30株大肠杆菌的泛基因组学特征分析

泛基因组(Pan-genome)是某一物种全部基因的总称,其中包括核心基因组(该物种所有个体中都存在的基因)和非必须基因组(只在部分个体中存在的基因,以及某个体特有的基因)。文章从泛基因组学角度比较分析了30株已经完成测序的大肠杆菌的基因、基因组成及其进化特征,结果表明核心基因只占据每株大肠杆菌全部基因数目的 50%左右,而平均每个菌株有146个特有基因,结果表明随着更多大肠杆菌菌株的基因组被测序,将会不断有新基因被发现。通过比较分析大肠杆菌不同菌株之间基因的保守性与基因的GC含量以及选择压力之间的关系,发现越保守的基因其GC含量变化范围越窄,同时在进化中受到的选择压力也越大。这些结果将有助于深入了解大肠杆菌基因组的进化特征及其基因组成的动态变化,并为预防和控制由致病性大肠杆菌引发的流行疾病提供理论依据,同时也为大规模病原菌基因组数据的分析方法提供借鉴。     

Comparisons among two fertile and three male-sterile mitochondrial genomes of maize

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding "native" mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.     

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes

Aims:  To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains.
Methods and Results:  Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes.
Conclusions:  This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes.
Significance and Impact of the Study:  ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.     

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.     

Conservation and variation of nucleotide sequences within related bacterial genomes: Escherichia coli strains.

Changes in the patterns produced by annealing restriction endonuclease digests of bacterial genomes with probe deoxyribonucleic acids (DNAs) containing small portions of a bacterial genome provide sensitive indicator of the degree of nucleotide sequence relatedness that exists in localized regions of the genomes of closely related bacteria. We have used five probe DNAs to explore the relatedness of parts of the genomes of six laboratory Escherichi coli strains. A range in in the amount of variability in the positions of restriction enzyme cleavage sites in the selected portions of the genomes was found. Portions of the genome that are believed to be inacative were more variable than portions that contained functional genes: the sites in and near regions of homology to phage lambda DNA in the genome showed the greatest variability. These regions probably represent remnants of cryptic prophages. Variability was assessed pairwise among four of the E. coli strains and ranged from 5 to > 25% base pair substitutions in the lambda-related regions. In contrast, the endonuclease cleavage sites in the trp, tna, lac, thy regions, and one other as-yet-unidentified segment of the genome were more highly conserved. It seems likely that these sites lie in genetic locations that are subject to functional constraints.     

Random PCR-based genome sequencing: a non-divide-and-conquer strategy.

We propose a genome sequencing strategy, which is neither divide-and-conquer (clone by clone) nor the shotgun approach. Random PCR-based and PCR relay sequencing constitute the basis of this novel strategy. Most of the genome is sequenced by the former process that requires only a set of non-specific primers and a template DNA. Random PCR-based sequencing reduces redundancy in sequencing by exploiting known sequence information. The number of primers required for random PCR was significantly diminished by using a combination of primers. The former process can be partially replaced by the shotgun method, if necessary. The gap-filling process can be effectively performed by way of PCR relay. The feasibility of this strategy was demonstrated using the Escherichia coli genome. This strategy enhances the global effort towards genome sequencing by being available through the Internet and by allowing the use of preexisting sequence data.     

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.     

Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities

The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.     

Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.     

Phylomark, a tool to identify conserved phylogenetic markers from whole-genome alignments

The sequencing and analysis of multiple housekeeping genes has been routinely used to phylogenetically compare closely related bacterial isolates. Recent studies using whole-genome alignment (WGA) and phylogenetics from >100 Escherichia coli genomes has demonstrated that tree topologies from WGA and multilocus sequence typing (MLST) markers differ significantly. A nonrepresentative phylogeny can lead to incorrect conclusions regarding important evolutionary relationships. In this study, the Phylomark algorithm was developed to identify a minimal number of useful phylogenetic markers that recapitulate the WGA phylogeny. To test the algorithm, we used a set of diverse draft and complete E. coli genomes. The algorithm identified more than 100,000 potential markers of different fragment lengths (500 to 900 nucleotides). Three molecular markers were ultimately chosen to determine the phylogeny based on a low Robinson-Foulds (RF) distance compared to the WGA phylogeny. A phylogenetic analysis demonstrated that a more representative phylogeny was inferred for a concatenation of these markers compared to all other MLST schemes for E. coli. As a functional test of the algorithm, the three markers (genomic guided E. coli markers, or GIG-EM) were amplified and sequenced from a set of environmental E. coli strains (ECOR collection) and informatically extracted from a set of 78 diarrheagenic E. coli strains (DECA collection). In the instances of the 40-genome test set and the DECA collection, the GIG-EM system outperformed other E. coli MLST systems in terms of recapitulating the WGA phylogeny. This algorithm can be employed to determine the minimal marker set for any organism that has sufficient genome sequencing.     

Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach

Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.     

Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis

The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach. Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method. The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments. The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information.     

Using genomic sequencing for classical genetics in E. coli K12

We here develop computational methods to facilitate use of 454 whole genome shotgun sequencing to identify mutations in Escherichia coli K12. We had Roche sequence eight related strains derived as spontaneous mutants in a background without a whole genome sequence. They provided difference tables based on assembling each genome to reference strain E. coli MG1655 (NC_000913). Due to the evolutionary distance to MG1655, these contained a large number of both false negatives and positives. By manual analysis of the dataset, we detected all the known mutations (24 at nine locations) and identified and genetically confirmed new mutations necessary and sufficient for the phenotypes we had selected in four strains. We then had Roche assemble contigs de novo, which we further assembled to full-length pseudomolecules based on synteny with MG1655. This hybrid method facilitated detection of insertion mutations and allowed annotation from MG1655. After removing one genome with less than the optimal 20- to 30-fold sequence coverage, we identified 544 putative polymorphisms that included all of the known and selected mutations apart from insertions. Finally, we detected seven new mutations in a total of only 41 candidates by comparing single genomes to composite data for the remaining six and using a ranking system to penalize homopolymer sequencing and misassembly errors. An additional benefit of the analysis is a table of differences between MG1655 and a physiologically robust E. coli wild-type strain NCM3722. Both projects were greatly facilitated by use of comparative genomics tools in the CoGe software package (http://genomevolution.org/).     

Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin): molecular cloning of the gene of Synechocystis sp. PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme.

The branched polypeptide multi-L-arginyl-poly-L-aspartic acid, also called cyanophycin, is a water-insoluble reserve material of cyanobacteria. The polymer is degraded by a specific hydrolytic enzyme called cyanophycinase. By heterologous expression in Escherichia coli, a gene encoding cyanophycinase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The gene, designated cphB, codes for a protein of 29.4 kDa. The high level of expression of active cyanophycinase in E. coli from the Synechocystis gene allowed for its purification to electrophoretic homogeneity. The enzyme, which appears to be specific for cyanophycin, hydrolysed the polymer to a dipeptide consisting of aspartic acid and arginine. Based on inhibitor sensitivity and primary sequence, cyanophycinase appears to be a serine-type exopeptidase related to dipeptidase E [Conlin, C.A., Haakensson, K., Liljas, A. & Miller, C.G. (1994) J. Bacteriol. 176, 166-172].     

The archaeal halophilic virus-encoded Dam-like methyltransferase M. phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli

The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M.Dam in DNA mismatch repair.     

In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction

We have developed a website, www.in-silico.com, which runs a software program that performs three basic tasks in completely sequenced bacterial genomes by in silico analysis: PCR amplification, amplified fragment length polymorphism (AFLP-PCR) and endonuclease restriction. For PCR, after selection of the genome and introduction of primers, fragment size, DNA sequence and corresponding open reading frame (ORF) identity of the resulting PCR product is computed. Plasmids of sequenced species may be included in the analysis. Theoretical AFLP-PCR analyzes similar parameters, and includes a suggestion tool providing a list of commercial restriction enzyme pairs yielding up to 50 amplicons in the selected genome. Endonuclease restriction analysis of complete genomes and plasmids calculates the number of restriction sites for endonucleases in a given genome. If the number of fragments is 50 or fewer, pulsed field gel electrophoresis image and restriction maps are illustrated. Other tools that have been included in this site are ORF search by name and DNA to protein translation as well as restriction digestion of user-defined DNA sequences. AVAILABILITY: This is a new molecular biology resource freely available over the Internet at http://www.in-silico.com