Yersinia genomics identifies features responsible for the colonization of specific host habitats and the horizontal transfer of virulence determinants.Comparative  Yersinia that cause invasive human diseases  had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments.New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats.We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the  Of the millions of species of bacteria that live on this planet, only a very small percentage cause serious human diseases . ComparaYersinia genus of the family Enterobacteriaceae. The first published sequencing studies on the Yersinia genus have focused exclusively on invasive human disease-causing species that included five Yersinia pestis genome sequences  ..Y. pestiYersinia species have been split off from Y. enterocolitica over the past 40 years based on biochemistry, serology and 16S RNA sequence [Y. ruckeri is an agriculturally important fish pathogen that is a cause of 'red mouth' disease in salmonid fish. The species has sufficient phylogenetic divergence from the rest of the Yersinia genus to stir controversy about its taxonomic assignment [Y. fredricksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. bercovieri, and Y. rohdei have been isolated from human feces, fresh water, animal feces and intestines and foods [Y. aldovae is most often isolated from fresh water but has also been cultured from fish and the alimentary tracts of wild rodents [Y. aldovae from human feces or urine [While most attention is focused on the three well-known human pathogens, several other, less familiar sequence ,29. Y. rsignment . Y. frednd foods . There hnd foods  and lethnd foods . Y. aldo rodents . There ior urine .Yersinia species. From these genome sequences, we were able to determine the core gene set that defines the Yersinia genus and to look for clues to distinguish the genomes of human pathogens from less virulent strains.Using microbead-based, massively parallel sequencing by synthesis  we rapidYersinia species  in Additional file Previous studies and our experience suggest that at this level of sequence coverage the assembly gaps fall in repeat regions that cannot be spanned by single-end sequence reads (average length 109 nucleotides in this study) . Fewer R package . SpecifiY. aldovae (4.22 to 4.33 Mbp) and Y. ruckeri (3.58 to 3.89 Mbp), have a substantially reduced total genome size compared with the 4.6 to 4.8 Mbp seen in the genus generally. The agreement between the optical maps and sequence-based estimates of genome sizes tallied with experimental evidence for the lack of large plasmids in the sequenced genomes . Furthermore, the non-pathogenic species with the most IS matches, namely Y. bercovieri (167 matches), Y. aldovae (143 matches) and Y. ruckeri (136 matches), have comparatively smaller genomes. We also searched for novel repeat families using a de novo repeat-finder [Yersinia genus , was abundant in all the Yersinia genomes except the fish pathogen Y. ruckeri. Insertion sequence IS1541C in the IS finder database, which has expanded in Y. pestis (to more than 60 copies), had only a handful of strong matches in Y. enterocolitica, Y. pseudotuberculosis, and Y. bercovieri and no discernable matches in the other Yersinia genomes.The number of IS elements per genome for the eight species 12 to 167 matches) discovered using the IS finder database  was mucht-finder  and collldovae 14 matches 7 matchesaldovae 1 matches Yersinia genus. The assay design process commonly starts by computationally identifying genomic regions that are unique to the targeted genus ('signatures') - an ideal signature is shared by all targeted pathogens but not found in a background comprising non-pathogenic near neighbors or in other unrelated microbes. While many pathogens are well characterized at the genomic level, the background set is only sparsely represented in genomic databases, thereby limiting the ability to computationally screen out non-specific candidate assays . As a result, many assays may fail experimental field tests, thereby increasing the costs of assay development efforts. To evaluate whether the new genomic sequences generated in our study can reduce the incidence of false positives in assay development, we computed signatures for the Y. pestis and Y. enterocolitica genera using the Insignia pipeline [V. cholerae [Y. pestis and Y. enterocolitica, respectively, that represent good candidates for the design of detection assays. In Y. pestis these regions tended to cluster around the origin of replication, whereas in Y. enterocolitica there was a more even distribution. The average G+C content of the regions for the unique sequences in both species was close to the Yersinia average (47%) and there was not a strong association with putative genome islands . Interestingly, 171 Y. pestis gene regions were spread over only 70 different genes, whereas the 96 Y. enterocolitica regions were found overlapping only 90 genes. There was no obvious trend in the nature of the genes harboring these putative signals except that many could be arguably classed as 'non-core' functions, encoding phage endonucleases, invasins, hemolysins and hypothetical proteins.The sequences generated in this study provide new background information for validating genus detection and diagnosis assays targeting pathogenic members of the pipeline , the syscholerae . We idenaverage 4% and theY. pestis-specific and 31 Y. enterocolitica-specific putative signatures have significant matches in the new genome sequence data  and obtained 98 locally collinear blocks  for the concatenated aligned region was 0.27, or an approximate genus-wide nucleotide sequence homology of 73%. As expected for a set of bacteria with this level of diversity, the alignment of the genomes shows evidence of multiple large genome rearrangements [We performed a multiple alignment of the 11 lgorithm   [Yersinia genome and to be nearly identical in length. We used ClustalW [Y. ruckeri; Y. pestis/Y. pseudotuberculosis; and the remainder of the 'enterocolitica'-like species. Y. kristensenii ATCC33638T was the nearest neighbor of Y. enterocolitica 8081. The outlying position of Y. ruckeri was confirmed further when we analyzed the contribution of the genome to reducing the size of the Yersinia core protein families set. If Y. ruckeri was excluded, the Yersinia core would be 2,232 protein families of N = 2 rather than 2,072  . We founClustalW  to alignClustalW   COGs in Y. ruckeri  hits  for eachi Figure .Yersinia proteomes were investigated for common clusters in the three high virulence species missing from the low human virulence genomes . Enhancin was originally identified as a factor promoting baculovirus infection of gypsy moth midgut by degradation of mucin [Y. pestis/Y. pseudotuberculosis linked with insect infection, the TccC and TcABC toxin clusters [Y. mollaretti. In Y. mollaretti the Tca and Tcc proteins show about 90% sequence identity to Y. pestis/Y. pseudotuberculsis and share identical flanking chromosomal locations. Further work will need to be undertaken to resolve whether the insertion of the toxin genes in Y. mollaretti is an independent horizontal transfer event or occurred prior to divergence of the species.The s Figure . Becauserculosis ,13,15 weof mucin . Other lclusters , were alY. enterocolitica and the other two pathogens was reduced to 12 and 13 for Y. pseudotuberculosis and Y. pestis, respectively  [ye, containing a type IV pilus gene cluster and other putative virulence determinants, such as arsenic resistance, is similar to a 99-kb YAPIpst that is found in several other serotypes of Y. pseudotuberculosis [Y. pestis and the serotype I Y. pseudotuberculosis strain IP32953 [Y. pseudotuberculosis and Y. enterocolitica and subsequent degradation to various degrees within the Y. pseudotuberculosis clade. However, the complete absence of YAPI from any of the seven species in the Y. enterocolitica branch  ,56,57, arculosis ,57 but i IP32953 . A modelh Figure , as wellcolitica , argues Y. enterocolitica in general and biotype 1B in particular turned out to have orthologs in the low human virulence species sequenced in this study. These included several putative biotype 1B-specific genes identified by microarray-based screening [Y. ruckeri, Y. mollaretii, and Y. aldovae. Y. aldovae and Y. mollaretii also had islands containing ysa type three secretion systems (TTSS) with 75 to 85% nucleotide identity to the homolog in Y. enterocolitica 1B. The ysagenes are a chromosomal cluster [Y. enterocolitica, at least, appears to play a role in virulence [Y. enterocolitica ysa genes are found in the plasticity zone  and 2 island . SpeciesY. enterocolitica genome was demonstrated further by two examples gleaned from comparison with the evolutionarily closest low human virulence genome, Y. kristensenii ATCC 33638T  locus responsible for non-specific surface binding. Y. kristensenii ATCC 33638T had an identical 13 gene tad locus in the same position, but the nucleotide sequence identity of the region to Y. enterocolitica 8081 was uniformly lower than that found for the rest of the genome, suggesting there had been either a gene conversion event replacing the tad locus with a set of new alleles in the recent history of Y. kristensenii or Y. enterocolitica or the locus was under very high positive selective pressure.The modular nature of the islands found in the T Figure . The YGIY. enterocolitica genome to Y. pestis and Y. pseudotuberculosis revealed some potentially significant metabolic differences that may account for varying tropisms in gastric infections [Y. enterocolitica 8081 alone contained entire gene clusters for cobalamin (vitamin B12) biosynthesis (cbi), 1,2-propanediol utilization (pdu), and tetrathionate respiration (ttr). In Y. enterocolitica and Salmonella typhimurium [Yersinia genus  is maintained. The loss of these genes in Y. pestis has been interpreted as a consequence of adaptation to an obligate host-dwelling lifecycle, where the availability of the sulfur-containing amino acids is not a nutritional limitation [s Figure  with theobiology   that colonize the mammalian gut. Y. pestis has lost many of these genes by deletion or disruption since its split from the enteric pathogen Y. pseudotuberculosis and adoption of an insect vector-mediated pathogenicity mode. The smaller Y. ruckeri chromosome does not appear to result from recent reductive evolution (as is the case of Y. pestis), evidenced by the relatively low number of frameshifts and pseudogenes, and the normal amount of repetitive contigs in the newbler genome assembly. Like Y. pestis, Y. ruckeri lacks urease, methionine salvage genes, and B12-related metabolism. The prevailing consensus is that the pathway of transmission of red mouth disease in fish is gastrointestinal yet the similarities of Y. ruckeri genome reduction to Y. pestis hint at an alternative mode of infection for Y. ruckeri.We cannot claim complete coverage of all the type strains of the  created -79 sinceE. coli. Bile salt hydrolase in pathogenic Brucella abortus has been shown to enhance bile resistance during oral mouse infections [Yersinia genome evolution rather than a formula specific to pathogens. Comparative genomic studies such as these can be used to enhance our ability to rapidly assess the virulence potential of a genome sequence of an emerging pathogen and we plan to continue to build more extensive databases of non-pathogenic Yersinia genomes that will allow us to draw conclusions with more statistical power possible than just 11 representative species.This comparative genomic study reaffirms that the distinguishing features of the high-level mammalian pathogens is the acquisition of a particular set of mobile elements: HPI, the pYV, pMT1 and pPCP plasmids, and the YADI island. However, the eight species sequenced in this study believed to have either low or zero potential for human infection, contain numerous, apparently horizontally transferred genes that would be considered putative virulence determinants if discovered in the genome of a more serious pathogen. Two examples are yaldo0001_40900  and yfred0001_36480, an ortholog of the TibA adhesin of enterotoxigenic fections . The lowfections  (Additiofections ). Addingfections  is, therYersinia species studied range in estimated size from 3.7 to 4.8 Mb. The nucleotide diversity (Π) of the conserved backbone based on large collinear conserved blocks was calculated to be 0.27. There were no orthologs of genes and predicted proteins in the virulence-associated plasmids pYV, pMT1, and pPla, and the HPI of Y. pestis in the genomes of the type strains - eight non- or low-pathogenic Yersinia speciesGenomes of the 11 Yersinia pathogen species  were not found on at least one of the other eight species. Therefore, our study is in agreement with the hypothesis that genes acquired by recent horizontal transfer effectively define the members of the Yersinia genus virulent for humans.Apart from functions encoded on the aforementioned plasmids, HPI and YAPI regions, only nine proteins detected as common to all three Yersinia species consists of approximately 2,500 proteins. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of COG families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogenases and B12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats.The core proteome of the 11 Y. ruckeri, a salmonid fish pathogen, is the earliest branching member of the genus and has the smallest genome (3.7 Mb). Like Y. pestis, Y. ruckeri lacks functional urease, methionine salvage genes, and B12-related metabolism. These losses may reflect adaptation to a lifestyle that does not include colonization of the mammalian gut.Y. enterocolitica clade' genomes likely indicates that YAPI was acquired independently in Y. enterocolitica and Y. pseudotuberculosis.The absence of the YAPI island in any of the seven 'Y. pestis and Y. enterocolitica, respectively, that represented potential candidates for the design of nucleotide sequence-based assays for unique detection of each pathogen.We identified 171 and 100 regions within the genomes of Yersinia species sequenced in this study (Table Y. ruckeri) on Luria media. DNA for genome sequencing was prepared from overnight broth cultures propagated from single colonies streaked on a Luria agar plate using the Promega Wizard Maxiprep System .Type strains of the eight dy Table  were acqde novo using the newbler program .Genomes were sequenced using the Genome Sequencer 20 Instrument  . LibrariAflII and NheI  were constructed by Opgen Inc. . The newbler assemblies for each genome were scaffolded using the optical maps and the SOMA package [-20) to identify sequences that could potentially be part of an extrachromosomal element.Optical maps  for each package  , compatible with most genome browsers . To find known insertion sequences, the genomes were scanned for matches using the IS finder web service [-10 (matches to known repeat contigs were counted as multiple matches based on the coverage of the contig). In addition, we searched for common repeat sequences in the genome using the RepeatScout program [-10 to record matches. The resultant figures for repeat content are estimations that may be lower than the true number found in the genomes.The presence of repeats is known to confound assembly programs and the  service  with a B program  after du program  . The accession numbers are: Yersinia genomes were aligned using the standard MAUVE [P by ± 5%  based on optical map data.Click here for fileE. coli strain with known plasmids was a positive control.An Click here for fileSequences of the detected repeat families.Click here for fileY. pestis CO92 signatures longer than 100 bp computed by the Insignia [Insignia  pipelineClick here for fileY. pestis CO92 signatures listed in Additional file Sequences of the new genomes that match  the Click here for fileY. enterocolitica signatures longer than 100 bp computed by the Insignia pipeline.Click here for fileY. enterocolitica signatures.Sequences of the new genomes that match  the Click here for fileY. pestis genome with the Insiginia-indentified repeats and genome islands identified using IslandViewer [ndViewer  plotted.ndViewer .Click here for fileY. enterocolitica genome with the Insiginia-indentified repeats and genome islands identified using IslandViewer [ndViewer  plotted.ndViewer .Click here for fileThe eight genomes sequenced in this study are represented as pseudocontigs, ordered by a combination of optical mapping and alignment to the closest completed reference genome.Click here for fileYersinia genomes in XMFA format [Whole genome multiple alignment produced by MAUVE of the 11 A format .Click here for fileYersinia_unclustered.out - list of all protein singletons that MCL did not group into a cluster ; PGL1_Yersinia_unique_locus_tags.txt - names of the 11 locus tag prefixes used for each genome; PGL1_unique_Yersinia.gff - mapping each Yersinia protein to a cluster in tab delimited GFF; PGL1_unique_Yersinia.sigfile - list of the longest protein in each cluster; PGL1_unique_Yersinia.summary - summary table of features of each of the clusters; PGL1_unique_Yersinia.table - summary table of each protein in the clusters. Within each cluster directory are the following files, where 'x' is the cluster name: PGL1_unique_Yersinia-x.faa - multifasta file of the proteins in the cluster; PGL1_unique_Yersinia-x.summary - summary of the properties of the proteins; PGL1_unique_Yersinia-x.matches - blast matches between the proteins of the cluster; PGL1_unique_Yersinia-x.muscle.fasta - muscle alignment of the proteins; PGL1_unique_Yersinia-x.muscle.fasta.gblo - gblocks output of muscle alignment ; PGL1_unique_Yersinia-x.muscle.fasta.gblo.htm - as above in html format; PGL1_unique_Yersinia-x.muscle.tree - treefile from muscle alignment; PGL1_unique_Yersinia-x.sif - matches between proteins in simple interaction format for display on graphing software.The top level directory consists of a directory called Additional_cluster_files and 5010 directories, one for each multi-protein cluster family. ; PGL1_Yersinia_unique_locus_tags.txt - names of the 11 locus tag prefixes used for each genome; PGL1_unique_Yersinia.gff - mapping each Yersinia protein to a cluster in tab delimited GFF; PGL1_unique_Yersinia.sigfile - list of the longest protein in each cluster; PGL1_unique_Yersinia.summary - summary table of features of each of the clusters; PGL1_unique_Yersinia.table - summary table of each protein in the clusters. Within each cluster directory are the following files, where 'x' is the cluster name: PGL1_unique_Yersinia-x.faa - multifasta file of the proteins in the cluster; PGL1_unique_Yersinia-x.summary - summary of the properties of the proteins; PGL1_unique_Yersinia-x.matches - blast matches between the proteins of the cluster; PGL1_unique_Yersinia-x.muscle.fasta - muscle alignment of the proteins; PGL1_unique_Yersinia-x.muscle.fasta.gblo - gblocks output of muscle alignment ; PGL1_unique_Yersinia-x.muscle.fasta.gblo.htm - as above in html format; PGL1_unique_Yersinia-x.muscle.tree - treefile from muscle alignment; PGL1_unique_Yersinia-x.sif - matches between proteins in simple interaction format for display on graphing software.The top level directory consists of a directory called Additional_cluster_files and 5010 directories, one for each multi-protein cluster family. ; PGL1_Yersinia_unique_locus_tags.txt - names of the 11 locus tag prefixes used for each genome; PGL1_unique_Yersinia.gff - mapping each Yersinia protein to a cluster in tab delimited GFF; PGL1_unique_Yersinia.sigfile - list of the longest protein in each cluster; PGL1_unique_Yersinia.summary - summary table of features of each of the clusters; PGL1_unique_Yersinia.table - summary table of each protein in the clusters. Within each cluster directory are the following files, where 'x' is the cluster name: PGL1_unique_Yersinia-x.faa - multifasta file of the proteins in the cluster; PGL1_unique_Yersinia-x.summary - summary of the properties of the proteins; PGL1_unique_Yersinia-x.matches - blast matches between the proteins of the cluster; PGL1_unique_Yersinia-x.muscle.fasta - muscle alignment of the proteins; PGL1_unique_Yersinia-x.muscle.fasta.gblo - gblocks output of muscle alignment ; PGL1_unique_Yersinia-x.muscle.fasta.gblo.htm - as above in html format; PGL1_unique_Yersinia-x.muscle.tree - treefile from muscle alignment; PGL1_unique_Yersinia-x.sif - matches between proteins in simple interaction format for display on graphing software.The top level directory consists of a directory called Additional_cluster_files and 5010 directories, one for each multi-protein cluster family. (This top level directory has been split into three data files for uploading purposes (Additional files Click here for fileYersinia.Complete protein sets for the 11 species of Click here for fileE. coli was used as an outgroup species.To evaluate node support, a majority rule-consensus tree of 1,000 bootstrap replicates was computed. Click here for fileE. coli was used as an outgroup species.To evaluate node support, a majority rule-consensus tree of 1,000 bootstrap replicates was computed. Click here for fileA curve showing the rate of decline in number of this set as more non-pathogen genomes are added is also included.Click here for fileYersinia and other enterobacteria species.Phylogeny of TTSS component YscN in Click here for fileYersinia genus determined using the Antibiotic Resistance Genes Database [Putative antibiotic resistance genes in the Database .Click here for fileYersinia core genomes.Calculations for the estimation of Π from aligned Click here for file


P. falciparum and P. vivax, respectively. Interestingly, non-immunosuppressive FK506 analogues also show antimalarial activities. This prompted us to attempt to develop potential small molecule antagonists that specifically target the parasite enzymes devoid of immunosuppressive activity. To this end, we have determined the three-dimensional structures of the FK506 binding domain (FKBD) of P. falciparum and P. vivax in unligand form and in complex with FK506.The structural studies reveal that the Plasmodium FKBPs have similar structural folds like the canonical FKBP folds . SRA exhibits a nanomolar inhibitory activity in both peptidylprolyl cis-trans isomerase (PPIase) assay and growth of


Arabidopsis thaliana, and compared it to data gathered in outcrossing Arabidopsis lyrata. We analyzed seven TE families in four natural populations of each species to tease apart the effects of mating system, demography, transposition, and selection in determining patterns of TE diversity.Transposable Elements (TEs) make up the majority of plant genomes, and thus understanding TE evolutionary dynamics is key to understanding plant genome evolution. Plant reproductive systems are diverse and mating type variation is one factor among many hypothesized to influence TE evolutionary dynamics. Here, we collected a large TE-display data set in self-fertilizing A. thaliana Ac elements differed significantly from that other TEs, perhaps signaling a lack of recent transposition. Across TE families, we estimated higher allele frequencies and lower selection coefficients on A. thaliana TE insertions relative to A. lyrata TE insertions.Measures of TE band differentiation were largely consistent across TE families. However, patterns of diversity in Arabidopsis species represents a synthesis of evolutionary forces that include the transposition dynamics of individual TE families and the demographic histories of populations. There are also species-specific differences that could be attributed to the effects of mating system, including higher overall allele frequencies in the selfing lineage and a greater proportion of among population TE diversity in the outcrossing lineage.The differences in TE distributions between the two  Arabidopsis thaliana genome [Transposable elements (TEs) are prevalent in plant genomes  and ubiqa genome . Moreovea genome ,11. ThesDrosophila melanogaster. These studies have revealed that there are far fewer TE insertions in the D. melanogaster genome than possible insertion sites [Population genetics has the potential to illuminate these forces, but our understanding of the population genetics of TEs has been based primarily on studies of on sites ,13 and ton sites -14. Bothon sites -18. Howeon sites . Purifyion sites ,19-23. Aon sites -28.higher TE copy numbers than outcrossing species. Conversely, the deleterious effects of recessive TE insertions are expected to be stronger in a homozygous selfer, which may result in more efficacious selection and fewer TEs in selfers [The mating system of host species is likely to be an important factor that shapes TE dynamics ,29,30. F selfers ,29,30. TNe) in a selfing species is expected to be half that of an otherwise identical outcrosser [Nes, where s is the strength of selection. It is thus not surprising that empirical studies suggest that shifts in Ne over time influence the number and frequency of TEs [Mating system influences the efficacy of selection against TEs in at least two other ways: First, the effective population size ; Helitron Basho TEs ("Basho"); CACTA; Gypsy-like ("Gypsy"); LINE-like ("LINE"); SINE-like I ("SINE"); and Tourist-like MITE ("MITE") elements. These TEs represent three RNA-mediated class I retrotransposons  and four class II DNA transposons . The primers used to generate Ac TE-display data were identical to those used by Wright et al. [A. lyrata populations: 11 individuals from Plech, Germany, 12 from Karhumäki, Russia, 12 from North America, and nine from Stubbsand, Sweden.We sampled four populations of ce (TAIR ). The sace  [PT, a statistic analogous to FST that measures genetic differentiation among populations. For our analyses, we used TE-display bands as genetic markers, and thus ΦPT became a measure of TE display band differentiation. Our analyses were performed with two different packages: GenAlEx 6 [PT between populations and the R package ade4 [PT among all populations.To measure levels of population differentiation in our  (AMOVA) . We focuenAlEx 6  was usedage ade4  was usedA. thaliana TE-display data to A. lyrata data by estimating TE allele frequencies and copy numbers in both species. We used estimates of the inbreeding coefficient (F) to estimate TE allele frequencies from dominant TE display data. For each A. lyrata population, F was estimated independently using (SNP) data in 77 loci [We compared our  77 loci  by , wheer locus , and  ispi is the frequency of the ith of k alleles, summed over the lth of m SNP loci [where SNP loci .F was estimated directly for these populations. However, they sampled two North American populations  that were combined to yield our North American sample. We thus average F for our North American sample. To estimate F in A. thaliana, we assumed the proportion of selfing (S) in A. thaliana was 0.99 [F = 0.98 from the relation F = S/(2 - S) [Ross-Ibarra et al.  sampled was 0.99  and esti/(2 - S) .F, we estimated pTE, the TE allele frequency, usingGiven estimates of z is the observed frequency of the null TE display band , q is the estimated null TE allele frequency, and q = 1 - pTE. We calculated allele frequency estimates both within populations and across entire species' samples., where znTE, the expected TE copy number of an individual, as:We calculated pi is TE allele frequency of the ith locus, summing over m TE loci. Ii is an indicator variable, where Ii = 1 when a TE band is present, and Ii = 0 when a TE band is absent, at the ith locus in a given individual. Bands fixed within our sample were included in our calculations of allele frequencies and nTE.where Nes) from our TE display data. Lockton et al. [Nes is compound parameter, but following Petrov et al. [Ne based on nucleotide polymorphism data. We used the point estimates of Ne inferred from demographic modeling of the same four A. lyrata populations - i.e., Germany Ne = 136,000; North America Ne = 11,000; Russia Ne = 12,000; and Sweden Ne = 12,000 . Species-wide A. lyrata Ne was calculated to be 250,000 by using estimates of θ from SNP data [μ) of 1.5 × 10-8 [A. thaliana, we also used estimates of θ from SNP diversity data [Ne, assuming μ = 1.5 × 10-8 [Ne was estimated to be 125,000; the UK population was 98,500; Germany, 83,000; and both New York and Indiana was 71,000. However, results did not differ qualitatively when Ne was assumed to be 125,000 in each A. thaliana population (data not shown).We used the Maximum Likelihood (ML) approach of Petrov et al. , with mon et al.  modifiedv et al.  we assumSNP data , and ass5 × 10-8 . For A. ity data  to estim5 × 10-8 . SpeciesA. thaliana across seven TE families. To compare, in A. lyrata, 274 bands were amplified in six TE families [Ac, CACTA, Gypsy, LINE, MITE, and SINE) there were more TE bands in outcrossing A. lyrata (n = 274) than in the self-fertilizing A. thaliana (n = 210). A sample of the bands amplified using TE family-specific primers were cloned, sequenced, and identified: 95% (20/21) of the A. thaliana bands were successfully identified as TEs belonging to their respective families (data not shown). The single unidentified A. thaliana Ac band showed strong sequence similarity to an "unknown protein"  in the A. thaliana genome sequence.We identified 267 TE display bands in families . Of the Basho bands in A. thaliana, but few strong bands in A. lyrata. The putative Basho bands that were amplified, cloned, and sequenced from A. lyrata could not be identified in TE databases using BLAST. Because of the uncertainty of the A. lyrata Basho data, they were not included in additional analyses. These empirical results are consistent with previous studies suggesting that some Basho subfamilies may be absent from A. lyrata [We readily identified 57 . lyrata .A. thaliana band differentiation between populations for each TE family  to estimate allele frequencies (pTE) from our TE band data (see Methods). This method intrinsically corrects for possibilities that A. lyrata may not always be obligately outcrossing , which form the basis for inferring the strength of selection [A. lyrata median pTE was 0.061, but the A. thaliana median pTE of 0.125 was substantially higher (Table p = 1.28 × 10-6). In addition to lower median frequencies, the A. lyrata SFS showed a skew towards lower frequency insertions in A. lyrata relative to A. thaliana  but doeelection ,66. We cana Fig. . This skana Fig. . The stat et al.  for Ac eF into the model [A. thaliana's Nes estimates for pooled TEs were not significantly different from zero, and individual TEs pooled across populations also yielded Nes estimates very close to zero  that are hypothesized to be more prevalent in an outcrosser, or to complicating factors in selfers  that slow selection [A. lyrata relative to A. thaliana may come from combining data from relatively more diverged populations , slightly positive for a third , and undefined (not estimable) for the fourth population  and A. thaliana has slightly higher grand average of 0.268 (sd ± 0.030). Similar trends are produced by taking the average of medians across populations: A. lyrata: 0.189 ± 0.050; A. thaliana: 0.198 ± 0.048. Thus, in considering individual populations - as opposed to 'species-wide' samples - there is a slight trend toward lower population frequencies of TEs in A. lyrata, consistent with the notion that selection against TEs is stronger in the outcrosser. However, the effect is much muted relative to the species-wide sample, suggesting that population differentiation and demography contributes to some of the differences between pooled, species-wide samples.These considerations make it difficult to determine whether there really is a systematic difference in SFS between the outcrosser and the inbreeder. However, nTE (see Methods). If selection is more effective in a heterozygous outcrosser, nTE is predicted to be lower in A. lyrata than in A. thaliana. Per-population nTE, summed over all six shared TE families (Table p = 0.649). Among A. thaliana TE families, median nTE varied per population, but nTE varied among populations more in A. lyrata  or by nTE  to copy number (nTE) could lead to a systematic underestimate of copy number in A. lyrata. Second, TE display bands represent TE sequence found on a limited range of band sizes (~50 to 1000 bases); if there are general differences in TE sizes between species then copy number comparisons may be inaccurate. There is reason to believe that this would trend toward an underestimate in A. lyrata: for example, early comparisons of gene structure between the congeners suggest that introns are generally larger in A. lyrata [A. thaliana we amplified 52 different MITE elements from the Tourist-like subfamily, whereas at least 818 MITEs have been found in the A. thaliana genome sequence [Overall, our study is like previous studies in that we detect apparent allele frequency differences between an outcrossing and an inbreeding species but no systematic differences in nTE ,40,42. H. lyrata  and may . lyrata . Finallysequence .A. thaliana) and an outcrosser (A. lyrata).The motivation for this study was to determine whether observed differences in the frequency and population dynamics of TEs can be attributed to species-wide effects, which presumably reflect differences between outcrossing and selfing mating systems, or are better attributed to factors like transposition dynamics and demographic history that may also differ between species. Our study is unique in that we sampled multiple TE families and multiple populations to compare population dynamics between a selfer , virtuan, e.g., ,40,42. SPT values (Fig. A. lyrata, demographic events perturb selection-transposition equilibria and influence the distribution and frequency of TEs [A. thaliana, although the magnitude of these effects is difficult to estimate with the present data.Our data also clearly demonstrate that demographic history shapes TE diversity, because pairwise comparisons involving geographically closer populations often have lower Φues Fig. . In A. ly of TEs ,33,47. PA. thaliana at both species-wide (Fig. Finally, some aspects of the data cannot be easily attributed to demography or transposition and thus may reflect differences due to host mating system. These include: the apportionment of diversity within and between populations Fig. ; an SFS ide Fig.  and popuide Fig. . GeneralSL carried out the molecular biology work, analyzed the data, and drafted the manuscript. BSG designed the study, assisted with data analysis, and helped draft the manuscript. Both authors read and approved the final manuscript.Nes maximum likelihood estimates. Maximum likelihood estimates of the strength of selection, including 95% confidence intervals, for TEs pooled across populations and across TE families in both species.Click here for file


P. knowlesi was the first Plasmodium species in which antigenic variation was observed. Variation was due to schizont infected cell agglutination (SICAvar) antigens expressed by the parasite and transported to the exposed surface of the host erythrocyte [P. falciparum's orthologue of P. knowlesi's SICA proteins [P. falciparum PfEMP1 is associated with infected erythrocytes binding to receptors such as ICAM-1 expressed on the endothelial cells of the host microvasculature. Here, we use a static protein assay [P. knowlesi infections can cause erythrocytes to bind to ICAM-1, VCAM-1 and CD36.throcyte . PfEMP1 proteins . In P. fin assay  to deterP. knowlesi infection at two well-established study sites in Sarawak, Malaysian Borneo. The samples were washed and cultured ex vivo until the majority of parasites had matured to late trophozoite/early schizont stages of development. The parasites were then assayed for their ability to bind potential endothelial ligands. Static assays were carried out with purified proteins, ICAM-1, VCAM-1 and CD36. P. falciparum (cloneHB3) was used as a positive control.Blood samples were collected after obtaining written consent from patients presenting with knowlesi-patient isolates compared with P. falciparum control assays will be presented.The relative binding of post-mortem - findings from a fatal case of P. knowlesi.The results of the binding assays will be presented and discussed in the context of recently described


Erwinia amylovora have either not been identified or have not yet been studied.Two-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive , negative , and intermediate regulators for swarming motility in E. amylovora were also identified.We used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.Our results demonstrated that TCSTs in  Prokaryotes use their small size and metabolic diversity to dominate every conceivable niche on earth. A large part of this success comes from the evolution of elaborate sensory systems to monitor and respond to dramatic fluctuations in their environment. Primary means of signal transduction in bacteria involve two-component signal transduction systems (TCSTs) ,2. The tEscherichia coli . The Gae review ,31, prote review , and type review ,29,33-35e review ,36. Most systems ,20. An aE. amylovora Ea273  identified a total of 46 TCST genes in E. amylovora including 17 sensor kinases, 20 response regulators, three hybrid HKs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases. A comparative genomic analysis of TCSTs was then conducted in related enterobacteria enabling classification of the TCSTs in E. amylovora. A total of 59 deletion mutants were generated, and their contribution to virulence, amylovoran biosynthesis, and swarming motility was characterized. Our findings suggested that TCSTs in E. amylovora played a major role in virulence and in the regulation of amylovoran biosynthesis and swarming motility. This indicated that networks of gene regulation existed in E. amylovora that respond to different environmental and host signals.In this study, our genome-wide analysis, using the recently closed genome sequence of E. amylovora. First, using a candidate gene approach, known HK and RR sequences from Es. coli K12 and P. carotovora pv. atroseptica SCRI1043 were used to search the closed genome sequence of E. amylovora . Second, based on conserved domains of known HKs and RRs, other putative HKs and RRs in E. amylovora were identified and confirmed by searching the complete genome sequences for proteins containing HK and RR domains using Pfam Hidden Markov Model (HMM) profiles and BLASTP. As a result, we identified 46 TCST and other related signal transduction genes in E. amylovora (Table cheABRWYZ). These putative regulatory genes represented 1.36% of the genome (3367 genes) [We utilized two approaches to identify HKs and RRs in the genome of 7 genes) . Among tE. amylovora have counterparts in related enterobacteria, including Dickeya dadantii (Erwinia chrysanthemi) 3937, P. carotovora subsp. atroseptica SCRI1043, Erwinia tasmaniensis Et1/99, and Es. coli K12 (Table ypdAB) in E. tasmaniensis is not present in E. amylovora Ea273  identity/similarity to those of E. tasmaniensis Et1/99, an epiphytic bacterium isolated from apple flowers in Australia [Erwinia pyrifoliae, a related blight pathogen of Asian pear . Classification of HKs is usually based on alignment of residues surrounding the H-box that contains the conserved His residue within the histidine kinase domain (HisKA) ,40. Multr groups  and Figur groups ). This iE. amylovora HKs, by either searching the Conserved Domain Databases (CDD) [, has revealed that all HKs in E. amylovora, except for HrpX and NtrB, contain one to three hydrophobic transmembrane (TM) helices within the N-terminal sensor region. This indicates that these HKs are periplasmic sensors. HrpX and NtrB contain two and one PAS domain , respectively, within the N-terminal sensor region, indicating that they are soluble and cytoplasmic proteins that sense intracellular signals [Analysis of the domain architecture of es (CDD)  or usinges (CDD) , has rev signals . The YehE. amylovora , one double (rcsBD), and one triple mutant (hrpXYS), were non-pathogenic on immature pears . Therefore, phenotypes of the 59 deletion mutant strains were evaluated using a swarming plate assay [E. amylovora WT and mutant strains, bacterial suspensions were plated onto swarming agar plates containing 0.3% agar, as previously described [grrA and grrS, exhibited substantially larger and lower density swarms than the WT . In contrast, envZ/ompR mutants showed reduced swarming even after 72 hr  for the complemented mutant strains was comparable to that of the WT strain (Data not shown). However, the swarming phenotype remained different between envZ/ompR mutants and those of flhDC and fliA mutants, which exhibited no movement and maintained the original size of 0.6 to 0.7 cm in diameter , as previously described [in vitro were identified  exhibited lack of amylovoran production  showed a dramatic increase in amylovoran production, about 20-fold, compared to that of the WT  operon, was measured using GFP as a reporter in two RR mutants (grrA and ompR). GFP intensity was measured in the WT and in mutants containing the amsG promoter-GFP fusion using flow cytometry [amsG gene was expressed at a basal level in the WT strain, with a GFP intensity value of 1.7 (geometric mean), compared to a geometric mean value of 1.5 for the control vector. The geometric mean value of the GFP intensity of the amsG promoter was 26.2 and 60.5 for ompR and grrA mutants, respectively  of E. amylovora is also the smallest among sequenced enterobacterial plant and mammalian pathogens [E. amylovora still maintains both enterobacterial-specific TCSTs (such as the Rcs system) [In this study, 46 TCSTs have been identified in athogens . However system)  and plan system) .E. amylovora has almost the same number of TCSTs as that found in E. tasmaniensis, a saprophytic bacterium isolated from apple flowers in Tasmania, Australia [E. tasmaniensis outside of Australia remains unknown. Surprisingly, we found that the majority of TCSTs in these two bacteria share a high level of aa similarity and identity. These results suggest that similar TCSTs might play important roles for the survival and proliferation among closely-related bacteria in similar plant niches.On the other hand, ustralia . Since bustralia . It is pE. amylovora, most HKs belong to four common HK groups, and are periplasmic sensors. Moreover, most RRs in this bacterium are OmpR-, NarL-, NtrC-, and LytR-like proteins, the four most common families of DNA-binding RRs found in prokaryotes [E. amylovora has maintained some basic signal transduction systems for the bacterium to survive. Phylogenetic and genomic analyses have revealed co-evolutionary relationships between cognate HKs and RRs. This seems obvious in E. amylovora as orphan HKs and RRs are rare.Currently, several thousands of TCSTs have been identified in sequenced bacterial genomes -57. Althkaryotes . These ret al. [X. campestris pv. campestris has revealed that two novel RRs are involved in virulence, thus facilitating future studies on signaling networks in this bacterium [E. amylovora, which will also provide valuable tools for future global gene expression assays using microarrays to deduce signaling networks in this bacterium.It is well understood that TCSTs are involved in regulating virulence gene expression in plant bacterial pathogens . Previouet al.  providesacterium . In thisE. amylovora, the Hrp T3SS, which delivers effector proteins into host plants, and the EPS amylovoran are two major virulence factors [hrp-T3SS gene expression. Recently, we have further demonstrated that the Rcs system is essential for virulence in E. amylovora and may play a role in the survival of the pathogen [hrpX, hrpY and hrpXY mutants remain virulent, and could induce a spotty weak hypersensitive response (HR) on tobacco  in tobacco is severely attenuated as showed in an HR assay  amended with 1% sorbitol [Bacterial strains and plasmids used in this study are listed in Table sorbitol ,75.[[TMHMM2 program [. Proteins without known domain were characterized by using PSI-BLAST of NCBI and methods described previously [Plasmid DNA purification, PCR amplification of genes, isolation of fragments from agarose gels, cloning, restriction enzyme digestion and T4 DNA ligation were performed using standard molecular procedures . DNA seq. Protein or the S[. Domain  program . Multipl program . Sequenceviously .E. amylovora stable mutants were generated by using the λ phage recombinases as previously described [E. amylovora Ea1189 was transformed with plasmid pKD46 expressing recombinases redα, β, and γ. The transformant Ea1189 (pKD46) was grown overnight at 28°C, reinoculated in LB broth containing 0.1% arabinose, and grown to exponential phase OD600= 0.8. Cells were collected, made electrocomponent, and stored at -80°C. Recombination fragments consisting of a kanamycin (kan) or chloramphenical (cat) gene with its own promoter, flanked by a 50-nucleotide (nt) homology arm, were generated by PCR using plasmid pKD13 or pKD3 as a template. The primers used for generating mutants are listed in Table S2 [see Additional file kan gene, cm1 and cm2 of the cat gene were used. For the resulting mutants, the majority of the coding region of each gene was replaced by the kan or cat gene, except for the first and last 50 nt. The resulting mutants were designated and listed in Table escribed -47. BrieE. amylovora WT strain which contains upstream and downstream sequences of the gene or operon, and PCR fragments were cloned into either pGEM T-easy vector through A-T ligation or into a low-copy number vector pWSK29. The final plasmids were listed in Table E. amylovora strain by electroporation. Transformants were selected on LB plates supplemented with Ap and Km or Cm. Their genotypes were confirmed by both enzymatic digestion and sequencing.For complementation of selected mutants, flanking sequences of genes or operon were used to design primers to amplify fragments of gene/or operon and their promoter sequences  with bacterial cells adjusted to concentrations of ~1 × 103 to 1 × 104 colony-forming units (CFU/μl) (OD600 = 0.1 and then diluted 100 times) in PBS. Immature fruits of pear (Pyrus communis L. cv. 'Bartlett') were surface-sterilized, and pricked with a sterile needle as described previously [For eviously ,46. WounE. amylovora WT, mutants, and complementation strains, bacterial suspensions were grown overnight in LB broth w/o appropriate antibiotics, harvested by centrifugation, and washed with PBS three times. After the final wash, the bacterial pellet was resuspended in 200 μl PBS. A total of 100 μl of bacterial suspension was inoculated into 10 ml MBMA medium with 1% sorbitol. One ml of bacterial cells was pelleted two to three days after inoculation at 28°C with shaking. Following centrifugation, 50 μl CPC at 50 mg ml-1 was added to one ml supernatant. After 10 min of incubation at room temperature, the amylovoran concentration was determined by measuring OD600 turbidity. The final concentration of amylovoran production was normalized for a cell density of 1.0. For each strain tested, the experiment was repeated at least three times.The amylovoran concentration in supernatants of bacterial cultures was quantitatively determined by a turbidity assay with cetylpyrimidinium chloride (CPC), as previously described ,52. BrieE. amylovora WT and mutant strains, bacterial suspensions were grown overnight in LB broth w/o appropriate antibiotics, harvested by centrifugation, washed with PBS once, and resuspended in 200 μl PBS. Then, bacterial suspensions were diluted 10 × in water, and 5 μl of the diluted bacterial suspension was plated onto the center of swarming agar plates  as previously described [For escribed ,51. SwarAmylovoran production and swarming motility data were undertaken a one-way ANOVA and Student-Newman-Keuls test to determine differences in means (P = 0.05) using SAS 9.1 program.gfp construct [in vitro amsG gene expression, WT and mutants containing the amsG promoter-GFP fusion plasmid were grown in LB overnight, harvested, and resuspended in water. Bacterial suspensions were re-inoculated in MBMA broth with 1% sorbitol and grown at 28°C with shaking for up to three days. Bacterial cultures were then harvested by centrifugation, washed once with PBS, and then resuspended in PBS for flow cytometry assay. Flow cytometry was performed on a BD LSRII 10 parameter multilaser analyzers . For both cases, data were collected for a total of 100,000 events and statistically analyzed by gating using flow cytometry software FCS Express V3 . A geometric mean was calculated for each sample. Each treatment was performed in triplicate and each experiment was repeated three times.The BD FACSCanto flow cytometer was used to monitor the GFP intensity of WT and mutant strains containing the corresponding promoter-onstruct . For in PER, ARNT, and SIM proteins; REC: receiver domain; RR: response regulator; TCST: two-component signal transduction system; T3SS: type III secretion system.CDD: Conserved Domain Databases; CPC: cetylpyrimidinium chloride; EAL: Type I c-di-GMP phosphodiesterase; EPS: extracellular polysaccharide; HisKA: histidine kinase domain; HK: histidine kinase; HMM: Hidden Markov Model; Hpt: histidine phosphotransfer; HTH: helix-turn-helix; GGDEF: diguanylate cyclase; PAS: initially found in YFZ designed and coordinated the project, YFZ, DPW, and SN performed the experiments and analyzed the data; GWS and SSK contributed reagents/materials, analyzed and discussed the results; YFZ wrote the manuscript and GWS and SSK critically read the manuscript. All authors have read and approved the final manuscript.E. amylovora and other related enterobacteria and primers used in this studyComparison and classification of TCSTs in . The file contains two tables. Table S1 compares and classifies TCSTs in E. amylovora and other related enterobacteria; Table S2 lists primers used in this study.Click here for fileStructural classification of histidine kinases, hypersensitive response of HrpXYS mutants in tobacco and schematic map of HrpXYS operon. The file contains two figures. Figure S1 shows structural classification of histidine kinases (HKs) based on phosphorylation sites (H-boxes); Figure S2 shows hypersensitive responses (HR) on tobacco for WT, ΔT3SS [hrpX, hrpY, hrpXY, and hrpXYS mutants  and a schematic map of the HrpXYS operons .T, ΔT3SS , hrpX, hClick here for file


Francisella tularensis, where F. tularensis subspecies holarctica has long been the cause of endemic disease in parts of northern Sweden. Despite this, our understanding of the natural life-cycle of the organism is still limited. During three years, we collected surface water samples (n = 341) and sediment samples (n = 245) in two areas in Sweden with endemic tularemia. Real-time PCR screening demonstrated the presence of F. tularenis lpnA sequences in 108 (32%) and 48 (20%) of the samples, respectively. The 16S rRNA sequences from those samples all grouped to the species F. tularensis. Analysis of the FtM19InDel region of lpnA-positive samples from selected sampling points confirmed the presence of F. tularensis subspecies holarctica-specific sequences. These sequences were detected in water sampled during both outbreak and nonoutbreak years. Our results indicate that diverse F. tularensis-like organisms, including F. tularensis subsp. holarctica, persist in natural waters and sediments in the investigated areas with endemic tularemia.Tularemia, caused by the bacterium  Francisella tularensis. At present, four subspecies of F. tularensis are suggested .http://www.smi.se/in-english/statistics/tularaemia/) , located 364 km south of Ljusdal, with a population of 274 121 (2005). Before 2000 only a handful of cases were reported from the county and limited numbers of cases occurred in 2001 and 2002. However, between 2003 and 2005, 229 human cases of tularemia were reported (raemia/) . The tulraemia/)  31]..http://wFor the first year sampling (2003), several sampling points were chosen , based on the knowledge of local physicians about the geographical distribution of human tularemia cases . In 2004F. tularensis cultivation and DNA preparation for polymerase chain reaction (PCR) analysis.Rodents were collected using live-traps , baited with a mixture of carrots, potatoes, oatmeal, and pieces of apples, from mid-May to mid-September. Trapping was performed in Ljusdal (on four occasions) and Örebro (two occasions) during 2003, and in Örebro (five occasions) during 2004. At each sampling during 2003, traps were set for five days, and in 2004 traps were set for two to five days. Traps were checked every 12 hours. Trapped rodents were anesthetized using halothane and euthanized through cervical dislocation. Carcasses were kept refrigerated during transportation to a local laboratory, where spleen and liver samples were prepared and deep-frozen (within four hours of euthanization) until further analysis. After thawing, spleen and liver samples were used for Francisella tularensis was cultured on modified Thayer-Martin agar plates . Moreover, Svensson et al. 2009, identified an association between disease clusters  and recreational areas adjacent to water in the Ljusdal and Örebro tularemia areas [Laboratory experiments have shown that r months . Howeverr months , 43, 44.acterium . Furtheracterium –29. The acterium . In suchacterium , 47. In  wetland . Altogetia areas . As statia areas  (BerglunF. tularensis and the environmental reservoir of the bacteria have long been subject to speculation. Our working hypothesis is that F tularensis subsp. holarctica persists in water and/or sediment between tularemia outbreaks. Data presented here support this hypothesis, although the factors promoting the spread of the bacterium to susceptible hosts remain to be revealed.The natural life-cycle of


Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage.P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters.The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.With the exception of its ability to degrade lactose,  Propionibacterium belongs to the class of high GC actinobacteria. All species of this genus produce propionic acid as a major metabolic end-product. However, taxonomical studies clearly distinguished two groups of propionibacteria: cutaneous and dairy. Over a century ago, P. freudenreichii was the first dairy species isolated from Emmental cheese P. acidipropionici, and P. thoenii have been found in milk and cheese, and sometimes also in various biotopes like silage, soil, rumen, and waste water The genus P. freudenreichii has received the American Generally Recognized as Safe (GRAS) status. It has also been granted the European Qualified Presumption of Safety (QPS) status. During ripening, this species drives the fermentation of lactate into propionate, acetate and CO2, via the Wood-Werkman cycle, resulting in the formation of the characteristic “eyes” and flavor of these cheeses. P. freudenreichii also plays an essential role in the production of other flavor compounds, such as free fatty acids released via lipolysis of milk glycerides, and branched-chain acids resulting from the catabolism of amino acids P. freudenreichii concerns production of vitamin B12, one of nature's most structurally complex small molecules. Most of the vitamin B12 pathway has been elucidated Due to its long documented use in cheese, and in particular in Swiss type cheeses, P. freudenreichii, as the bifidogenic compound it produces, 1,4-dihydroxy-2-naphthoic acid (DHNA) P. freudenreichii adapt very well to gastric and bile salt stresses in vivo in the rat or human gut In vitro, P. freudenreichii produces beneficial metabolites, including short chain fatty acids, and conjugated linoleic acid; some strains like P. freudenreichii JS also exhibit immunomodulatory activity Finally, there is increasing interest in the probiotic activity of Propionibacterium genus is that of the commensal cutaneous species P. acnesThe only completely sequenced, fully annotated and publicly available genome within the P. freudenreichii subsp. shermanii CIRM-BIA1T have been studied by several teams around the world. Genome sequencing will provide the molecular basis of traits important for cheese or probiotic applications, and will allow a better assessment of the “distance” between the two main groups of propionibacteria: dairy and cutaneous.The physiology and technological properties of P. freudenreichii CIRM-BIA1T consists of a circular chromosome of 2,616,384 base pairs (bp) with 67% GC content. The genome contains 2 rRNA operons and 45 tRNAs  belonging to four different families  according to the nomenclature at http://www-is.biotoul.fr : (1) ISPfr21 contains a transposase gene and two other genes, one coding for PFREUD_08140, a transcription factor belonging to the TetR regulator family, and the other coding for PFREUD_08150, a protein of unknown function similar (42%) to a protein of Sinorhizobium sp.; (2) a mobile insertion cassette (MIC) with the same termini as ISPfr11 but without the transposase gene is located between 371,214 and 372,936 bp. This MIC contains a gene encoding a protein of unknown function, PFREUD_03020, similar (52%) to a protein of Kineococcus radiotolerans. In most cases, the IS insertion has no obvious impact because very few genes are present.The transposase gene is mutated in ISPfr8: a guanidine insertion created a frameshift observed in all three copies rendering it non-functional. IS Aar30 was also found in P. freudenreichii can induce phenotypic changes. The first example is provided by an IS upstream the gtf gene, PFREUD_19370. The gtf gene encodes a polysaccharide synthase involved in the synthesis of capsular exopolysaccharides (EPS). While the presence of capsular EPS in P. freudenreichii is strain-specific, all strains tested were shown to possess one gtf copy. Recently, we demonstrated that the presence of an IS element within the putative promoter region of gtf enhanced gtf transcription, and was associated to the capsular phenotype P. freudenreichii CIRM-BIA1T is able to degrade lactose but this trait is strain-dependent. In the genome, the lactose locus consists of three genes, PFREUD_02370, PFREUD_02360 and PFREUD_02350, encoding a β-galactosidase, LacZ, a galactoside transporter, GalP, and an UDP-glucose isomerase, GalE1, respectively. These three genes are surrounded by integrases (PFREUD_24460) and transposases (PFREUD_24470) (Leifsonia xylii) to GalE of other actinobacteria, but LacZ and GalP are similar to those of several species of Mannheimia succiniciproducens and of Clostridium . These two species, like propionibacteria, are present in the cow rumen ecosystem. These observations strongly suggest that the Lac genes may have been acquired through a horizontal transfer event mediated by phage infection, which could explain why this ability is strain dependent. As lactose fermentation is one of the two phenotypic criteria used to distinguish the subspecies P. freudenreichii subsp. shermanii (positive for lactose fermentation) from P. freudenreichii subsp. freudenreichii (negative for lactose fermentation), the justification of these subspecies should be further investigated. Since no difference in GC content was observed in these regions relative to the rest of the genome, this locus may have been acquired a long time ago.However, the transposable elements of D_24470) . GalE isattP and attB sites are unknown. One temperate phage closely related to the L5 Mycobacterium sp.), several proteins of unknown function, and a phage integrase (PFREUD_04210) with 45% similarity with the integrase of mycobacteriophage L5 (Swissprot entry VINT_BPML5).The phages in the genome could not be precisely located because the Several traces of prophages were also identified in the genome. They are evidenced by the presence of 31 integrase loci, scattered throughout the chromosome, but with most (22/31) clustered less than 100 kb from each other. Six regions of the chromosome have clusters of integrases: (1) between 124,000 and 129,000 bp, two copies of the PFREUD_24770; (2) from 233,000 to 291,000 bp, three integrases PFREUD_24990, PFREUD_24980 and PFREUD_24460 at the lactose locus; (3) from 427,000 to 509,000, three integrases ; (4) from 1,825,318 to 1,836,629 bp, PFREUD_24770 and PFREUD_24830; (5) from 2,288,000 to 2,295,000 bp PFREUD_24990, PFREUD_24980; and, lastly, (6) from 2,432,000 to 2,482,000, PFREUD_21190, PFREUD_22610 to PFREUD_22650, PFREUD_25160, and PFREUD_25070. These regions also have a lower GC content . This ilP. freudenreichii ITG P18 and the presence of prophage was detected using hybridization experiments with phi101 in many species, including CIRM BIA1 Streptococcus thermophiluscas (CRISPR associated) genes. Three CRISPR loci were identified in the P. freudenreichii genome using the CRISPR finder tool  sequence GCCTCAATGAAGGGCCCCTCCAGAAGGAGGGGCAAT and 34 spacer sequences of 35 to 40 bp. Blast results on the EMBL phage database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the third spacer displays a strong similarity (32/39 nt identity) to the Stenotrophomonas phage S1 (EU849489), and that the 26th and 27th spacers shared a high level of identity with orf6 and orf9 of the Propionibacterium phage phiB5, a filamentous ssDNA inovirus CAAGCGCCCTGCTGTGTTCGTTT and only one spacer, without homology. The second one, located between 2,215,874 and 2,216,038, contains a 24 bp DR sequence CTTCTTCGCCGCCGGCTTCTTGGC and three spacer sequences without homology in the database. The PFREUD_03690 gene for cas1/cas4 associated CRISPR proteins was found near the replication origin between 399 and 2000 bp but no CRISPR was associated with it. The presence of CRISPR loci indicates that P. freudenreichii has been in contact with phages, and strongly suggests that they may contribute to its resistance to phage attacks.Phage attacks are frequently reported during cheese manufacture using lactic acid bacteria, but not for dairy propionibacteria. However, lysogeny was fully demonstrated for P. freudenreichii was illustrated by the formation of a capsular EPS and the ability to ferment lactose. The presence of the CRISPR loci may protect P. freudenreichii against phage attacks.In conclusion, IS and prophages are a source of plasticity and adaptability of the bacteria to various environments, and the influence of transposable elements on two phenotypic traits in P. acnes is the only species within the Propionibacterium genus with a complete genome sequence publicly available. A comparison of this species with P. freudenreichii CIRM-BIA1T was carried out. P. acnes has a 2.5 Mb and 60% GC genome composed of 2333 putative genes P. acnes is a major inhabitant of human skin and is considered to be an opportunistic pathogen, while P. freudenreichii has GRAS status. The two species, however, share some similarities. Both species can grow under microaerophilic and anaerobic conditions, and produce propionic acid as the main fermentation product. To compare gene colinearity, we aligned the genomes at the protein level because the nucleotide sequences were too divergent  were found in P. freudenreichii . These proteins were annotated in P. freudenreichii as “cell-wall peptidase, NlpC/P60 family secreted protein”, due to their high similarity (73% and 76%) to S. coelicolor Q9KY71_STRCO and Q9KY68_STRCO, respectively, and the presence of a signal peptide and Pfam 00877 domain.Belonging to the cutaneous group, ivergent . A relatest hits , no simiP. freudenreichii and P. acnes because lipolytic activity is an important feature in both species. P. freudenreichii has a major role in the hydrolysis of milk glycerides during cheese ripening P. acnes lipases are thought to degrade human skin lipids such as sebum. Free fatty acids resulting from this activity could promote bacterial adherence and colonization of the sebaceous follicle P. acnes and P. freudenreichii showed that the two species shared a common pool of intracellular esterases, but differed in secreted esterases/lipases. P. acnes produces a secreted lipase, GehA, and another lipase with 41% identity to GehA at the protein level, encoded by the genes PPA2105 and PPA1796, respectively P. freudenreichii. P. freudenreichii was recently shown to produce a secreted lipase active on milk fat, encoded by PFREUD_04340 P. acnes. Of the 11 putative esterases identified in the P. freudenreichii genome, six predicted cytoplasmic proteins and one potentially surfaced exposed protein P. acnes.We also compared the putative esterases/lipases in P. acnes was identified in P. freudenreichii. This finding is consistent with the GRAS and QPS status of P. freudenreichii.In conclusion, no pathogenicity factor from P. freudenreichii is able to grow, under anaerobic conditions, in a minimal medium containing a carbon source, ammonium as the sole nitrogen source, minerals, and two to four vitamins. Like most other P. freudenreichii strains, CIRM-BIA1T is able to use a variety of carbon substrates, including sugars , alcohols , and acids  P. freudenreichii  encoding a ribulokinase is a pseudogene in the CIRM-BIA1T strain. At least one other araB gene is present at another locus of the genome (PFREUD_22370) and may complement the pseudogene. However, no transporter for arabinose was found, which may explain why CIRM-BIA1T does not catabolize L-arabinose. The ability to use propanediol, a less common growth substrate, was reported for P. freudenreichii subsp. freudenreichii ATCC 6207 T possesses a complete pdu (propanediol utilization) operon (from PFREUD_08980 to PFREUD_09150), with predicted proteins similar to those of Salmonella enterica serovar Typhimurium Salmonella, an integrase-coding gene was identified at the end of the locus, suggesting that this operon could have been acquired through horizontal transfer.Experimentally, nreichii . For exaP. freudenreichii is prototrophic for all amino acids and nucleotides. The complete biosynthetic pathways of all amino acids were reconstituted .Regarding nitrogen requirements, p-aminobenzoic acid in addition T, with the exception of pantothenate and biotin. In our study, it was shown  of one strain of P. freudenreichii was previously described in detail E. coli and were sequenced. The organization of genes for vitamin B12 synthesis in CIRM-BIA1T is in agreement with the results reported for this strain  and hemD) were discovered and revealed another locus putatively responsible for cobalt transport (PFREUD_20190 and 20200). The complete description of vitamin B12 synthesis pathway in P. freudenreichii opens up new prospects for genetic engineering and for the screening of highly productive strains using molecular tools.Regarding vitamins, all propionibacteria strains require pantothenate (vitamin B5) and biotin (vitamin H). Some strains require thiamine (B1) and hown see  that pans strain . HoweverP. freudenreichii and Pelobacter propionicusBacteroïdes fragilisVeillonella parvulaVeillonella gazogenesClostridium propionicumP. freudenreichii at biochemical P. freudenreichii is a transcarboxylation reaction without the involvement of free CO2. The enzyme catalyzing this reaction is a methylmalonyl-CoA carboxytransferase, transferring a carboxyl group from methylmalonyl-CoA to pyruvate to form oxaloacetate and propionyl-CoA  of the Swiss-Prot entry Q8GBW6, and would be a functional but truncated protein, as in the P. acnes genome P. acnes but do not agree with the crystallographic data of the 12S subunit in P. freudenreichiiPropionic acid is the major end product of fermentation in propionibacteria and confers their typical flavor to Swiss-type cheeses. Two pathways of propionate formation have been described in bacteria. The first pathway, known as Wood-Werkman cycle, involves succinyl-CoA and methylmalonyl-CoA as intermediates. It was first described in onyl-CoA . The enz2. A partial TCA cycle was also reconstituted, but lacked a succinyl-CoA hydrolase (E.C.3.1.2.3)  . This acaspA (PFREUD_16320 and PFREUD_16330), fumarate is then converted to succinate, with regeneration of oxidized co-enzymes and ATP. The intensity of this conversion depends on the conditions and on the P. freudenreichii strain 2 at the expense of pyruvate, which is reduced to propionate via the Wood-Werkman cycle. In addition to this aspartate catabolism pathway, other genes encoding aspartate-converting enzymes were found in the CIRM-BIA1T genome: aminotransferases , L-aspartate oxidase, ) and adenylosuccinate synthetase . Each enzymatic activity can impact intracellular aspartate concentration and can modulate CO2 production, consequently affecting the size of the eyes that form in cheese.The co-metabolism of aspartate/asparagine and lactate was previously reported P. freudenreichii is usually grown under anaerobic or microaerophilic conditions, it possesses all the genes required for aerobic respiration: genes encoding NADH dehydrogenase , succinate dehydrogenase , cytochrome bd complex (cydA and cydB), ATPase  and the complete pathway for heme synthesis (hem genes). Under anaerobic conditions, the electron acceptor in P. freudenreichii can be sulfate, fumarate, menaquinone (vitamin K2), or the pool of ferrous iron and humic acid in the soil T has no denitrification capacity. In fact, the gene corresponding to the beta subunit of nitrate reductase is a pseudogene due to a frameshift. The ability to reduce nitrate is the second criterion used to distinguish the subspecies shermanii (negative) from freudenreichii (positive).Although P. freudenreichii displays numerous features which allow its long-term survival, including the accumulation of energy and carbon storage compounds, the accumulation of compatible solutes, and the induction of a multi-tolerance response under carbon starvation.P. freudenreichii is able to accumulate inorganic polyphosphate (polyP) as an energy reserve whereas most bacteria utilize ATP. Only bacteria particularly adapted to extreme environments are able to use polyP P. freudenreichii strain ST33 grown on lactate accumulates large amounts of long-chain polyP, up to 3% of the cell dry weight ppk mutants are unable to survive during stationary phase P. freudenreichii CIRM-BIA1T, the ppk gene is followed by genes coding for NUDIX (a kind of pyrophosphohydrolase) and for an ABC phosphate transporter. Several polyP or pyrophosphate using enzymes were found in the genome  pathway and catabolised by trehalose synthase (TreS). The genes otsA, otsB, and treS were previously identified in strain NIZO B365 T and the corresponding proteins showed 99%, 99% and 100% similarity, respectively, to the previously reported sequences.P. freudenreichii is also known to accumulate glycine betaine. In addition to osmotic stress adaptation dha1, PFREUD_01860).P. freudenreichii CIRM-BIA1T survives for a very long time at room temperature even under conditions of carbon starvation, and is considered a non-lytic strain. Starvation and stationary phases induce a multi-tolerance response in P. freudenreichiiKoccuria sp, Mycobacterium sp. and Rhodoccocus sp P. freudenreichii genome, PFREUD_06100 encodes a protein similar (50%) to the Rpf (resuscitation promoting factor) protein from Mycobacterium smegmatis ATCC 700084. Rpf is an essential protein for the growth of dormant cells from different actinobacteria and is propably involved in long-term survival of P. freudenreichii.P. freudenreichii strains in long-term stationary phase conditions. The long-term survival ability is favorable for both probiotic and cheese starter applications of dairy propionibacteria.In conclusion, several genes involved in phosphate, glycogen, and trehalose metabolism and a gene encoding an Rpf protein are good candidates to explain the survival of a majority of groSL operon , the clpB ATP-dependent protease , the endopeptidase clpP , the dnaKJ operon , and grpE  and 3 copies of the surface serine protease htrA  and the hsp20 chaperone  were found in the genome. The redundancy and inducibility of this chaperone and protease machinery in P. freudenreichiiGeneral stress adaptation genes, key factors of probiotic capacity, are multicopy and stress-induced in P. freudenreichii. Two copies of the P. freudenreichii genome reveals a complete atpBEFHAGDC operon, whose gamma chain  is induced in P. freudenreichii by acid and bile salts nifJ, PFREUD_01840) and succinate dehydrogenase , which are involved in electron transport and ATP synthesis, as well as glutamate decarboxylase  and aspartate ammonia-lyase , which are involved in intracellular pH homeostasis. Proteins involved in protection and repair of DNA are crucial for survival. Genome analysis demonstrated the presence of members of the SOS response including lexA, recA and uvrABC in P. freudenreichii. Moreover, the helix-destabilizing Ssb protein (PFREUD_23460), which is involved in DNA recombination and repair, as well as Dps (PFREUD_02870), which protects DNA against oxidative stress, are stress-induced in P. freudenreichiiddlA, PFREUD_13250) and the UDP- MurNAc-pentapeptide synthetase  murF gene localizes to the same locus as the entire set of genes corresponding to peptidoglycan biosynthesis in P. freudenreichii, suggesting co-regulation. Stress-induced genes include the branched-chain aminoacid transferase IlvE (PFREUD_13350), which is involved in the synthesis of branched-chain fatty acids that are important for stress tolerance in S. aureusP. freudenreichii possesses an arsenal of genes for disulfide-reduction and elimination of reactive oxygen species. The P. freudenreichii genome encodes a redundant thioredoxin system  and two peptide-methionine-S-oxide reductases . Moreover, in response to bile salts, P. freudenreichii over-expressed the iron/manganese superoxide dismutase , catalase , Glutathione S-transferase , two cysteine synthases  and S-adenosylmethionine synthetase . The occurrence of a sodium/bile acid symporter, PFREUD_14830, reflects adaptation to the gut environment. Moreover, we identified four genes encoding multidrug resistance transporters  indicating an ability to cope with toxic compounds. Two genes encoding heavy metal translocating P-type ATPases  further suggest adaptation to toxic environments. The genome suggests a significant ability to sense changes in the environment. Eleven two-component regulatory system histidine kinases were detected . Finally, stress-induced genes pnpA, PFREUD_14570) and the inosine-5-monophosphate dehydrogenase , suggesting the ability to synthesize the alarmone ppGpp in response to stress.Stress-induced genes reflect the ability to cope with digestive (acid and bile) stresses. Regulation of intracellular pH is crucial for survival. Analysis of the P. freudenreichii maintains an active metabolism in animal and human guts iol operon, mostly detected in inhabitants of soil but also in L. casei BL23 P. freudenreichii, consistent with utilization of myo-inositol by this bacterium. Finally, the preferred carbon source for propionibacteria, lactic acid, is one of the end products of colic fermentation by indigenous bacteria including lactic acid bacteria and bifidobacteria.The genome reveals adaptation to the nutritional environment of the gut. P. freudenreichii genome. The protein was identified by proteomics. Moreover, PFREUD_05930 exhibited 53% sequence similarity to the Bifidobacterium bifidum surface lipoprotein BopA, which is involved in adhesion to colonocytes and immunomodulation gtf gene (PFREUD_19370), responsible for the biosynthesis of a P. freudenreichii surface polysaccharide was discovered. A - β-D-glucan capsule has been reported to affect immunomodulation in Pediococcus parvulusP.freudenreichii surface proteins in adhesion Interaction with the host gut is strongly suggested by the presence of genes encoding key surface proteins. Analysis of the genome using SurfG+ in vivo during transit through the digestive tract P. freudenreichii is able to release in vivo short chain fatty acids known for their beneficial effects on colon epithelial cells T genome. DHNA is synthesized by the naphtoate synthase (or dihydroxynaphtoic acid synthethase) in P. freudenreichii encoded by menB (PFREUD_07540).Some genes support production of beneficial metabolites. The methylmalonyl-CoA carboxytransferase operon is induced by acid and bile salt stress and is also induced 9 colony-forming units (cfu)/g. However, P. freudenreichii shows poor growth in milk, even for the subspecies shermanii which is able to ferment lactose. P. freudenreichii is able to grow in Swiss cheese, mainly due to its ability to ferment lactic acid under anaerobic conditions, the abundance of peptides due to the activity of the lactic acid starter bacteria  induced by stress and starvation in P. freudenreichii . L-lactate enters the cells due to the expression of a L-lactate permease (PFREUD_18660). No gene encoding a D-lactate permease was found in the genome. The two isomers of lactate are converted into pyruvate by specific lactate dehydrogenases, two NAD-dependent L-lactate dehydrogenases encoded by two paralogs (PFREUD_11570 and PFREUD_12840), and one FAD-dependent D-lactate dehydrogenase (PFREUD_16710).P. freudenreichii also plays a key role in the formation of other cheese flavor compounds. It releases free fatty acids through milk fat lipolysis and short branched-chain fatty acids through amino acid catabolism E. coli, and five putative esterases were also found in the P. freudenreichii genome P. freudenreichii strain JS, estAT strain. The sequences flanking estA were close to the lactose locus . This result shows that P. freudenreichii cannot be considered a milk-adapted species.In lactic acid bacteria, adaptation to the rich dairy niche is associated with gene decay, leading to metabolic simplification and auxotrophy. In contrast, P. freudenreichii genome reveals the hardiness of the bacterium, its ability to cope with different stresses , to withstand phage attack, to accumulate glycogen and polyphosphate under favorable conditions, to mobilize these compounds during starvation conditions and, lastly, to synthesize most vitamins and amino acids. Unlike lactic acid bacteria such as L. bulgaricus, Propionibacteria do not seem to be over-adapted to cheese conditions. The presence of beta galactosidase probably resulting from a horizontal transfer event seems to be the unique adaptive trait revealed by our study. The genetic basis of the P. freudenreichii capacity to produce aromatic compounds was in agreement with those described at the biochemical level: the Wood-Werkman cycle producing propionic acid, pathways for production of acetic acid and CO2, production of branched-chain fatty acids from leucine and isoleucine, and discovery of an extracellular lipolytic esterase. Regarding probiotic activity, the complete pathway for synthesis of a bifidogenic compound was reconstructed and a large number of surface proteins involved in adhesion and immunomodulatory activity, as in the Lactobacillus and Bifidobacterium genera, were identified. Functional validations should now be investigated using transcriptomic and inactivation-complementation approaches. These investigations should take into account the large panel of P. freudenreichii phenotypes for aromatic and probiotic properties among the thousands of strains stored and described in microbiological resource centers for this species. The versatility and the high adaptability of the species could also include non-alimentary perspectives.Annotation of the P. freudenreichii subsp. shermanii CIRM-BIA1T .The strain used for sequencing was P. freudenreichii subsp. shermanii CIRM-BIA1T (Genbank accession number FN806773) was determined after genomic DNA fragmentation by mechanical shearing or BamHI partial digest for the construction of plasmid and large insert libraries, respectively. The 3 kb (A), 10 kb (B) and 25 kb (C) fragments were cloned onto pCNS  and (B)) and pBBc (pBeloBac11 derived (C)). Vector DNAs were purified and end-sequenced (9216 (A), 29184 (B) and 5376 (C)) using dye-terminator chemistry on ABI3730 sequencers. A pre-assembly was made without repeat sequences as described by Vallenet et al. www.phrap.org). The finished genome sequence was achieved by primer walking using a GC sequence finishing kit  and transposition bombs. A complement of 4004 sequences  was needed for gap closure and quality assessment .The complete sequence of Automatic and manual annotations were conducted with the AGMIAL platform For the vitamin B12 synthesis pathway, the gene nomenclature from P. acnes. The homology of P. freudenreichii predicted proteins with proteins putatively involved in the degradation of host molecules or in the mediation of inflammation was determined by bidirectional best hits with an e-value less than 10−3 and covering over 80% of the shortest sequence.The Promer program from the Mummer package P. freudenreichii in a chemically defined medium, as previously described Vitamin requirements were evaluated by growing Fermentation of various carbohydrates was tested using the Biolog plate test  and api50CH , according to manufacturer's instructions.Table S1Propionibacterium freudenreichii genome by 2D-LC MSMS. Three different experimental setups were used and are given in the last column. Setup “a” is nanoLC-ESI/MS/MS, “b” is MALDI/MS/MS and “c” is 2DLC-ESI/MS/MS (first dimension being cation exchange chromatography and second dimension being reverse phase liquid chromatography).List of identified proteins from the (0.09 MB PDF)Click here for additional data file.Table S2Insertion sequences occurence.(0.00 MB PDF)Click here for additional data file.Table S3P. freudenreichii proteins and P. acnes proteins. No P. acnes protein putatively involved in the degradation of host molecules or in the mediation of inflammation was found in P. freudenreichii.Bidirectional best hits results between (0.15 MB PDF)Click here for additional data file.Table S4Propionibacterium freudenreichii carbon substrate degradation.(0.03 MB PDF)Click here for additional data file.Table S5P. freudenreichii CIRM-BIA1.Results of test of vitamin requirements by (0.04 MB PDF)Click here for additional data file.Table S6List of pyrophosphate and polyphosphate enzymes.(0.03 MB PDF)Click here for additional data file.


Mycoplasma haemofelis–like infection in an HIV-positive patient co-infected with Bartonella henselae.Hemotrophic mycoplasmas infect a variety of mammals. Although infection in humans is rarely reported, an association with an immunocompromised state has been suggested. We report a case of a  Mycoplasma spp. are host specific. However, there are occasional reports of infection in an animal species not perceived as primary hosts. These infections may have a pathologic effect, particularly when predisposing conditions, such as immunodeficiency, are present , and viral load was 38,100 copies/mL. Additional laboratory findings included anemia, hematocrit 29% (reference range 38%–50%); leukopenia, 3,300 leukocytes/μL ; thrombocytopenia, 108,000 platelets/µL ; aspartate aminotransferase 66 U/L (15–40 U/L), alanine aminotransferase 79 U/L (10–40 U/L), and lactate dehydrogenase 657 U/L (240–480 U/L). Blood cultures yielded no bacterial growth; sputum cultures were negative for acid-fast bacilli, bacteria, and fungi. Test results were negative for hepatitis B and C, human T lymphotropic virus type 1, syphilis, chlamydia, and cryptococcus infections. Bone marrow and lymph node biopsy and culture results were negative for mycobacterial or fungal infections.At admission, his CD4 cell count was 286 cells/mmBartonella spp. were observed by Warthin-Starry staining, and antibodies against B. henselae (titer 256) were detected in serum. Treatment with doxycycline was initiated and symptoms subsequently subsided. The patient was discharged and instructed to continue antiretroviral therapy and oral doxycycline.Abdominal computed tomography showed hepatomegaly, splenomegaly, and hypoechoic lesions on the spleen. Transesophageal echocardiography showed no lesions compatible with infective endocarditis. An inguinal node biopsy showed granuloma with necrosis suggesting cat-scratch disease, and no signs of acid-fast bacilli or fungi. Many bacilli suggestive of Bartonella spp. infection. His symptoms disappeared and an echocardiogram 17 days later showed resolution of mitral vegetations. He was discharged and instructed to continue antiretroviral therapy and oral doxycycline for Bartonella spp. infection. Ten months after discharge, the patient returned for a follow-up visit while taking recommended therapy. He had no clinical signs and his laboratory findings were improved.In June 2007, the patient was hospitalized with fever, malaise, weight loss, and lymphadenomegaly. Echocardiography showed mitral vegetations. Multiple hepatic hypoechoic lesions were found by abdominal computed tomography. Lymph node biopsy specimens showed tiny bacilli by Warthin-Starry staining. The patient had prematurely discontinued antiretroviral and antimicrobial drug treatment, which may have predisposed him to endocarditis and hepatic peliosis. He was treated with doxycycline and gentamicin for Bartonella spp. infection . These bacteria infect cats and possible infection of this patient was investigated. Positive controls for M. haemofelis included DNA extracted from naturally (GenBank accession no. EU930823) and experimentally infected cats  were used. Negative controls included ultrapure water and DNA extracted from blood of a healthy person and a noninfected cat. All negative controls were negative by PCR.During the patient’s first hospitalization in 2006, blood was collected into tubes containing EDTA and 2 aliquots of DNA were extracted  at the Hospital de Clínicas de Porto Alegre. DNA was tested by PCR for Bartonella spp. and M. haemofelis PCRs. The 393-bp PCR product for M. haemofelis . PCR product amplified from the patient’s blood was purified and sequenced. A 1,214-bp sequence was submitted to GenBank (accession no. EU888930); it was 99% homologous with the sequence for M. haemofelis.Amplicons of the expected size were obtained in emofelis  was puriM. haemofelis and all 5 cats were positive for Bartonella spp. The cats were not tested for other infectious agents.The possibility that the patient’s cats might be involved in zoonotic transmission was also investigated. Two weeks after the patient’s first blood collection, blood was collected from the 5 cats, and DNA was extracted at the Veterinary Hospital of the Universidade Federal do Rio Grande do Sul, Porto Alegre. Two of the cats were positive by PCR for Bartonella spp. but negative for hemoplasmas.PCRs for hemoplasmas were performed in duplicate at 2 laboratories  by using split aliquots. PCR results were reproducible. During the patient’s second hospitalization, the same PCRs were used and the patient was positive for Hemoplasma infections may occur more frequently than is generally recognized, given that these organisms fail to grow in culture and only a few laboratories are equipped to detect and identify hemoplasmas (M. haemofelis (M. suis and M. wenyonii (96%–100%) and only 75% homologous to M. haemofelis ; C. felis may be involved in transmission of M. haemofelis among cats (M. haemofelis and B. henselae (Epidemiologic studies have linked cat bites and scratches and flea-infested cats with transmission of Mycoplasma spp., hemoplasmas might act as a cofactor in HIV infection, contributing to acceleration of the course of the disease (M. haemofelis.As with other