Physcomitrella patens (Hedw.) Bruch & Schimp. is an important experimental model system for evolutionary-developmental studies. In order to shed light on the evolutionary history of Physcomitrella and related species within the Funariaceae, we analyzed the natural genetic diversity of the Physcomitrium-Physcomitrella species complex.The moss BRK1 reveals that three Physcomitrium species feature larger genome sizes than Physcomitrella patens and encode two expressed BRK1 homeologs , indicating that they may be allopolyploid hybrids. Phylogenetic analyses of BRK1 as well as microsatellite simple sequence repeat (SSR) data confirm a polyphyletic origin for three Physcomitrella lineages. Differences in the conservation of mitochondrial editing sites further support hybridization and cryptic speciation within the Physcomitrium-Physcomitrella species complex.Molecular analysis of the nuclear single copy gene Physcomitrella patens into three distinct species, namely Physcomitrella patens, Physcomitrella readeri and Physcomitrella magdalenae. We argue that secondary reduction of sporophyte complexity in these species is due to the establishment of an ecological niche, namely spores resting in mud and possible spore dispersal by migratory birds. Besides the Physcomitrium-Physcomitrella species complex, the Funariaceae are host to their type species, Funaria hygrometrica, featuring a sporophyte morphology which is more complex. Their considerable developmental variation among closely related lineages and remarkable trait evolution render the Funariaceae an interesting group for evolutionary and genetic research.We propose a revised classification of the previously described four subspecies of  Physcomitrella patens (Hedw.) Bruch & Schimp., the first bryophyte with completely sequenced and well-annotated nuclear, plastid and mitochondrial genomes ; other accessions were measured up to four times if in doubt of measurement quality.Gametophores, pre-cultured for four weeks, were chopped using a razor blade in buffer , three accessions from P. eurystomum  and two accessions from P. collenchymatum  were reverse transcribed using Superscript III . The reverse primer BRK1_rev (CACCGTTAGCTTCTCGTTCA) was used for priming the first strand synthesis. The resulting cDNA was then further amplified using both BRK1 primers, BRK1_for (GACAATCGCCATTTTTCGAG) and BRK1_rev, and subsequently Sanger sequenced . The primers used were selected based on multiple sequence alignment of clonal sequences.Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit  according to the manufacturer’s instructions, followed by on-column DNAse digestion. The resulting RNA samples from P. collenchymatum (BRK1_for: GACAATCGCCATTTTTCGAG and BRK1_rev CACCGTTAGCTTCTCGTTCA). Each qPCR was carried out using the SensiMix HRM kit  on a LightCycler480  with the following parameters: 95 °C for 10 min, followed by 50 cycles of 95 °C, 60 °C and 72 °C, for 15 s each. High resolution melting analysis was subsequently carried out from 65 °C to 95 °C with a ramp rate of 0.02 °C/s. Melting curve data were analyzed using the Gene Scanning software  and a sensitivity setting of 0.4 with auto-grouping. Pre-melt and post-melt temperatures were chosen in the range of 77 °C and 84 °C respectively.Total RNA was extracted and cDNA was synthesized as described above. cDNA synthesis was verified by PCR. Primers for quantitative Real-Time PCR (qPCR) were designed to specifically amplify a 129 bp long fragment of the exon containing polymorphisms in BRK1 we amplified this gene from selected Funariaceae, including the genera Aphanorrhegma, Physcomitrella, Physcomitrium and Funaria  cluster with the P. patens  accession from Africa , whereas P. pyriforme, P. eurystomum and P. collenchymatum have a larger size . These higher amounts of DNA, together with the BRK1 homeologs, indicate that P. pyriforme, P. collenchymatum and P. eurystomum are recent allopolyploids.Flow cytometric measurements were performed for all Funariaceae for which the ze Table , albeit BRK1 are expressed in P. collenchymatum and P. eurystomum, RT-PCR and quantitative Real-Time PCR (qPCR) were performed using primers that cover polymorphisms in the exon  was used as a reference. Both P. collenchymatum accessions show two distinct expressed transcripts of BRK1, whereas all three P. eurystomum and Physcomitrella yielded only one product in this analysis  with their single polymorphism at the exon level, while the two polymorphisms in the exon of P. collenchymatum  all led to essentially the same topology, separating the Funaria sequences from four well supported clades , Physcomitrella from Australia and Japan  and P. patens ssp. magdalenae from Africa are distinct in the BRK1-based phylogeny  actually belong to two different clades. Moreover, A. serratum clusters in a clade together with P. patens ssp. patens and may therefore be considered to belong to the Physcomitrium-Physcomitrella complex as well.Based on the high conservation of BRK1  with small genetic distances, a second clade is formed by the Japanese and the Australian accessions , while the African P. patens ssp. magdalenae is on a separate branch  loci  were anah Figure . The genk Figure  suggestsP. patens from Gransden (Europe) was checked for a broad set of accessions across the Physcomitrium-Physcomitrella species complex  showed editing at both positions, in accessions of Physcomitrella from Australia and Japan  and P. patens ssp. magdalenae from Africa a T was already encoded at the appropriate positions, making editing obsolete. This supports the independent speciation of at least two Physcomitrella lineages, and the assignment of accessions of P. patens ssp. californica into different Physcomitrella lineages.Requirement for RNA editing at eight positions in the mitochondrial genome known to be edited in rometrica, were foA. serratum to the P. patens clade, as shown in the BRK1-based phylogeny, was confirmed by the same editing requirements of A. serratum and P. patens ssp. patens in contrast to Funaria . However, consistent with the European accessions, the leaflets of the Californian accession develop a costa in most cases, albeit less pronounced and extending to only three quarters of the leaflets  at four nuclear loci . However, each haploid individual generally contained either the sphaericum or pyriforme-like allele, but never both. This finding was consistent with either homoploid hybrid speciation, or allopolyploid speciation, followed by a loss of one homeolog at each of these loci (fractionation). The one exception to this pattern was the ho1 locus where four European isolates of P. pyriforme contained two divergent copies of this locus.Previously,  reportedP. eurystomum, P. collenchymatum, and five European isolates of P. pyriforme have genome sizes larger than those of their putative parent lineages, while they also contain two divergent paralogs of BRK1. For these data to be explained by a homoploid hybrid speciation event, we would have to assume both a massive expansion of transposable elements  from Gransden, Europe; (B)Physcomitrella patens [patens ssp. patens] from Lviv, Europe; (C)Physcomitrella patens [patens ssp. patens] from Illinois, USA; (D)Physcomitrella patens [patens ssp. california] from California, USA; (E)Physcomitrella readeri [patens ssp. readeri] from Australia; (F)Physcomitrella magdalenae [patens ssp. magdalenae] from Rwanda, Africa; (G)Physcomitrella readeri [patens ssp. californica] from Okayama, Japan; (H)Physcomitrella readeri [patens ssp. californica] from Kumamoto, Japan; (I)Physcomitrella readeri [patens ssp. californica] from Saitama, Japan.Habitus of gametophytes. Click here for filePhyscomitrella accessions grown under standardized in vitro conditions on solid mineral medium. (A) Physcomitrella patens [Physcomitrella patens ssp. patens] from Gransden, Europe; (B) Physcomitrella patens [patens ssp. patens] from Lviv, Europe; (C) Physcomitrella patens [patens ssp. patens] from Illinois, USA; (D) Physcomitrella patens [patens ssp. california] from California, USA; (E) Physcomitrella readeri [patens ssp. readeri] Australia; (F) Physcomitrella magdalenae [patens ssp. magdalenae] from Rwanda, Africa; (G) Physcomitrella readeri [patens ssp. california] Okayama, Japan; (H) Physcomitrella readeri [patens ssp. californica] from Kumamoto, Japan; (I) Physcomitrella readeri [patens ssp. california] from Saitama, Japan.Habitus of gametophores and leaflets. Click here for filePhyscomitrella patens [Physcomitrella patens ssp. patens] from Gransden, Europe; (B) Physcomitrella patens [patens ssp. californica] from Del Valle Lake, California, USA; (C) Physcomitrella readeri [patens ssp. californica] from Japan, Okayama; (D) Physcomitrella magdalenae [patens ssp. magdalenae] from Rwanda, Africa. Both accessions from Europe and North America reveal a costa, in contrast to those from Okayama, Japan. Physcomitrella from Rwanda, Africa has orbiculate leaflets in comparison to the lanceolate leaflets of the other accessions.Leaflet details. Leaflets of (A) Click here for file


Caenorhabditis elegans [C. elegans endeavor has been tough to repeat, even with the advent of new high-throughput DNA sequencing technologies. For example, the first plant-parasitic nematode (PPN) genomes were published ten years after the C. elegans genome [Genomic data acquisition is now trivial for biologists. Yet, moving from millions of sequence reads to an assembled and annotated genome continues to pose a daunting challenge. The first animal genome sequenced arose from the free-living model nematode  elegans . This ves genome ,3, and os genome –6.The term “genome skimming” was recently coined –9 to desAnguina agrostis, Globodera ellingtonae, Pratylenchus neglectus, P. penetrans, P. thornei, and Xiphinema americanum. Five of these species are in the “top ten” list of nematode plant pathogens [X. americanum, whose relative in the family Longidoridae, Longidorus kuiperi, also has a small genome size estimate of 56.5 Mb [We applied our genome skimming strategy  to six Pathogens . Our appathogens ,12. We n 56.5 Mb . The N50 56.5 Mb ,12, the  56.5 Mb .C. elegans, in which sequenced DNA came from highly inbred lab populations. Modern pathogen genomics, however, often requires analysis of natural populations in which numerous factors can lead to deviations from the genomic uniformity of an inbred lab culture. For example, pathogens may display population-level genetic variation, within-individual heterozygosity, and other deviations . These pose potential challenges but also opportunities for discovery. Interspecies hybridization and associated genome admixture is of increasing relevance to natural parasite populations [Meloidogyne incognita, the world’s most devastating PPN species, evolved through between-species hybridization, as evidenced by recent phylogenomic analyses and the complex ploidy state of its nuclear genome [Early genome sequencing initiatives focused on model organisms such as ulations . Meloidor genome ,16. The C. elegans and G. rostochiensis , with small variances in copy number among the 65 genes . This small variation likely reflects some small genetic variation among the nematodes sequenced and/or the occurrence of lineage-specific duplicates for some of the orthologs. The median number of orthologs detected was 2 for all 3 Pratylenchus species. For the P. penetrans sample, it was known that nematodes from many field populations were combined in the sample used for the Illumina run, and thus, this genomic diversity is reflected in the high variance in ortholog copy number calculated for this species (4.46). The sequenced DNA samples for P. neglectus and P. thornei, however, each came from single nematode populations. The variances for these two species  were similar to those calculated for A. agrostis and X. americanum. The median value of two copies per ortholog for P. neglectus and P. thornei, combined with their low variance, suggests possible tetraploidy in these species. This hypothesis is supported by cytological evidence collected nearly 50 years ago [P. neglectus and diploidy for P. penetrans.We developed a simple BLASTN-based method to quickly screen for evidence of genomic variation, using a list of 65 conserved single copy orthologs found in the genomes of chiensis  and our observed . The medears ago  suggestiX. americanum, in which only 3 hits were observed. These 3 genes  were found in all 5 of the other species studied; a previous study revealed evidence for an expressed endoglucanase effector in X. index [X. americanum. The 3 X. americanum hit e-values (averaging 7.1 E-30) and hit lengths (averaging 459 bp) were larger and shorter, respectively, compared to averages for these 3 genes in the other 5 species . The addition of a simple single BLAST step to our genome skimming strategy quickly revealed the presence of numerous putative effector genes in the PPN species, though follow-up experimentation and analysis remains necessary to evaluate whether or not bona fide effectors are encoded by the DNA sequences identified.Discovery and functional characterization of effector genes, whose products directly engage in attacks on host defenses, is a central aim of any pathogen genome project. Protein sequences for 10 effectors, well characterized in other PPN species , were usX. index , a congeWolbachia spp., are well known and widespread components of diverse arthropods. Genome sequencing efforts in filarial nematode species revealed the presence of Wolbachia, which functions as an obligate mutualist in these pathogens of animals and humans [Bacterial endosymbionts, such as d humans ,20.X. americanum analysis, evidence for its known endosymbiont Xiphinematobacter sp. [Xiphinematobacter sp. genome. Follow-up bioinformatics, functional genomics, and fluorescence in situ hybridization (FISH) microscopy work supported this hypothesis and suggested that the endosymbiont functions as a nutritional mutualist with its nematode host [We combined “Blob plot” approaches  with BLActer sp.  was obseode host .P. penetrans, in which 1,593 contigs matched bacterial DNA of diverse origins. Although many of these sequences contained high %GC, which were likely environmental contaminants . Nonetheless, our genome skimming pilot experiment provided quick and exciting biological insights and community genomic resources, essentially doubling the number of PPN species for which published genome sequence resources are available. How might our understanding of nematode pathogens change if genome skimming were applied to 600 PPN species instead of 6?S1 Text(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Helicobacter pylori, is an invariably commensal resident of the gut microbiome associated with gastric ulcer in adults. In addition, these patients also suffered from a low grade inflammation that activates the immune system and thus increased shunting of energy to host defense mechanisms. To assess whether a H. pylori infection could affect growth in early life, we determined the expression levels of selected metabolic gut hormones in germ free (GF) and specific pathogen-free (SPF) mice with and without the presence of H. pylori. Despite H. pylori-infected (SPFH) mice display alteration in host metabolism  compared to non-infected SPF mice, their growth curves remained the same. SPFH mice also displayed increased level of eotaxin-1. Interestingly, GF mice infected with H. pylori (GFH) also displayed increased levels of ghrelin and PYY. However, in contrast to SPFH mice, GFH showed reduced weight gain and malnutrition. These preliminary findings show that exposure to H. pylori alters host metabolism early in life; but the commensal microbiota in SPF mice can attenuate the growth retarding effect from H. pylori observed in GF mice. Further investigations of possible additional side effects of H. pylori are highly warranted. It harbors approximately 100 trillion 101 bacteriaHelicobacter pylori, is a key member of the human gastric microbiome. It co-evolved with the human race and our association with H. pylori could be traced as early back as 60,000 years agoH. pylori, gut microbiota, the host and the environment, may contribute to the development of H. pylori-associated diseases.The ancient gastric pathogen, H. pylori and normal gut microbiota during early stages of life using GF and SPF mice models. In order to understand the influences of H. pylori and normal gut microbiota on the development of metabolic and immune systems, we assessed the changes of gut metabolic hormones and cytokines/chemokines in response to H. pylori colonization in both GFH and SPFH mice.In this study, we aimed to investigate the interplay between H. pylori from inoculated SPFH and GFH mice at 2-, 8- and 16-weeks post-colonization. This showed that H. pylori strain 298 successfully colonized the stomach of the mice inoculated with this organism. On the other hand, none of the SPF and GF mice was positive for H. pylori.We recovered H. pylori-colonized SPFH, control GF and H. pylori-colonized GFH mice at 2-, 8- and 16-weeks post-colonization (H. pylori colonization caused significant reduced weight gain. GF mice weighed significantly less compared to their SPF counterparts (p < 0.01), which is consistent with previous reports from other research groups12H. pylori and its interaction with normal gut microbiota have not been studied.In this study, we measured the weight of the control SPF, nization . At the H. pylori colonized   was originally isolated from the stomach, but ghrelin has also been identified in other peripheral tissues, such as the pancreas, ovary and adrenal cortexolonized . Therefo < 0.05) . Normal H. pylori colonization caused significantly increased leptin production in SPFH mice compared to SPF mice (p < 0.01), which became obvious by 8-week post-colonization  and glucagon-like peptide-1 (GLP-1) belong to a class of molecules referred as incretins. L-cells in the ileum secrete GLP-1 postprandialH. pylori or normal gut microbiota alone or together) down-regulated the expression of host GLP-1 or accelerated the degradation of incretins. However, insulin level in our study did not correlate with levels of the incretins. Fasting plasma insulin was low in GF mice despite higher levels of incretins  when measured at 8- and 16-weeks post-colonization . A similncretins . FastingH. pylori appeared to up-regulate PYY production, such that fasting level was significantly higher in SPFH and GFH mice compared to SPF (p < 0.05) and GF (p < 0.01) mice.Peptide YY (PYY) is found in L cells in the mucosa of gastrointestinal tract, especially in the ileum and colon. In addition, a small amount of PYY, about 1–10%, is found in the esophagus, stomach, duodenum and jejunumFasting PYY level was highest among GFH mice followed by those of GF mice . High PYH. pylori on the composition and function of the normal gut microbiome has not been fully elucidated. Nevertheless, in this study, our preliminary results demonstrated that H. pylori may alter the energy homeostasis of its host, and that the normal gut microbiome could make a difference on the effect of H. pylori on its host.The impact of H. pylori and obesity prevalence rates in random population samples of more than 100 human subjects and discovered there was an inverse correlation between prevalence of the bacteria and rate of overweight/obesity in developed countriesH. pylori eradication in human volunteers with an associated increase in body mass intex (BMI) providing evidence that H. pylori colonization might regulate ghrelin and leptin levels, which affects body morphometryH. pylori colonization in young GF mice was shown to result in reduced gain in body mass.A meta-analysis reporting on H. pylori-associated up-regulation of PYY stimulated the host to produce more insulin and leptin in response, thus sustaining normal body mass. However, the long-term implications of higher fasting PYY, insulin and leptin remain to be determined. On the other hand, GF and GFH mice had higher ghrelin, lower leptin and lower insulin than SPF and SPFH mice. In addition, 16-week GFH mice had higher PYY level than GF mice. All these hormonal differences between GF and GFH mice in comparison to SPF and SPFH mice might be linked to their body masses. Interestingly, the effect of H. pylori on body mass was most obvious in GFH mice in the absence of normal gut microbiota.SPFH mice had significantly higher fasting leptin, insulin and PYY than SPF mice. Despite the differences in hormonal levels, SPFH mice had normal body mass. We postulate that The relationship between body mass and different gut metabolic hormones/eotaxin-1 was determined . Leptin H. pylori infection in GFH mice was associated with high plasma PYY level. This may signify high energy expenditure and high rate of metabolism in these mice, which translate to rapid loss of body mass. However, the presence of normal gut microbiota in SPFH mice might provide buffering against the direct adverse impact of H. pylori on body mass. On the other hand, high level of leptin in SPFH mice might suppress food intake and thereby preventing the mice from becoming obese over long term. Results on SPFH mice from this study is consistent with the observation in human subjects that H. pylori eradication resulted in increased BMIH. pylori regulating metabolic hormones may play a role in keeping obesity at bay.In our study, et al. reported that the same GF mice used in this study demonstrated increased motor activity and reduced anxiety compared to SPF miceet al.Heijtz H. pylori and/or gastritis and Parkinson's diseaseH. pylori–induced gastritisH. pylori and normal gut microbiota may intensify chronic inflammation. Therefore, it is possible that H. pylori exposure during early developmental stages will have long-term implication on brain development. Unfortunately, we did not carry out any behavioral study or motor control tests in this study for confirmation.Increased eotaxin-1 level in blood plasma is also associated with aging in mice and humansH. pylori imply that the gastric pathogen, H. pylori, interacts with normal gut microbiota, which alters metabolic and inflammatory pathways on SPFH mice compared to SPF mice  for 4 days at 37°C in a humidified 10% CO2 incubator. The bacteria count was adjusted to the concentration of 109 bacteria per ml to be used for animal inoculation.The C57BL/6 mice-adapted H. pylori were fasted overnight and fed intragastrically with 0.1 ml of bacterial culture three times with a 1-day interval in between. Control animals were inoculated with sterile BHYC media in the same way. Subsequently, mice were sacrificed 2 weeks (N = 3 per group), 8 weeks (N = 5) and 16 weeks (N = 5) post-colonization. Mouse gastrics were collected for H. pylori culturing, and plasma was used for hormone and cytokine analysis.Male SPF and GF C57BL/6 mice of 4–6 weeks old were used for experiments. Male mice were used because they have been reported to show a higher incidence of intestinal-type gastric carcinoma than femalesAll experimental procedures were approved and carried out in accordance to the regulations and guidelines of the SingHealth Institutional Animal Care and Use Committee  and National Cancer Centre Singapore Research Biosafety Committee/SingHealth Institutional Biosafety Committee .Blood was drawn into pre-chilled EDTA-coated tubes and mixed gently by inversion. Blood was transferred into a chilled Eppendorf tube containing 2 M 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride  to a final concentration of 0.2 M and mixed gently. The tube was centrifuged at 3,000 × g for 3 min (4°C). The resultant plasma was added to a cryotube containing 1N HCl to give a final concentration of 0.1N and was stored at −80°C until analysis.H. pylori from mouse gastric stomach was homogenized and inoculated onto chocolate agar plates supplemented with 7% lysed horse blood and, in the case of selective media, trimethoprim (5 μg/ml), vancomycin (10 μg/ml), nalidixic acid (20 μg/ml) and amphotericin B (5 μg/ml), were added. All the antibiotics were from Sigma-Aldrich Corporation . The agar plates were simultaneously incubated at 37°C under humidified conditions with 10% carbon dioxide. H. pylori-like colonies were confirmed by positivity with urease, oxidase and catalase tests.Culturing Milliplex MAP Kit for Mouse Metabolic Magnetic Bead Panel  was used to quantify eight gut hormones that are important regulators of food intake, energy expenditure and body mass: acylated (active) ghrelin, leptin, active amylin, insulin, active glucagon-like peptide-1 (GLP-1), total gastric inhibitory polypeptide (GIP), total peptide YY (PYY) and pancreatic polypeptide (PP). Mouse Cytokine/Chemokine Magnetic Bead Panel  was used to measure a panel of 32 different mouse cytokines/chemokines. Plasma was used and assays were carried out according to manufacturer's instructions. Measurement was performed on Luminex 200™ system with xPONENT™ software, version 3.1 .Statistical analyses were performed using the IBM SPSS version 22.0 software. One-way ANOVA  and Tukey's honestly significant difference (HSD) post-hoc test were performed. Parametric Pearson correlation was carried out. P-value of < 0.05 was considered significant.Y.K., W.Y.W., Q.H.P. and I.M.D.S.: Carried out the lab investigation and data analysis. S.W.S., J.O., A.A.A. and R.M.B.: Animal study. S.P., M.F.L., J.V., K.H.C. and T.L.T.: Planned the experiments.


Hearing screening before discharge was negative. Bilateral deafness was diagnosed at 3 months of life.Intraventricular hemorrhage (IVH) occurs rarely in term infant, since subependymal area is a transient structure in fetal life. IVH in term infant indicates generally that it happened prenatally. Congenital cytomegalovirus (CMV) infection is frequent, occurring in 1% of live births. It is a severe infection leading to developmental defects, especially sensorineural deafness. The diagnosis of congenital CMV infection is rarely evoked in term eutrophic newborn. We report a term male neonate born to a 32-year-old mother gravida 2, para 2. Pregnancy was uneventful. Ultrasound follow-up was unmarked. The newborn was eutrophic, birth weight was 3400g, length was 49cm, and head circumference 33cm. Neonatal examination showed no anomalies. On the first day of life, blood cells count performed for suspected materno-fetal infection discovered fortuitous thrombocytopenia at 30 x 109/L. Within the second day of life, platelet level dropped to 20 x 109/L. Management of thrombocytopenia included multiple platelet transfusion. Cranial ultrasound on day one of life showed bilateral subependymal hemorrhage with cysts (A) and hydrocephaly (B), signs of prenatal occurrence. Platelet phenotypage rules out the diagnosis of allo-immune thrombocytopenia. Laboratory testing for rubella was negative. Blood PCR CMV was positive. Liver function tests noted the absence of hepatic cytolysis and cholestasis. The newborn received intravenous Ganciclovir


E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains.By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify  E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants , was published up to now for akazakii . HoweverE. sakazakii strains from various sources  and of E. sakazakii type strain ATCC 51329 were determined. This sequence data has been deposited in the GenBank . Thereafter, a sequence comparison was performed between the 16S rRNA genes of the fourteen E. sakazakii strains and the E. sakazakii type strain ATCC 29544 by calculation of a distance matrix using the ARB programme. Thirteen E. sakazakii strains used in the study exhibited 99.4 to 100% sequence similarity to the E. sakazakii type strain ATCC 29544 [GenBank: AB004746]. Meanwhile similarity between strain ATCC 51329 and the strain ATCC 29544 was significantly lower (97.9%).As a starting point, the full-length 16S rRNA sequences of thirteen E. sakazakii species. In figure E. sakazakii type strain ATCC 29544, whereas the E. sakazakii strain ATCC 51329 forms a second branch within this group. The subtree was calculated using the TREEPUZZLE tool within the ARB package exhibiting a "consensus tree" from different calculation methods. The value on each branch is the percent occurrence of the branching order in 500 bootstrapped trees. The 92% bootstrap value for the E. sakazakii ATCC 51329 branch strongly supports the theory about the second lineage.Phylogenetic affiliation of the newly obtained sequences to tree_jul04_1450 comprising >28.000 almost full-length (>1449 nucleotides) 16S rRNA gene sequences revealed the presence of two phylogenetically distinct lineages within the E. sakazakii 16S rRNA gene . Within these regions substitutions ranging from two to seven bases are present in all strains investigated and are most pronounced in E. sakazakkii strain ATCC 51329, thus providing the bases for the formation of the second phylogenetic branch. In table E. sakazakii strains investigated in the study in relation to the E. sakazakii typestrain ATCC 29544, E. cloacae typestrain ATCC13047 and E. coli type strain ATCC 11775. Recently, a number of E. sakazakii partial sequences were deposited into the public database , E. sakazakii 1084 fruit powder isolate [GenBank: AY803192], E. sakazakii 954 fruit powder isolate [GenBank: AY752938], E. sakazakii 858 fruit powder isolate [GenBank: AY752936], E. sakazakii 759 fruit powder isolate [GenBank: AY752939], E. sakazakii 236 fruit powder isolate [GenBank: AY752943], E. sakazakii FSM393 baby food isolate [GenBank: AY752941], E. sakazakii FSM33 milk isolate [GenBank: AY752940], E. sakazakii 265 milk powder isolate [GenBank: AY803191], E. sakazakii FSM468 production environment isolate [GenBank: AY752942], E. sakazakii 266 production environment isolate [GenBank: AY803190], E. sakazakii ES4 human isolate [GenBank: AY803186], E. sakazakii ES11 human isolate [GenBank: AY803187], E. sakazakii ES Vo7/24922 human isolate [GenBank: AY803189].AL and TT carried out the experimental part of the study. RS carried out the conception of the study. All authors participated in production and approval of the manuscript.


PLoS Biology, volume 2, issue 5:In Mre11 Assembles Linear DNA Fragments into DNA Damage Signaling ComplexesVincenzo Costanzo, Tanya Paull, Max Gottesman, Jean Gautier10.1371/journal.pbio.0020110DOI: Because of a labeling error, the size of the DNA fragment used throughout the experiments was reported incorrectly (reported as 1 kb). The actual size of the DNA fragment was 131 bp. The fragment corresponds to the M13 DNA sequence from nucleotide 5656 to nucleotide 5787. This difference in fragment size does not affect any of the conclusions of the paper.10.1371/journal.pbio.0020229.This correction note may be found online at DOI:


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


Bacillus licheniformis ATCC 14580 genome was determined, revealing 4,208 predicted protein-coding genes, 7 rRNA operons and 72 tRNA genes.The complete sequence of the  Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.We determined the complete nucleotide sequence of the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.Despite the unmistakable organizational similarities between the  Bacillus licheniformis is a Gram-positive, spore-forming bacterium widely distributed as a saprophytic organism in the environment. This species is a close relative of Bacillus subtilis, an organism that is second only to Escherichia coli in the level of detail at which it has been studied. Unlike most other bacilli, which are predominantly aerobic, B. licheniformis is a facultative anaerobe, which may allow it to grow in additional ecological niches. Certain B. licheniformis isolates are capable of denitrification; the relevance of this characteristic to environmental denitrification may be small, however, as the species generally persists in soil as endospores [dospores .B. licheniformis and its extracellular products. The species has been used for decades in the manufacture of industrial enzymes including several proteases, α-amylase, penicillinase, pentosanase, cycloglucosyltransferase, β-mannanase and several pectinolytic enzymes. The proteases from B. licheniformis are used in the detergent industry as well as for dehairing and bating of leather [B. licheniformis are deployed for the hydrolysis of starch, desizing of textiles and sizing of paper [B. licheniformis strains are also used to produce peptide antibiotics such as bacitracin and proticin in addition to a number of specialty chemicals such as citric acid, inosine, inosinic acid and poly-γ-glutamic acid [B. licheniformis isolates can mitigate the affects of fungal pathogens on maize, grasses and vegetable crops [There are numerous commercial and agricultural uses for  leather ,3. Amylamic acid . Some B.le crops . As an eB. licheniformis can be differentiated from other bacilli on the basis of metabolic and physiological tests [B. licheniformis is closely related to B. subtilis and Bacillus amyloliquefaciens on the basis of comparisons of 16S rDNA and 16S-23S internal transcribed spacer (ITS) nucleotide sequences [et al. [B. licheniformis chromosome using a PCR approach, and established a number of regions of colinearity where gene content and organization were conserved with the B. subtilis genome.al tests ,7; howevequences . Lapidus [et al.  recentlyB. licheniformis is an industrial organism used for the manufacture of enzymes, antibiotics, and chemicals, important in nutrient cycling in the environment, and a species that is taxonomically related to B. subtilis, perhaps the best studied of all Gram-positive bacteria, we derived the complete nucleotide sequence of the B. licheniformis type strain (ATCC 14580) genome. With this data in hand, functional and comparative genomics studies can be initiated that may ultimately lead to new strategies for improving industrial strains as well as better understanding of genome evolution among the species within the subtilis-licheniformis group.Given that B. licheniformis ATCC 14580 consists of a circular chromosome of 4,222,336 base-pairs (bp) with an average G+C content of 46.2% . Among the list of hypothetical and conserved hypothetical gene products, 683 (52%) have protein motifs contained in PFAM . There are 72 tRNA genes representing all 20 amino acids and seven rRNA operons.The genome of B. subtilis and other bacteria. These included co-localization of four genes  near the origin, GC nucleotide skew ((G-C)/(G+C)) analysis, and the presence of multiple dnaA-boxes and AT-rich sequences immediately upstream of the dnaA gene [BstBI restriction site between the rpmH and dnaA genes to be the first nucleotide of the B. licheniformis genome. The replication termination site was localized near 2.02 megabases (Mb) by GC skew analysis. This region lies roughly opposite the origin of replication  in B. licheniformis. The Bacillus halodurans genome also lacks an obvious rtp function [B. subtilis acquired the rtp gene following its divergence from B. halodurans and B. licheniformis.The likely origin of replication Figure  was idennaA gene -12. On tfunction ; therefoB. licheniformis ATCC 14580 contains nine identical copies of a 1,285 bp insertion sequence element termed IS3Bli1 [IS3 family elements [IS3Bli1 encodes two predicted overlapping CDSs, designated orfA and orfB in relative translational reading frames of 0 and -1. The presence of a 'slippery heptamer' motif, AAAAAAG, before the stop codon in orfA may indicate that programmed translational frameshifting occurs between these two coding sequences, resulting in a single gene product [orfB gene product harbors the DD(35)E(7)K motif, a highly conserved pattern among insertion sequences. Eight of these IS3Bli1 elements lie in intergenic regions, and one interrupts the comP gene as noted previously [-11) to one identified in the Thermoanaerobacter tengcongensis genome [B. halodurans [Oceanobacillus iheyensis [Streptococcus agalactiae [Streptococcus pyogenes [The genome of  IS3Bli1 . This seelements  includineviously . In addis genome ; howeverlodurans , Oceanobheyensis , Streptoalactiae  and Strepyogenes .B. subtilis prophages PBSX and φ-105), were discovered by noting the presence of nearby genes that code for the large subunit of terminase, a signature protein that is highly conserved among prophages [B. subtilis phage SPP1); however, its absence may be the result of genome deterioration during evolution. Interestingly, we observed that regions in which the G+C content is less than 39% usually encoded proteins that have no B. subtilis ortholog and share identity only with hypothetical and conserved hypothetical genes. Two of these AT-rich segments correspond to the NZP2 and NZP3 prophages.The presence of several bacteriophage lysogens or prophage-like elements was revealed by Smith-Waterman comparisons to other bacterial genomes and by their AT-rich signatures Figure , Table 2rophages . InteresB. licheniformis. The first gene encodes a protein of 1,277 amino acids for which Interpro predicts 16 collagen triple-helix repeats, and the amino acid pattern TGATGPT is repeated 75 times within the polypeptide. The second CDS is smaller, and encodes a protein with 11 collagen triple-helix repeats; the same TGATGPT motif recurs 56 times. The primary translation products from these genes do not contain canonical signal peptides for secretion, and they do not contain motifs for the twin-arginine or sortase-mediated translocation pathways. Therefore, it is not likely that they are exported to the cell surface or the extracellular medium. Interestingly, the chromosomal region (19 kb) adjacent to these genes is clearly non-colinear with the B. subtilis genome, and virtually all of the predicted genes encode hypothetical or conserved hypothetical proteins. There are a number of bacterial proteins listed in PIR that also contain collagen triple-helix repeat regions, including two from Mesorhizobium loti (accession numbers NF00607049 and NF00607035) and three from B. cereus . These putative orthologs share 53-76% amino-acid sequence identity with their counterparts in B. licheniformis, and their functions are unknown.An isochore plot Figure  also revBacillus licheniformis genome, 689 of the 4,208 gene models have signal peptides forecast by SignalP [B. licheniformis ATCC 14580 genome that are not found in B. subtilis 168. In accordance with its saprophytic lifestyle, the secretome of B. licheniformis encodes numerous secreted enzymes that hydrolyze polysaccharides, proteins, lipids and other nutrients.In the  SignalP  and 134 B. licheniformis, and there are no counterparts in B. subtilis 168. The enzymes encoded by the first gene cluster include two putative endoglucanases belonging to glycoside hydrolase families GH9 and GH5, a probable cellulose-1,4-β-cellobiosidase of family GH48, and a putative β-mannanase of family GH5. The β-mannanase (GH5) and endoglucanase (GH9) both harbor carbohydrate-binding motifs. With the exception of the cellulose-1,4-β-cellobiosidase (GH48), all of the gene products encoded in this cluster have secretory signal peptides, and all have homologs in Bacillus species other than B. subtilis. The overall G+C content of this cluster (48%) does not appear to differ appreciably from that of the genome average (46%). The second gene cluster encodes a putative β-glucosidase (GH1) and three components of a cellobiose-specific PTS transport complex. A second β-glucosidase (GH3) gene is present at an unlinked locus in the genome. Collectively, the genes in these two clusters should enable B. licheniformis to utilize cellulose as a carbon and energy source, converting it to cellobiose and ultimately glucose. In this regard, we have confirmed that B. licheniformis ATCC 14580 is capable of growth on carboxymethyl cellulose as a sole carbon source (not shown). The chromosome of B. licheniformis ATCC 14580 encodes a number of additional carbohydrase activities that may allow the organism to grow on a broad range of polysaccharides. These include xylanase, endo-arabinase and pectate lyase that may be involved in degradation of hemicellulose, α-amylase and α-glucosidase for starch hydrolysis, chitinases for the breakdown of chitooligosaccharides from fungi and insects, and levanase for utilization of β-D-fructans (levans). Several of these activities are marketed as industrial enzymes.Cellulose is the most abundant polysaccharide on Earth, and microorganisms that hydrolyze cellulose contribute to the global carbon cycle. Interestingly, two gene clusters involved in cellulose degradation and utilization were discovered in B. licheniformis ATCC 14580 possesses the ability to acquire nitrogen from exogenous proteins, peptides, amino acids, ammonia, nitrate and nitrite. Like B. subtilis, the repertoire of extracellular proteases produced by B. licheniformis includes serine proteases , metalloprotease (mpr), and an assortment of endo- and exopeptidases , yfxM, yuiE, yusX, ywaD, pepT). However, B. licheniformis also has the capacity to produce a number of additional proteases and peptidases that are not encoded in the B. subtilis genome. These include a clostripain-like protease, a zinc-metallopeptidase, a probable glutamyl endopeptidase, an aminopeptidase C homolog, two putative dipeptidases and a zinc-carboxypeptidase.Saprophytic organisms must utilize a variety of nitrogenous compounds as nutrients for growth and metabolism. On the basis of the information encoded in its genome, B. licheniformis also has the ability to utilize amino and imino nitrogen from arginine, asparagine and glutamine via arginine deiminase, arginase, asparaginase and glutaminase activities. Interestingly, there appear to be two genes each for arginase, asparaginase and glutaminase. Presumably, the arginine deiminase activity is expressed during anaerobic growth on arginine, whereas the arginase activities are predominant during aerobic growth. The occurrence of putative arginase genes is somewhat of an enigma in B. licheniformis, because there are no genes encoding urease activity for the hydrolysis of urea that is generated by the arginase reaction. In addition to the absence of urease gene homologs (ureABC) in B. licheniformis, the glutamine ABC transporters  are also lacking.B. licheniformis and B. subtilis owing to the presence of key genes such as glnA, glnR, tnrA and nrgA in both species. Likewise, the pathways for nitrate/nitrite transport and metabolism in B. licheniformis appear to be analogous to the corresponding pathways in B. subtilis as suggested by the presence of nasABC (nitrate transport), narGHIJ (respiratory nitrate reductase), and nasDEF (NADH-dependent nitrite reductase) genes. Unlike B. subtilis, B. licheniformis evidently possesses the capability for anaerobic respiration using nitric oxide reductase. Moreover, the gene encoding this activity lies in a cluster that includes CDSs for narK (nitrite extrusion protein), two putative fnr proteins , and a dnrN-like gene product (nitric oxide-dependent regulator). These observations are consistent with previous findings that certain B. licheniformis isolates are capable of denitrification [B. licheniformis may be small as the species exists predominantly as endospores in soil [It appears that nitrogen assimilation and transport pathways may be coordinated similarly in fication . While d in soil .N-carbamoyl amino acids. Hydantoinase activities have been detected in a variety of bacterial genera, and a cluster of six genes in B. licheniformis appears to confer a similar capability. This gene cluster encodes N-methylhydantoinase (ATP-hydrolyzing), hydantoin utilization proteins A and B (hyuAB homologs), a possible transcriptional regulator (TetR/AcrR family), a putative pyrimidine permease, and a hypothetical protein that contains an Interpro domain (IPR004399) for phosphomethylpyrimidine kinase.Microbial D-hydantoinase enzymes have been applied to the industrial production of optically pure D-amino acids for synthesis of antibiotics, pesticides, sweeteners and therapeutic amino acids . This enet al. [B. subtilis 168. This list includes several chaperonins, signal peptidases, components of the signal recognition particle and protein translocase complexes. All members of this list have B. licheniformis counterparts. In addition to the Sec pathway, some B. subtilis proteins are directed into the twin-arginine (Tat) export pathway, possibly in a Sec-independent manner. Curiously, the B. licheniformis genome encodes three tat gene orthologs , but two others (tatAC and tatAD) are conspicuously absent. Furthermore, specific proteins may be exported to the cell surface via lipoprotein signal peptides or sortase factors. Lipoprotein signal peptides are cleaved with a specific signal peptidase (Lsp) encoded by the lspA gene in B. subtilis. An lspA homolog can be found in B. licheniformis as well, suggesting that this species may possess the ability to export lipoproteins via a similar mechanism. Lastly, surface proteins in Gram-positive bacteria are frequently attached to the cell wall by sortase enzymes, and genome analyses have revealed that more than one sortase is often produced by a given species. In this regard, three possible sortase gene homologs were detected in the genome of B. licheniformis ATCC 14580. Together these observations suggest that the central features of the protein export machinery are principally conserved in B. subtilis and B. licheniformis.Kunst et al.  listed 1et al. [B. licheniformis. These six exceptions (spsABCEFG) comprise an operon involved in synthesis of a spore coat polysaccharide in B. subtilis. In addition, the response regulator gene family (phrACEFGI) appears to have a low level of sequence conservation between B. subtilis and B. licheniformis.From the list of 139 sporulation genes tabulated by Kunst et al. , all butB. licheniformis strains [B. licheniformis genome encodes all of the late competence functions ascribed in B. subtilis , it lacks an obvious comS gene, and the comP gene is punctuated by an insertion sequence element (IS3Bli1), suggesting that the early stages of competence development have been pre-empted in B. licheniformis ATCC 14580. Whether these early functions can be restored by introducing the corresponding genes from B. subtilis is unknown. In addition to an apparent deficiency in DNA uptake, two type I restriction-modification systems were discovered that may also contribute to diminished transformation efficiencies. These are distinct from the ydiOPS genes of B. subtilis, and could participate in degradation of improperly modified DNA from heterologous hosts used during construction of recombinant expression vectors. Each of these loci in B. licheniformis (designated as BliI and BliII) encode putative HsdS, HsdM and HsdR subunits that share significant amino-acid sequence identity to type I restriction-modification proteins in other bacteria. Curiously, the G+C-content for these loci (37%) is substantially lower than the overall genome average (46%) which may hint that they are the result of gene acquisitions. Lastly, the synthesis of a glutamyl polypeptide capsule has also been implicated as a potential barrier to transformation of B. licheniformis strains [B. subtilis usually do not produce significant capsular material, the genome sequence of B. subtilis 168 indicates that they may harbor the genes required for synthesis of polyglutamic acid. In contrast, many B. licheniformis isolates produce copious amounts of capsular material, giving rise to colonies with a wet or slimy appearance. Six genes were predicted (ywtABDEF and ywsC orthologs) that may be involved in the synthesis of polyglutamic acid capsular material in B. licheniformis.Natural competence (the ability to take up and process exogenous DNA in specific growth conditions) is a feature of few  strains . The rea strains . While lB. licheniformis isolates [bac operon [bac operon is not present in the type strain (ATCC 14580) genome. Seemingly, the only non-ribosomal peptide synthase operon encoded by the B. licheniformis type strain genome is that responsible for lichenysin biosynthesis. Lichenysin structurally resembles surfactin from B. subtilis [B. licheniformis counterparts for the pps (plipastatin synthase) and polyketide synthase (pks) operons of B. subtilis. Collectively, these two regions represent sizeable portions  of the chromosome in B. subtilis, although they are reportedly dispensable [Bacitracin is a cyclic peptide antibiotic that is synthesized non-ribosomally by some isolates . While tc operon . Interessubtilis , and thepensable .Bacillus sp. strain HIL-Y85/54728 [B. licheniformis may be an attractive candidate for the development of such an expression system.Unexpectedly, a cluster of 11 genes was found encoding a lantibiotic, with its associated modification and transport functions. We designated this peptide of 75 amino acids as lichenicidin, and its closest homolog is mersacidin from 85/54728 . Lantibi85/54728 . Like me85/54728 . HoweverB. subtilis 168, the B. licheniformis ATCC 14580 chromosome harbors a siderophore biosynthesis gene cluster (dhbABCEF), and the organization of the cluster is similar to the corresponding chromosomal segment in B. subtilis. In addition, the B. licheniformis genome contains a second gene cluster of four genes (iucABCD) that show significant similarity to proteins involved in aerobactin biosynthesis in E. coli. Surprisingly, a gene encoding the receptor protein (iutA homolog) was not found in B. licheniformis. The B. halodurans genome also contains genes that are homologous to iucABCD, but like B. licheniformis, no iutA homolog could be found using BLAST or Smith-Waterman searches.Like B. licheniformis ATCC 14580 gene models were compared to the list of essential genes in B. subtilis [B. subtilis have orthologs in B. licheniformis, and most are present in a wide range of bacterial taxa. In pairwise BLAST comparisons, 66% of the predicted B. licheniformis genes have orthologs in B. subtilis, and 55% of the gene models are represented by orthologous sequences in B. halodurans .The subtilis . Predictet al. [B. licheniformis and B. subtilis .The organism ; however. cereus . Cereuli. cereus . ImportaB. licheniformis, Manachini et al. [B. licheniformis. Similarly, De Clerck and De Vos [B. licheniformis isolates.In a comparison of the genotypic and phenotypic characteristics among 182 soil isolates of i et al.  observedd De Vos  proposedB. licheniformis ATCC 14580 was sequenced by a combination of the whole-genome shotgun method [MboI-digested genomic DNA that was enriched for fragments of 2-3 kb by preparative agarose gel electrophoresis. Approximately 49,000 random clones were sequenced using dye-terminator chemistry (Applied Biosystems) with ABI 377 and ABI 3700 automated sequencers yielding approximately 6× coverage of the genome. A combination of methods was used for gap closure, including sequencing on fosmids [The genome of n method  and fosmn method . Plasmid fosmids  and prim fosmids .AAGGAG . This motif concurs with the consensus Shine-Dalgarno sequence for B. subtilis (AAAGGAGG) [B. subtilis homologs were also used to determine the incidence of specific genes. During the gene-finding process, possible errors and frameshifts were detected by both visual inspection of the CDSs to look for interrupted or truncated genes and by deploying FrameD software [IS3Bli1 elements). It is not known at present whether these represent pseudogenes or instances of programmed translational frameshifting. The positions of rRNA operons in the genome assembly were confirmed by long-range PCR amplification using primers that annealed to genes flanking the rRNA genes. These PCR fragments were sequenced to high redundancy and the consensus sequences were manually inserted into the assembly. Among the seven rRNA operons, the nucleotide sequences of 16S and 23S genes are at least 99% identical, differing by only one to three nucleotides in pairwise comparisons. Protein-coding sequences were annotated in an automated fashion with the following software applications. Predicted proteins were compared to the nonredundant database PIR-NREF [B. subtilis genome [-5. InterProScan was used to predict putative function [B. licheniformis genes that shared significant homology with B. subtilis counterparts were named using the nomenclature in the SubtiList database [Protein-coding regions in the assembled genome sequence were identified using a combination of previously described software tools including EasyGene , GlimmerAAGGAGG) . RBSFINDsoftware . FrameshPIR-NREF  and the s genome  using BLfunction . The Intfunction , TIGRFAMfunction , Interprfunction  signal pfunction  and tranfunction . These Cfunction  with manfunction ,55. Phagfunction . B. lichdatabase  with upddatabase  and UniPdatabase  databaseB. licheniformis, B. subtilis and B. halodurans genomes with precomputed BLAST results stored in a local database.VisualGenome software  was used for comparisons of ortholog distribution among B. licheniformis ATCC 14580 genome is CP000002. An interactive web portal for viewing and searching the assembled genome based on the generic genome browser developed by Stein et al. [The GenBank accession number for the n et al.  is availn et al. .


Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.The development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 10 The use of adeno-associated viruses (AAV) for the targeting of the central nervous system (CNS) has revolutionized gene therapy for neurological disorders. However, AAV-vectors still face significant challenges in achieving broad and efficient CNS biodistribution and transduction. Although currently in clinical use, AAV9-mediated gene delivery requires high viral load to achieve relatively limited transduction throughout the CNS. Recent development of a Cre-dependent in vivo platform for the selection of target-specific AAV capsids led to the discovery of novel engineered AAV9-derived variants, AAV.PHP.eB and AAV.PHP.B , 2. BothSignificant evidence, primarily with AAV9, shows that more efficient global CNS penetration of AAVs can be achieved when delivered into the cerebrospinal fluid (CSF) –10. IntrHere, we aimed to address whether the AAV.PHP.eB virus improves CSF-mediated biodistribution in comparison to its parent serotype, AAV9, particularly for use in rat models of CNS disorders. We report significant enhancement of CNS viral transduction via delivery of AAV.PHP.eB into the CM of aged rats compared AAV9. Using a GFP reporter transgene to characterize viral biodistribution, we observed enhanced GFP expression consistently throughout neocortical and subcortical brain regions, including regions distal to the CM. Efficient transduction was also observed in regions without direct interface to CSF. We demonstrate significant increases in AAV.PHP.eB-mediated viral transduction compared to AAV9 with expected neuronal tropism and no indications of regional inflammation due to virus or transgene expression. These data demonstrate the value of using AAV.PHP.eB in CNS targeting for gene therapy studies in large rodents and highlight the potential of intracisternal (ICM) injections of this serotype clinically.All husbandry and procedural use of animals was approved by the Institutional Animal Care and Use Committee (IACUC) at Rush University Medical Center. Thirty middle-aged (6–9 month old), female Sprague–Dawley rats (Charles River Laboratories) were maintained at 25 °C and 50% humidity. Animals were housed in 12-hour light/dark cycle and fed chow and water ad libitum. All animals used in the study were acclimatized in the vivarium and handled for two weeks prior to initiation of study.http://n2t.net/addgene:37825; RRID:Addgene_37825). Animals were anesthetized with sodium ketamine hydrochloride/xylazine hydrochloride solution (100 mg/kg and 10 mg/kg) and loaded into a stereotactic frame with the snout of the animal depressed by the nose bar to elevate the occipital crest , or AAV.PHP.eB-GFP  at an injection rate of 10 µL/min with a 10-minute post-injection needle dwell time . At 1-month post injection, animals were deeply anesthetized with ketamine/xylazine cocktail and transcardially perfused with ice-cold PBS. Brains were removed and hemisected with one hemisphere immersion fixed overnight in 4% paraformaldehyde and the other hemisphere dissected to separate forebrain, midbrain, cerebellum, and spinal cord. CNS regions, as well as liver samples, were snap frozen and stored at −80 °C for molecular processing.Viral preps of AAV-CAG-GFP were a gift from Edward Boyden  with Sidak’s post-hoc test for multiple comparisons. All data represented as Mean ± SEM with *p < 0.05 and **p < 0.01.All statistical analysis was performed using Prism software . Stereological estimations and immunoblot data were analyzed with two-tailed, unpaired Here we report results from a rapid and streamlined intrathecal protocol that describes single-administration, high-volume dosing into the CM irrespective of adult animal age or weight Fig. . The metWe first qualitatively evaluated the relative transduction profiles of AAV.PHP.eB-GFP and AAV9-GFP following equititer, high-dose ICM injection. Rats injected with AAV.PHP.eB-GFP showed significantly higher expression of GFP throughout the rostracaudal extent of the rat brain Fig. . GFP hisp = 0.016), low-dose AAV.PHP.eB , and low-dose AAV9  , somatosensory cortex , and substantia nigra  of AAV.PHP.eB-treated animals versus AAV9-treated.To quantify AAV.PHP.eB- and AAV9-mediated biodistribution, unbiased stereological estimates of GFP + neurons were performed across four primary regions . High-dose AAV.PHP.eB significantly improved cerebellar transduction compared to high-dose AAV9  Figs. , S1a. Li1) Figs. . All subp = 0.049), ~14-fold higher in the midbrain (p = 0.049), and ~4-fold higher in the cerebellum (p = 0.011) in AAV-PHP.eB-treated rats  , indicating transduction efficiency increases remain limited to the CNS and not peripheral tissue  Fig. . Liver esue Fig. .The use of ICM delivery may provide a platform for effective pan-CNS treatment of neurodegenerative diseases. To this end, we aimed to evaluate transduction levels across regions-of-interest pertinent to rodent models of Parkinson’s Disease (PD) , 12. SixThe generation of AAV.PHP.B and AAV.PHP.eB, as well as recent advances in capsid engineering , 14, hasIn this study, we evaluated a head-to-head comparison of equititer doses of AAV9 and AAV.PHP.eB to determine whether the superior CNS transduction phenomena observed in AAV.PHP.eB-injected mice were maintained in rats. Using a CAG promoter to drive GFP expression in both vectors, we observed superior transduction throughout the brain of AAV.PHP.eB-infused animals. Transduced soma were a prominent source of the increased AAV.PHP.eB biodistribution, as determined by stereological analysis and observed densities of GFP+ parikariya. However, visual inspection of AAV.PHP.eB-treated rat brains revealed marked increases in neuritic GFP expression, indicating a significant fraction of GFP-stained tissue sourced from neuronal processes compared to that of AAV9. Higher GFP staining was also detected in the neuropil surrounding the areas such as molecular layer of the dentate gyrus.11 vg/animal) were ~3 times lower than the minimum doses utilized in comparable rat studies investigating intravascular delivery [We incorporated several factors into our study to address the challenges in high-fidelity neurological disease modeling and intervention. First, viral penetration across the BBB is less effective in adult subjects compared to neonatal rodents –21. We udelivery , 5. Intrdelivery . We alsodelivery . The levThe enhanced BBB transport mechanism for AAV.PHP vectors has recently been linked to the GPI-tethered receptor lymphocyte antigen six complex (locus A), Ly6A , 24. TheAlthough AAV.PHP.eB exhibited improved CNS transduction efficiency compared to AAV9, ICM viral diffusion resulted in moderate levels of intra-cohort variability in GFP expression. One potential source of variability may have been the use of equivalent volumes of injectant across animals, regardless of weight. However, linear regression analyzing cerebellar GFP+ cell estimates and body weight showed conflicting data between high-dose and low-dose cohorts on whether animal size is a critical factor for predicting biodistribution. Use of animal brain, weight, or CSF volume may provide better standards for comparison to predict biodistribution for follow-up studies. Although AAV.PHP.eB improved transduction efficiency via ICM injection over AAV9, the improvement was modest compared to efficiency spikes observed through intravascular comparisons in mice . In addiIntrathecal administration of AAV.PHP.eB successfully achieves widespread transduction throughout the neuraxis with a simplified ICM injection paradigm that can be used in older rats with relatively low viral titers and minimal risk of adverse effects on subjects. These data show the relative utility of AAV.PHP.eB vectors compared to AAV9 for development of gene therapies in rat models of neurodegenerative diseases, with potential for more effective clinical therapeutics.SUPPLEMENTARY INFORMATION - Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9


S. gallolyticus is one of the pathogenic agents of endocarditis, and mitral valve aneurysm is a rare but potentially devastating complication. We present a case of S. gallolyticus aortic valve endocarditis with concomitant anterior mitral valve leaflet aneurysm. Patient underwent surgery before aneurysm perforation, and postoperative course was uneventful. Time of surgery is crucial to avoid severe complications due to aneurysm rupture. Streptococcus gallolyticus is one of the etiologic factors of infective endocarditis (IE); it is also associated with colonic neoplasia. Rarely, aortic valve (AV) infection may lead to a specific scenario involving mitral valve (MV) and causing MV anterior leaflet aneurysm. Infection can spread to MV through direct contact between aortic vegetation and MV leaflet, contiguous infection through mitral-aortic fibrosa, and/or regurgitant infectious flow from aortic valve [S. gallolyticus and intraoperative evidence of anterior MV leaflet aneurysm.ic valve , 2. MV aic valve . Accordiic valve . In caseic valve , 3, 5–14S. gallolyticus. Treatment with ceftriaxone was started, and imaging found no sign of embolization. Patient had been reevaluated 4 weeks later, finding worsened aortic regurgitation (3 + /4+) with regurgitant flow directed to the anterior MV leaflet and confirming the vegetation-like formation on A2 (suspecting now a pseudoaneurysm) with worsened MV regurgitation (3 + /4+) , moderate aortic regurgitation, and moderate ascending aorta dilation. In November 2021, patient experienced dyspnea, high-rate AF, and low-grade fever; he had been admitted in a regional hospital and diagnosis was acute congestive heart failure (HF). Transesophageal echocardiogram (TEE) revealed a tricuspid aortic valve with mild regurgitation  and thin fibrotic striae on the arterial side of the cusps. It also revealed an isoechogenic vegetation-like formation (10 mm) on the atrial side of A2 scallop of the MV, associated with trivial-mild MV regurgitation (1 + −2+/4+). Left ventricular ejection fraction (EF) was preserved. Blood cultures were positive for After 8-week intravenous antibiotic therapy, patient was hemodynamically stable and his symptoms improved, but vegetations did not. Patient was then transferred to our center, where he underwent surgery on January. Intraoperatively, a prominent aneurysm was noted on the atrial aspect of A2, with no signs of rupture ; apart fS. gallolyticus-related native aortic valve infective endocarditis with concomitant anterior mitral valve leaflet aneurysm. Fortunately, patient underwent surgery before aneurysm rupture. We hypothesize that regurgitant infectious flow from aortic valve played a major role in the present case. Incidence of MV aneurysms is low, and echocardiographic diagnosis is made in just 0.2-0.3% of overall cases. Mostly, aneurysm is related to IE [S. gallolyticus related to colon cancer and colon polyps [S. gallolyticus-related IE.This is a case of ed to IE , 8, 11. ed to IE . Few dozed to IE . Despiteed to IE . The imped to IE ; our pated to IE . Due to n polyps , 18, anyS. gallolyticus, colonoscopy should be done to rule out colon cancer.Mitral valve aneurysm is rare but mostly associated with infective endocarditis. Transesophageal echocardiogram is crucial to determine vegetation size and mobility but also signs of concomitant abscess, perforation, and valve aneurysm. If aneurysm is present or suspected, surgery is strictly suggested due to high risk of rupture with subsequent deterioration of hemodynamics  and systemic embolization. If blood culture is positive for