质粒RSF1010寄主范围特性的遗传学分析

采用Tn5插入诱变、限制性核酸内切酶作图以及DNA转化等方法,对广泛寄主范围型质粒SF 1010的衍生体－pKT 2 40进行研究。证实质粒的寄主围决定于它在遗传背景不同的寄主中复制并保存自身的能力,而repA,rcpB和repC基因为该质拉复制所必需。     

Control of replication of the broad host range plasmid RSF1010: The incompatibility determinant consists of directly repeated DNA sequences

Summary A 500 bp DNA fragment located in the vicinity of the origin of replication of plasmid RSF1010 was cloned into the plasmid vector pBR322 and shown to exhibit incompatibility against parental RSF1010. The rightmost region of this fragment contains three perfect 20 pb direct repeats and a fourth half-repeat of 11 bp, as shown by DNA sequencing. Delection of the four repeats from the cloned fragment resulted in complete loss of incompatibility whereas partial deletion of the repeated sequence resulted in a concurrent decrease in the expression of incompatibility. We conclude that the incompatibility determinant of RSF1010 is defined by the four repeats and also that the incompatibility expressed is not very strong, since the presence of about 1.5 times as many copies of the repeated sequence as are normally in a cell does not cause a total switch off of RSF1010 replication, but only a 40% reduction in the rate of replication.Abbreviations kb kilobase pairs - bp base pairs - Km^r, Tc^r resistance to kanamycin and tetracycline, respectively     

A base-paired hairpin structure essential for the functional priming signal for DNA replication of the broad host range plasmid RSF1010.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.     

Transfer and expression of broad host range plasmids in Zymomonas mobilis

Summary The broad host range vectors, pKT210, pKT212, pKT248, pKT240, pGSS33 were mobilized into Zymomonas mobilis with the help of conjugative plasmids belonging to various incompatibility groups. The vectors pKT210, pKT212, pKT248, pGSS33 were stably maintained in Zymomonas mobilis in contrast to the conjugative plasmids. Based on these results we suggest the potential usefulness of these vectors as cloning vehicles in Zymomonas mobilis.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Evaluation of Arabidopsis thaliana as a model host for Xylella fastidiosa

The bacterium Xylella fastidiosa causes a number of plant diseases of significant economic impact. To date, progress determining mechanisms of host-plant susceptibility, tolerance, or resistance has been slow, due in large part to the long generation time and limited available genetic resources for grape, almond, and other known hosts of X. fastidiosa. To overcome many of these limitations, Arabidopsis thaliana has been evaluated as a host for X. fastidiosa. A pin-prick inoculation method has been developed to infect Arabidopsis with X. fastidiosa. Following infection, X. fastidiosa multiplies and can be detected by microscopy, polymerase chain reaction, and isolation. The ecotypes Van-0, LL-0, and Tsu-1 all allow more growth of strain X. fastidiosa Temecula than the reference ecotype Col-0. Affymetrix ATH1 microarray analysis of inoculated vs. noninoculated Tsu-1 reveals gene expression changes that differ greatly from changes seen after infection with apoplast-colonizing bacteria such as Psuedomonas syringae pvs. tomato or syringae. Many genes responsive to oxidative stress are differentially regulated, while classic pathogenesis-related genes are not induced by X. fastidiosa infection.     

Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions.

The broad-host-range vectors pSUP104, pSUP106, pSUP204, pSUP304, and pSUP404 are based on conventional Escherichia coli vectors (such as pBR325 and pACYC184) which have been modified to include the mobilization and broad-host-range replication functions of the IncQ plasmid RSF1010. These vector plasmids now can be maintained in a wide range of bacterial genera including Rhizobium, Agrobacterium, and Pseudomonas. They are efficiently mobilized by RP4 and thus are of particular interest for bacteria refractory to transformation. They offer the selection markers and cloning sites characteristic of the basic E. coli vectors. Therefore, they can be applied and adapted to a variety of cloning strategies. However, the cloning of very large fragments (e.g., in cosmid hybrids of pSUP106) was found to affect the stability of the recombinant molecules in a Rec+ background. This instability was not observed with smaller inserts of about 5 kilobases.     

Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.     

Influence of Xylella fastidiosa infection of citrus on host selection by leafhopper vectors

Infection of plants by pathogens can influence their attractiveness and suitability to insect vectors and other herbivores. Here we examined the effects of Citrus sinensis (L.) Osbeck (Rutaceae) infection by the bacterium Xylella fastidiosa, which causes citrus variegated chlorosis (CVC), on the feeding preferences of two sharpshooter vectors, Dilobopterus costalimai Young and Oncometopia facialis (Signoret) (Homoptera: Cicadellidae). Experiments were performed inside observation chambers, in which a healthy plant and an infected one (with or without CVC symptoms) were supplied to a group of 40 sharpshooters. The number of insects that selected each treatment was recorded at several time intervals in 48 h. In another experiment, the ingestion rate on healthy and infected (symptomatic or not) plants was evaluated by measuring the liquid excretion of sharpshooters that were confined on branches of each plant for 72 h. Both sharpshooter species preferred healthy plants to those with CVC symptoms. However, O. facialis did not discriminate between healthy citrus and symptomless infected plants. Feeding by D. costalimai was markedly reduced when confined on CVC‐symptomatic plants, but not on asymptomatic infected ones. The ingestion rate by O. facialis was not affected by the presence of CVC symptoms. The results suggest that citrus trees with early (asymptomatic) infections by X. fastidiosa may be more effective as inoculum sources for CVC spread by insect vectors than those with advanced symptoms.     

Transfer and replication of RSF1010-derived plasmids in several cyanobacteria of the generaSynechocystis andSynechococcus

RSF1010-derived plasmids are most efficiently transferred by conjugation to the unicellular cyanobacteriaSynechocystis strains sp. PCC6803 and PCC6714 andSynechococcus strains sp. PCC7942 and PCC6301, where they replicate autonomously, even though they contain no cyanobacterial DNA. These results are especially important in the case of the facultative heterotrophic strainSynechocystis PCC6714, which is not transformable [Mol Gen Genet 204:185, 1986]     

The interaction of RepC initiator with iterons in the replication of the broad host-range plasmid RSF1010.

The replication origin of the broad host-range plasmid RSF1010 contains 3.5 copies of a 20mer iteron sequence that bind specifically to the plasmid-encoded initiator, RepC. Here we demonstrated that even a single iteron was bent upon binding of RepC. Moreover, the bending angle seems to become larger along with the increment of the number of iterons. In a mutational analysis of the iteron sequence, we isolated seven kinds of base-substitution mutants of iterons, and estimated the replication activity of these mutants in vivo. We found that each of the subsections in the 20mer iteron sequence made a distinct contribution to the initiation of RSF1010 DNA replication. With the binding assay of RepC and mutated iterons in vitro, we found that the formation of a productive RepC-iteron complex was required for the initiation of plasmid DNA replication.     

Mapping analysis of the Xylella fastidiosa genome

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.fastidiosa sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.     

Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids

Single-stranded DNA (ssDNA) promoters are the key components of the single-strand origins (ssos) of replication of rolling-circle (RC) replicating plasmids. The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging-strand synthesis. This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts. A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram-positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-containing plasmids. Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram-positive bacteria, S. aureus and B. subtilis. We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology. Our data support the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.