A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.     

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solancacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter. and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.     

Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene

The trimethoprim-resistant dihydrofolate reductase associated with the R plasmid R388 was isolated from strains that over-produce the enzyme. It was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. The purified enzyme is composed of four identical subunits with molecular weights of 8300. A 1100 bp long DNA segment which confers resistance to trimethoprim was sequenced. The structural gene was identified on the plasmid DNA by comparing the amino acid composition of the deduced proteins with that of the purified enzyme. The gene is 234 bp long and codes for 78 amino acids. No homology can be found between the deduced amino acid sequence of the R388 dihydrofolate reductase and those of other prokaryotic or eukaryotic dihydrofolate reductases. However, it differs in only 17 positions from the enzyme associated with the trimethoprim-resistance plasmid R67.     

Occurrence of insertion sequence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications.

Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.     

Stringent and relaxed recognition of oriT by related systems for plasmid mobilization: implications for horizontal gene transfer

The plasmids R1162 and pSC101 have origins of conjugative transfer (oriTs) and corresponding relaxases that are closely related. The oriTs are made up of a highly conserved core, where DNA is cleaved by the relaxase prior to transfer, and an inverted repeat that differs in size and sequence. We show that in each case the seven base pairs adjacent to the core and within one arm of the inverted repeat are sufficient to determine specificity. Within this DNA there are three AT base pairs located 4 bp from the core. Mutations in the AT base pairs suggest that the relaxase makes essential contacts at these locations to the minor groove of the DNA. The remaining four bases are different for each oriT and are both necessary and sufficient for stringent recognition of oriT by the pSC101 mobilization proteins. In contrast, the R1162 mobilization proteins have a much more relaxed requirement for the base sequence of this specificity region. As a result, the R1162 mobilization proteins can initiate transfer from a variety of sites, including those derived from the chromosome. The R1162 mobilization proteins could therefore contribute to the horizontal gene transfer of DNA from diverse sources.     

Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes.

Using transposon Tn5-mediated mutagenesis, an essential Rhizobium meliloti nitrogen fixation (nif) gene was identified and located directly downstream of the regulatory gene nifA. Maxicell and DNA sequence analysis demonstrated that the new gene is transcribed in the same direction as nifA and codes for a 54-kilodalton protein. In Klebsiella pneumoniae, the nifBQ operon is located directly downstream of a gene which is structurally and functionally homologous to the R. meliloti nifA gene. The DNA sequences of the K. pneumoniae nifB and nifQ genes (which code for 51- and 20-kilodalton proteins, respectively) were determined. The DNA sequence of the newly identified R. meliloti gene was approximately 50% homologous to the K. pneumoniae nifB gene. R. meliloti does not contain a gene homologous to nifQ directly downstream of nifB. The R. meliloti nifB product shares approximately 40% amino acid homology with the K. pneumoniae nifB product, and 10 of the 12 cysteine residues of the R. meliloti nifB product are conserved with 10 of the 17 cysteine residues of the K. pneumoniae nifB product.     

Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product.

An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti. A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis. The fixF gene was found to be related to K. pneumoniae nifN and was therefore renamed as the R. meliloti nifN gene. Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110). By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon. The control region of this operon contains a nif promoter and also the putative nifA-binding sequence. For the deduced amino acid sequence of the nifN gene product a striking homology to the R. meliloti nifK protein was found. One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R. meliloti nifK, R. meliloti nifN, and K. pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.     

Plasmid R6K DNA replication: I. Complete nucleotide sequence of an autonomously replicating segment

A 1565-base segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and the structural gene pir for a protein, designated π, that is required for the initiation of R6K DNA replication. The nucleotide sequence of this 1565 base-pair replicon was determined. From the sequence and the analysis of proteins produced by minicells of E. coli strains carrying the wild-type pir gene and a deletion of the pir gene, it can be concluded that the π structural gene encodes for a protein of a molecular weight of approximately 35,000.On the basis of the nucleotide sequence, the π protein is the only protein containing more than 50 amino acid residues that is encoded by this 1583 base-pair replicon. The nucleotide sequence also contains eight 22 base-pair direct repeats. Seven of the direct repeats are in tandem and located in the origin region, while the eighth repeat is near or part of the promoter for the π structural gene. This eighth repeat may play a role in the autoregulated expression of the π structural gene.     

Presence of STRA-STRB linked streptomycin-resistance genes in clinical isolate of Escherichia coil 2418

The streptomycin resistance of Escherichia coli 2418 strain has been shown to be associated with a 1.2-kb DNA fragment found in the naturally occurring plasmid R2418S. Here, nucleotide sequence analysis of the 1.2-kb DNA fragment revealed the presence of the strB gene which is located immediately downstream of the strA gene. Both sequences are identical to those of strA and strB genes in plasmid RSF1010. Thus, the observed resistance in the clinical isolate is due to the presence of strA-strB genes encoding streptomycin-modifying enzymes. The sequence downstream of strB gene showed a perfect homology with that of RSF1010. In addition, it contained the right inverted repeat of the transposon Tn5393 that has been suggested to be a relic of this transposon found in DNA plasmids isolated from human- and animal-associated bacteria.     

Evolutionary meta-analysis of solanaceous resistance gene and solanum resistance gene analog sequences and a practical framework for cross-species comparisons

Cross-species comparative genomics approaches have been employed to map and clone many important disease resistance (R) genes from Solanum species-especially wild relatives of potato and tomato. These efforts will increase with the recent release of potato genome sequence and the impending release of tomato genome sequence. Most R genes belong to the prominent nucleotide binding site-leucine rich repeat (NBS-LRR) class and conserved NBS-LRR protein motifs enable survey of the R gene space of a plant genome by generation of resistance gene analogs (RGA), polymerase chain reaction fragments derived from R genes. We generated a collection of 97 RGA from the disease-resistant wild potato S. bulbocastanum, complementing smaller collections from other Solanum species. To further comparative genomics approaches, we combined all known Solanum RGA and cloned solanaceous NBS-LRR gene sequences, nearly 800 sequences in total, into a single meta-analysis. We defined R gene diversity bins that reflect both evolutionary relationships and DNA cross-hybridization results. The resulting framework is amendable and expandable, providing the research community with a common vocabulary for present and future study of R gene lineages. Through a series of sequence and hybridization experiments, we demonstrate that all tested R gene lineages are of ancient origin, are shared between Solanum species, and can be successfully accessed via comparative genomics approaches.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

Molecular genetics of disease resistance in cereals

AIMS: This Botanical Briefing attempts to summarize what is currently known about the molecular bases of disease resistance in cereal species and suggests future research directions. SCOPE: An increasing number of resistance (R) genes have been isolated from rice, maize, wheat and barley that encode both structurally related and unique proteins. This R protein diversity may be attributable to the different modus operandi employed by pathogen species in some cases, but it is also a consequence of multiple defence strategies being employed against phytopathogens. Mutational analysis of barley has identified additional genes required for activation of an R gene-mediated defence response upon pathogen infection. In some instances very closely related barley R proteins require different proteins for defence activation, demonstrating that, within a single plant species, multiple resistance signalling pathways and different resistance strategies have evolved to confer protection against a single pathogen species. Despite the apparent diversity of cereal resistance mechanisms, some of the additional molecules required for R protein function are conserved amongst cereal and dicotyledonous species and even other eukaryotic species. Thus the derivation of functional homologues and interacting partner proteins from other species is contributing to the understanding of resistance signalling in cereals. The potential and limit of utilizing the rice genome sequence for further R gene isolation from cereal species is also considered, as are the new biotechnological possibilities for disease control arising from R gene isolation. CONCLUSIONS: Molecular analyses in cereals have further highlighted the complexity of plant-pathogen co-evolution and have shown that numerous active and passive defence strategies are employed by plants against phytopathogens. Many advances in understanding the molecular basis of disease resistance in cereals have focused on monogenic resistance traits. Future research targets are likely to include less experimentally tractable, durable polygenic resistances and nonhost resistance mechanisms.     

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.     

拟南芥NPR1基因的克隆与表达载体的构建

NPR1基因为植物抗病基因表达和系统获得性抗性中的一个关键基因。该文以DNA PCR扩增的方法,从拟南芥基因组DNA中克隆出NPR1基因,通过序列分析,所克隆的 NPR1 基因与报道的基因序列完全一致。将其构建成植物表达载体,为今后植物抗病基因工程的开展奠定了基础。     

Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato

Rx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.     

Excision of a DNA sequence determining kanamycin resistance from a ColE1-Km recombinant plasmid

Summary A 4.8×10⁶ dalton ECoRI-generated fragment of the R-factor R6-5 carrying the gene for kanamycin resistance (Km) was joined in vitro to ECoRI-treated ColE1 plasmid DNA. Transformation ofE. coli with the ColE1-Km recombinant plasmid yielded clones, which were immune to colicin E1, resistant to kanamycin and failed to produce colicin E1. During multiplication of this recombinant plasmid in the presence of chloramphenicol, cells expressed an increased resistance to kanamycin. Transformation studies with the recombinant DNA molecule showed very frequent loss of Km resistance in those cells harbouring a preexisting F'gal plasmid. Since colicin immunity is not affected and the col^- phenotype is still present, one has to test for a remaining DNA sequence further existing in ColE1 DNA by cleaving the plasmid DNA with the ECoRI restriction endonuclease. The full length of ColE1 DNA (6.2 kb) was restored, which confirmed that no deletion of ColE1 DNA sequences had occured. The remaining DNA sequence was identified as a 2.0 or 2.2 kb segment. On the basis of the length of the excised fragment it is proposed that the insertion sequence IS1 and a part of the inverted repeat sequence with coordinates 21.0 to 22.0 of the R6-5 DNA are recognised by a nucleolytic function.     

R391: a conjugative integrating mosaic comprised of phage,plasmid, and transposon elements

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.     

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.