hop_database.xls was blasted on protein level versus the annotation of the E. pyrifoliae DSM 12163 T genome.For the detailed analysis of the E. pyrifoliae DSM 12163T has been deposited at EBI and received the accession numbers EMBL:FN392235 (chromosome), EMBL:FN392238 (pEP36), EMBL:FN392239 (pEP5), EMBL:FN392237 (pEP3) and EMBL:FN392236 (pEP2.6).The genome sequence of THMS and SJ conducted the genome assembly. THMS performed the annotation, comparative genomics and wrote the manuscript. SJ, AG, FR and TK participated in the genome annotation, pathway identification and in writing the manuscript. JEF contributed to the project conception. BD conceived of and supervised the project and participated in writing the manuscript. All authors read and approved the final manuscript.


Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance.The Solanaceae family includes several economically important vegetable crops. The tomato  was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%.http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom  Solanum lycopersicum) is a member of the Solanaceae, and it has served as a model system for fruit development .fasta" dataset obtained from the NONCODE download site nload.htmusing BLAt2genome . HTCs dehttp://iant.toulouse.inra.fr/FrameDP/[http://www.kazusa.or.jp/tomato/. CDSs with the smallest E-value against proteins registered in either nr or SBM proteins were selected. If CDS with the smallest E-value did not cover full-length protein, CDS with the second smallest E-value were selected. If E-values of all CDSs were the same, the longest CDSs were selected. Whether or not HTCs have full-length CDS was assessed by checking if the amino acid sequences contain both start and stop codons. Resulting set of 11,502 non-redundant HTCs encoding full-length proteins was referred to as 'nrFLcDNA.'Amino acid sequences were predicted in sense orientation using three methods. First, the CDS coding the longest amino acid sequence was predicted. Second, CDSs were predicted using FrameDP /FrameDP/. Paramet/FrameDP/. The modUTRs were identified according to the selected CDS. 5'-UTRs were defined as the nucleotide sequence upstream of the start codon. 3'-UTRs were defined as the nucleotide sequence downstream of the stop codon to the poly(A) tail. Nucleotide composition analysis was performed essentially by counting A, T, C, G, N, and X using an in-house Perl program.ftp://ftp.sgn.cornell.edu/unigene_builds), and the DFCI Tomato Gene Index  using BLASTN with threshold E-value of 1e-50. Sequences with similarity to a given target nrFLcDNA were grouped into a multi-sequence group. To detect introns, pair-wise alignments of each target non-redundant HTCs with members of that multi-sequence group were carried out using est2genome. Intron detection was performed bi-directionally, that is, nrFLcDNA/TC/unigene and a target nrFLcDNA were set as "genome" and "est," and vice versa. A BAC sequence file named "bacs.v374.seq.20081128091837" was downloaded from the SGN ftp site http://sgn.cornell.edu/bulk/input.pl?mode=ftp. The target HTCs were mapped onto the BAC sequence with the threshold E-value of 1e-50.After removing poly(A) tails, a set of 12,106 non-redundant HTC sequences were subjected to similarity searches against non-redundant HTCs, the SGN tomato unigenes , and rice protein sequences  using the BLASTX and BLASTP. To estimate the similarity to tomato unigenes, nrFLcDNA sequences were queried against the unigene/TC datasets from SGN unigene sequences  and the DFCI Tomato Gene Index  using the BLASTN. Searches for the protein domains in the amino acid sequences predicted from six open reading frames were performed using InterProScan [http://www.arabidopsis.org/tools/bulk/go/index.jsp. GO annotation documents for InterProScan domains including "interpro2go," "entry.list," and "ParentChildTreeFile.txt" were obtained at the InterPro public ftp site ftp://ftp.ebi.ac.uk/pub/databases/interpro/ in May 2009. Data files of Arabidopsis transcription factors (file named AtTFDB.zip) were downloaded from AGRIS AtTFDB http://arabidopsis.med.ohio-state.edu/AtTFDB/. The difference in the frequency of GO terms was tested by Fisher's exact probability test (P < 0.01).Functional annotations for the nrFLcDNAs were provided according to sequence similarity with public sequence datasets. To estimate the similarity in a protein sequence, nrFLcDNA sequences were queried against protein datasets from NCBI nr, UniProt rProScan . Gene onrProScan  for Arabftp://ftp.sgn.cornell.edu/unigene_builds/) with the highest similarity to each nrFLcDNAs was searched for using BLASTN, and then SGN tomato unigenes that matched nrFLcDNAs at the threshold E-value of < 1e-180 were assigned to tomato metabolic pathways according to LycoCyc version 1.0.1.1 ftp://ftp.sgn.cornell.edu/pathways/lycocyc.dump.txt. Pathway hierarchy was retrieved on May 1, 2009 from "Hierarchical pathway ranking for Solanum lycopersicum" http://solcyc.sgn.cornell.edu/LYCO/hierarchical.html. The version of LycoCyc used in this study provides pathway annotations to 1532 SGN unigenes. The difference in the frequency of pathway annotation terms was tested by Fisher's exact probability test (P < 0.05).The SGN tomato unigene , Triticum aestivum (wheat), Zea mays (maize), Pinus (pine), Picea (spruce), Populus (poplar), Lotus japonicus, Medicago truncatula, Glycine max (soybean), Citrus sinensis (orange), Malus × domestica (apple), Vitis vinifera (grape), Nicotiana tabacum (tobacco), and Solanum tuberosum (potato) . Tomato genome scaffold sequences with the highest similarity to a given nrFLcDNA sequence were identified using BLASTN . Each pair of scaffold and nrFLcDNA sequences was then submitted to exon and intron prediction using the est2genome program [Poly(A) tails of the nrFLcDNA sequences were excised, and this set of nrFLcDNAs without poly(A) tails was used for similarity searches against the Prerelease of Tomato Genome Shotgun Sequence : high throughput cDNA sequence; (nrFLcDNA): non-redundant full-length cDNA; (CDS): coding sequence; (UTR): untranslated region; (DFCI): Dana-Farber Cancer Institute; (SGN): SOL genomics network; (TC): tentative consensus.This study was conceived and directed by KA and DS. RNA samples were prepared by NS, KS, TT, MW, HT, YW, ME, MK, YI, MK, SF, AO, TA, YS, KY, SS, TO, and HE. cDNA libraries were constructed by KA, SS, HE, and DS. Sequencing was carried out by KS, AK, TS, TT, MW, MT, TN, TS, and YK. Assembly of the full-length sequences was performed by KS, AK, TS, and MW under the direction of KY. Sequence analyses and other bioinformatics were directed by KY, and performed by AS, SK, NY, and KA. Database construction was directed by KY and performed by AS, SK, and KO. KA wrote the paper. All authors approved the final manuscript.Tom tissues used for RNA preparation. Detailed description of Micro-Tom tissues used for RNA preparation including tissue positions, treatments, and age of plants when tissues were harvested.Click here for fileClone number list. Lists of clone numbers of nrFLcDNAs-sets appeared in the textClick here for filecDNA deived from non-coding RNA. Clone numbers and annotations of nrFLcDNAs that matched to non-coding RNAs registered in NONCODE database.Click here for filePredicted CDS of 11,597 non-redundant HTCs. A list of amino acid sequences predicted for 11,597 non-redundant HTCs and results of BLASTP search against protein datasets of nr and tomato SBM database http://www.kazusa.or.jp/tomato/.Click here for fileDatasets used for comparative analysis with other plants. Name and version of protein- and tentative consensus-datasets used for comparison of nrFLcDNAs with gene of other plants.Click here for file


Bartonella as an endocarditis agent in cattle. Bartonella bovis was identified by PCR, gene sequences analysis, and specific internal transcribed spacer amplicon product size in 2 bovine endocarditis cases with high antibody titers, which demonstrates that B. bovis is a pathogen for cattle.This study aimed to determine the role of  Arcanobacterium pyogenes (up to 90% of the strains), Streptococcus sp., and numerous Enterobacteriaceae  led to the identification of many noncultivable or poorly cultivable bacteria as agents of human endocarditis, such as Twenty-two cases of bovine endocarditis were diagnosed in adult cows  at the School of Veterinary Medicine, Toulouse, France, from September 2004 through June 2006. Eighteen cows were hospitalized for poor condition, anorexia, weight loss, wasting syndrome, and abnormal cardiac auscultation. Endocarditis was diagnosed at physical examination. Lesions of the cardiac valves were confirmed at necropsy for all 18 animals. Four additional cases of endocarditis were identified at necropsy after an apparent sudden death. Most of the damaged valves of these 22 animals had large, cauliflower-like lesions.For each cow, fragments of the vegetative valve and of 1 normal-appearing valve were collected. DNA extraction from each valve sample was performed by using Nucleospin Tissue extraction kit  according to the supplier’s recommendation.gltA) Bartonella sp. DNA detection. Additional PCR amplification was performed on gltA-positive valves for the following Bartonella-specific genes or genomic region: rpoB, ribC, groEL, internal transcribed spacer (ITS) of 16S–23S rRNA , B. chomelii (CIP 107869 T), and B. schoenbuchensis (NCTC 13165T), respectively, were used as antigens.Serology by indirect fluorescent antibody assay (IFA) was performed as reported elsewhere  were the 2 oldest cows . ITS 16–23 rRNA amplification was obtained only for the damaged valve of cow 04–927; the size of the amplicon product was ≈190 bp, which was identical to the size of the product obtained with the B. bovis reference strain , ribC , gltA , groEL  were partially sequenced. Sequence identities were respectively 100% with B. bovis (gltA) and 99% with B. bovis . The sequence obtained with 16S rRNA (accession no. EF432054) had a 99% identity with B. bovis.The 22 vegetative valves included 8 pulmonary valves, 7 tricuspid valves, 6 aortic valves, and 1 mitral valve. The only vegetative mitral valve and 1 of the 6 aortic vegetative valves showed positive results for e strain . AmplifiBartonella isolates. The 2 PCR-positive cows had high IFA titers , whereas the 20 PCR-negative cows had low or negative titers  shared 99% identity with B. bovis genes. ITS amplification of 1 vegetative valve gave a fragment of ≈190 bp, which is the size expected for B. bovis  of the 22 endocarditis cases were carditis , which sB. bovis, and diseases attributed to infection with this Bartonella species in cows are scarce (B. bovis is a potential bovine pathogen and that B. bovis can induce endocarditis in the animal reservoir host, as previously shown for B. quintana, B. henselae, and B. vinsonii subsp. berkhoffii in humans, cats, and dogs, respectively (B. bovis can cause endocarditis in cows like B. henselae and B quintana in their respective feline and human reservoirs.Cattle are the main reservoir for


Rickettsia typhi  and R. felis  DNA were detected in rat fleas (Xenopsylla cheopis) collected from mice on Oahu Island, Hawaii. The low prevalence of R. typhi on Oahu suggests that R. felis may be a more common cause of rickettsiosis than R. typhi in Hawaii. Rickettsia typhi. The classic view is that R. typhi circulates among rats (Rattus rattus or R. norvegicus) and rat fleas (Xenopsylla cheopis) , black rat (R. rattus), Norway rat (R. norvegicus), and house mouse (Mus musculus) (Herpestes auropunctatus) was also identified as a potential reservoir; however, its role has not yet been evaluated. We conducted a molecular survey of fleas in Oahu to characterize the prevalence and identity of rickettsiae in Hawaii.The most recent outbreak of murine typhus in Hawaii occurred in 2002 with 47 cases reported on 5 islands  for further analyses. Mice were humanely killed; only brains were removed and frozen.and 2007 . Fleas wDNA was isolated from each flea by using the Biomek 2000 Laboratory Automation workstation  and reagents from the Wizard Prep kit  (R. felis and R. typhi DNA was conducted by using a TaqMan assay for the citrate synthase (gltA) gene of Rickettsia spp.  and species-specific probes A(C)TA(C)A(A)AG(A)T(T)G(C)T(C)A-BHQ2-3′; R. felis: 5′-Cy5-CTA(C)GGA(G)AATT(G)CCA-BHQ3-3′); locked nucleic acid bases, shown in parentheses, were incorporated to improve probe binding. Specificity of probes was tested by using DNA of R. prowazekii, R. typhi, and 23 spotted fever group rickettsial isolates. The Brilliant Q PCR core reagent kit  and an iCycler  were also used. Positive control plasmids contained a 265-bp target fragment from R. typhi strain Wilmington and R. felis strain LSU.Detection of X. cheopis fleas was examined, including 122 fleas collected from 61 mice trapped in 2004, 84 fleas from 55 mice trapped in 2006, and 4 fleas from 2 mice trapped in 2007  fleas contained only R. felis DNA. The amplicon sequences were identical to homologous sites of R. typhi gltA (AE017197) or R. felis gltA (CP000053). One flea contained R. felis and R. typhi DNA. This result was confirmed by cloning of 4 replicate amplicons and sequencing of 24 randomly selected clones. Both DNAs were confirmed to be present in each amplicon. The highest rates of fleas infected with rickettsial agents were detected in Makaha , Waianae , and Makakilo  during the 2004 collection. R. typhi was detected in 4 sites . All DNAs extracted from brain tissues of 55 mice collected in 2006 were PCR negative for R. felis and R. typhi.Four fleas  contained only R. typhi associated with rat fleas (X. cheopis) collected from house mice (M. musculus) in western Oahu, Hawaii. R. felis, the etiologic agent of cat flea rickettsiosis, was also found associated with rat fleas collected from house mice.We report molecular detection and identification of R. typhi, respectively  as demonstrated here and in a recent report from Indonesia   are not agent specific and, as for other rickettsioses, include fever, headache, and rash. Antibodies against R. felis variably cross-react with R. typhi, R. rickettsii, and other spotted fever group rickettsia antigens (R. felis infection in humans can be misdiagnosed or missed without R. felis antigen testing. Only a handful of cases of CFR have been reported worldwide, and only 8 cases have been specifically confirmed by PCR (Murine typhus caused by R. felis DNA, this pathogen may pose a serious risk to human health in Oahu. Further studies are warranted to establish the true human prevalence of murine typhus and cat flea rickettsiosis in Hawaii, to define the clinical spectrum of these infections with more specific confirmatory diagnostic tests, and to establish the role of fleas and different rodents in the epidemiology of the 2 diseases.Since 25% of the fleas removed from mice were positive for only


Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms ). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden–Meyerhoff–Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used  and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes. Mycoplasma hominis, M. genitalium, and Ureaplasma parvum are human pathogenic bacteria that colonize the urogenital tract. They have minimal genomes, and thus have a minimal metabolic capacity. However, they have distinct energy-generating pathways and distinct pathogenic roles. We compared the genomes of these three human pathogen minimal species, providing further insight into the composition of hypothetical minimal gene sets needed for life. To this end, we sequenced the whole M. hominis genome and reconstructed its energy-generating pathways from gene predictions. Its unusual major energy-producing pathway through arginine hydrolysis was confirmed in both genome analyses and in vivo assays. Our findings suggest that M. hominis and U. parvum underwent genetic exchange, probably while sharing a common host. We proposed a set of genes likely to represent a minimal genome. For M. hominis, this minimal genome, not including cytadherence and virulence-related genes, can be defined comprising the 247 genes shared by the three minimal genital mollicutes, combined with a set of nine genes needed for energy production for cell metabolism. This study provides insight for the synthesis of artificial genomes. Mycoplasma hominis is an opportunistic human mycoplasma species which resides, as a commensal, in the lower urogenital tract. However, it can also cause pelvic inflammatory disease and postpartum or postabortion fevers, and has been associated with bacterial vaginosis M. genitalium and Ureaplasma urealyticum, the latter having been recently separated into two species U. parvum and U. urealyticumM. hominis. However, these species have overlapping but distinct pathogenic roles M. genitalium and Ureaplasma spp., but not M. hominis, are involved in male urethritis. M. genitalium is also the only mycoplasmal species involved in cervicitis, whereas Ureaplasma spp. are significantly associated with prematurity, low birth weight and chronic lung disease in infants Mollicutes: M. genitalium and Ureaplasma spp. belong to the Pneumoniae group and M. hominis belongs to the Hominis group. They possess the smallest genomes among self-replicating free living organisms. The M. genitalium genome is the smallest of these, comprising 580 Kbp, with a capacity to encode only 482 genes M. genitalium genome triggered particular interest in this organism and several studies have addressed the concept of a minimal cell Mycoplasma laboratoriumM. hominis is slightly larger than that of M. genitalium, with pulsed-field gel electrophoresis revealing a chromosome size of 696 Kbp U. parvum  is the largest of the three species, encompassing 751 Kbp M. genitalium is a glycolytic species, whereas M. hominis and Ureaplasma spp. are both nonglycolytic species, producing energy through arginine degradation or urea hydrolysis, respectively Two other mollicute species, M. hominis was the only one that had not been sequenced. We therefore sequenced the whole genome of the M. hominis PG21 type strain. We examined its energy-generating pathways and investigated the essential role of its arginine dihydrolase pathway in vivo. To provide further insight into the composition of hypothetical minimal gene sets and of the associated energy-generating pathways, we carried out whole genome comparisons of the three pathogenic mycoplasmas, M. hominis, M. genitalium and U. parvum, sharing the same urogenital niche and displaying near minimal genomes. This analysis resulted in the identification of a set of shared genes representing the core genome. Other genes include those that are involved in the energy-yielding pathways specific to each mollicute and those that play a role in the interaction with the host, such as cytadherence and virulence.Of all the mycoplasma species with a known pathogenic role in humans, M. hominis PG21 genome and their comparison with those of M. genitalium G37 and U. parvum serovar 3 are shown in M. hominis PG21 genome is a single, circular chromosome of 665,445 bp with an overall G+C content of 27.1%. It contains 537 putative coding DNA sequences (CDSs), representing a 89.8% gene density, and 14 pseudogenes were found. Function could be predicted for 345 of the CDSs, whereas 86 were conserved hypothetical proteins (CHP) and 106 were hypothetical proteins (HP). A minimal but complete set of 33 tRNA genes was identified. The M. hominis genome contained two copies of rRNA genes, as previously described M. arthritidisThe general features of the M. hominis genome, including three ABC transporter substrate-binding proteins , two predicted nucleases  and one predicted peptidase (MHO_4970). Interestingly, the lipobox sequence, located upstream from the cleavage site, was highly conserved in five of the predicted lipoproteins, MHO_1730, MHO_2100, MHO_2340, MHO_2440, MHO_2620, with the consensus motif PLVAAGC present in all of them. In contrast to these conserved regions, the other regions of the proteins were very different in length and sequence, suggesting their rapid and divergent evolution from a common ancestral gene.A set of 43 lipoproteins was predicted from the M. arthritidis genome oriC was experimentally demonstrated to be functional. We did not detect any significant inversion in the GC skew for M. hominis. The synteny rnpA → rpmH → dnaA → dnaN was present, as previously observed in M. arthritidis, M. capricolum subsp. capricolum and M. mycoides subsp. mycoides SC dnaA gene, but none between dnaA and dnaN. These boxes showed sequence identity at seven of the nine positions of the Escherichia coli consensus sequence TTATCCACA dnaA gene in M. mobileM. arthritidisM. hominis.We assigned the origin of replication based on sequence homology with the M. hominis CDSs that show a best Blast hit (BBH) in species other than those belonging to the Hominis phylogenetic group. From the 537 CDSs predicted from the M. hominis chromosome, 59 had a BBH in a mollicute belonging to the phylogenetic groups Pneumoniae, Spiroplasma or Phytoplasma and 12 had a non-mollicute BBH. We then examined each of the candidate CDSs for potential HGT, using phylogenetic reconstructions and synteny analysis. Five CDSs seemed to be likely candidates for HGT between M. hominis and non-mollicute bacteria (M. arthritidis (85% similarity), but the deduced protein was truncated in N-terminal by a third of its length. A putative DDAH was also proposed in M. penetrans (MYPE1510) but it appeared very different from MHO_2540 (45% similarity) and from other bacterial orthologs, suggesting a specific evolution and/or origin. Except in M. arthritidis, closest homologs of MHO_2540 were found in non-mollicute bacteria including the human pathogens Eggerthella lenta (76% similarity), Atopobium vaginae (72% similarity) and Pseudomonas aeruginosa (71% similarity). Although phylogenetic reconstructions remained unclear, this suggests that MHO_2540 and its truncated ortholog in M. arthritidis might have a non-mollicute origin.We identified genes that may have been exchanged through horizontal gene transfer (HGT), by searching for bacteria . Their cM. mycoides subsp. mycoides SC  and U. parvum (UU476) homologs with a bootstrap support value of 100%, suggesting that HGT occurred between the two species. In both species, the modification methylase-encoding gene was split into two by a frameshift that occurred within tracts of repeated nucleotides. In MHO_0130/MHO_0140, a simple deletion of one nucleotide within a tract of 11 guanosines restored the complete ORF, suggesting that the expression of the encoded methylase may be switched on and off by a phase variation mechanism, similar to that previously described for another type III restriction/modification system in M. pulmonisFive clusters of genes from the ae group . The firae group . CloselyhsdR, hsdS (2 genes) and hsdM genes and encoded the components of the system. In contrast, the second cluster only contained the two hsdS genes and an hsdM gene that was split into two by a frameshift mutation. Nucleotide sequences of the genes within the two clusters were nearly identical, indicative of a probable duplication event. Phylogenetic trees inferred from the protein sequences of the M. hominis R and M subunits showed a close relationship with their homologs in U. parvum (UU095 to UU100).The two gene clusters, MHO_5210 to MHO_5240 and MHO_3220 to MHO_3250, encoded predicted type I restriction/modification systems . The firU. parvum (UU374/UU373/UU372) than with any other transposase gene. Moreover, sequence alignments revealed more than 90% similarity for the predicted proteins and extensive regions in which the nucleotide sequences were strictly identical. These data strongly suggest recent exchange of this mobile element between the two species.A fourth cluster included CDSs MHO_2520 and MHO_2530, two fragments of a transposase-encoding gene from an IS1138 element . PhylogeU. parvum encoded seven conserved hypothetical proteins and two additional ATP synthase subunits (MHO_3120 to MHO_3200) (The fifth group of genes that may have been exchanged with HO_3200) . As descU. parvum, no HGT was predicted with the urogenital pathogen M. genitalium, or with M. penetrans, another human mollicute reported in the urogenital tract It should be noted that, whereas five clusters of genes may have been exchanged with the M. hominis genome was compared to the genomes of M. genitalium and U. parvum, the two other urogenital mollicute species that infect humans. Orthology relationships between the three genomes were computed using a bidirectional best hit method. M. hominis, 172 CDSs specific to M. genitalium and 280 CDSs specific to U. parvum. The M. hominis genome shared 24 CDSs only with M. genitalium and 46 CDSs only with U. parvum, whereas 41 CDSs found in both M. genitalium and U. parvum were not found in the M. hominis genome.The M. genitaliumM. arthritidisM. pulmonisThe 247 orthologous genes shared by the three mollicutes encoded proteins involved in major cellular functions, including DNA metabolism, protein synthesis, nucleotide synthesis, transport and binding of substrates, and fatty acid and phospholipid metabolism . InteresM. hominis coded for: (i) the lipoprotein P120 (MHO_3660) and the protein P120' (MHO_3800), which are M. hominis-specific surface-exposed proteins displaying antigenic variation M. hominisM. hominis also included OppA (MHO_1510), an oligopeptide permease substrate-binding protein. OppA is a multifunctional lipoprotein involved in cytadherence, but is probably also the main ecto-ATPase at the surface of the M. hominis cell M. hominis virulence factor M. hominis-specific genes. The eight Lmp proteins represent a family of proteins characterized by a predicted N-terminal transmembrane helix and several repeat sequences. In particular, using the MEME method, we identified a 57 amino-acid motif, [QK][QK]L[DKQ][DN]LI[DK]S[NQE][DE][AG][KQ][DKN][VI]D[KT][SQ]K[EA][TN][QDN][IS][LF][NQ]N[TN]N[LVI][DT][AG][SKN][SD][LT][IT][KD][DQ]I[EKV][SN][KA][TI][KN][TE]I[EK][DK]A[IT][QEK][SD]L[TQ]K[KL]I[ND], repeated between one and 15 times, in all proteins except MHO_0530 and MHO_4280.Many of the species-specific genes were associated with cytadherence and virulence . Genes fM. genitalium and U. parvum genomes included several CDSs associated with cytadherence and virulence , the MgpC protein (MG 192), the adhesin P32 (MG318), the protein P200 (MG386) and the cytadherence accessory proteins Hmw1, Hmw2 and Hmw3 . Of the 280 CDSs specific to U. parvum, genes associated with cytadherence and virulence included those encoding the MBA protein (UU375) — a major antigen recognized during infection of humans — and the five MBA N-terminal paralogs . Genes encoding eight iron transporters , nine putative ABC substrate-binding protein-iron  and two hemin transporters  were also found in the U. parvum genome, but not in M. hominis or M. genitalium. It should be noted that the virulence-related gene msrA, encoding the peptide methionine sulfoxide reductase, was found in both M. genitalium (MG408) and U. parvum (UU289), but not in M. hominis. In M. genitalium, inactivation of the msrA gene reduces adherence and abolishes virulence Similarly, genes specific to irulence . The 172M. genitalium, which is a glycolytic species, several genes encoding components of the carbohydrate, pyruvate and glycerol metabolic pathways were found among its 172 specific CDSs, including carbohydrate transporter genes (fruK (MG063), fruA (MG062) and pstG (MG069) genes of the fructose and glucose phosphoenolpyruvate-dependent sugar phosphotransferase transport system (PTS) were found specifically in M. genitalium. Moreover, the four genes encoding the pyruvate dehydrogenase complex, pdhA (MG274), pdhB (MG273), pdhC (MG272) and pdhD (MG271), the glycerol kinase gene gplK (MG038) and the glycerol uptake facilitator gene glpF (MG033) were predicted in the M. genitalium genome only, and not in the M. hominis or U. parvum genomes. In U. parvum, which generates ATP through urea hydrolysis, the seven components of the urease complex (UU428 to UU434), the ammonium transporters Amt-1 and Amt-2  and the atypical delta subunits of the FoF1-ATPase  probably involved in this unique energy production pathway M. hominis and M. genitalium  pathway were present except 6-phosphofructokinase  in bacteria, were present in M. genitalium and U. parvum; but the phosphotransacetylase gene was absent from the M. hominis genome  was present in both M. hominis and U. parvum. This enzyme could lead to glyceraldehyde 3-phosphate production via the second part of the EMP pathway involving triosephosphate isomerase , ornithine carbamoyltransferase  and carbamate kinase  — were found to be specific among the three species discussed here. Moreover, a putative dimethylarginine dimethylaminohydrolase  was predicted in M. hominis but not in U. parvum and M. genitalium. In P. aeruginosa, DDAH (EC 3.5.3.18) catalyzes the conversion of the Nω-alkylated arginine residues to citrulline. A more detailed overview of the M. hominis energy-generating pathway was obtained by searching for specific transporter genes in the genome. The results are graphically displayed in arcABC, in the M. penetrans genome M. hominis, MHO_5040 was not located in close proximity to the arcABC cluster. In addition, it should be noted that no ammonia/ammonium transporters was identified in the M. hominis genome.In  pathway  — arginiM. hominis, we studied the growth of M. hominis PG21 in the presence of arginine and arginine analogs (M. hominis culture reached 107 color changing units (CCU)/ml and the pH increased from 7.0 to 7.2 after 48 hours. In medium supplemented with 10 mM arginine, the titer after 48 hour growth was unchanged, but the pH was higher than that observed without addition of arginine, increasing from 7.0 to over 8.1. A total loss of viability was observed after 120 hours under these conditions. The effect on growth of adding horse serum was checked by inoculating media with or without 2.5% horse serum. Growth was independent of serum content in medium supplemented with 10 mM arginine. However, a non-supplemented medium lacking horse serum was growth-limiting, with a 10-fold lower titer of organisms after 48 hours (data not shown). Thus, the serum-containing Hayflick modified medium supplied the cells with nutrients that were necessary to reach a high cell titer after 48 hours. Furthermore, in the absence of horse serum, the exogenously added arginine provided energy for cell growth. The effect on growth of canavanine, an arginine analog that interferes with the arginine uptake and metabolism was evaluated. Canavanine is an ADI-specific competitive inhibitor 5 CCU/ml over 120 hours. Addition of 23 mM arginine at 48 hours was sufficient to displace the competitive inhibitor and restore growth  could be used as a substrate by DDAH and could serve as an energy source for protein synthesis in M. hominis, growth was evaluated in the presence of ADMA with ADI activity suppressed by canavanine. The maximum concentration of ADMA that could be assessed was 0.5 mM, because of the poor solubility and dissolution rate of the arginine analog in aqueous solutions. Under these conditions, in presence of 2.5% horse serum in the medium  contains four putative cell-surface proteins that may be involved in the interaction between the mycoplasma and its human host. This is the first report of HGT among human mollicutes. We did not find any evidence of HGT between M. hominis and M. genitalium, or M. penetrans. M. hominis and U. parvum are both frequent commensals of the human urogenital tract and they are commonly detected together in healthy individuals. In contrast, M. penetrans is very rarely detected in the urogenital tract M. genitalium, which has a low prevalence in the general population In this study, we first sequenced, annotated and analyzed the genome of M. hominis. These included the genes encoding P120, P120', Vaa, LMP1 and LMP3, which have been previously identified as surface proteins displaying size, sequence, and antigenic variation M. pneumoniae P1 adhesin gene M. genitalium mgpB adhesin gene M. hominis to further elucidate the role of LMPs. Size variation among these cell envelope proteins could also explain the heterogeneity in genome size within the M. hominis species. Indeed, genome size was previously estimated for 15 strains using pulsed-field gel electrophoresis, and was found to vary by more than 15%, ranging from 696 to 825 Kbp M. hominis pathogenic mechanisms that are yet to be described, so the characterization of genes specific to M. hominis could help to identify unknown adhesion or virulence factors. Indeed, such factors could be searched for among the 220 M. hominis-specific CDSs, particularly among the hypothetical proteins, although virulence factors may also be shared with other mycoplasma species.We also compared the whole genomes of the three human pathogenic mycoplasmas. Two major sets of genes could be identified among species-specific genes. The first group contained genes involved in energy-generating mechanisms, with the second group containing genes involved in cytadherence and virulence. Thus, these three mycoplasma species have their own specific genetic equipment underlying their pathogenic roles. We found several CDSs specific to M. hominis has been described as a nonglycolytic species that generates ATP through arginine hydrolysis M. arthritidis, the mycoplasma most closely related to it for which the genome sequence is also available M. hominis genome revealed that the gene sets of the pentose phosphate pathway and of the glucose and fructose transport PTS were incomplete, as has been observed for other sequenced glycolytic and nonglycolytic mollicutes M. hominis were confirmed in silico by the lack of the corresponding gene U. parvumM. hominis metabolizes hexoses such as glucose or fructose. The presence of a gene encoding glycerol 3-phosphate dehydrogenase in M. hominis suggests that glycerol 3-phosphate may be oxidized into dihydroxyacetone phosphate, which could then enter glycolysis  is particularly notable, given the possible role of this enzyme in ATP generation in M. hominisM. hominis genome but is absent from the U. parvum and M. genitalium genomes. The presence of several gaps in the M. hominis glycolysis and pyruvate pathways supports the notion that these energy-generating pathways have a low efficiency in this species. Consistent with this, we observed that the growth of M. hominis in Hayflick modified broth supplemented with glucose M. hominis species-specific genes included a complete set of genes coding for the enzymes of the arginine dihydrolase pathway, which can generate energy in the form of ATP for growth. Considering that alternative energy-yielding pathways have been demonstrated for several arginine-utilizing mollicutes M. hominis growth requirements. Arginine is considered an essential nutrient for initiation of growth of M. hominis2 production, cell growth and ADI activity, as well as the delayed induction of ADI relative to the cell cycle, led Fenske and Kenny M. hominis used the arginine pathway only as an alternative energy source. In this study, we showed arginine to be sufficient for cell growth in a growth-limiting medium. Interestingly, the pH of the medium after 48 hours of growth varied with the initial composition of the medium. Supplementation of the basal medium with arginine resulted in an increase of the pH, probably due to a higher rate of ammonium production via the arginine dihydrolase pathway. This increase in pH could have deleterious effects on cell survival in vitro, as suggested by the loss of viability after 120 hours in the presence of arginine. Moreover, canavanine, a specific competitive inhibitor of ADI from M. arthritidisM. hominis, suggesting that ADI activity is essential for its growth. For its metabolism, arginine can be transported into M. hominis cells in its free-form M. hominis genome, arginine-containing oligopeptides should be considered as possible alternative source of arginine. Currently, a considerable limitation on in vivo metabolic studies is the absence of a defined medium for growing M. hominis. An additional in silico genome-scale metabolic reconstruction of the metabolic pathways could help to elucidate the true biochemical capacity of M. hominis, as recently described for M. genitaliumycolysis . This wos genome . The revM. hominis PG21 genome. This suggests that a secondary citrulline-producing pathway, involving the catalytic conversion of ADMA, may occur in M. hominis. However, addition of a maximum soluble concentration of ADMA to a medium supplemented with canavanine was not sufficient to promote detectable growth. The ADMA concentration was probably not high enough in our experiments to allow a significant increase in bacterial cell concentration. Thus, the potential production of additional citrulline through metabolism of ADMA in M. hominis remains uncertain but cannot be excluded. ADMA is naturally present in biological fluids and inhibits nitric oxide synthase in humans P. aeruginosaM. hominis could indirectly increase host NO production leading to tissue damage. Consistent with this hypothesis, M. hominis can stimulate nitric oxide synthase production by host macrophages M. hominis and its truncated ortholog found in the closely related M. arthritidis may have been acquired by HGT from non-mollicute bacterial species, notably from A. vaginae which belongs to the same urogenital niche than M. hominis and is also involved in bacterial vaginosis We identified a gene encoding a putative DDAH, an enzyme structurally related to ADI, in the M. hominis and its host through ammonia formation. This compound is also generated during the hydrolysis of urea in U. parvum. As both species are mainly found in the acidic female genital tract, these pathways could be required to protect them from the deleterious effects of the acidic environment and may also promote their pathogenicity. Moreover, M. hominis may be able to form biofilms since it was identified in microbial biofilms involved in intraamniotic infection Staphylococcus aureusS. aureus, the expression levels of the deiminase pathway were higher in biofilm cells than in planktonic cells.The arginine utilization pathway may have implications for the relationship between et al. used a global transposon mutagenesis strategy in M. genitalium. They found 382 of the 482 protein-coding genes to be essential under laboratory conditions M. hominis protein-coding genes were compared with this set, 147 (38%) were absent from the M. hominis genome (data not shown). These 147 genes absent from M. hominis mainly encoded proteins involved in carbohydrate transport and metabolism  or in cytadherence and virulence . It is therefore possible that at least two whole gene sets involved in cytadherence/virulence and in energy-generating carbohydrate metabolism may be replaced by two other whole gene sets facilitating similar functions in another minimal species  and the phosphoketolase- (MHO_3010) genes, which are specific to M. hominis, and three genes absent from the core genome but present in M. hominis and in one other species   genes. This gene list could be useful for the synthesis of artificial genomes using the recently reported complete chemical synthesis procedure To identify a minimal gene set essential for cell viability, Glass  species . Thus, iogenase)  should bM. hominis PG21 has the second smallest genome among self-replicating free-living organisms. This species hydrolyses arginine as its major energy-source and possesses its own set of cell-surface proteins as the basis of its urogenital pathogenicity. We have described a 247 gene core genome based on whole genome comparisons between M. hominis and the two other human urogenital pathogens with minimal genomes, M. genitalium and U. parvum. An additional set of nine genes may be required for the energy generation in M. hominis. Moreover, a set of 220 M. hominis-specific genes were identified, which may harbor unknown virulence genes.In conclusion, M. hominis type strain PG21 (ATCC 23114) was chosen and grown in Hayflick modified medium supplemented with arginine The M. hominis PG21 genome. For plasmid libraries, genomic DNA was mechanically sheared and 3 kb fragments were cloned into pNAV(A) and pCNS vectors (B) . Large inserts (25 kb), generated by HindIII partial digestion, were introduced into pBeloBac11  (C). Vector DNA was purified and end-sequenced using dye-terminator chemistry on ABI3730 sequencers . The Phred/Phrap/Consed software package (www.phrap.com) was used for the sequence assembly. A complement of 91 sequences was needed for gap closure and quality assessment.We constructed three libraries to determine the complete sequence of the http://www.ebi.ac.uk/swissprot/index.html), TrEMBL (http://www.ebi.ac.uk/embl/index.html), and MolliGen (http://cbi.labri.fr/outils/molligen/). Sequence similarity and start codons were determined as previously described The genome annotation was performed using the CAAT-Box platform Lipoproteins were predicted using the PROSITE prokaryotic membrane lipoprotein lipid attachment site motif (PROKAR_LIPOPROTEIN).−8. CDSs displaying a BBH from a species not belonging to the Hominis phylogenetic group were further investigated by phylogenetic analyses. Protein phylogenies were determined using the MEGA4 package Putative HGTs were identified as previously described M. hominis PG21, M. genitalium G37 and U. parvum serovar 3 (ATCC 700970) were determined using the bi-directional best hit method followed by manual curation.Genome analysis and comparisons were conducted using MolliGen, a database dedicated to the mollicute genomes M. hominis PG21 was grown in a basal medium containing heart infusion broth (Difco), yeast extract (Bio-Rad), and 2.5% horse serum (Difco), with or without 10 mM arginine (Fluka), 10 mM canavanine (Sigma) or 0.5 mM Nω,Nω-dimethyl-L-arginine (ADMA) (Sigma) in 4% dimethylsufoxide, individually or in combination. Identical experiments were conducted with a basal medium without horse serum to limit the arginine content. The pH of the media was adjusted to 7 before inoculation with M. hominis. The initial free arginine concentration of 2.5% serum-containing Hayflick media was measured at 1.4 mM by HPLC (High Performance Liquid Chromatography) after deproteinisation. Incubation time was 48 and 120 hours and growth was measured in CCU per ml −12 in Hayflick modified broth medium supplemented with 23 mM arginine. M. hominis PG21 strain were submitted to the EMBL/GenBank/DDBJ databases under accession number FP236530. All data are also available from the MolliGen database (http://cbib1.cbib.u-bordeaux2.fr/outils/molligen/home.php).The genome sequence and related features of the Figure S1M.hominis genomic region MHO_0120-MHO_0140. (A) Schematic of the homologous genomic regions encoding a Type III restriction-modification system in U. parvum (UU), M. hominis (MHO), and M. pulmonis (MYPU). Homologous genes are connected by dashed lines. (B) Partial amino acid and nucleotide multiple alignments corresponding to the C-terminal region of MHO_0140. Repeats that may be involved in phase variation are written in blue; stop codons are in red. (C) Phylogenetic tree inferred from amino acid sequences of the restriction component of the systems. Bootstrap values are indicated on nodes. MAG, M. agalactiae; MHP7448, M. hyopneumoniae 7448; mhp, M. hyopneumoniae 232; MHJ, M. hyopneumoniae J; MYPU, M. pulmonis; MYPE, M. penetrans; MMOB, M. mobile. Numbers after the strain name indicate mnemonics.Analysis of the (0.87 MB TIF)Click here for additional data file.Figure S2M. hominis genomic regions MHO_3220-MHO_3250 and MHO_5210-MHO_5240 encoding Type I restriction-modification systems. (A) Schematic of the homologous genomic regions in U. parvum (UU) and M. hominis (MHO). Homologous genes are connected by dashed lines; the locus is recombined in U. parvum. Phylogenetic trees inferred from amino acid sequences of the MHO_5230 homologs (S subunit) and MHO_5210 homologs (M subunit) are presented in (B) and (C), respectively. Bootstrap values are indicated on nodes. MPN, M. pneumoniae; MAG, M. agalactiae; SPICI, Spiroplasma citri; MGA, M. gallisepticum; MYPU, M. pulmonis; MMOB, M. mobile. Numbers after the strain name indicate mnemonics.Analysis of the (0.71 MB TIF)Click here for additional data file.Figure S3M. hominis genomic region MHO_2520-MHO_2530. (A) Schematic of the homologous genomic regions encoding IS1138 transposase gene in U. parvum (UU), M. hominis (MHO) and M. pulmonis (MYPU). The transposase encoding gene is fragmented in M. hominis and U. parvum. (B) Partial nucleotide alignment showing the nearly identical sequences of MHO_2530, MHO_2560, and UU372. (C) Phylogenetic tree inferred from amino acid sequences of the IS1138 transposases. Bootstrap values are indicated on nodes. MSC, M. mycoides subsp. mycoides SC; SPICI, S. citri; AYWB, ca. Phytoplasma asteris Aster Yellows Witches Broom; PAM, ca. Phytoplasma asteris Onion Yellows. Numbers after the strain name indicate mnemonics.Analysis of the (0.83 MB TIF)Click here for additional data file.Figure S4M. hominis genomic region MHO_3120-MHO_3200. (A) Schematic of the homologous genomic regions in U. parvum (UU) and M. hominis (MHO). Homologous genes are connected by dashed lines. Phylogenetic trees inferred from amino acid sequences of the MHO_3120 homologs and MHO_3200 homologs are presented in (B) and (C), respectively. Bootstrap values are indicated on nodes. MSC, M. mycoides subsp. mycoides SC; MCAP, M. capricolum subsp. capricolum; MAG, M. agalactiae; mhp, M. hyopneumoniae; MGA, M. gallisepticum; MYPU, M. pulmonis; MS53, M. synoviae; MMOB, M. mobile. Numbers after the strain name indicate mnemonics.Analysis of the (0.73 MB TIF)Click here for additional data file.Table S1M. hominis, M. genitalium and U. parvum. These data are presented in an excel file to facilitate analysis. Orthology was determined using a bidirectional best hit method, followed by manual curation. The columns are as follows: (A) M. hominis gene locus (mnemonic); (B) M. hominis gene name; (C) M. hominis gene product; (D) M. genitalium gene locus (mnemonic); (E) M. genitalium gene name; (F) M. genitalium gene product; (G) U. parvum gene locus (mnemonic); (H) U. parvum gene name; (I) U. parvum gene product.Orthology between the genomes of (0.21 MB XLS)Click here for additional data file.Table S2M. hominis, M. genitalium and U. parvum:transposon insertions in orthologs from M. pulmonis, M. arthritidis, and M. genitalium and orthologs in other human and animal mycoplasma sequenced genomes. The columns are as follows: (A) Gene name, (B) Product, (C) Locus in M. hominis PG21 (mnemonic), (D) Locus in M. genitalium G37 U. parvum serovar 3 M. pulmonis according to M. arthritidis according to M. genitalium according to M. capricolum subsp. capricolum, M. mycoides subsp. mycoides SC, M. gallisepticum, M. genitalium, M. pneumoniae, M. penetrans, U. parvum, M. agalactiae, M. arthritidis, M. hyopneumoniae 232, M. mobile, M. pulmonis, M. synoviae, respectively. (V) Presence in all species.The 247 genes shared by (0.16 MB XLS)Click here for additional data file.


Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, B. cinerea and E.c. carotovora were found to readily infect Physcomitrella gametophytic tissues and cause disease symptoms. Treatments with B. cinerea spores or cell-free culture filtrates from E.c. carotovoraSCC1 (CF(SCC1)), resulted in disease development with severe maceration of Physcomitrella tissues, while CF(SCC3193) produced only mild maceration. Although increased cell death was observed with either the CFs or B. cinerea, the occurrence of cytoplasmic shrinkage was only visible in Evans blue stained protonemal cells treated with CF(SCC1) or inoculated with B. cinerea. Most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. CF treatments and B. cinerea inoculation induced the expression of the defense-related genes: PR-1, PAL, CHS and LOX.B. cinerea and E.c. carotovora elicitors induce a defense response in Physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. Since cytoplasmic shrinkage is the most common morphological change observed in plant PCD, and that harpins and B. cinerea induce this type of cell death in vascular plants, our results suggest that E.c. carotovora CFSCC1 containing HrpN and B. cinerea could also induce this type of cell death in Physcomitrella. Our studies thus establish Physcomitrella as an experimental host for investigation of plant-pathogen interactions and B. cinerea and elicitors of E.c. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant. PCR fragments were purified using Qiaquick columns (Qiagen), and were labelled with [α32P]-dCTP using Rediprime II Random Prime labelling system (Amersham Biosciences). After hybridization, membranes were washed twice for 30 min at 65°C with 5 × SCC, 0.1% SDS and twice 30 min with 2 × SCC, 0.1% SDS. Subsequently, membranes were exposed on autoradiography film. The amount of RNA loaded was verified by addition of ethidium bromide to the samples and photography under UV light after electrophoresis.Total RNA was isolated from control and treated plant tissue corresponding to 64 moss colonies, using standard procedures based on phenol/chloroform extraction followed by LiCl precipitation. Ten micrograms of total RNA separated by denaturing agarose-formaldehyde gels was transferred to a nylon membrane (Hybond N) following standard procedures . MembranIPDL participated in the Northern blot analysis and the microscopic studies, designed this study, drafted and edited the manuscript. JPO carried out the analysis of symptom development and all the microscopic studies. AC and MB participated in the cell death analysis by Evans blue staining. CG participated in the Northern blot analysis. SV helped to draft the manuscript. All authors read and approved the final manuscript.


To the Editor: Abortion in ruminants is of worldwide economic importance. Moreover, several abortigenic agents have a zoonotic potential, i.e., Brucella abortus, Coxiella burnetii, and Chlamydophila abortus.C. abortus, which causes ovine enzootic abortion, may also infect pregnant women who have had contact with C. abortus–infected sheep and goats, and such infection can lead to miscarriage . These specimens had previously been investigated for several abortigenic agents  by using a modified version of Everett’s PCR  as described , as reported (Parachlamydiaceae (Protochlamydia spp./Neochlamydia hartmannellae). To confirm positive results, another specific PCR, which targeted the tlc gene, was performed  and a specific mouse polyclonal antibody against Parachlamydia spp. was used as described  for the Parachlamydia antibody, respectively. Double immunohistochemical labeling was performed on the sheep abortion specimen identified as simultaneously infected with Chlamydiaceae and Parachlamydia spp. Immunohistochemical analysis for both pathogens was performed subsequently by using diaminobenzidine as substrate for the Chlamydiaceae antibody (brown labeling) and by using 3-amino-9-ethylcarbazole as substrate for the Parachlamydia antibody (red labeling). Specificity of PCR and immunohistochemical tests for Chlamydiaceae and Parachlamydia spp., respectively, was assessed by using negative control placentas taken from 2 healthy ruminants .Paraffin sections from specimens positive in real-time PCR were further examined by immunohistochemical tests. A Chlamydiaceae. All 55 cases could be identified as C. abortus by ArrayTube Microarray (Clondiag). Of these, 42 (76.4%) could be confirmed by immunohistochemical analysis with the anti-Chlamydiaceae antibody.Results of real-time PCR showed that 55 (26.1%) of 211 specimens were positive for Parachlamydia spp. by real-time PCR, and both cases could be confirmed by immunohistochemical testing with the parachlamydial antibody. These 2 specimens were negative for other common abortigenic agents such as Toxoplasma gondii, C. burnetii, and border disease virus (data not shown). One case was recorded among the 144 goat samples investigated. This placenta displayed necrotizing placentitis and was positive for Parachlamydia spp. by 16S rRNA-specific real-time PCR (cycle threshold [Ct] 40.5) and immunohistochemical testing, but negative for Chlamydiaceae. Results of this PCR was confirmed by another PCR, targeting the tlc gene (Ct 36.7), which excluded false-positive results because of amplicon contamination.Of the 211 specimens, only 2 (0.9%) were positive for A), interstitial pneumonia (B), and mixed cellular periportal hepatitis. Fetal liver was negative by parachlamydial 16S rRNA real-time PCR and immunohistochemical analysis, but the fetal lung was positive by parachlamydial 16S rRNA real-time PCR (Ct 40.7) and immunohistochemical tests (C), but negative with the tlc PCR. Fetal lung and liver were positive by real-time PCR for Chlamydiaceae (mean Ct for both organs 36.7), but negative by immunohistochemical tests. The placenta was positive for Chlamydiaceae by immunohistochemical tests and real-time PCR (mean Ct 23.3), and C. abortus was identified by ArrayTube Microarray. Brown (Chlamydiaceae) and red (Parachlamydia spp.) granular reaction was demonstrated within the necrotic lesions of the placenta by double immunohistochemical labeling (D).The second case was identified among the 86 sheep investigated. Placenta and fetal lung and liver exhibited necrotizing placentitis and vasculitis , panel Aneumonia , panel Bal tests , panel Clabeling , panel DParachlamydia infection in small ruminant abortion. C. abortus and Parachlamydia spp. were simultaneously present in an aborted sheep placenta. Parachlamydia spp. could be further detected in the lung of the aborted sheep fetus by real-time PCR and immunohistochemistry. Parachlamydia was also detected in a goat placenta. Thus, Parachlamydia spp. should be considered as a new abortigenic agent in sheep and goats. Persons in contact with small ruminants should be informed about the zoonotic potential of these abortigenic agents.We report


P. ginseng is currently found only in the Russian Primorye and the populations are extremely exhausted and require restoration. Analysis of the genetic diversity and population structure of an endangered species is a prerequisite for conservation. The present study aims to investigate the patterns and levels of genetic polymorphism and population structures of wild P. ginseng with the AFLP method to (1) estimate the level of genetic diversity in the P. ginseng populations in the Russian Primorsky Krai, (2) calculate the distribution of variability within a population and among populations and (3) examine the genetic relationship between the populations.The natural habitat of wild P. ginseng populations were investigated with Amplified Fragment Length Polymorphism (AFLP) markers. The genetic relationships among P. ginseng plants and populations were delineated.Genetic variability and population structure of ten P. ginseng. The partition of genetic diversity with AMOVA suggested that the majority of the genetic variation (64.5%) was within populations of P. ginseng. The inter-population variability was approximately 36% of the total variability. The genetic relationships among P. ginseng plants and populations were reconstructed by Minimum Spanning tree (MS-tree) on the basis of Euclidean distances with ARLEQUIN and NTSYS, respectively. The MS-trees suggest that the southern Uss, Part and Nad populations may have promoted P. ginseng distribution throughout the Russian Primorye.The mean genetic variability within populations was high. The mean level of polymorphisms was 55.68% at the population level and 99.65% at the species level. The Shannon's index ranged between 0.1602 and 0.3222 with an average of 0.2626 at the population level and 0.3967 at the species level. The analysis of molecular variances (AMOVA) showed a significant population structure in P. ginseng populations in the Russian Primorye are significant in genetic diversity. The high variability demonstrates that the current genetic resources of P. ginseng populations have not been exposed to depletion.The  Panax ginseng C.A. Meyer  is a representative species of the Panax L. genus which is a relic of the Araliacea family [P. ginseng spread over a vast territory including the Russian Primorsky Krai, Korea and China. Currently, wild P. ginseng can only be found in Russia; however, its populations are extremely exhausted and restoration is needed [P. ginseng is listed in the Red Book of Primorsky Krai as an endangered species [a family . Their na family . At the s needed . P. gins species .Analysis of the genetic diversity and population structure of an endangered species is a prerequisite for conservation . GeneticP. ginseng [Panax quinquefolius  [P. quinquefolius in the United States [P. quinquefolius population from Ontario, Canada, has a polymorphism level of about 46% estimated with RAPD analysis [A high level of polymorphism of a marker is a basic condition that must be assessed population genetics studies . A study ginseng . Allozymginseng) -16. Theranalysis .Miscanthus, three fluorescently labeled AFLP primer pairs generated 998 polymorphic markers, as opposed to only 26 polymorphic markers produced by two ISSR [As a reproducible and robust technique, AFLP  generatetwo ISSR .P. ginseng with the AFLP method to (1) estimate the level of genetic diversity in the P. ginseng populations in the Russian Primorsky Krai, (2) calculate the distribution of variability within a population and among populations and (3) examine the genetic relationship between the populations.The present study aims to investigate the patterns and levels of genetic polymorphism and population structures of wild P. ginseng individuals were collected from the ten administrative areas of Primorsky Krai , 19 from the Spassk area (Spa), 16 from the Ussuriisk area (Uss), 13 from the Dalnerechensk area (Drech), 16 from the Dalnegorsk area (Dgor), 15 from the Olginsk area (Olg), 15 from the Pozharsk area (Pozh), 24 from the Nadezhdinsk area (Nad), 19 from the Partizansk area (Part) and 10 from the Yakovlevsk area (Yak).One hundred and sixty-seven 167) 67 P. ginet al. [Total genomic DNA was extracted from fresh leaf tissue according to Echt et al. . The extet al. .et al. [EcoRI and MseI restriction enzymes. Pre-amplification reactions utilized AFLP primers with two selective nucleotides. EcoRI and MseI selective amplification primers contained three and four selective nucleotides, respectively (Table EcoRI-NNN selective primers were labeled with fluorescent 6-carboxy fluorescein (6-FAM) at the 5' end. The AFLP fragments were analyzed on an ABI Prism 3100 automated capillarity system with GeneScan Analysis Software . All unambiguous peaks including monomorphic peaks between 50-500 base pairs (bp) were analyzed and the scoring results were exported as a presence/absence matrix.AFLP genotyping was performed according to Vos et al.  using Ecly Table . AFLP adIS) [FST) between pairs of populations  were calculated. Gene flow between pairs of populations (Nm = (1-FST)/4FST) was calculated from FST values [P. ginseng and populations from a matrix of squared Euclidean distances using ARLEQUIN  and NTSYS  respectively.Parameters of genetic variability and genetic mutual relations of populations were calculated with the POPGEN32   and ARLEIS)  was usedIS)  was condT values . We recoEco(ACG)/Mse(CCTC), Eco(ACG)/Mse(CCTT), Eco(ACA)/Mse(CCTG), Eco(ACA)/Mse(CCGG), Eco(ACA)/Mse(CCAC), Eco(ACT)/Mse(CCGA), Eco(ACT)/Mse(CCTA), Eco(ACC)/Mse(CCAG), and Eco(ACC)/Mse(CCGC). Using two of the primer pairs Eco (ACA)/Mse(CCTG) and Eco(ACA)/Mse(CCGG)  were found in the Uss and Dgor populations. The mean level of polymorphisms was 55.68% at the population level and 99.65% at the species level. The Shannon's index ranged between 0.1602 and 0.3222 with an average of 0.2626 at the population level and 0.3967 at the species level. The intra-population genetic polymorphisms ranged from 38.65% (Uss) to 69.15% (Chu) with an average of 55.68% (Table Nine (9) AFLP primer pairs were tested, namely G) Table , we deteFST between populations, obtained with AMOVA, were significant (P = 0.0000) and varied from 0.09180 (Pozh-Nad) to 0.60506 (Drech-Uss) (Table Nm) between populations based on AMOVA (FST = 0.355) was 0.45.All pair wise s) Table . The nons) Table . The aveP. ginseng plants , whereas the Olg population was characterized by the highest value (36.5 units). The average value of Euclidean genetic distances between the plants of different populations (28.78 units) was higher than that of intra-population genetic distances (26.35 units)  by allozyme and RAPD [and RAPD ,27-29 whP. ginseng was revealed by the AFLP method. While genetic diversity is theoretically higher in large populations, the Uss population was small in size but appeared to have suffered from the loss of a genetic diversity more than other populations. Several populations  were distinguished by having higher levels of variability. For these populations, the average value of polymorphisms was 65.39%. At the species level, the percentage of polymorphisms was 99.65%. The high level of variability may be due to cross-pollination; however, P. ginseng's capability for cross-pollination is yet to be established [P. ginseng inflorescences are potential pollinators [Panax notoginseng, four pairs of fluorescently labeled AFLP primers produced 312 fragments, of which 240 (76.9%) were polymorphic [Panax stipuleanatus, the same primers revealed 346 loci, of which 334 (96.5%) were polymorphic [High genetic variability in ablished . A largelinators . In PanaP. ginseng. FST, estimates of inter-population variability, varied from 0.09180 to 0.60506  was 0.45; therefore, wild P. ginseng has a relatively high genetic differentiation value among populations and a relatively low level of gene flow. In cultivated P. ginseng, inter-population RAPD variability ranged from 1.77% to 42.01% [P. stipuleanatus was among the populations [P. ginseng' FST values are consistent with estimates of inter-population variability, which were obtained with AMOVA and AFLP markers for plant species with mixed type of propagation (FST = 0.35) [P. ginseng is a species with mixed type of propagation. The ability of P. ginseng to produce seeds via autogamy, out-crossing or agamospermy without pollination was demonstrated earlier [P. ginseng suggest that human activities  are the major contributor that threatens the survival of the wild P. ginseng populations.Analysis of molecular variance (AMOVA) of the AFLP data showed a significant population pattern of the wild Russian 06 Table , indicatty Table . The valo 42.01%  and was o 42.01% . The fluulations . P. gins = 0.35) . Accordi = 0.35) , P. gins earlier . The higUss, Part, Olg, Yak, Dgor and Drech) clustered together and four populations  were partially mixed with other populations , suggesting the ancestral status of the Uss population. The Part population, also at the central position on the MS-tree, may have the same ancestral status as the Uss population  that belongs to the n Figure ; Nad andl Figure . The abse Figure  may be eUss, Part and Nad populations may have promoted P. ginseng distribution throughout the Russian Primorye. This result supports the assumption that Sikhote-Alin was re-colonized by P. ginseng when thermophilic plants spread from the south to the north during the early Holocene warm period [The MS-trees suggest that the southern m period .P. ginseng and whether P. ginseng is adapted for heterogeneous conditions; (2) whether a positive correlation between genetic and geographical distances among P. ginseng populations may be established; and (3) using the multi-locus mating system program (MLTR) to estimate the level of inbreeding and cross-pollination in wild P. ginseng populations.Future studies may focus on (1) using AMOVA to investigate whether genetically differentiated regions exists for P. ginseng populations in the Russian Primorye contain a significant level of genetic diversity and are essentially differentiated. The gene flow of the populations was less than one (Nm = 0.45) which indicates continued divergence among populations [P. ginseng populations have not been exposed to depletion.The ulations . The curChu: Chuguevsk area; Spa: Spassk area; Uss: Ussuriisk area; Drech: Dalnerechensk area; Dgor: Dalnegorsk area; Olg: Olginsk area; Pozh: Pozharsk area; Nad: Nadezhdinsk area; Part: Partizansk area; Yak: Yakovlevsk area; bp: base pairs; AMOVA: Analysis of molecular variance; MS-tree: Minimum Spanning tree; 6-FAM: 6-carboxy fluoresceinAFLP: Amplified Fragment Length Polymorphism; ISSR: Inter Simple Sequence Repeat; AFLP: Amplified Fragment Length Polymorphism; The authors declare that they have no competing interests.YNZ and GDR designed the research. GDR and ILK performed the research and analyzed the data. TIM collected the plants. GDR wrote the manuscript. AAB contributed to the data acquisition. YNZ helped in writing the manuscript and coordinating the study. All authors read and approved the final version of the manuscript.


To the Editor:Bartonella are fastidious, aerobic, gram-negative, facultative, intracellular bacteria that infect erythrocytes, erythroblasts, endothelial cells, monocytes, and dendritic cells, and are transmitted by arthropod vectors or by animal scratches or bites  from North Carolina coastal waters. Reptilian erythrocytes are nucleated, and commercial lysis methods clogged filtration columns because of the high DNA content of whole blood. Consequently, DNA was extracted from frozen whole blood by using a modified alkaline lysis method adapted from an avian cell culture DNA extraction method  region  of 37 sea turtle blood samples tested, including samples from 13 free-ranging and 3 rehabilitated turtles. Pap31 PCR was performed for Bartonella ITS-PCR–positive samples. Pap31 amplicons were obtained from 5 samples of which 3 were successfully sequenced. Amplification and sequencing of the 16S-23S ITS region detected 2 Bartonella species: a B. henselae–like organisms and 1 more similar to B. vinsonii subsp. berkhoffii. The 3 Pap31 amplicons successfully sequenced confirmed B. henselae infection. Sequences obtained from 1 sample matched B. henselae strains H1-like, the B. henselae SA2-like strain, and B. vinsonii subsp. berkhoffii genotypes II and IV, which suggests that this turtle was co-infected with multiple Bartonella spp. and strains. Three other samples yielded amplicons 99%–100% identical with B. henselae strain SA2, and 3 yielded sequences most similar to B. vinsonii subspecies berkhoffii genotypes II and IV. Two samples contained an ITS region sequence most similar to B. henselae SA2, but with a 15-bp deletion beginning 617 bases downstream from the 16S rRNA gene. Whether these ITS sequence differences represent distinct strains or nonrandom translocation events is uncertain.The purpose of this study was to determine whether sea turtles are infected with ) region . BartoneB. vinsonii subsp. berkhoffii. However these amplicons were much shorter than expected for B. vinsonii subspecies berkhoffii genotype II and genotype IV sequences in GenBank. Because Pap31 gene amplification was unsuccessful for these samples, it is unclear whether small amplicons represent a species related to B. vinsonii subsp. berkhoffii or a new Bartonella sp.Four sea turtle blood samples contained partial ITS sequences most similar to Bartonella spp. DNA in sea turtle blood represents the first molecular evidence of Bartonella infection in nonmammalian vertebrates. B. henselae infection, now reported in porpoises and sea turtles, may represent an emerging infection of marine animals. According to previous studies, immune status appears to affect disease severity, variation in clinical manifestations, the pattern of histopathologic features, and the relative ease of diagnostic detection of the organism (Bartonella spp. in blood of sea turtles is a function of prior immunosuppression induced by stressors is not known. Such stressors could include mechanical injury, malnutrition, environmental toxins, parasites, or concurrent bacterial or viral infections. Alternatively, sea turtles may be a natural marine reservoir for B. henselae or for a Bartonella sp. genetically related to B. vinsonii subsp. berkhoffii.To our knowledge, detection of B. henselae and an organism genetically similar to B. vinsonii subsp. berkhoffii in the blood of loggerhead sea turtles provides evidence that this genus is not ecologically limited to terrestrial reservoirs. The geographic distribution, prevalence of infection, carrier potential, mode of transmission, and pathogenicity of bloodborne Bartonella spp. in sea turtles await additional studies.In summary, documentation of