大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.     

Does experimental evolution reflect patterns in natural populations? E. coli strains from long-term studies compared with wild isolates

Our results show that experimental evolution mimics evolution in nature. In particular, only 1000 generations of periodic recombination with immigrant genotypes is enough for linkage disequilibrium values in experimental populations to change from a maximum linkage value to a value similar to the one observed in wild strains of E. coli. Our analysis suggests an analogy between the recombination experiment and the evolutionary history of E. coli; the E. coligenome is a patchwork of genes laterally inserted in a common backbone, and the experimental E. coli chromosome is a patchwork where some sites are highly prone to recombination and others are very clonal. In addition, we propose a population model for wild E. coli where gene flow (recombination and migration) are an important source of genetic variation, and where certain hosts act as selective sieves; i.e., the host digestive system allows only certain strains to adhere and prosper as resident strains generating a particular microbiota in each host. Therefore we suggest that the strains from a wide range of wild hosts from different regions of the world may present an ecotypic structure where adaptation to the host may play an important role in the population structure. This revised version was published online in August 2006 with corrections to the Cover Date.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Stationary-state mutagenesis inEscherichia coli: A model

Stationary-phase mutagenesis in nondividingE. coli cells exposed to a nonlethal stress was, a few years ago, claimed to be a likely case of a Lamarckian mechanism capable of producing exclusively useful mutations in a directed manner. After a heated debate over the last decade it now appears to involve a Darwinian mechanism that generates a transient state of hypermutagenesis, operating on a large number of sites spread over the entire genome, at least in a proportion of the resting cells. Most of the studies that clarified this position were on the reversion of a frameshift mutation present in alacI-lacZ fusion inE. coli strain FC40. Several groups have extensively examined both the sequence changes associated with these reversions and the underlying genetic requirements. On the basis of our studies on the genomic sequence analysis, we recently proposed a model to explain the specific changes associated with the reversion hotspots. Here we propose a more detailed version of this model that also takes into account the observed genetic requirements of stationary-state mutagenesis. Briefly, G:T/U mismatches produced at methylatable cytosines are preferentially repaired in nondividing cells by the very short patch mismatch repair (VSPMR) mechanism which is itself mutagenic and can produce mutations in very short stretches located in the immediate vicinity of these cytosine methylation sites. This mechanism requires a homologous or homeologous strand invasion step and an error-prone DNA synthesis step and is dependent on RecA, RecBCD and a DNA polymerase. The process is initiated near sequences recognized by Dcm and Vsr enzymes and further stimulated if these sequences are a part of CHI or CHI-like sequences, but a double-strand-break-dependent recombination mediated by the RecBCD pathways proposed by others seems to be nonessential. The strand transfer step is proposed to depend on RecA, RuvA, RuvB and RuvC and is opposed by RecG and MutS. The model also gives interesting insights into the evolution of theE. coli genome.     

Ectopic Gene Conversions in Four <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> Genomes: Increased Recombination in Pathogenic Strains

We characterized the ectopic gene conversions in the genomes of the K-12 MG1655, O157:H7 Sakai, O157:H7 EDL933, and CFT073 strains of E coli. Compared to the three pathogenic strains, the K-12 strain has a much smaller number of gene families, its gene families contain fewer genes, and gene conversions are less frequent. Whereas the three pathogenic strains have gene conversions covering hundreds of nucleotides when their flanking regions have as little as 50% similarity, flanking region similarity of at least 94% on both sides of the converted region is required to observe conversions of more than 87 nucleotides in the K-12 strain. Recombination is therefore more frequent and requires less sequence similarity in the three pathogenic strains than in K-12. This higher recombination level might be due to mutations in some of their mismatch-repair genes. In contrast with the gene conversions present in the yeast genome, the gene conversions found in the E. coli genomes do not occur more frequently between duplicated genes that are close to one another than between duplicated genes that are far apart and are randomly distributed along the length of the genes. In E. coli, gene conversions are not more frequent near the origin of replication. However, they do occur more frequently near the terminus of replication of the Sakai genome, where multigene family members are more abundant. This suggests that, in E. coli, gene conversions occur randomly between genes located in different chromosomal locations or located on different copies of the multiple chromosomes found in E. coli cells.     

The uvp1 gene of plasmid pR cooperates with mucAB genes in the DNA repair process

Summary We show that a DNA fragment that contains the uvp1 gene of the plasmid pR directs the synthesis in Escherichia coli minicells of a protein of apparent molecular weight 20 kDa. Inspection of the nucleotide sequence of the region reveals an open reading frame that has the capacity to encode a protein of 198 amino acids. The uvp1 gene product has been found, in two different systems, to enhance the recombination activity of E. coli cells. We have also observed a striking similarity to resolvase and invertase proteins. The significance of this finding for the function of the uvp1 gene product requires further investigation. We conclude that the uvp1 gene encodes a 20 kDa protein which appears to be responsible for enhancement of both UV survival and recominational activity in E. coli.     

Genotypic exclusion: a novel relationship between the ribitol-arabitol and galactitol genes of E. coli

Summary Genetic studies indicate that the E. coli C chromosomal genes which are responsible for catabolism of the pentitol sugars, ribitol and D-arabitol, are not present in the closely related E. coli K12 strains (Reiner 1975). Molecular studies of these tightly linked genes reveal that they are surrounded by 1.4 kilobase inverted repeats of imperfect homology (Link and Reiner 1982). Here we report that E. coli C lacks genes for catabolism of the hexitol sugar galactitol, genes which are present in E. coli K12. Furthermore, the ribitol-arabitol and galactitol genes, which show no mutual homology, are mutually exclusive when exchanged (by homologous recombination) between E. coli C and K12. Physical characterization of specialized transducing phages carrying the ribitol-arabitol or galactitol genes demonstrates that this exclusion results because these genes have identical locations in their respective chromosomes. This novel type of allelic relationship between nonhomologous genes has not been previously described in prokaryotes. Analysis of the catabolic capabilities of a collection of natural E. coli strains suggests that this exclusion relationship extends to strains in the natural E. coli population. We suggest an insertion/deletion model to account for the origins of this unusual gene arrangement.     

The history and evolution of Escherichia coli O157 and other Shiga toxin-producing E. coli

Shiga toxin-producing Escherichia coli (STEC) O157 is a formidable human pathogen with the capacity to cause large outbreaks of gastrointestinal illness. The known virulence factors of this organism are encoded on phage, plasmid and chromosomal genes. There are also likely to be novel, as yet unknown virulence factors in this organism. Many of these virulence factors have been acquired by E. coli O157 by transfer from other organisms, both E. coli and non-E. coli species. By examination of biochemical and genetic characteristics of various E. coli O157 strains and the relationships with other organisms, an evolutionary pathway for development of E. coli O157 as a pathogen has been proposed. E. coli O157 evolved from an enteropathogenic E. coli ancestor of serotype O55:H7, which contained the locus of enterocyte effacement containing the adhesin intimin. During the evolutionary process, Shiga toxins, the pO157 plasmid and other characteristics which enhanced virulence were acquired and other functions such as motility, sorbitol fermentation and β-glucuronidase activity were lost by some strains. It is likely that E. coli O157 is constantly evolving, and changes can be detected in genetic patterns during the course of infection. A variety of mechanisms may be responsible for the development of the virulent phenotype that we see today. Such changes include uptake of as yet uncharacterised virulence factors, possibly enhanced by a mutator phenotype, recombination within virulence genes to produce variant genes with different properties, loss of large segments of DNA (black holes) to enhance virulence and possible adaptation to different hosts. Although little is known about the evolution of non-O157 STEC it is likely that the most virulent clones evolved in a similar manner to E. coli O157. This revised version was published online in November 2006 with corrections to the Cover Date.     

An improved recombineering approach by adding RecA to λ Red recombination

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the λ phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redα, a 5′ to 3′ exonuclease, Redβ, an annealing protein, and Redλ, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance thestabulity of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total numer of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC 101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double-or single-stranded DNA, published to date.     

Vegetative recombination of bacteriophage Mu-1 in Escherichia coli

Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec⁺ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination.The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.Research Fellow from the Laboratory of Genetics, State University, Leiden, The Netherlands.     

Insights into the biology of Escherichia coli through structural proteomics

Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.     

Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 × 10^–5 per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.     

Role of Intraspecies Recombination in the Spread of Pathogenicity Islands within the Escherichia coli Species

Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI–encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI–flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli.     

Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination

Summary The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. Multimerization, promoted by homologous recombination, leads to plasmid loss. Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination. One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96–97 min region of the E. coli map.     

Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes

Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10^1–3 in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.     

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

Phage cocktail strategies for the suppression of a pathogen in a cross-feeding coculture

Cocktail combinations of bacteria-infecting viruses (bacteriophages) can suppress pathogenic bacterial growth. However, predicting how phage cocktails influence microbial communities with complex ecological interactions, specifically cross-feeding interactions in which bacteria exchange nutrients, remains challenging. Here, we used experiments and mathematical simulations to determine how to best suppress a model pathogen, E. coli, when obligately cross-feeding with S. enterica. We tested whether the duration of pathogen suppression caused by a two-lytic phage cocktail was maximized when both phages targeted E. coli, or when one phage targeted E. coli and the other its cross-feeding partner, S. enterica. Experimentally, we observed that cocktails targeting both cross-feeders suppressed E. coli growth longer than cocktails targeting only E. coli. Two non-mutually exclusive mechanisms could explain these results: (i) we found that treatment with two E. coli phage led to the evolution of a mucoid phenotype that provided cross-resistance against both phages, and (ii) S. enterica set the growth rate of the coculture, and therefore, targeting S. enterica had a stronger effect on pathogen suppression. Simulations suggested that cross-resistance and the relative growth rates of cross-feeders modulated the duration of E. coli suppression. More broadly, we describe a novel bacteriophage cocktail strategy for pathogens that cross-feed.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

The Interaction of Protein Structure, Selection, and Recombination on the Evolution of the Type-1 Fimbrial Major Subunit (fimA) from Escherichia coli

Fimbrial adhesins allow bacteria to interact with and attach to their environment. The bacteria possibly benefit from these interactions, but all external structures including adhesins also allow bacteria to be identified by other organisms. Thus adhesion molecules might be under multiple forms of selection including selection to constrain functional interactions or evolve novel epitopes to avoid recognition. We address these issues by studying genetic diversity in the Escherichia coli type-1 fimbrial major subunit, fimA. Overall, sequence diversity in fimA is high (π= 0.07) relative to that in other E. coli genes. High diversity is a function of positive diversifying selection, as detected by d _N/d _S ratios higher than 1.0, and amino acid residuces subject to diversifying selection are nonrandomly clustered on the exterior surface of the peptide. In addition, McDonald and Kreitman tests suggest that there has been historical but not current directional selection at fimA between E. coli and Salmonella. Finally, some regions of the fimA peptide appear to be under strong structural constraint within E. coli, particularly the interior regions of the molecule that is involved in subunit to subunit interaction. Recombination also plays a major role contributing to E. coli fimA allelic variation and estimates of recombination (2N _e c) and mutation (2N _eμ) are about the same. Recombination may act to separate the diverse evolutionary forces in different regions of the fimA peptide. Received: 13 April 2000 / Accepted: 28 October 2000