细菌中基因组岛的转移与调控机制研究进展

基因水平转移可导致细菌不同种属间个体DNA的交换,从而使细菌对环境的适应性增强,是细菌进化的重要途径之一。基因组岛是基因水平转移的重要载体,可移动的基因组岛能够整合到宿主的染色体上,并在特定的条件下切除,进而通过转化、接合或转导等方式转移到新的宿主中。基因组岛具有多种生物学功能,如抗生素抗性、致病性、异源物质降解、重金属抗性等。基因组岛的转移造成可变基因在不同种属细菌间的广泛传播,例如毒力和耐药基因的传播导致了多重耐药细菌的产生,威胁人类健康。基因组岛由整合酶介导转移,同时在转移的过程受到多种不同转录因子的调控。本文对细菌中基因组岛的结构特点、转移和调控机制以及预测等方面进行了综述,并最终阐明基因组岛的转移及其调控机制是遏制基因组岛传播的重要策略。     

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.     

The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival.

Pathogenicity islands are chromosomal clusters of horizontally acquired virulence genes that are often found at tRNA loci. The selC tRNA locus of Escherichia coli has served as the site of integration of two distinct pathogenicity islands which are responsible for converting benign strains into uro- and enteropathogens. Because virulence genes are targeted to the selC locus of E.coli, we investigated the homologous region of the Salmonella typhimurium chromosome for the presence of horizontally acquired sequences. At this site, we identified a 17 kb DNA segment that is both unique to Salmonella and necessary for virulence. This segment harbors a gene, mgtC, that is required for intramacrophage survival and growth in low Mg2+ media. The mgtC locus is regulated by the PhoP/PhoQ two-component system, a major regulator of virulence functions present in both pathogenic and non-pathogenic bacterial species. Cumulatively, our experiments indicate that the ability to replicate in low Mg2+ environments is necessary for Salmonella virulence, and suggest that a similar mechanism is responsible for the dissemination and acquisition of pathogenicity islands in enteric bacteria.     

Bacterial genomic islands: organization, function, and role in evolution

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are extended DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA genes, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.     

Assembly of large genomic segments in artificial chromosomes by homologous recombination in Escherichia coli

We developed a method for the reconstruction of a 100 kb DNA fragment into a bacterial artificial chromosome (BAC). The procedure makes use of iterative rounds of homologous recombination in Escherichia coli. Smaller, overlapping fragments of cloned DNA, such as cosmid clones, are required. They are transferred first into a temperature-sensitive replicon and then into the BAC of choice. We demonstrated the usefulness of this procedure by assembling a 90 kb genomic segment into an E.coli-STREPTOMYCES: artificial chromosome (ESAC). Using this procedure, ESACs are easy to handle and remarkably more stable than the starting cosmids.     

Bacterial Genomic Islands: Organization,Function, and Evolutionary Role

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are long DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA gene loci, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.     

Mode of insertion of the broad-host-range plasmid RP4 and its derivatives into the chromosome of Myxococcus xanthus

The mode of insertion of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus strain DZ1 has been analyzed. The plasmid integrated in numerous sites of the chromosome and generated insertional mutations. There is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the EcoRI site of the plasmid. In the absence of this segment the insertion can, however, take place, but much less efficiently. The presence of transposable elements on the plasmid decreases severely the insertion frequency. Once integrated, RP4 could be transferred back to Escherichia coli, either by precise excision or with a segment of the Myxococcus chromosome. The role of site-specific recombination in RP4 integration is discussed.     

In vivo excision and amplification of large segments of the Escherichia coli genome.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.     

Regulation of expression and biochemical characterization of a β-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7

The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element ( parDE ). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens . To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.     

A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50-to 100-kb genomic fragments for direct DNA sequencing

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRTelements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392–2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid.We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriVinsertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriVsites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites.To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the P_tet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRTsites (in lacZ and in phoB), and (ii) its amplification.     

Approaches to Half-Tetrad Analysis in Bacteria: Recombination between Repeated, Inverse-Order Chromosomal Sequences

In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant type is recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion), or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Since two recombinant products remain in the chromosome of a recombinant with an inversion, one can, in principle, approach the capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences (40 kb) recombine, conversion frequently accompanies exchange of flanking sequences. (b) When short sequences (5 kb) recombine, conversion rarely accompanies exchange of flanks. (c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombining sequences in the chromosome. The results are presented with the assumption that inversions occur by simple interaction of two sequences in the same circular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which could also apply to standard fungal systems.     

Structure of Recombinants from Conjugational Crosses between Escherichia Coli Donor and Mismatch-Repair Deficient Salmonella Typhimurium Recipients

To get more insight into the control of homologous recombination between diverged DNA by the Mut proteins of the long-patch mismatch repair system, we have studied interspecies Escherichia coli/Salmonella typhimurium recombination. Knowing that the same recombination pathway (RecABCD) is responsible for intraspecies and interspecies recombination, we have now studied the structure (replacement vs. addition-type or other rearrangement-type recombinants) of 81 interspecies recombinants obtained in conjugational crosses between E. coli donor and mutL, mutS, mutH, mutU or mut(+) S. typhimurium recipients. Taking advantage of high interspecies sequence divergence, a physical analysis was performed on one third of the E. coli Hfr genome, which was expected to be transferred to S. typhimurium F(-) recipients during 40 min before interruption of the mating. Probes specific for each species were hybridized on dot blots of genomic DNA, or on colonies, and the composition of the rrn operons was determined from purified genomic DNA. With very few exceptions, the structure of these interspecies recombinants corresponds to replacements of one continuous block of the recipient genome by the corresponding region of the donor genome.     

Transfer of the methicillin resistance genomic island among staphylococci by conjugation

Methicillin resistance creates a major obstacle for treatment of Staphylococcus aureus infections. The resistance gene, mecA, is carried on a large (20 kb to > 60 kb) genomic island, staphylococcal cassette chromosome mec (SCCmec), that excises from and inserts site‐specifically into the staphylococcal chromosome. However, although SCCmec has been designated a mobile genetic element, a mechanism for its transfer has not been defined. Here we demonstrate the capture and conjugative transfer of excised SCCmec. SCCmec was captured on pGO400, a mupirocin‐resistant derivative of the pGO1/pSK41 staphylococcal conjugative plasmid lineage, and pGO400::SCCmec (pRM27) was transferred by filter‐mating into both homologous and heterologous S. aureus recipients representing a range of clonal complexes as well as S. epidermidis. The DNA sequence of pRM27 showed that SCCmec had been transferred in its entirety and that its capture had occurred by recombination between IS257/431 elements present on all SCCmec types and pGO1/pSK41 conjugative plasmids. The captured SCCmec excised from the plasmid and inserted site‐specifically into the chromosomal att site of both an isogenic S. aureus and a S. epidermidis recipient. These studies describe a means by which methicillin resistance can be environmentally disseminated and a novel mechanism, IS‐mediated recombination, for the capture and conjugative transfer of genomic islands.     

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.     

Genomic cleavage map of Salmonella typhi Ty2.

The genomic cleavage map of Salmonella typhi Ty2, 4,780 kb in size, was determined through digestion of the genomic DNA with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 33, 26, 7, and 35 sites for the enzymes XbaI, BlnI, I-CeuI, and SpeI, respectively. The fragments were arranged around the chromosome through excision of fragments from the gel, redigestion with a second enzyme, and labelling with 32P, and reelectrophoresis and named in alphabetical order. Tn10 transposons inserted in 82 different genes of Salmonella typhimurium were transduced by phage P22 into S. typhi, and the location of Tn10, and thus of the gene, was mapped through the XbaI and BlnI sites of Tn10. All seven I-CeuI sites (in rrl genes for 23S rRNA) were conserved, and the gene order within the I-CeuI fragments resembles that of S. typhimurium LT2, but the order of I-CeuI fragments is rearranged from ABCDEFG in S. typhimurium LT2 to AGCEFDB in S. typhi. In addition, there is a 500-kb inversion which covers the terminus region. Comparisons of lengths of segments between genes showed that S. typhi has segments which differ in size from those in S. typhimurium. The viaB locus, for synthesis of the Vi antigen of S. typhi, was shown to be within a 118-kb loop (a segment of DNA with no homolog in most other Salmonella species) between mel and poxA on the chromosome.     

病原菌毒力岛研究进展

毒力岛作为基因组岛的一种亚类,是细菌染色体上具有特定结构和功能特征的可移动基因大片段,经基因水平转移（转导、接合或转化）获得,可使细菌基因组进化在短期内发生“量的飞跃”,直接或间接增强细菌的生态适应性,与病原菌的致病性密切相关。毒力岛存在于多种动植物病原细菌中,对于细菌的毒力变异、遗传进化甚至新病原亚种形成有重要意义。简要综述了病原菌毒力岛的研究进展,介绍了毒力岛的结构、功能特征及其在病原菌进化中作用。     

Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system

The process of DNA donation for natural transformation of bacteria is poorly understood and has been assumed to involve bacterial cell death. Recently in Neisseria gonorrhoeae we found that mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. To better characterize the GGI and the process of DNA donation, the 57 kb genetic island was cloned, sequenced and subjected to insertional mutagenesis. DNA sequencing revealed that the GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. The GGI was found to be incorporated near the chromosomal replication terminus at the dif site, a sequence targeted by the site-specific recombinase XerCD. Using a plasmid carrying a small region of the GGI and the associated dif site, we demonstrated that this model island could be integrated at the dif site in strains not carrying the GGI and was spontaneously excised from that site. Also, we were able to delete the entire 57 kb region by transformation with DNA from a strain lacking the GGI. Thus the GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. We made mutations in six putative type IV secretion system genes and assayed these strains for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Our data indicate that N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response.     

Identification and sequence of a gene required for a developmentally regulated DNA excision in Anabaena

Vegetative cells of the cyanobacterium Anabaena contain an 11 kb DNA element within the coding region of the nifD gene. This element is excised by site-specific recombination between directly repeated 11 bp sequences at each of its ends during differentiation of nitrogen-fixing cells called heterocysts. Site-specific recombination, leading to the same rejoined nifD gene, was observed during propagation in E. coli of a fragment containing the 11 kb element and flanking sequences. An assay for excision of the element in E. coli was developed, based on mini-Mu-lac transposition into the element. Since the 11 kb element lacks an origin of replication, its excision results in loss of lac and conversion of blue colony-forming cells to white on X-gal plates. Insertion and deletion mutagenesis identified a region of the element needed for excision. Mutations in this region could be complemented by a 6 kb fragment containing an open reading frame that runs counter to those of the nif genes, beginning 240 bp from the recombination site.