Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs).The genome of gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer , involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit.On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of  Parachlamydia acanthamoebae is an obligate intracellular bacterium naturally infecting free-living amoebae [Acanthamoeba spp. recovered from the nasal mucosa of healthy volunteers [Parachlamydiaceae have been found within about 5% of Acanthamoeba spp. and once within Hartmanella vermiformis [Parachlamydiaceae are about 14% different from those of both genera Chlamydophila and Chlamydia [Chlamydophila sp. and Chlamydia sp. is 6% only, it clearly appears that the speciation between the two latter occurred after the divergence between Parachlamydiaceae and Chlamydiaceae.First described in 1997,  amoebae . It was miformis ,3. The 1hlamydia ,3. SinceChlamydiales, Parachlamydiaceae can present two developmental stages: the reticulate body, a metabolically active dividing form, and the elementary body, an infective stage; the crescent body is another infective form, not observed in Chlamydiaceae [Like other ydiaceae . Differeydiaceae . Dependiydiaceae .Parachlamydiaceae [Parachlamydia serologies were shown to be associated with a febrile epidemic [Parachlamydia-related bacteria as agents of inhalation pneumonia is further suggested by the temperature-dependent release of the bacteria from their amoebal reservoir [Parachlamydia [Chlamydia-like organisms within human macrophages [There is a growing evidence of the human pathogenicity of ydiaceae . For insepidemic , communiepidemic , and inhepidemic . The roleservoir . PCR amphlamydia -12. The rophages  is an adet al. [Parachlamydia-related UWE25 contributed much to the understanding of the evolution of chlamydiae. Indeed, they demonstrated that major virulence mechanisms of Chlamydiaceae such as the Type Three Secretion System (TTSS) and the Chlamydial Protease-like Activity Factor (CPAF) are also encoded by the chromosome of the evolutionary early-branching Parachlamydiaceae UWE25. Genome analysis of the parachlamydial endosymbiont also identified Open Reading Frames (ORFs) homologous to Type Four Secretion Systems (TFSS) and characterized by a high G+C content, suggesting that they result from an horizontal transfer. Based on their annotation revealing the apparent absence of genes necessary for DNA transfer, Horn et al. [Horn et al. , by sequn et al.  proposedTo date, numerous genomic islands (GIs) were already identified along whole chromosomal sequences of various bacterial species. For instance, 140 GIs are described in the Islander database, including GIs of proteobacteria, firmicutes, actinobacteria and cyanobacteria . Thus, wloci are generally used as insertion sites by GIs for their chromosomal integration [GIs are genetic elements which length vary from 10 to 200 kb and are inserted in a chromosome after a lateral transfer occurring, in some instances, between phylogenetically-distant microorganisms. Their heterologous origins are generally evidenced by a G+C content different from that of the remaining bacterial chromosome and by the presence of various mobility genes , that are occasionally source of GI instability ,17. Theyegration -18. SincParachlamydia-related UWE25 chromosome, we observed a G+C-rich region  according to the nomenclature used in the Islander database [Using standard G+C content analyses of n Figure  and 1B, e et al. . Using te et al. , we weret Figure , Table 1database . Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content Figure , Table 2gly-tRNAs in tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 . Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a Chlamydiaceae ORF , four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene).Characterized by a low G+C content (34.1%), the 2rd module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as tra genes by Horn et al. [tra operons encoding the TFSS previously described in proteobacterial genomes  is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. [Presenting a low G+C content (33.4%), the 4n et al.  as encodh module 0 kb is cth module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein.A single large protein is encoded along the 5th module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase.The 6th module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7th module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis.The 7tra genes showed a genetic unit unable to transfer DNA [tra genes, and compared the general organization of this tra unit with other genetic elements encoding TFSS genes [tra unit displays a striking colinearity with tra operons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of Escherichia coli and Novosphingobium aromaticivorans, respectively  and involved in sex pilus retraction and mating pair stabilization [tra unit are devoted to DNA transfer. With the only exception of traG, these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25 tra unit and their F-like plasmids counterparts.The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the sfer DNA . Using dSS genes . The UWEy Figure . All homlization  are prestra unit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree . An omit test performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure p-distances  of nucleotide sequences of the concatenated traA, traK, traB, traV, and traC genes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected p-distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on p-distance of the whole coding sequences of the concatenated tra genes . Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the tra genes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial tra unit could not be precisely defined by this first approach.Our BLAST analyses  reveal tgly-tRNA at the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the Islander database of Mantri et al. [gly-tRNA (14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a gly-tRNA gene. Again, a precise proteobacterial origin could not be proposed, because the distribution of gly-tRNA genes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of gly-tRNA genes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively.The presence of i et al. ,22 insertra units also failed in assigning a precise origin to the UWE25 tra unit since it branched near the alpha- and gamma-proteobacterial tra operons  on similarity analyses demonstrating the presence of all genes encoding proteins used during a DNA transfer, (ii) on phylogenetic analyses of tra unit genes and, (iii) on comparison of gene order. These analyses clearly demonstrate that the UWE25 tra unit is strongly more related to F-like conjugative system than to P-like and I-like secretion systems. The significant bootstraps of all trees obtained by standard gene phylogeny and their congruent topologies with others obtained by the gene order breakpoint analysis not biased by codon usage homing, strongly support the validity of these analyses confirming the F-like conjugative nature of the parachlamydial tra unit. Thus, our model significantly differs from the other proposed by Horn et al. [traA, traL, traK, traV, and concluded that the UWE25 tra unit is involved in protein export, and not in DNA transfer.The 3n et al. , who didth module presents a nucleotide composition similar to the tra unit and is composed of a single high G+C 6-kb gene, whose product is similar to the human Nod3 protein. The Nod (Nucleotide-binding oligomerization domain) proteins are members of a family that also includes the apoptosis regulator Apaf1 (Apoptotic protease activating factor 1) and plant disease-resistance gene products [The 5products . The funnd, 4th, and 6th modules are similar, explaining the observed similar negative slope of the residual G+C curves. Moreover, these three modules encode phage-related proteins and proteins involved in DNA metabolism. These modules probably involved in mobility might have a common origin, the ancestral single phage module being currently separated in three pieces by the presence of the tra unit and of the Nod3-like protein encoding gene.The nucleotide G+C composition of the 2th module echoes those of the tra unit (3rd module) and of the Nod3-like protein (5th module). The 7th module encodes a transposition resolvase and three transposases similar to gamma-proteobacterial homologs. With the only exception of the phage-related Doc protein, that has an homolog at the beginning of the sixth module, and that might be located there after transposition, the 7th module appears thus to have a different origin than the 2nd, 4th and 6th modules, though also encoding mobility genes.The positive slope in the G+C analysis of the 7tra unit along the sequences of UWE25 is the first evidence of a putative conjugative system in chlamydiae. If conjugation occurs, it most probably takes place within free-living amoebae, that may contain several hundreds of Parachlamydia bacteria tightly packed in their vacuoles [The presence of a F-like vacuoles . Such a tra units/operons in the parachlamydial UWE25 and in proteobacteria could be explained by an emergence of this unit in a common ancestor of both clades, and by its subsequent loss in Chlamydiaceae. Another evolutionary scenario is that the tra unit was acquired from a proteobacteria by a Parachlamydiaceae in a common amoebal vacuole. Since the tra unit is absent from all sequenced Chlamydiaceae genomes, this transfer would have occurred after the divergence of Parachlamydiaceae and Chlamydiaceae, at a time when Parachlamydia was already an intracellular bacteria. An intra-amoebal transfer of this GI is supported by the permissivity of free-living amoebae to proteobacteria [tra, further analyses have to confirm whether this GI module was acquired from an alpha-, beta-, or gamma-proteobacteria unit. We hypothesize that the F-like parachlamydial tra unit has been brought by a lateral transfer from a proteobacterial genome. This hypothesis is strongly supported by the cumulative GC skew analysis [Chlamydiales. Sequencing additional genomes of environmental chlamydiae, that present a large biodiversity [The presence of bacteria , and by analysis -30 produanalysis . The genParachlamydiaceae bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired by a Parachlamydiaceae from phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia [Chlamydiales.We showed that a GI present on the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of neumonia , the PamParachlamydia-related endosymbiont UWE25, and thus not to the Acanthamoeba sp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered [et al. recently proposed UWE25 as the type strain of a new bacterial species: Protochlamydia amoebophila [The genome sequence of UWE25   = knn is the position of the center of the nth window. The residual cumulative G+C content curve GC' can then be drawn as a function of the position of each window center:where GC'n = GCn - knZhang and Zhang  recentlytra genes previously identified by Horn et al. [The perfect tandem repeats identification was first performed using the EQUICKTANDEM software   on a 200n et al. . The dupn et al. , with thet al. [traA, traL, traK, traV, re-annotation of the UWE25 tra unit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following tra genes, i.e. traA, traK, traB, traV, traC, and ii) the corresponding ORFs of P- and I-like plasmids [trbC/VirB2, trbG/VirB9, trbI/VirB10, trbH/VirB7, trbE/VirB4 of P-plasmids and traX, traN, traO, traI, traU of I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW ([p-distances (the proportion p of nucleotide sites at which two sequences compared are different) and Kimura corrected p-distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1.Since Horn et al.  did not plasmids , i.e. trLUSTALW  by Blanchette et al. [tra unit of UWE25 and the tra operons reviewed by Lawley et al. [Oi and Oj containing homologous genes proposed by Blanchette et al. [tra operons: instead of counting the number of minimal breakpoints existing between two tra operons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1.To quantify the inversion and transposition events leading to the current organization of e et al.  was usedy et al. . The dise et al.  was sligOi encodes sequentially four genes  and the operon Oj, six genes , the gene order breakpoint analysis reveals that two gene pairs are conserved: ab and cd. The dissimilarity distances existing between the operons i) Oi and Oj, and ii) Oj and Oi would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively.For instance, if the operon omit test [From the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an mit test  was perftra unit and performed all BLAST analyses. FC and LG delimited the GI by direct repeat analyses. GG drew the phylogenetic trees of the concatenated tra genes. After developing the residual cumulative G+C content analysis used for a software development, LG performed the G+C content analyses and the gene order comparison of tra units by the gene order breakpoint analysis. CAR established the correlation existing between the tRNA genes and the GIs according to taxonomy and coordinated the team work. GG wrote the first draft of the paper. All authors improved the manuscript and approved its final version.GG and CAR initiated the project. GG and FC reannotated the parachlamydial Chlamydiaceae are presented. Direct repeats (DR), gly-tRNA genes and limits of each modules of the GI are highlighted.Supplementary table. Results of BLAST ,36 analyClick here for filetra unit is phylogenetically closely related to F-like DNA conjugative tra operons: (A) UPGMA-, (B) Neighbor-joining-, and (C) minimum evolution-trees comparing p-distances and Kimura corrected p-distances of nucleotide sequences of the concatenated traA, traK, traB, traV, and traC genes (the UPGMA tree comparing the Kimura corrected p-distances of tra genes, shown in Figure p-distances, the tra unit of UWE25 is clustered with tra operons of gamma-proteobacterial F-like plasmids: bootstrap values of 96% and 92%, respectively, support the node separating the concatenated tra genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmids, from their closest relative R27 plasmid. Similarly, in neighbor-joining and minimum evolution analyses of the Kimura corrected p-distances, bootstrap values of 94% and 91%, respectively, support the node separating the concatenated tra genes of the chromosomal UWE25 and the R27 plasmid, another gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids.Supplementary figure. Phylogenetic analyses suggest that the UWE25 Click here for file


Soy protein isolate (SPI) in the diet may inhibit colon tumorigenesis. We examined azoxymethane (AOM)-induced aberrant crypt foci (ACF) in male rats in relation to lifetime, pre-weaning, or post-weaning dietary exposure to SPI and also within the context of fetal alcohol exposure. Pregnant Sprague Dawley rats were fed AIN-93G diets containing casein  or SPI (20%) as the sole protein source starting on gestation day 4 (GD 4). Progeny were weaned on postnatal day (PND) 21 to the same diet as their dams and were fed this diet until termination of the experiment at PND 138. Rats received AOM on PND 89 and 96. Lifetime (GD 4 to PND 138) feeding of SPI led to reduced frequency of ACF with 4 or more crypts in the distal colon. Progeny of dams fed SPI only during pregnancy and lactation or progeny fed SPI only after weaning exhibited similarly reduced frequency of large ACF in distal colon. Number of epithelial cells, in the distal colon, undergoing apoptosis was unaffected by diet. SPI reduced weight gain and adiposity, but these were not correlated with fewer numbers of large ACF. Lifetime SPI exposure similarly inhibited development of large ACF in Sprague Dawley rats whose dams were exposed to ethanol during pregnancy. In summary, feeding of SPI to rat dams during pregnancy and lactation suppresses numbers of large ACF in their progeny, implying a long-term or permanent change elicited by the maternal diet. Moreover, results support the use of ACF as an intermediate endpoint for elucidating effects of SPI and its biochemical constituents in colon cancer prevention in rats. Colorectal cancer is the third leading cause of cancer-related deaths in the U.S.; an estimated 147,500 new cases of this disease will have occurred in the U.S. during 2003 . Howeveret al., [et al. [Soy foods are the main dietary sources of isoflavones, which are implicated in cancer prevention ,5,9. Soyet al., , feeding [et al.  formulatThe picture is also unclear when tumors rather than ACFs are used as the end point. In an early study, soybean protein did not differ from beef protein in terms of relative numbers of colon tumors in DMH-treated rats . DefatteThe above studies used diets made with soy protein isolates or soy components prepared in different ways and supplemented to varying levels. Furthermore, diets usually were fed just prior to or concurrent with the chemical carcinogen in order to focus on initiation or progression of tumorigenesis. Some studies did not account for the fact that commercial rodent diets can include soy protein; moreover there is significant maternal-fetal transfer of isoflavones in rats fed soy-containing diets . Thus, pDiets contained either casein or SPI as the sole protein source (200 g/Kg diet) and their formulation has been previously described ,28. Casead libitum access to diet and water. Animal rooms had constant humidity and a 12-h light-dark cycle.Animals were housed in an AAALAC-approved animal facility at the Arkansas Children's Hospital Research Institute; animal use protocols were approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Animals were housed in polycarbonate cages and allowed Pregnant Sprague Dawley dams from Harlan, Inc.  were received at gestation day (GD) 4 and immediately assigned in random fashion to casein or SPI diet. At postnatal day (PND) 2, each litter was culled to 5 males and 5 females . At weaning, animals were divided into four diet groups: lifetime (GD 4 to PND 138) casein, n = 25; lifetime SPI, n = 25; casein to SPI, n = 25; and SPI to casein, n = 25. Diet switchovers were performed at PND 21 and all rats were given AOM on PND 89 and 96. Rats were injected subcutaneously with AOM (Midwest Research Institute) in saline, 15 mg/kg body weight. Animals were weighed weekly from birth and were euthanized at PND 138 for ACF determination or TUNEL assay.ad libitum. At PND 2, each litter was culled to 5 males and 5 females . Progeny were weaned to the same diet as for their dam. Number of male progeny allocated to casein and SPI diets was 24 and 29, respectively.Pregnant rats at GD 5 were surgically implanted with an intra-gastric cannula as previously described . Dams weFifteen animals of each diet group in Experiments I and II were used for ACF determination. Colon contents were removed by flushing from the cecal end with ~20 ml of PBS via syringe. Each colon was slid onto a 2 ml pipette and was fixed in this position for 10 min in 10% neutral buffered-formalin. The colon was opened, laid flat and placed between sheets of labeled filter paper in fixative in the cold. Tissue was removed from formalin, divided into proximal and distal halves, and stained in 0.2% methylene blue in PBS for 5–7 min or until the tissue had a uniform blue appearance. Tissues were rinsed with PBS for ~1 min and stored in 0.4% formalin-PBS at 4°C. Aberrant crypt foci were viewed immediately after staining using a Nikon AMZ800 stereoscope at 40× magnification with side illumination. Proximal and distal colon halves were reviewed along their entire lengths and all ACF were counted. Aberrant crypt foci were categorized according to crypt complexity . All colons were scored in blinded fashion by a single observer. Colons that failed to yield useable data due to poor fixation and/or staining were excluded from statistical analysis.Colons not used for ACF determination  were divided into proximal and distal halves and the midpoints of each half taken for fixation. Tissue was fixed in 10% neutral buffered-formalin, processed through a graded series of ethanol and xylene washes, embedded in paraffin, 4 μm sections obtained, and these were subjected to TUNEL assay. The TdT-FragEL DNA Fragmentation Detection kit  was used for this purpose. Approximately 200 crypt columns were examined from each of 4–5 animals in each diet group.Expt. I) for DXA analysis. Percentage of global fat (% fat) was determined.Body composition data were obtained on anesthetized rats by dual energy x-ray absorptiometry (DXA) using a Hologic QDR 4500A instrument . Five rats (at 33 days post-AOM treatment) were randomly chosen from casein and SPI diet groups  and hence were not normally distributed. We took the natural logarithm of all data points since log-transformed values were less skewed than the original data and more amenable to analysis. However, for simplicity, figures and tables present the original (not log-transformed) data. To examine diet effects, we used the SAS System's  PROC GLM procedure. Unpaired t tests were also used to examine differences between proximal and distal colon for several endpoints. Incidence of large ACF (with 5 or more crypts per focus) was analyzed using Fisher's Exact Test . For all analyses, a P value less than 0.05 was considered significant, while 0.05 < P < 0.1 was deemed marginal. Body weights and body fat content were compared by t-test (SigmaStat).In casein-fed animals, the distal half of the colon had 2–3 fold more ACF of each size class than did the proximal half Fig. . In ratsTo examine the possibility that dietary exposure to SPI over the period encompassing fetal and neonatal development could mimic effects of lifetime SPI, we performed diet switchovers of Sprague Dawley rats at weaning and surveyed their colons six weeks after AOM administration, relative to animal's lifetime-fed SPI diet concurrently. The opposite switchover, from casein to SPI at weaning, was carried out in parallel to examine effects of SPI, from post-weaning through to adulthood.ACF frequency for each diet switchover generally mimicked that for lifetime SPI Fig. . AnalysiAt six weeks, post-AOM, there were no observable differences in the relative apoptotic state of total colonic epithelium in distal colons of CAS, SPI, CAS/SPI and SPI/CAS groups Table . HoweverSPI-fed animals weighed less than corresponding casein-fed counterparts Fig. . The bodWe confirmed the inhibitory effect of SPI on occurrence of large ACF in a second model, namely, progeny of Sprague Dawley dams that received ethanol + casein or ethanol + SPI by total enteral nutrition (TEN) during gestation and which were weaned to the same diet as their dams  elucidate effects of SPI on colon ACF frequency in rat models, b) study effects of dietary SPI on somatic growth and adiposity in view of their potential interactions with colon ACF incidence, c) examine developmental and lifetime 'exposures' to dietary SPI and consequent effects on ACF indices, and d) correlate ACF data with our previously published colon tumor data obtained with animals lifetime fed SPI . ACF mayet al. [ACF have been identified on the colon luminal surface in rats and mice treated with chemical carcinogens  and in humans with and without overt colon carcinoma ,32. AOM et al.  reportedet al. -46. Moreet al. ,47-51. Pet al. . In our et al. -46.A diet switching paradigm examined whether the suppressive effect of SPI on larger ACF required lifetime exposure or could be mimicked with a shorter developmental period of SPI feeding. We also questioned if 'protective' effects could be transferred maternally to the offspring. The results of this experiment are of interest for a number of reasons. The observation that SPI elicited reduced growth by day 10, postnatal, may point to a 'maternal effect' of SPI, during gestation or lactation or both that is transmitted to progeny to affect their growth. The exact nature of this effect awaits further clarification. Both diet switchovers generally mimicked the effects of lifetime SPI on ACF incidence, although the casein/SPI regimen appeared to be slightly more effective than the SPI/casein dietary treatment in this regard. We therefore surmise that SPI can manifest long lasting effects on ACF incidence and that at least some of these effects can be transmitted to the offspring through feeding of SPI to pregnant and/or lactating dams.Obesity is considered by some to be a promoter of oncogenesis in rat models of chemically induced colon cancer ,53 and oKállay et al.  observedSPI inhibited the development of large ACF, some of which may be tumor precursors. Feeding of SPI to rat dams during pregnancy and lactation led to a suppression in numbers of large ACF in their progeny, implying a long-term or permanent anti-carcinogenic effect elicited by the SPI-based diet. Body weight and body composition were differentially affected by soy protein isolate or casein in the diet. ACF may be a valid intermediate endpoint for elucidating effects of SPI and its biochemical constituents in tumor prevention in the colons of Sprague Dawley rats.ALL performed the whole-mount and TUNEL assays, performed data analysis, and assisted with tissue collection. RX performed data analysis, assisted with tissue collection, and prepared the final figures. JGP and PMS performed statistical analyses. TMB participated in the design of the study and oversaw the animal and diet manipulations. FAS conceived of the study, participated in its design, interpreted the data and prepared the manuscript. All authors read, modified and approved the final manuscript.


Open Access publishing is a valuable resource for the synthesis and distribution of essential health care information. This article discusses the potential benefits of Open Access, specifically in terms of Low and Middle Income (LAMI) countries in which there is currently a lack of informed health care providers – mainly a consequence of poor availability to information. We propose that without copyright restrictions, Open Access facilitates distribution of the most relevant research and health care information. Furthermore, we suggest that the technology and infrastructure that has been put in place for Open Access could be used to publish download-able manuals, guides or basic handbooks created by healthcare providers in LAMI countries. This basic information would be most useful if it is informed by relevant research, produced locally, and made available in the local language. The potential benefits of Open Access in terms of access to the research literature in general, and to research from low- and middle-income (LAMI) countries in particular, have been well described elsewhere.We would like to introduce a new dimension into this debate: Open Access has an untapped potential to enhance the synthesis and distribution of essential healthcare knowledge. Open Access, as opposed to free access, allows readers the right to use the article without restriction. Local publishers can therefore filter, reproduce and distribute the most relevant research and healthcare information from any and all Open Access journals. In essence, they can create journals focused on local issues based on content from a variety of journals. These "local journals" can be circulated in print – a medium that remains essential in countries with limited computer and Internet access. To our knowledge, this has yet to be done, although we are hoping someone will exploit this opportunity soon.In the future, we imagine the technology and infrastructure that has been developed for Open Access could be used to publish download-able manuals, guides or basic handbooks created by healthcare providers in these countries. These free resources could then be accessed worldwide and, where necessary, reproduced within local communities in the optimal medium. In an "author-pays" Open Access model the charges would be standard and could be covered by a national government organization or charity.Open Access will increase the availability of research and, in doing so, stimulate researchers in LAMI countries to develop their own research and practices. With research published in the Open Access medium it also becomes possible for producers of healthcare materials to optimize the use of research produced from their own and other countries. Thus, Open Access will optimize the distribution of local healthcare information, with potential benefits worldwide.CELS is an employee of BioMed Central, an Open Access publisher that is funded through article-processing charges levied on accepted manuscripts. CELS receives a fixed salary, which is unaffected by the amount of money received by BioMed Central from article-processing charges.World Journal of Surgical Oncology, an Independent journal published by BioMed Central.MP is the Editor-in-Chief of


The Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can also induce the expression of pro-angiogenic factors in fibroblasts postnatally. Misregulation of the Hh pathway has been implicated in a variety of different types of cancer, including pancreatic and small-cell lung cancer. Recently a putative antagonist of the pathway, Hedgehog-interacting protein (HIP), was identified as a Hh binding protein that is also a target of Hh signaling. We sought to clarify possible roles for HIP in angiogenesis and cancer.Inhibition of Hh signaling by HIP was assayed by measuring the induction of Ptc-1 mRNA in TM3 cells treated with conditioned medium containing Sonic hedgehog (Shh). Angiogenesis was assayed in vitro by EC tube formation on Matrigel. Expression of HIP mRNA was assayed in cells and tissues by Q-RT-PCR and Western blot. HIP expression in human tumors or mouse xenograft tumors compared to normal tissues was assayed by Q-RT-PCR or hybridization of RNA probes to a cancer profiling array.We show that Hedgehog-interacting protein (HIP) is abundantly expressed in vascular endothelial cells (EC) but at low or undetectable levels in other cell types. Expression of HIP in mouse epithelial cells attenuated their response to Shh, demonstrating that HIP can antagonize Hh signaling when expressed in the responding cell, and supporting the hypothesis that HIP blocks Hh signaling in EC. HIP expression was significantly reduced in tissues undergoing angiogenesis, including PC3 human prostate cancer and A549 human lung cancer xenograft tumors, as well as in EC undergoing tube formation on Matrigel. HIP expression was also decreased in several human tumors of the liver, lung, stomach, colon and rectum when compared to the corresponding normal tissue.These results suggest that reduced expression of HIP, a naturally occurring Hh pathway antagonist, in tumor neo-vasculature may contribute to increased Hh signaling within the tumor and possibly promote angiogenesis. Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). All three Hedgehog (Hh) proteins function by binding to the transmembrane receptor, Patched-1 (Ptc-1), leading to the de-repression of the membrane-bound inhibitor, Smoothened [The Hedgehog family of genes encodes secreted signaling molecules that regulate cell proliferation and cell fate determination. In mammals, there are three such genes: Sonic hedgehog (oothened . This reoothened .Hedgehog-interacting protein (HIP) was discovered by screening a mouse cDNA expression library for proteins that bound to Shh . HIP bin-/- mice can initiate vasculogenesis and hematopoiesis but are defective in vascular remodeling to form blood vessels [in vitro, were unable to respond to Shh, even though they express the receptor Ptc-1 [During gastrulation in the mouse, Ihh is secreted by the endoderm and is sufficient to activate hematopoiesis and vasculogenesis . In addi vessels . In the or Ptc-1 .Misregulation of the Hh signaling pathway has been implicated in several different types of cancer, including basal cell carcinomas (BCCs), medullablastomas, and gliomas  using LipofectAMINE 2000 and OptiMem reduced-serum medium (Invitrogen). Three to five hours after transfection, cells were re-fed with serum-containing growth medium, and three or four days later conditioned medium was collected and filtered through 0.2-μm cellulose acetate filters.For RNA isolation, human aortic endothelial cells (HAEC) were cultured in 12-well plates with or without Matrigel (BD Biosciences) for 16 hours, then cells were lysed, digested with proteinase K, and total RNA was extracted using the RNeasy Mini Kit (Qiagen). HIP expression was assayed by real-time quantitative reverse transcriptase PCR (Q-RT-PCR) and normalized to 18s rRNA (see below).2. Cells were transfected in 12-well or 24-well plates at 80–90% confluence using LipofectAMINE 2000 transfection reagent and OptiMem reduced-serum medium (Invitrogen). For conditioned medium (CM) assays, cells were transfected with the plasmid MycHIP-pCDNA3 , which encodes full-length mouse HIP, or empty vector (mock). Mock or Shh CM was added to cells 36 hours after transfection, then RNA was harvested 36–48 hours later.The mouse testicular epithelial cell line, TM3, was grown in complete medium  at 37°C and 5% COPC3 human prostate cancer cells were implanted subcutaneously into male nude mice (strain nu/nu), and on day 39 the resulting tumors were excised, weighed, placed on dry ice, then stored at -80°C prior to RNA isolation. A549 lung cancer cells were implanted subcutaneously into female nude mice and harvested as described above on day 9. Normal livers or skin from 5- to 6-week-old male or 4- to 5-week-old female nude mice were excised immediately following cervical dislocation and frozen in liquid nitrogen, then stored at -80°C.RNA was extracted from cultured cells using the RNeasy kit (Qiagen), including on-column DNase I digestion, according to the manufacturer's instructions. To extract RNA from frozen normal mouse tissues and tumors, hard-frozen tissue samples of 100–300 μg were pulverized over dry ice, placed in denaturing buffer, and disrupted using a Mixer Mill (Retsch) or rotor-stator homogenizer . RNA was isolated from the homogenate either by phenol-chloroform extraction followed by precipitation with isopropanol and washing with 70% ethanol  or by using the RNeasy kit (Qiagen). All RNA samples were treated with DNase I (Qiagen) prior to quantitative RT-PCR. Concentrations of RNAs were determined by reading the absorbance at 260 nm.Quantitative RT-PCR was performed using the ABI PRISM™ 7700 Sequence Detection System and TaqMan™ chemistry. Reactions contained 0.3 μM of each primer, 0.1 μM probe, 0.25 U/ml MultiScribe reverse transcriptase, 0.4 U/ml RNase inhibitor, and 1x PCR master mix (Applied Biosystems). Primers and probes used to quantitate the mRNA level of various genes were as follows: mouse Ptc-1 , mouse HIP , mouse vWF , mouse CD146 , human HIP , human vWF . Mouse and human GAPDH and ribosomal RNA probe and primer sets were purchased from Applied Biosystems. RNA quantities were determined from a standard curve of serially diluted total cellular or tissue RNA run in parallel with each set of reactions. Standard curves had a slope between -3.1 and -3.3 and correlation coefficients of 0.98 or greater.Total cell lysates were made by lysing cells in 2% SDS, 10% glycerol, and 0.063 M Tris-HCl (pH 6.8) containing a cocktail of protease inhibitors (Pierce). Lysates were heat denatured at 95°C for 10 minutes, passed through a 26G needle to shear DNA, and centrifuged at 10,000 g for 30 minutes to remove insoluble material. Protein concentrations were determined using the BCA assay (Pierce). Conditioned medium samples were diluted 1:1 in Laemmli sample buffer (Sigma). 50 μg of cell lysate or 20 μl of conditioned medium/sample buffer was resolved on 4–15% polyacrylamide gels (BMA) and transferred to nitrocellulose (Invitrogen). Even loading of protein samples was verified by staining with Ponceau S. After overnight blocking with 5% nonfat dry milk, 0.05% Tween-20 in phosphate-buffered saline, blots were incubated with antibodies against Shh , Myc tag (Invitrogen), or HIP . Antibody-antigen complexes were visualized using horseradish peroxidase-conjugated secondary antibodies  and SuperSignal West Pico chemiluminescent substrate (Pierce).32P-dUTP by in vitro transcription using the MAXIscript kit (Ambion). The array was pre-hybridized in Clontech ExpressHyb solution with 100 μg/ml boiled, sheared salmon testis DNA (SS-DNA) for 2–12 hours at 68°C with rotation. Pre-hybridization solution was replaced with fresh hybridization solution with SS-DNA, plus a total of 1.5–1.7 × 107 cpm of 32P-labelled HIP or vWF probe. Hybridization of the probe proceeded for 16–18 hours at 68°C, followed by four washes in 2X SSC/0.5% SDS at 68°C, one wash in 0.2X SSC/0.5% SDS at 68°C, and one wash in 2X SSC at room temperature. The array was exposed to a phosphor-screen for 24–72 hours and scanned in a Storm phosphor-imager at a resolution of 50 μm. After hybridization to the HIP probe, the same array was stripped by boiling in 0.5% SDS and re-scanned to verify the absence of residual probe before hybridization to the vWF probe. Relative levels of HIP and vWF expression were determined using ImageQuant software (Molecular Dynamics).Linearized templates containing a 716-bp fragment of human HIP cDNA (bases 521–1236 of human HIP coding sequence) or a 777-bp fragment of human vWF cDNA (bases 7301–8077 of human vWF coding sequence) were radiolabelled with All results are expressed as mean ± standard deviation unless otherwise noted. Statistical significance of differences was determined using a two-tailed Student's t-test.-3 copies per cell based on the typical sensitivity of this assay. Hedgehog-interacting protein (HIP) binds Hh proteins with an affinity equal to that of Ptc-1 and can function as an antagonist of Ihh signaling in vivo [Ptc-1 and Smo mRNA are expressed in human aortic endothelial cells (HAEC) at levels comparable to that of GAPDH, as determined by comparing the threshold cycle numbers obtained by quantitative RT-PCR (Q-RT-PCR) (data not shown). Thus HAEC express components of the Hh pathway that are known to mediate a response to Hh ligands. However, when HAEC or human umbilical vein endothelial cells (HUVEC) were treated with conditioned medium containing functional Shh, the Hh pathway was not activated as determined by measuring the levels of mRNA for Ptc-1 and Gli-1, two genes known to be responsive to Hh signaling (data not shown). Shh mRNA levels in HAEC and HUVEC were near the limits of detection by the Q-RT-PCR assay and could therefore be estimated at less than 10 in vivo . Thus weConditioned media from 293 cells co-transfected with Shh and full-length (membrane-anchored) HIP contained a reduced amount of Shh and had a reduced ability to induce differentiation in C3H10T1/2 cells compared to cells transfected with Shh alone, demonstrating that HIP can sequester Shh . HoweverShh has been shown to induce angiogenesis , and HIPAngiogenesis is a common feature of tumor growth  containing cDNA from 154 tumor and corresponding normal tissues from individual patients was used to compare the expression of HIP in a larger set of samples. A Mouse xenograft tumors resulting from transplantation of PC3 (human prostate cancer cell line) or A549 (human lung cancer cell line) cells into nude mice are known to have high levels of neovascularization as a result of angiogenesis, which is required for their continued growth ,19, so wOur demonstration that HIP functions to inhibit Ptc-1 upregulation in mouse testicular epithelial cells (TM3) exposed to Shh-containing conditioned media is the first direct evidence to date that Shh signaling is attenuated in cells that express full-length, membrane-bound HIP. The decrease in Ptc-1 induction caused by transfection with HIP is consistent with previous reports indicating that HIP can bind and reduce the availability of Shh and presumably prevent it from signaling through Ptc-1 ,5. In huSeveral lines of evidence support the hypothesis that HIP expression is decreased in EC participating in angiogenesis. Firstly, we describe that HIP is downregulated in HAEC forming tubes on Matrigel. In contrast, Ptc-1 mRNA levels were increased in EC forming tubes on Matrigel, suggesting that the decrease in HIP mRNA under these conditions does not reflect a general downregulation of Hh-responsive genes. Given that transcription of HIP and Ptc-1 are both activated by Hh signaling, it is likely that the decrease in HIP expression in EC on Matrigel is mediated by a pathway independent of Hh signaling. Secondly, we have observed that HIP mRNA levels are decreased in human tumors and xenograft human tumors grown in mice, situations in which angiogenesis occurs. Previous studies have demonstrated a requirement for Shh in angiogenesis in the ischemic mouse limb and embryonic yolk sac ,9. The pThe Shh pathway has been implicated in several different types of cancer, where upregulation of signaling, associated with mutations in Patched or amplification of Gli, is often the suspected cause of malignancy ,12,24-26Xenotransplantation of PC3 or A549 cells into immunocompromised mice is a commonly used animal model for prostate or lung cancer, respectively. Extensive neovascularization occurs in the resulting tumors, and it has been demonstrated that angiogenesis inhibitors slow their growth ,31. The Angiogenesis has been well-characterized as a prerequisite to continued tumor growth and metastasis ,17,32, aWe have shown that HIP is expressed predominantly in EC but at low or undetectable levels in other cell types, and that the high expression of HIP in EC may be responsible for their inability to respond to Shh. Expression of HIP in TM3 cells attenuated their response to Shh, demonstrating that HIP antagonizes Hh signaling when expressed in the responding cell. The reduction of HIP in a variety of human tumor samples as well as PC3 and A549 xenograft tumors, and in EC undergoing tube formation on Matrigel, suggests that modulation of HIP may play a role in tumor angiogenesis.None declared.CLO participated in experimental design and carried out CM production and assays, tissue and RNA processing, Q-RT-PCR, Western blot, and cancer array hybridization. PPH participated in the design of the study and performed tube formation assays, tissue and RNA processing, Q-RT-PCR, and Western blot. JG participated in initial characterization of HIP and provided supporting data. GMR was involved in the planning and organization of the study and reviewed the manuscript. ARB initiated, planned, and supervised the entire study.The pre-publication history for this paper can be accessed here:


Campylobacter including C. lari RM2100, C. upsaliensis RM3195, and C. coli RM2228 has revealed major structural differences that are associated with the insertion of phage- and plasmid-like genomic islands, as well as major variations in the lipooligosaccharide complex. Poly G tracts are longer, are greater in number, and show greater variability in C. upsaliensis than in the other species. Many genes involved in host colonization, including racR/S, cadF, cdt, ciaB, and flagellin genes, are conserved across the species, but variations that appear to be species specific are evident for a lipooligosaccharide locus, a capsular (extracellular) polysaccharide locus, and a novel Campylobacter putative licABCD virulence locus. The strains also vary in their metabolic profiles, as well as their resistance profiles to a range of antibiotics. It is evident that the newly identified hypothetical and conserved hypothetical proteins, as well as uncharacterized two-component regulatory systems and membrane proteins, may hold additional significant information on the major differences in virulence among the species, as well as the specificity of the strains for particular hosts.Sequencing and comparative genome analysis of four strains of  Campylobacter jejuni yielded some insight, comparison of multiple species of the same genus elucidated greater understanding of virulence mechanismsAlthough the single genome sequence of the human  Campylobacter jejuni is commensal in cattle, swine, and birds . Th. ThC. jeTC 11168  and S2.  genomes . In contC. jejuni RM1221 protein sequences with those of other fully sequenced members of the ɛ-Proteobacteria revealed 540 shared protein sequences, many of which are proposed to have “house-keeping” functions   B. ElevenC. jejuni RM1221 with a p-value less than or equal to 10−5, identity of 35% or more, and match lengths of at least 75% of the length of both query and subject sequence. Not surprisingly, C. jejuni NCTC 11168 had the highest average protein percent identity  with C. jejuni RM1221 proteins. C. coli RM2228 was second, with 1,399 proteins averaging 85.81% identity. Surprisingly, C. upsaliensis RM3195 had the third highest average protein percent identity with C. jejuni RM1221 , followed by C. lari RM2100 with 1,251 proteins having 68.91% average identity. This was surprising since a 16S rRNA tree depicts C. upsaliensis to be more dissimilar to C. jejuni, C. coli, and C. lari ); 66% (35/53) of predicted proteins have BLASTP matches   suggests that they represent either intact prophages with novel head morphogenesis proteins, satellite phages, or nonfunctional prophages or genomic islands.In contrast to CMLP1, y ≥ 30%) . CLIE1 iC. coli RM2228 megaplasmid (pCC178) , many are similar to proteins encoded within the 71-kb H. hepaticusATCC 51449 genomic island (HHGI1), suggesting this genomic island could also be plasmid-derived [The absence of any phage-related ORFs within CJIE3 (located within an arginyl-tRNA), suggests that CJIE3 is not a prophage but rather a genomic island or integrated plasmid. Seventy-three percent (45/62) of the CJIE3 predicted proteins are similar to predicted proteins encoded on the Campylobacter prophages (CMLP1) has been shown to be inducible, we cannot predict whether the other putative prophages or plasmid-like element can be excised. Because the majority of ORFs that lie within prophage regions are hypothetical proteins, we are unable to deduce any putative functions from them; however, we cannot rule out possible functions that either directly impact virulence or increase the fitness of the host in a particular environment.Bacteriophages are vehicles for the lateral or horizontal movement of genes that can increase bacterial fitness ,46. AddiC. coli RM2228 and C. lari RM2100 each contain a single plasmid , whereas C. upsaliensis RM3195 contains two plasmids  has a defined plasmid replication region. The single-stranded binding (Ssb) proteins are conserved among all of the plasmids, alluding to a common evolutionary origin; however, the nickase proteins on the plasmids are not conserved, suggesting that nickase may be specific to the plasmid or strain.ogenesis . The codCampylobacter plasmids is the presence of a Type IV secretion system (T4SS), possibly involved in conjugative plasmid transfer or secretion of virulence factors [C. jejuni species are most similar to each other based on synteny and amino acid identity; however, they share only synteny with the T4SS encoded by pVir or the Agrobacterium tumefaciens Ti plasmid [C. jejuni plasmid T4SSs may be involved in conjugation rather than secretion of virulence factors because they are more similar to T4SSs known to mobilize DNA than to T4SSs that secrete effectors [Campylobacter plasmids encode proteins similar to VirB2 of the Ti plasmid, which is responsible for pilus formation [One conserved feature of all of the large  factors   elements. With the exception of one copy of a degenerate transposase resembling IS605, located between the tonB gene and a 5S rRNA gene, their genomes are devoid of IS elements. In contrast, C. coli RM2228 contains five copies of an IS element (ISCco1 of the IS605 family) at three positions in the chromosome and at least two positions in the megaplasmid pCC178, hinting at recent acquisition and transposition competence. Both the C. upsaliensis RM3195 and C. lari RM2100 pseudomolecules lack the tonB–5S rRNA locus; however, since these are not closed genomes, we cannot accurately assess the status of the IS605 family in these genomes.Both Campylobacter strains in this study were examined for the presence or absence of clustered regularly interspaced short palindromic repeats (CRISPRs) in intergenic regions. A strain was considered CRISPR-positive if it contained two or more direct repeats of a 21-bp or larger DNA segment separated by unique spacer sequences of a similar size. We identified CRISPR elements in only C. jejuni NCTC 11168 and C. jejuni RM1221. However, a previous study found that CRISPR elements are sometimes detectable in C. coli [C. jejuni examined here can be differentiated by both the unique sequence of the spacer sequences (C. jejuni NCTC 11168 and four in C. jejuni RM1221). It is noteworthy that the previous study did not include C. lari or C. upsaliensis, which appear not to contain CRISPR elements, unless they are in a different region of the genome from the C. jejuni CRISPRs and are in unsequenced areas. This further demonstrates the limited utility of CRISPRs in genotyping studies of Campylobacter species.The chromosomes of all five  C. coli . Also coequences  and the C. jejuni strains have been characterized previously [C. jejuni, C. coli, and C. lari strains sequenced in this study, the C. upsaliensis RM3195 genome is predicted to contain at least three Type I RM loci  loci from 65 eviously . In cont RM loci . C. upsaelements .Campylobacter RM systems has implications for Campylobacter biology, specifically DNA uptake and phage infection. Campylobacter spp. are naturally competent [C. jejuni [Campylobacter [Diversity within the ompetent , and hor. jejuni . Naturallobacter ,57. RM sCampylobacter spp., but they are known to have a respiratory type of metabolism, with some strains growing under both aerobic and anaerobic conditions [C. jejuni RM1221, C. coli RM2228, C. lari RM2100, and C. upsaliensis RM3195 revealed that these species have very similar metabolic profiles, with the main variation being the presence of a complete or partial tricarboxylic acid cycle . All four sequenced strains have pathways for the metabolism and biosynthesis of a number of amino acids  protein export pathways for the secretion of proteins across the inner/periplasmic membrane. In addition, Campylobacter has the signal recognition particle pathway. We have found no evidence for chromosomally encoded lol, Type III, or Type IV secretion systems other than the flagellar export apparatus [C. coli RM2228 (CCO0190), C. lari RM2100 (CLA0150), and C. jejuni NCTC 11168 (Cj0975); however, CCO1305 in C. coli and CJE0841–CJE0843 and CJE1056 in C. jejuni RM1221 are disrupted , emphasizing the importance of bacterial motility and adhesion for virulence [Motility is the major factor that has been implicated directly in intestinal colonization . Of the tudy see C, 27 ORFirulence .Campylobacter spp.: CJE0968–CJE0969, CJE1357–CJE1358, CJE1361–CJE1362, racR–racS (CJE1397– CJE1398), and CJE1664–CJE1665. In addition, another four putative response regulator genes  and one putative histidine kinase gene (CJE0884) could be found in the finished C. jejuni genomes. Brás et al. [Campylobacter pathogenicity, not surprising given the likely exposure of the bacteria to temperature stress during the infectious process.Two-component regulatory (TCR) systems are used commonly by bacteria to respond to specific environmental signals. We identified five TCR systems (pairs of adjacent histidine kinase and response regulator genes) that appear to be conserved across the s et al.  showed tC. jejuni to epithelial cells is mediated by multiple adhesins, including CadF (CJE1651), PEB1 (CJE0997–CJE1000), JlpA (CJE1065), and a 43-kDa major outer membrane protein (CJE1395). Fibronectin (FN) has been implicated in C. jejuni adherence to epithelial cells via the protein CadF [Campylobacter strains. The FN host cell-surface receptor is the α5β1 integrin. In intact epithelia, α5β1 integrins are restricted to the basolateral membrane and thus are not available for interaction with luminally positioned microbial pathogens [C. jejuni were greatly increased by exposure of cellular basolateral surfaces, and that FN was the receptor [C. jejuni invasion preferentially occurs via a paracellular route, rather than via an intracellular route. Additionally, inspection of loci adjacent to putative TpsB proteins revealed two intact filamentous hemagglutinin (FHA)–like adhesions: in C. lari RM2100, CLA0151, and in C. coli RM2228, CCO1312. The regions upstream of the remaining TpsB-like proteins have fragmented adhesion-like ORFs (C. lari RM2100 has both an undisrupted TpsB-like transporter (CLA0150) and an adjacent putative FHA-like adhesion (CLA0151), which, if functional, could enable C. lari RM2100 to attach to cell surfaces.Adherence of ein CadF . In addiathogens . Howeverreceptor . This suike ORFs . Only C.Escherichia coli have been shown to disrupt the barrier function of host intestinal epithelial tight junctions [Campylobacter strains. In addition, C. lari RM2100 encodes a single peptide (CLAA0034) in pCL46 that is similar to the Yersinia invasin proteins that enable Yersinia to penetrate host cells [C. lari strain might also have the ability to penetrate host cells.Cytolethal distending toxins from enteropathogenic unctions . The thrst cells , suggestC. upsaliensis RM3195 sequence revealed a putative licABCD (CUP0277–CUP0274) locus with varying, but significant, identity to genes present in Haemophilus influenzae [Neisseria species [Streptococcus pneumoniae [licABCD genes in these microorganisms encode proteins involved in the acquisition of choline , synthesis of phosphorylcholine (PCho) , and transfer of PCho  to LOS or teichoic/lipoteichoic acids to facilitate attachment to host cells [C. upsaliensis from South Africa also contain licA (unpublished data). It is noteworthy that licA expression in Haem. influenzae is regulated by variation in the number of intragenic tandem tetranucleotide repeats (CAAT) at the 5′ end, resulting in translational on/off synthesis of PCho and expression on LOS [licA gene (bp 132–146) of C. upsaliensis RM3195 probably regulates synthesis of PCho and decoration of LOS by a similar mechanism.Examination of the fluenzae , commenseumoniae . licABCDC. jejuni NCTC 11168 has been described [Campylobacter strains, a number of other phenomena related to these repetitive regions were observed. First, when a homopolymeric repeat region was associated with a potential coding region, the base mostly included in the repeated region on the coding strand was G, resulting in poly-glycine, not poly-proline, in the peptide. Secondly, the C. upsaliensis RM3195 genome contains nearly three times as many variable homopolymeric repeats (22) as C. jejuni RM1221 (8), seven times as many as C. lari RM2100 (3), and 22 times as many as C. coli RM2228 (1)  are strain-specific  . These vC tracts  as C. jec Tables  and S6. ted ORFs , which ifluenzae , and hasC. jejuni that function in the interactions of the organism with the environment. Interesting aspects of C. jejuni LOSs are their molecular mimicry of host gangliosides and their presumed roles in evasion of host immune responses and autoimmunity [C. jejuni 81–176 has been reported to have a role in increasing serum resistance, invasion of cell lines, and surface hydrophilicity [LOSs and EPs are important surface structures in immunity , decreasimmunity , and attimmunity . The capCampylobacter spp. are organized as previously observed in other C. jejuni strains [waaC and waaF, that surround regions exhibiting significant variation in ORF content. Thus, these organisms likely synthesize novel LOS structures [C. jejuni RM1221 is distinct from the LOS of NCTC 11168, as seen on polyacrylamide gels, in that it possesses three LOS bands while NCTC 11168 possesses only one (unpublished data). Two LOS genes from C. jejuni RM1221 possess homopolymeric G:C tracts that may explain the additional bands. Comparison of the LOS genes from the sequenced Campylobacter spp. with those from C. jejuni strains that produce ganglioside mimics [N-acetylneuramic  acid or the associated sialic acid transferase, and are not likely to produce ganglioside mimics. Within the LOS loci of C. lari RM2100 and C. upsaliensis RM3195, there are ORF clusters that have homologs in NCTC 11168 that are unrelated to LOS biosynthesis. It is unclear what role this genomic reorganization plays in the biosynthesis of LOS.The LOS biosynthesis loci of all sequenced  strains . At eithructures . In parte mimics  demonstrC. jejuni RM1221, C. coli RM2228, and C. lari RM2100 possess kps orthologs like the EP locus of C. jejuni NCTC 11168 that are involved in polysaccharide export; however, many putative EP biosynthesis genes from C. jejuni RM1221 and C. coli RM2228 are unique to these strains. The kps orthologs are present in C. upsaliensis RM3195, but they are not clustered with other polysaccharide biosynthetic genes as observed in the other strains. Specifically, there are three clusters of EP genes: CUP0615–CUP0619, CUP1248–CUP1270, and CUP1328–CUP1329. The second cluster contains many ORFs that are unique to C. upsaliensis (C. jejuni strains (Cj1428c and CJE1612) and C. upsaliensis RM3195 encode this enzyme. Of these GDP-fucose synthetases, only CUP1257 was shown to contain variable poly G tracts , which was found as an intact single copy in NCTC 11168 (Cj0299), RM1221 (CJE0344), and RM2100 (CLA0304). The corresponding sequence in C. coli RM2228 may reside in unsequenced regions. Only C. lari RM2100 was resistant to a broad range of quinolone/fluoroquinolone antibiotics ((gyrA) that changed codon 86 from threonine to valine [C. coli RM2228. This is likely due to a mutation in all three copies of the 23S rRNA (A2122G), corresponding to position 2,143 of the H. pylori sequence [C. coli RM2228 has acquired resistance to the aminoglycosides kanamycin and neomycin, tetracycline, oxytetracycline, minocycline, and presumably hygromycin B (but not gentamicin) from the megaplasmid pCC178  [C. lari RM2100, but was not due to the classic mutations in the β subunit of RNA polymerase [The sequenced sistance  and S2. ter spp. . Every sibiotics . This bro valine . The macsequence . C. colid pCC178 . It is pce genes , and thexic acid  and novoxic acid . There w [(folP)  to explalymerase .Campylobacter genomes has provided the core genetic blueprint of the genus. Although the blueprint reveals obvious differences in genome structure and content, additional epidemiological data are needed to correlate these differences, and other, more elusive differences (e.g. differences in regulation and point mutations), with differences in virulence. Some obvious differences were the presence of drug resistance genes that may have been the result of adaptation in the animal production environment, where antibiotics are frequently used to eliminate bacterial infections. It is anticipated that the analysis of the Campylobacter genomes presented here will lay the foundation for the development of systems for fingerprinting strains for phylogenetics, epidemiology, and source tracking, as well as the development of alternative treatments for controlling Campylobacter in food production and in human infection.The comparison of five sequenced C. jejuni strain RM1221 (ATCC BAA-1062) was isolated from the skin of a retail chicken using methods modified from those described previously for isolation of Campylobacter from chicken products [C. coli strain RM2228 (ATCC BAA-1061) was isolated from a chicken carcass obtained from an inspected slaughter plant. A rinse sample was streaked on 5% sheep blood agar plates, and the plates were incubated at 37 °C for 48 h under an atmosphere of 5% O2, 10% CO2, and balance N2. An isolated single colony was picked and maintained on sheep blood agar plates. Three rounds of mixing and sonication of single colony picks were done as described [C. lari strain RM2100 (ATCC BAA-1060) is a human isolate obtained from the Centers for Disease Control and Prevention, Atlanta, Georgia, United States  laked horse blood . Three rounds of mixing and sonication of single colony picks were done as described [C. upsaliensis strain RM3195 (ATCC BAA-1059) was obtained from the feces of a 4-y-old boy confirmed clinically to have Guillain-Barré syndrome. The isolation procedure involved a filtration method with selection of Campylobacter cells in diluted feces by their migration through a 0.6-μm membrane filter and subsequent growth on nonselective medium [products . C. coliescribed . C. lari“case 6” ). The stescribed . C. upsae medium .Campylobacter were sequenced by the random shotgun method [C. jejuni RM1221 was sequenced to closure, whereas the genomes of strains C. lari RM2100, C. coli RM2228, and C. upsaliensis RM3195 were sequenced to 8-fold coverage of an estimated 1.8-Mbp genome. Briefly, one small insert plasmid library (1.5–2.5 kb) and one medium insert plasmid library (10–12 kb) were constructed for each strain  by random nebulization and cloning of genomic DNA. In the random sequencing phase, 8-fold sequence coverage was achieved from the two libraries . The sequences from the respective strains were assembled separately using TIGR Assembler [C. jejuni RM1221 were closed by editing the ends of sequence traces, primer walking or transposon-primed sequencing [C. jejuni RM1221 were confirmed by sequencing PCR products that spanned each repeat unit. Pseudomolecules for the draft sequences were constructed using NUCmer [The four species of n method . The genssembler  or Celerssembler . All seqquencing  on plasmg NUCmer  and BAMBg NUCmer ,94 as prg NUCmer .http://www.tigr.org/software/autoeditor/) [The ambiguity rate for the unfinished genomes was determined using the following procedure. First, the consensus of the contigs was recalled using the consensus caller included in the AutoEditor package (editor/)  by execuC. jejuni RM1221 [An initial set of ORFs that likely encode proteins was identified using GLIMMER , and thoi RM1221 . Two seti RM1221 .Campylobacter genomes and C. jejuni NCTC 11168 [http://blast.wustl.edu) [Campylobacter genomes plus three Helicobacter genomes  and Gnuplot version 4.0 (http://www.gnuplot.info/).For the identification of species-specific  and straTC 11168  were seastl.edu) . To idenri 26695 , H. pyloDSMZ1740  were coms Tables  and S9. C. jejuni RM1221 was determined by searching the nucleotide sequences of aspartate ammonia-lyase , glutamine synthetase type I , citrate synthase , serine hydroxymethyltransferase , phosphoglucosamine mutase , transketolase , and ATP synthase F1 alpha subunit  on the PubMLST Web site (http://pubmlst.org/) [C. jejuni RM1221 FlaA SVR was found by searching the flaA (CJE1528) nucleotide sequence using the sequence of primers FLA242FU and FLA625RU [flaA allele database (http://phoenix.medawar.ox.ac.uk/flaA/) to elucidate the FlaA SVR type [The MLST of st.org/) . The seqFLA625RU . This nuSVR type ,102.http://evolution.genetics.washington.edu/phylip.html, http://fink.sourceforge.net/) [Desulfovibrio vulgaris subsp. vulgaris strain Hildenborough sequences [The programs SEQBOOT, DNAML, PROML, and CONSENSE are part of the PHYLIP version 3.62 package (ge.net/) . Both thequences . One hunhttp://35.8.164.52/cgis/phylip.cgi, which aligns user-supplied 16S rRNA sequences against the Ribosomal Database Project alignment. The produced alignment was trimmed and gaps removed using an in-house PERL (http://www.perl.org) script. Maximum-likelihood trees were generated using DNAML .16S rRNA trees were generated by first creating a multiple alignment using the “PHYLIP Interface” option of the Ribosomal Database Project release 8.1 (http://www.cgb.ki.se/cgb/groups/sonnhammer/Belvu.html). Each organism's aligned sequences were concatenated using an in-house PERL script. Maximum-likelihood trees were generated using PROML .Protein trees were generated from concatenated multiple alignments of 12 conserved proteins . The seventh circle denotes tRNA, rRNA, and sRNA (tmRNA and 4.5S RNA) loci.Each concentric circle represents genomic data and is numbered from the outermost to the innermost circle. Refer to the key for details on color representations. The first and second circles represent predicted ORFs on the plus and minus strands, respectively. The third circle shows the GC-skew. The fourth circle depicts genetic loci with characteristics or functions of interest: CRISPRs, DNA competence, EP, LOS, prophage and genomic island regions, motility, repeats, and Type I restriction/modification regions. The fifth circle demarcates (2.6 MB EPS).Click here for additional data file.Figure S2(274 KB PDF).Click here for additional data file.Figure S3H. hepaticusATCC 51449 HHGI1 (bottom line) against pCC178 megaplasmid of C. coli RM2228 (middle line). Colors of ORFs are indicated in the key by putative function. Connecting lines represent those ORFs whose protein sequences match at a BLASTP of 30% identity or better. These lines do not indicate the coordinates of match, merely that there is a match.CJIE3 (top linear figure) and (76 KB PDF).Click here for additional data file.Figure S4(A) shows a conceptual diagram indicating where each of the proteins thought to be involved in the T4SS interact. Each corresponding loci is color-coded in each of the plasmids.Campylobacter plasmids; however, the non–C. jejuni plasmids contain a structure that is more similar to the Agrobacterium tumefaciens T4SS. .(B) The T4SS in each of the plasmids demonstrates that a number of the core proteins are conserved in all of the (5.3 MB EPS).Click here for additional data file.Figure S5n = 4) and NCTC 11168 (n = 5). The characters in roman typeface indicate regions flanking the repeat region that are identical in the two strains.The characters in italics indicate the 32-bp spacer sequences that are unique to the two strains; the spacer sequences for NCTC 11168 are 1 bp longer than presented by others . The bol(20 KB DOC).Click here for additional data file.Figure S6The tricarboxylic (TCA) cycle has major variations based on comparative analysis across the strains (please refer to text). Differences in substrate respiration based on an analysis of Biolog data and species-specific pathways are also presented in the text.(51 KB PPT).Click here for additional data file.Figure S7Bordetella pertussis FHA across the outer membrane, was used as the query for BLASTP searches against a database containing Campylobacter protein sequences. Fragments of single accessory proteins were found as matches in the Campylobacter match table .Click here for additional data file.Table S1(19 KB XLS).Click here for additional data file.Table S2(22 KB XLS).Click here for additional data file.Table S3(22 KB XLS).Click here for additional data file.Table S4(16 KB XLS).Click here for additional data file.Table S5(238 KB XLS).Click here for additional data file.Table S6(57 KB XLS).Click here for additional data file.Table S7(31 KB XLS).Click here for additional data file.Table S8(647 KB XLS).Click here for additional data file.Table S9(894 KB XLS).Click here for additional data file.Table S10(155 KB XLS).Click here for additional data file.C. jejuni RM1221 has been deposited at the DNA Data Bank of Japan  under accession number CP000025. The whole-genome shotgun projects for the genomes of C. lari RM2100, C. coli RM2228, and C. upsaliensis RM3195 that were sequenced to at least 8-fold coverage were deposited at DDBJ, EMBL, and GenBank under accession numbers AAFK00000000, AAFL00000000 and AAFJ00000000, respectively. The versions described in this paper are the first versions, AAFK01000000, AAFL01000000 and AAFJ01000000, respectively. Additionally, all sequence traces and assemblies were deposited at the National Center for Biotechnology Information assembly archive (http://www.ncbi.nlm.nih.gov/Traces/assembly). The contig separator that was used to create the pseudomolecules for the unfinished genomes is NNNNNTTAATTAATTAANNNNN.The nucleotide sequence for the closed genome of