The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage

Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.     

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis,Dublin and Gallinarum

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.     

Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup

We have determined the genome sequences of two closely related lytic bacteriophages, SP6 and K1-5, which infect Salmonella typhimurium LT2 and Escherichia coli serotypes K1 and K5, respectively. The genome organization of these phages is almost identical with the notable exception of the tail fiber genes that confer the different host specificities. The two phages have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized. The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats. About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage. The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved. Low levels of nucleotide similarity between genomes in the T7 and SP6 groups suggest that they diverged a long time ago but, on the basis of this conservation of genome organization, they are expected to have retained similar developmental strategies.     

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.     

Genome plasticity and ori-ter rebalancing in Salmonella typhi

Genome plasticity resulting from frequent rearrangement of the bacterial genome is a fascinating but poorly understood phenomenon. First reported in Salmonella typhi, it has been observed only in a small number of Salmonella serovars, although the over 2,500 known Salmonella serovars are all very closely related. To gain insights into this phenomenon and elucidate its roles in bacterial evolution, especially those involved in the formation of particular pathogens, we systematically analyzed the genomes of 127 wild-type S. typhi strains isolated from many places of the world and compared them with the two sequenced strains, Ty2 and CT18, attempting to find possible associations between genome rearrangement and other significant genomic features. Like other host-adapted Salmonella serovars, S. typhi contained large genome insertions, including the 134 kb Salmonella pathogenicity island, SPI7. Our analyses showed that SPI7 disrupted the physical balance of the bacterial genome between the replication origin (ori) and terminus (ter) when this DNA segment was inserted into the genome, and rearrangement in individual strains further changed the genome balance status, with a general tendency toward a better balanced genome structure. In a given S. typhi strain, genome diversification occurred and resulted in different structures among cells in the culture. Under a stressed condition, bacterial cells with better balanced genome structures were selected to greatly increase in proportion; in such cases, bacteria with better balanced genomes formed larger colonies and grew with shorter generation times. Our results support the hypothesis that genome plasticity as a result of frequent rearrangement provides the opportunity for the bacterial genome to adopt a better balanced structure and thus eventually stabilizes the genome during evolution.     

Toward a more robust assessment of intraspecies diversity, using fewer genetic markers

Phylogenetic sequence analysis of single or multiple genes has dominated the study and census of the genetic diversity among closely related bacteria. It remains unclear, however, how the results based on a few genes in the genome correlate with whole-genome-based relatedness and what genes (if any) best reflect whole-genome-level relatedness and hence should be preferentially used to economize on cost and to improve accuracy. We show here that phylogenies of closely related organisms based on the average nucleotide identity (ANI) of their shared genes correspond accurately to phylogenies based on state-of-the-art analysis of their whole-genome sequences. We use ANI to evaluate the phylogenetic robustness of every gene in the genome and show that almost all core genes, regardless of their functions and positions in the genome, offer robust phylogenetic reconstruction among strains that show 80 to 95% ANI (16S rRNA identity, >98.5%). Lack of elapsed time and, to a lesser extent, horizontal transfer and recombination make the selection of genes more critical for applications that target the intraspecies level, i.e., strains that show >95% ANI according to current standards. A much more accurate phylogeny for the Escherichia coli group was obtained based on just three best-performing genes according to our analysis compared to the concatenated alignment of eight genes that are commonly employed for phylogenetic purposes in this group. Our results are reproducible within the Salmonella, Burkholderia, and Shewanella groups and therefore are expected to have general applicability for microevolution studies, including metagenomic surveys.     

RS-SNP: a random-set method for genome-wide association studies

Background

Common carp is one of the most important aquaculture teleost fish in the world. Common carp and other closely related Cyprinidae species provide over 30% aquaculture production in the world. However, common carp genomic resources are still relatively underdeveloped. BAC end sequences (BES) are important resources for genome research on BAC-anchored genetic marker development, linkage map and physical map integration, and whole genome sequence assembling and scaffolding.

Result

To develop such valuable resources in common carp (Cyprinus carpio), a total of 40,224 BAC clones were sequenced on both ends, generating 65,720 clean BES with an average read length of 647 bp after sequence processing, representing 42,522,168 bp or 2.5% of common carp genome. The first survey of common carp genome was conducted with various bioinformatics tools. The common carp genome contains over 17.3% of repetitive elements with GC content of 36.8% and 518 transposon ORFs. To identify and develop BAC-anchored microsatellite markers, a total of 13,581 microsatellites were detected from 10,355 BES. The coding region of 7,127 genes were recognized from 9,443 BES on 7,453 BACs, with 1,990 BACs have genes on both ends. To evaluate the similarity to the genome of closely related zebrafish, BES of common carp were aligned against zebrafish genome. A total of 39,335 BES of common carp have conserved homologs on zebrafish genome which demonstrated the high similarity between zebrafish and common carp genomes, indicating the feasibility of comparative mapping between zebrafish and common carp once we have physical map of common carp.

Conclusion

BAC end sequences are great resources for the first genome wide survey of common carp. The repetitive DNA was estimated to be approximate 28% of common carp genome, indicating the higher complexity of the genome. Comparative analysis had mapped around 40,000 BES to zebrafish genome and established over 3,100 microsyntenies, covering over 50% of the zebrafish genome. BES of common carp are tremendous tools for comparative mapping between the two closely related species, zebrafish and common carp, which should facilitate both structural and functional genome analysis in common carp.     

A Novel Construction of Genome Space with Biological Geometry

A genome space is a moduli space of genomes. In this space, each point corresponds to a genome. The natural distance between two points in the genome space reflects the biological distance between these two genomes. Currently, there is no method to represent genomes by a point in a space without losing biological information. Here, we propose a new graphical representation for DNA sequences. The breakthrough of the subject is that we can construct the moment vectors from DNA sequences using this new graphical method and prove that the correspondence between moment vectors and DNA sequences is one-to-one. Using these moment vectors, we have constructed a novel genome space as a subspace in R^N. It allows us to show that the SARS-CoV is most closely related to a coronavirus from the palm civet not from a bird as initially suspected, and the newly discovered human coronavirus HCoV-HKU1 is more closely related to SARS than to any other known member of group 2 coronavirus. Furthermore, we reconstructed the phylogenetic tree for 34 lentiviruses (including human immunodeficiency virus) based on their whole genome sequences. Our genome space will provide a new powerful tool for analyzing the classification of genomes and their phylogenetic relationships.     

Dynamics of actin evolution in dinoflagellates

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.     

Chromatin domain boundary element search tool for Drosophila

Chromatin domain boundary elements prevent inappropriate interaction between distant or closely spaced regulatory elements and restrict enhancers and silencers to correct target promoters. In spite of having such a general role and expected frequent occurrence genome wide, there is no DNA sequence analysis based tool to identify boundary elements. Here, we report chromatin domain Boundary Element Search Tool (cdBEST), to identify boundary elements. cdBEST uses known recognition sequences of boundary interacting proteins and looks for 'motif clusters'. Using cdBEST, we identified boundary sequences across 12 Drosophila species. Of the 4576 boundary sequences identified in Drosophila melanogaster genome, >170 sequences are repetitive in nature and have sequence homology to transposable elements. Analysis of such sequences across 12 Drosophila genomes showed that the occurrence of repetitive sequences in the context of boundaries is a common feature of drosophilids. We use a variety of genome organization criteria and also experimental test on a subset of the cdBEST boundaries in an enhancer-blocking assay and show that 80% of them indeed function as boundaries in vivo. These observations highlight the role of cdBEST in better understanding of chromatin domain boundaries in Drosophila and setting the stage for comparative analysis of boundaries across closely related species.     

Variability in occurrence of multiple prophage genes in Salmonella Typhimurium strains isolated in Slovak Republic

Lysogenic bacteriophages are a significant source of variability in closely related Salmonella strains. In this study, screening for diversity of 152 Salmonella Typhimurium strains was performed using PCR detection of selected prophage regions derived from phages P22, Gifsy-1, Gifsy-2, Fels-1, ST104 and SopEPhi. A high degree of variability was observed in the presence of specific genes. Based on the presence of particular prophage genes, we divided strains into 37 different PCR-prophage profiles; 20 of them were represented by only a single strain. Using multilocus variable number tandem repeats analysis (MLVA), 152 Salmonella strains were separated into 82 MLVA strings. Similar grouping of Salmonella strains was observed in the case of PCR-prophage detection and MLVA and the results corresponded well with the phage type of strains. However, several Salmonella strains were detected, which were closely related according to MLVA; yet, they differed in PCR phage profiles. The observations support a view that integration/excision of bacteriophages in Salmonella strains are frequent events shaping the bacterial genome.     

Human-specific sequences: isolation of species-specific DNA regions by genome subtraction

Uniqueness is fundamental to the individuality of species, and this in turn is based on the uniqueness of their genomes. For the purpose of resolving the genetic basis of human uniqueness, we describe here the isolation of human-specific sequences using the technique of genome subtraction, i.e., competitive reassociation of genomic DNAs between two very closely related species. One such sequence, HS5, was found to be present only in the human genome and absent in the genomes of non-human primates including chimpanzees, the species most closely related to humans.     

Gene decay in archaea

The gene-dense chromosomes of archaea and bacteria were long thought to be devoid of pseudogenes, but with the massive increase in available genome sequences, whole genome comparisons between closely related species have identified mutations that have rendered numerous genes inactive. Comparative analyses of sequenced archaeal genomes revealed numerous pseudogenes, which can constitute up to 8.6% of the annotated coding sequences in some genomes. The largest proportion of pseudogenes is created by gene truncations, followed by frameshift mutations. Within archaeal genomes, large numbers of pseudogenes contain more than one inactivating mutation, suggesting that pseudogenes are deleted from the genome more slowly in archaea than in bacteria. Although archaea seem to retain pseudogenes longer than do bacteria, most archaeal genomes have unique repertoires of pseudogenes.     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。     

Genome-wide detection of spontaneous chromosomal rearrangements in bacteria

Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal bacterial populations are still poorly characterized. Here we used 454 pyrosequencing technology and a 'split mapping' computational method to identify unique junction sequences caused by spontaneous genome rearrangements in chemostat cultures of Salmonella enterica Var. Typhimurium LT2. We confirmed 22 unique junction sequences with a junction microhomology more than 10 bp and this led to an estimation of 51 true junction sequences, of which 28, 12 and 11 were likely to be formed by deletion, duplication and inversion events, respectively. All experimentally confirmed rearrangements had short inverted (inversions) or direct (deletions and duplications) homologous repeat sequences at the endpoints. This study demonstrates the feasibility of genome wide characterization of spontaneous genome rearrangements in bacteria and the very high steady-state frequency (20-40%) of rearrangements in bacterial populations.     

Analysis of papaya BAC end sequences reveals first insights into the organization of a fruit tree genome

Papaya (Carica papaya L.) is a major tree fruit crop of tropical and subtropical regions with an estimated genome size of 372 Mbp. We present the analysis of 4.7% of the papaya genome based on BAC end sequences (BESs) representing 17 million high-quality bases. Microsatellites discovered in 5,452 BESs and flanking primer sequences are available to papaya breeding programs at . Sixteen percent of BESs contain plant repeat elements, the vast majority (83.3%) of which are class I retrotransposons. Several novel papaya-specific repeats were identified. Approximately 19.1% of the BESs have homology to Arabidopsis cDNA. Increasing numbers of completely sequenced plant genomes and BES projects enable novel approaches to comparative plant genomics. Paired BESs of Carica, Arabidopsis, Populus, Brassica and Lycopersicon were mapped onto the completed genomes of Arabidopsis and Populus. In general the level of microsynteny was highest between closely related organisms. However, papaya revealed a higher degree of apparent synteny with the more distantly related poplar than with the more closely related Arabidopsis. This, as well as significant colinearity observed between peach and poplar genome sequences, support recent observations of frequent genome rearrangements in the Arabidopsis lineage and suggest that the poplar genome sequence may be more useful for elucidating the papaya and other rosid genomes. These insights will play a critical role in selecting species and sequencing strategies that will optimally represent crop genomes in sequence databases.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Chun Wan J. Lai and Qingyi Yu have contributed equally to this work.     

Isolation and characterization of a hepatitis B virus endemic in herons.

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.     

Sequence heterogeneity of TT virus and closely related viruses

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.