Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10⁵ transformants/g DNA) was achieved. Correspondence to: J. Nevera     

Functional subsets of the virB type IV transport complex proteins involved in the capacity of Agrobacterium tumefaciens to serve as a recipient in virB-mediated conjugal transfer of plasmid RSF1010

The virB-encoded type IV transport complex of Agrobacterium tumefaciens mediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, between Agrobacterium strains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups of virB genes revealed that translation of virB6 is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient in virB-mediated conjugal RSF1010 transfer.     

The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2

Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.     

vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.     

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders.

Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.     

Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.

The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

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。     

Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis.

The plasmid RSF1010 belongs to a class of plasmids (IncQ) that replicate in a range of bacterial hosts. Although non-self-transmissible, it can be mobilized at high frequency between different gram-negative bacterial species if transfer functions are supplied in trans. We report the transfer of RSF1010 by conjugation from Escherichia coli to the gram-positive actinomycetes Streptomyces lividans and Mycobacterium smegmatis. In its new hosts, the plasmid was stable with respect to structure and inheritance and conferred high-level resistance to streptomycin and sulfonamide. This is the first reported case of conjugative transfer of a naturally occurring plasmid between gram-negative and gram-positive bacteria.     

A relative of the broad-host-range plasmid RSF1010 detected in Erwinia amylovora.

Streptomycin- and sulfonamide-resistant Erwinia amylovora CA3R from California contained an 8.7-kb plasmid, pEa8.7, with a sulII-strA-strB resistance region; furthermore, PCR, sequencing, hybridization, and restriction analyses showed that pEa8.7 was closely related or identical to broad-host-range plasmid RSF1010. Although RSF1010 has been found in a variety of bacteria, this is the first report of its presence in plant pathogenic bacteria.     

Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12.

Replicating DNA molecules of the nonconjugative R plasmid RSF1010 (Smr Sur) were cleaved with the EcoRI restriction endonuclease and examined with the electron microscope. Results of this analysis indicated that replication is initiated from an origin located at about 19% of total genome size from one of the EcoRI ends. Replication proceeded either unidirectionally or bidirectionally with equal frequency. Results of the analysis of replicative intermediates of RSF1010 containing the Apr-transposable sequence (Tn) are also presented.     

A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity.     

RSF1010 plasmid as a potentially useful vector in Pseudomonas species.

RSF1010 plasmid DNA was introduced into Pseudomonas putida and P. aeruginosa cells and maintained stably, suggesting the potential usefulness of this plasmid as a vector in Pseudomonas species. The number of copies of RSF1010 was 43 per chromosome equivalent in P. putida cells.     

A multi-resistance plasmid isolated from commensal Neisseria species is closely related to the enterobacterial plasmid RSF1010

pFM739, an R plasmid from Neisseria sicca that encodes penicillin, streptomycin and sulphonamide resistance, and the enterobacterial IncQ(P-4) plasmid RSF1010, which encodes streptomycin and sulphonamide resistance, were incompatible, and were mobilized by the same conjugative plasmids. Restriction mapping confirmed a high degree of similarity between both R plasmids; pFM739 carried DNA fragments corresponding to the known replication and resistance regions of RSF1010. pFM739 also carried an extra segment with the same restriction map as that described for the beta-lactamase-coding region of transposon Tn3. It is suggested that the R plasmids isolated from commensal Neisseria sp. could have resulted from transposition of a Tn3-like genetic element to an RSF1010-like plasmid, and that they contain deletion derivatives of transposon Tn3.     

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.