Functional organization of MobB, a small protein required for efficient conjugal transfer of plasmid R1162

MobB is a small (molecular weight = 15,097) protein encoded by the broad-host-range plasmid R1162 and is required for its efficient transfer by conjugation. The C-terminal half of the protein contains a membrane domain essential for transfer. This region can be replaced by a putative membrane domain from another, unrelated protein, and thus is likely to function independently from the rest of MobB. The other, functionally active region of MobB, identified by mutagenesis, is at the N-terminal end. One mutation affecting this region inhibits replication, suggesting that this part of the protein is contacting and sequestering the relaxase-linked primase. The overall organization reflects a multimeric and bipolar organization, with molecules of MobB anchored in the membrane at one end and engaging the relaxase at the other. This arrangement could increase the transfer frequency by raising the probability of contact between the relaxase and the membrane-embedded, coupling protein for type IV secretion.     

Molecular handcuffing of the relaxosome at the origin of conjugative transfer of the plasmid R1162

The assembly of plasmid-encoded proteins at a unique site (oriT) on the plasmid R1162, to form a complex called the relaxosome, is required for conjugative transfer of the plasmid and for negative regulation of neighboring promoters. Two-dimensional chloroquine gel electrophoresis was used to show that oriTs are physically coupled at the relaxosome. This interaction requires all the relaxosome proteins, which are assembled into a structure resulting in a decrease in the average linking number of the plasmid DNA in the cell. Molecules with higher superhelical densities are preferentially selected for assembly of the relaxosome. Genetic data obtained earlier indicate that the molecular coupling reported here is a ‘handcuffing’ reaction that contributes to the regulation of adjacent plasmid promoters. However, although these promoters affect the expression of the genes for replication, plasmid copy-control is regulated independently. This is the first time ‘handcuffing’ has been observed at an oriT, and its possible significance for transfer is discussed.     

MobB protein stimulates nicking at the R1162 origin of transfer by increasing the proportion of complexed plasmid DNA.

An essential early step in conjugal mobilization of R1162, nicking of the DNA strand that is subsequently transferred, is carried out in the relaxosome, a complex of two plasmid-encoded proteins and DNA at the origin of transfer (oriT). A third protein, MobB, is also required for efficient mobilization. We show that in the cell this protein increases the proportion of molecules specifically nicked at oriT, resulting in lower yields of covalently closed molecules after alkaline extraction. These nicked molecules largely remain supercoiled, with unwinding presumably constrained by the relaxosome. MobB enhances the sensitivity of the oriT DNA to oxidation by permanganate, indicating that the protein acts by increasing the fraction of complexed molecules. Mutations that significantly reduce the amount of complexed DNA in the cell were isolated. However, plasmids with these mutations were mobilized at nearly the normal frequency, were nicked at a commensurate level, and still required MobB. Our results indicate that the frequency of transfer is determined both by the amount of time each molecule is in the nicked form and by the proportion of complexed molecules in the total population.     

The primase of broad-host-range plasmid R1162 is active in conjugal transfer.

The broad-host-range plasmid R1162 is conjugally mobilized at high frequency by the IncP-1 plasmid R751 but is poorly mobilized by pOX38, a derivative of the F factor. In both cases, the origin of transfer (oriT) and the Mob proteins of R1162 are required, indicating that these plasmids are mobilized by similar mechanisms. R1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of MobA, one of the R1162 mobilization proteins (P. Scholz, V. Haring, B. Wittman-Liebold, K. Ashman, M. Bagdasarian, and E. Scherzinger, Gene 75:271-288, 1989). When R751 is the mobilizing vector, the primase is not required for mobilization of plasmids containing cloned mob-oriT R1162 DNA. However, detectable mobilization of such plasmids by pOX38 requires both the primase and its cognate initiation site, oriented for synthesis of the complement to the transferred strand. The long form of the primase is required for optimal transfer: R1162 replicons lacking this form also are not transferred detectably by pOX38 and are less well mobilized by R751. The distance between oriT and the primase initiation site affects the frequency of mobilization, and this effect is polar in the direction of transfer. Our results indicate that the R1162 primase is active in mobilization of R1162 and suggest that the MobA-linked form is an adaptation increasing its effectiveness during transfer.     

Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization. Evidence for genetically distinct events at oriT

A segment of R1162 DNA containing genes for conjugative mobilization (Mob) and the origin of transfer (oriT) was integrated into the Escherichia coli chromosome. Bacterial genes were transferred in a polar fashion during conjugative mobilization of the integrated segment by a self-transmissible plasmid vector. The direction of transfer, together with the properties of mutated derivatives of oriT, indicate that initial cleavage of oriT, and subsequent religation after transfer, entail different mechanisms that can be distinguished genetically.     

The MobA-linked primase is the only replication protein of R1162 required for conjugal mobilization

Cells newly transformed with plasmid R1162 DNA were used as donors in conjugal matings to determine if the plasmid replication genes are necessary for transfer. An intact system for vegetative replication is not required for transfer at normal frequency, but the plasmid primase, in the form linked to the nickase, must be present in donor cells.     

The R1162 relaxase/primase contains two, type IV transport signals that require the small plasmid protein MobB

The relaxase of the plasmid R1162 is a large protein essential for conjugative transfer and containing two different and physically separate catalytic activities. The N-terminal half cleaves one of the DNA strands at the origin of transfer (oriT) and becomes covalently linked to the 5' terminal phosphate; the C-terminal half is a primase essential for initiation of plasmid vegetative replication. We show here that the two parts of the protein are independently transported by the type IV pathway. Part of the domain containing the catalytic activity, as well as an adjacent region, is required in each case, but the required regions do not physically overlap. Both transport systems contribute to the overall frequency of conjugative transfer. MobB is a small protein, encoded within mobA but in a different reading frame, that stabilizes the relaxase at oriT. MobB is required for efficient type IV transport of both the complete relaxase and its two, separate functional halves. MobB inserts into the membrane and could thus stabilize the association between the relaxase and the type IV transfer apparatus.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer

The frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50-fold if donor cells lack MobC, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer ( oriT  ). The absence of MobC has several different effects on oriT DNA. Site- and strand-specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome-induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single-stranded substrate required for cleavage by MobA.     

Inhibition of conjugal transfer of R plasmids by nitrofurans

Nifurzide is a nitrofuran with antibacterial activity. As nitrofurans have been reported to interact with DNA, we tested the ability of nifurzide to inhibit plasmid transfer. Inhibition of plasmid transfer between Escherichia coli strains was obtained for ten plasmids belonging to nine incompatibility groups. The same effect was observed when plasmid RP4 was harboured in six different members of the Enterobacteriaceae. Inhibition depended on the reduction of the -NO2 group of nifurzide and was obtained with four other nitrofuran derivatives.     

Origin and fate of the 3' ends of single-stranded DNA generated by conjugal transfer of plasmid r1162

During conjugation, a single strand of DNA is cleaved at the origin of transfer (oriT) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5'-to-3' direction, with the 3' end likely extended by rolling-circle replication. The resulting, newly synthesized oriT must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at oriT, with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3' ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved oriT within the strand. A model is presented where these internal oriTs bind and trap the relaxase molecule covalently bound to the 5' end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer.

R1162 is efficiently comobilized during conjugative transfer of the self-transmissible plasmid R751. Bacteriophage M13 derivatives that contain two directly repeated copies of oriT, the site on R1162 DNA required in cis for mobilization, were constructed. Phage DNA molecules underwent recombination during infection of Escherichia coli, with the product retaining a single functional copy of oriT. Recombination was strand specific and depended on R1162 gene products involved in mobilization, but did not require the self-transmissible plasmid vector. Two genes were identified, one essential for recombination and the other affecting the frequency of recombination. Recombination of bacteriophage DNA could form the basis of a simple model for some of the events occurring during conjugation without the complexity of a true mating system.     

The exonuclease and host shutoff functions of the SOX protein of Kaposi's sarcoma-associated herpesvirus are genetically separable

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.     

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.     

Two domains at the origin are required for replication and maintenance of broad-host-range plasmid R1162.

Two domains at the replicative origin of broad-host-range plasmid R1162 are required in cis for plasmid maintenance in Escherichia coli and for plasmid DNA replication in cell extracts. Increasing the distance between the domains reduces replication in vitro, without substantially changing plasmid DNA content or stability in vivo.     

Regulation of conjugal transfer by Lrp and Dam methylation in plasmid R100.

Conjugal transfer of the F-like plasmid R100 occurs at higher frequencies in the absence of DNA adenine methylation. Lower levels of R100-encoded FinP RNA were found in a Dam host, suggesting that Dam methylation regulates FinP RNA synthesis. Lack of the leucine-responsive regulatory protein (Lrp) causes a decrease in R100 plasmid transfer, indicating that Lrp is an activator of R100-mediated conjugation. Hence, host-encoded regulators previously described for the Salmonella virulence plasmid (pSLT) seem to play analogous roles in R100. Repression of conjugal transfer in rich medium is an additional trait shared by R100 and pSLT. DNA sequence comparisons in regulatory loci support the view that R100 and pSLT are closely related.     

Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA.

Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter.     

The corals Acropora palifera and Acropora cuneata are genetically and ecologically distinct

Two of the most abundant corals of the Great Barrier Reef Acropora cuneata and Acropora palifera, can be distinguished reliably by conventional taxonomic methods only if large specimens of both species are collected from the same uniform biotope. In this study gross and microscopic examination of the corallite structures of large coralla, collected from a reef crest biotope, were used to confirm the assignment of these specific names to two electrophoretically distinct groups of corals. Identifications based on genetic and morphological data gathered from small fragments of coralla collected from five different sites were in agreement for two sites within which skeletal data were adequate for taxonomic purposes (i.e. one reef crest and one outer lagoon site). However, specimens collected from the other three sites could be identified reliably only by using the electrophoretic data. These results confirm that small fragments from diverse habitats may not be adequate for morphologically based identification. The application of electrophoretic identifications to the results of an earlier electrophoretic survey of 76 sites on 11 reefs revealed that, although A. palifera and A. cuneata frequently co-occur, they have significantly different habitat distributions. Acropora cuneata is more commonly associated with high energy environments such as the reef crest, whereas A. palifera occurs most commonly in the sheltered reef lagoons and on the deeper reef slopes.     

Tritrichomonas foetus from domestic cats and cattle are genetically distinct

The genetic relationship amongst Tritrichomonas foetus isolated from domestic cats and cattle was investigated by DNA sequencing of the internal transcribed region of the ribosomal DNA unit and the TR7/TR8 variable-length repeat. The results reject the hypothesis that T. foetus from domestic cats is genetically identical to T. foetus in cattle. We suggest recognition of a ‘cat genotype’ and a ‘cattle genotype’ of T. foetus. Review of public nucleotide repositories revealed that the ‘cat genotype’ has not been isolated from cattle nor the ‘cattle genotype’ recovered from cats. However, at least one cat isolate has been shown to induce disease in experimentally infected cattle. We conclude that these genotypes fall within the single species T. foetus.