Genomic characterization of the Yersinia genus

Background

New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments.

Results

We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats.

Conclusions

Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.     

Genomic characterization of the Taylorella genus

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus.     

Genomic and functional characterization of StCDPK1

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4–8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (β7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the β7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.     

On the sensitivity of intact cells to perturbation by ethanol

A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range (100 - 200 mM). Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations (greater than 250 mM) to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol-induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM1 sensitized the liposomes to ethanol-induced perturbation.     

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.     

Genomic characterization of the S-adenosylmethionine decarboxylase genes from soybean

A full-length gene GmSAMDC1, encoding the S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in polyamine biosynthesis, was identified from soybean expressed sequence tags and was characterized. GmSAMDC1 encoded a peptide of 355 amino acids. When compared with other plant SAMDCs, the GmSAMDC1 protein had several highly conserved regions including a putative pro-enzyme cleavage site and a PEST sequence. The 5 leader sequence of the the GmSAMDC1 mRNA contained two additional open reading frames (ORFs), which may regulate the translational process. The genomic sequence of the GmSAMDC1 gene contained three introns in the 5 leader sequence, but no intron in the 3-UTR or the main pro-enzyme ORF. A simple sequence repeat (SSR) was found in intron 2, and the GmSAMDC1 gene was mapped to linkage group D1 using this SSR. The genomic organization of the GmSAMDC1 gene in the subgenus Glycine and the subgenus Soja was found to be different by Southern-blot and PCR analysis. A pseudogene, GmSAMDC2, was also identified. This gene contained no intron and lost its two uORFs. Northern-blot analysis showed that the GmSAMDC1 gene expression was induced by salt, drought and cold, but not induced by wounding; suggesting that the gene was implicated in response to multiple-stress conditions.Communicated by H. F. Linskens     

Genomic characterization and high prevalence of bocaviruses in swine

Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ～2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45-75% and 55-70% respectively, with 30-47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses.     

Genomic characterization of the RUNX2 gene of Fugu rubripes

Inducibility of the mouse gene imap38 in the spleen has been recently described to correlate with resistance to Plasmodium chabaudi malaria. Here, we characterize the human ortholog gene himap1. The HIMAP1 34 kDa protein is localizable at the endoplasmic reticulum in transfected cells. It contains a GTP-binding domain, but it does not bind GTP, in contrast to mouse IMAP38. The himap1 gene belongs to a gene family clustered on chromosome 7q32-36 within a region highly syntenic to the mouse imap38 locus on chromosome 6B. The himap genes 1, 2, 3, and 4 display a conserved intron/exon structure. The mRNA of the himap1 gene is predominantly expressed in the spleen, in lymph nodes to a lesser extent, and only at very low levels in diverse cancer cell lines. In accordance, imap-like genes in mice and plants are associated with proliferative and apoptotic events suggesting a role in the control of cell death/survival.     

Genomic characterization of two Staphylococcus epidermidis bacteriophages with anti-biofilm potential

ABSTRACT: BACKGROUND: Staphylococcus epidermidis is a commensal bacterium but can colonize the hospital environment due to its ability to form biofilms favouring adhesion to host tissues, medical devices and increasing resistance to antibiotics. In this context, the use of phages to destroy biofilms is an interesting alternative. RESULTS: The complete genomes of two Staphylococcus epidermidis bacteriophages, vB_SepiSphiIPLA5 and vB_SepiS-phiIPLA7, have been analyzed. Their genomes are 43,581 bp and 42,123 bp, and contain 67 and 59 orfs. Bioinformatic analyses enabled the assignment of putative functions to 36 and 29 gene products, respectively, including DNA packaging and morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, regulation, modification and replication. A point mutation in vB_SepiS-phiIPLA5 lysogeny control-associated genes explained its strictly lytic behaviour. Comparative analysis of phi- IPLA5 and phi-IPLA7 genome structure resembled those of S. epidermidis phiPH15 and phiCNPH82 phages. A mosaic structure of S. epidermidis prophage genomes was revealed by PCR analysis of three marker genes (integrase, major head protein and holin). Using these genes, high prevalence (73%) of phage DNA in a representative S. epidermidis strain collection consisting of 60 isolates from women with mastitis and healthy women was determined. Putative pectin lyase-like domains detected in virion-associated proteins of both phages could be involved in exopolysaccharide (EPS) depolymerization, as evidenced by both the presence of a clear halo surrounding the phage lysis zone and the phage-mediated biofilm degradation. CONCLUSIONS: Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have a mosaic structure similar to other widespread S. epidermidis prophages. Virions of these phages are provided of pectin lyase-like domains, which may be regarded as promising anti-biofilm tools.     

Genomic characterization ofCampylobacter jejuni by field inversion gel electrophoresis

The genome ofCampylobacter jejuni was characterized by field inversion gel electrophoresis (FIGE) after digestion with three rare-cutting restriction endonucleases. The restriction enzymesSac II (5-CCGCGG),Sal I (5-GTCGAC), andSma I (5-CCCGGG) were found to produce 13, 5, and 8 fragments respectively from theC. jejuni genome. The fragment sizes ranged from 1.6 kb to 1300 kb, which gaveC. jejuni a genome size of approximately 1900 kb. Furthermore, thegly A and rRNA genes ofC. jejuni were localized to specific fragments by use of Southern analysis, and thegly A gene was shown to be closely linked to one of the three rRNA genes.     

Genomic characterization of Pinzgau cattle: genetic conservation and breeding perspectives

A genome-wide scan of Slovak Pinzgau cattle was prepared for the first time in order to estimate their genetic diversity at a more detailed level compared to previously published studies. The aim of this study was to describe the genetic diversity based on the runs of homozygosity (ROH_s), linkage disequilibrium (LD) and effective population size (N_eLD) using genome-wide data. Moreover, Bayesian clustering algorithms and multivariate methods were used to detect the population structure, potential admixture level and relationship between Austrian and Slovak Pinzgau cattle with respect to a large meta-population consisting of 15 European cattle breeds. The proportion of ROH segments ranged from 0.43 to 1.91% in Slovak Pinzgau, depending on the minimum size of an ROH. The genomic inbreeding coefficients were higher than the pedigree ones possibly due to the limited number of available generations in pedigree data. The observed N_eLD was close to the limit value characterizing the endangerment status, based both on genomic and pedigree data. Population structure within analyzed breeds based on the Wright’s F_ST index, Nei’s genetic distances, and unsupervised as well as supervised analysis has been established. Overall, these analyses clearly distinguished populations based on their origin. A detailed analysis of the introgression of each breed into the Pinzgau breeds prepared using a Bayesian approach showed that the contribution of Holstein cattle in Austrian as well as Slovak Pinzgau was larger than contribution of beef breeds. A possible reason is the recent usage of Holstein sires to increase milk production. There are considerable differences between well-defined regions that clearly distinguish Austrian and Slovak Pinzgau, despite their close common history. Generally, the breeding program of Austrian Pinzgau is more focused on meat production than Slovak Pinzgau, which was clearly reflected in the obtained autozygosity islands. Considering the genetic establishment of Slovak Pinzgau population the genetic potential of the breed is insufficiently used. On a long term, more global breeding program including very close populations will be more efficient providing higher genetic progress and diversity. Established methodology how to distinguish genealogically close populations on high-throughput molecular information based of Slovak and Austrian Pinzgau can be proposed as general for analysis of differences in all highly related breeds.