Compound microsatellites in complete Escherichia coli genomes

Compound microsatellites consisting of two or more repeats in close proximity have been found in eukaryotic genomes. So far such compound microsatellites have not been investigated in any prokaryotic genomes. We have therefore examined compound microsatellites in 22 complete genomes of Escherichia coli, which is one of the ideal model organisms to analyze the nature and evolution of prokaryotic compound microsatellites. Our results indicated that about 1.75-2.85% of all microsatellites could be accounted as compound microsatellites with very low complexity, and most compound microsatellites were composed of very different motifs. Compound microsatellites were significantly overrepresented in all surveyed genomes. These results were dramatically different from those in eukaryotes. We discussed the possible reasons for the observed divergence.     

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。     

Improved generation of recombinant baculovirus genomes in Escherichia coli

An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene. Recombinant bacmids can be generated at a frequency of ~10⁷/µg of donor vector with a negligible background. This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombinant baculoviruses as well as a more convenient way to produce single viruses. The introduced selection scheme is also useful for the construction of other vectors by transposition in E.coli.     

Long W tracts are over-represented in the Escherichia coli and Haemophilus influenzae genomes.

The occurrence of DNA tracts of the three binary base combinations: R.Y, K.M and W;S has been mapped in the complete genomes of Haemophilus influenzae and Escherichia coli. A highly significant over-representation of W tracts is observed in both bacteria. The excess of W tracts is particularly striking in the 10% intercoding regions. Subdivision of intercoding regions into divergent (promoting), convergent (terminating) and sequential subregions shows that the excess of W tracts is most concentrated in the promoter regions. A particularly high excess of W tracts is observed in the first 200 bases 5' upstream of coding start sites. The data suggest that W tracts have a role in promoter function. A function as unwinding centers, analogous to the role of R.Y tracts in eukaryotes, is proposed. R.Y and K.M tracts are only modestly over-represented in the two bacteria.     

Accessory DNA in the genomes of representatives of the Escherichia coli reference collection

Different strains of the Escherichia coli reference collection (ECOR) differ widely in chromosomal size. To analyze the nature of the differential gene pool carried by different strains, we have followed an approach in which random amplified polymorphic DNA (RAPD) was used to generate several PCR fragments. Those present in some but not all the strains were screened by hybridization to assess their distribution throughout the ECOR collection. Thirteen fragments with various degrees of occurrence were sequenced. Three of them corresponded to RAPD markers of widespread distribution. Of these, two were housekeeping genes shown by hybridization to be present in all the E. coli strains and in Salmonella enterica LT2; the third fragment contained a paralogous copy of dnaK with widespread, but not global, distribution. The other 10 RAPD markers were found in only a few strains. However, hybridization results demonstrated that four of them were actually present in a large selection of the ECOR collection (between 42 and 97% of the strains); three of these fragments contained open reading frames associated with phages or plasmids known in E. coli K-12. The remaining six fragments were present in only between one and four strains; of these, four fragments showed no similarity to any sequence in the databases, and the other two had low but significant similarity to a protein involved in the Klebsiella capsule synthesis and to RNA helicases of archaeal genomes, respectively. Their percent GC, dinucleotide content, and codon adaptation index suggested an exogenous origin by horizontal transfer. These results can be interpreted as reflecting the presence of a large pool of strain-specific genes, whose origin could be outside the species boundaries.     

Efficient cloning and engineering of entire mitochondrial genomes in Escherichia coli and transfer into transcriptionally active mitochondria

We have devised an efficient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques. In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modified genomes into E.coli. We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers. When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone. We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT-PCR. This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a first step towards true in vivo engineering of mammalian mitochondrial genomes.     

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

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.     

Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.     

Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes

Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase. However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells. We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences. The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E. coli genes. Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA. This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.     

Mining mammalian genomes for folding competent proteins using Tat‐dependent genetic selection in Escherichia coli

Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin‐arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N‐terminal Tat export signal and a C‐terminal selectable marker, namely β‐lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.     

Functional interactions of the genomes of Shigella sonnei phages and Escherichia coli phage T4 in mixed infection]

A comparative study of Shigella sonnei phages U and G and Escherichia coli phage T4 has shown that enzymes coded for by the Sh. sonnei phages can functionally substitute for some T4-coded products. This finding in indicative of an evolutionary relationship between T-even phages and disenteric phages U and G. The U phage is uncapable to compensate amber mutants for the genes that control the conversion of cytosine into 5-hydroxymethyl cytosine (5-HMC) and the glucosylation of the latter, which agrees with our earlier finding that the U phage DNA contains no 5-HMC. U and G phages are also found to exclude the T4 phage in the course of mixed infection.     

Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli.

Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid. Evidence that plasmid-like structures are formed in H. polymorpha and that their formation is stimulated by DNA damage is presented in this study.     

Mesosomes in Escherichia coli

When Escherichia coli was grown in a synthetic medium and fixed with osmium, sections of the cells revealed clearly defined mesosomes. These mesosomes appeared to develop, in dividing cells, as coiled infoldings of the cytoplasmic membrane. Mature mesosomes formed a link between the cytoplasmic membrane and the nucleus of the cell. The arrangement of the mesosomes in dividing cells led to the hypothesis that division of the nucleus in these cells is accomplished by two separate polar mesosomes. One mesosome is derived from the parent cell and is present at one pole of the daughter cell. The other is freshly synthesized at or near the newly forming pole of the daughter cell. While the old mesosome remains attached to the chromosome received from the parent cell, the newly synthesized mesosome becomes attached to and initiates replication of the new chromosome. As the cell grows and elongates, the two mesosomes, attached to their respective chromosomes move apart, thus effecting nuclear division.