The role of the T-pilus in horizontal gene transfer and tumorigenesis.

The T-pilus is a flexuous filamentous appendage that is essential for Agrobacterium tumefaciens virulence. T-pilus subunits are derived from a VirB2-processing reaction that generates cyclized polypeptide subunits. The T-pilus filament has a diameter of 10 nm and contains a lumen approximately 2 nm in diameter. Biogenesis of the T-pilus requires all 11 VirB proteins, but not the VirD4 protein, which is used in conjugal plasmid transfer. VirB4 and VirB11 are two ATPases that may form homohexameric rings within the transport apparatus, which is composed of VirB6-10 proteins.

A single amino acid substitution beyond the C2H2-zinc finger in Ros derepresses virulence and T-DNA genes in Agrobacterium tumefaciens.

Ros is a chromosomally-encoded repressor containing a novel C2H2 zinc finger in Agrobacterium tumefaciens. Ros regulates the expression of six virulence genes and an oncogene on the Ti plasmid. Constitutive expression of these genes occurs in the spontaneous mutant 4011R derived from the octopine strain Ach-5, resulting in T-DNA processing in the absence of induction, and in the biosynthesis of cytokinin. Interestingly, the mutation in 4011R is an Arg to Cys conversion at amino acid residue 125 near the C-terminus well outside the zinc finger of Ros. Yet, Ros bearing this mutation is unable to bind to the Ros-box and is unable to complement other ros mutants.

Antirestriction protein Ard (Type C) encoded by IncW plasmid pSa has a high similarity to the "protein transport" domain of TraC1 primase of promiscuous plasmid RP4.

The IncW plasmid pSa contains the gene ard encoding an antirestriction function that is specific for type I restriction and modification systems. The nucleotide sequence of ard was determined and an appropriate polypeptide of about 33 kDa was identified in Escherichia coli T7 expression system. Analysis of deduced amino acid sequence of Ard encoded by pSa revealed that this protein has no significant similarities with the known Ard proteins (ArdA and ArdB types) except the "antirestriction" motif (14 amino acid residues in length) conserved for all known Ard proteins. This finding suggests that pSa Ard may be classified as a new type of Ard proteins which we designated ArdC. The remarkable feature of ArdC is that it has a high degree of similarity (about 38 % identity) to the N-terminal region of RP4 TraC1 primase which includes about 300 amino acid residues and seems to be essential for binding to the single-stranded DNA and TraC1 protein transport to the recipient cells during the conjugal transfer of plasmid DNA. ArdC also binds to single-stranded DNA. In addition, this protein is able in vitro to protect the single-stranded but not double-stranded plasmid DNA against the activity of type II restriction endonuclease HhaI that cleaves both single and double-stranded DNA. We suggest that like TraC1, ArdC would be transported as a result of their interaction with the single-stranded DNA of transferred plasmid strand during conjugative passage through the cell envelope to the recipient bacterium. Such properties of ArdC protein might be useful to protect immediately the incoming single-stranded DNA from the host endonucleases.

Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation.

Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.

pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export.

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.

Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens.

Previous studies have implicated the obligatory requirement for the vir regulon (or "virulon") of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19 degreesC as compared with 28 degreesC, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107-1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the "T pilus" of A. tumefaciens.

Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt.

Virulence genes of Agrobacterium tumefaciens are under the control of positive and negative transcriptional regulators. We found that the transcriptional regulator Ros controls expression of the plant oncogene ipt, which encodes isopentenyl transferase, in A. tumefaciens. This enzyme is involved in biosynthesis of the plant growth hormone cytokinin in the host plant. An ipt promoter::cat reporter gene fusion showed a 10-fold increase in ipt promoter activity in A. tumefaciens ros mutant strains when compared with wild type. Also, increased levels (10- to 20-fold) of isopentenyl adenosine, the product of the reaction catalyzed by isopentenyl transferase, were detected in ros mutant strains. In vitro studies using purified Ros showed it binds directly to the ipt promoter. Analysis of the deduced Ros amino acid sequence identified a novel type of C2H2 zinc finger. In Ros the peptide loop spacing of the zinc finger is 9 amino acids as opposed to the invariant 12 amino acids in the classical C2H2 motif. Site-directed mutagenesis of Cys-82 and His-92 in this motif showed that these residues are essential for Zn2+ and DNA binding activities of Ros. The existence of such a regulator in Agrobacterium may be due to horizontal interkingdom retrotransfer of the ros gene from plant to bacteria.

Characterization of a new Agrobacterium tumefaciens strain from alfalfa.

Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar.

Origin and evolution of plasmids.

Studies on the origin and evolution of plasmids may provide valuable insights on the promiscuous nature of DNA. The first examples of the selfish nature of nucleic acids are exemplified by primordial oligoribonucleotides which evolved into primitive replicons. The propagation of these molecules were likely patterned after the current viral RNA ribozymes, which have been recently shown to possess RNA synthesizing and template mediated polymerizing capabilities in the absence of proteins. The parasitic nature of nucleic acids is depicted by satellite nucleic acid molecules associated with viruses. The satellite of adenovirus and tobacco ringspot virus serve as established examples: they contain no open reading frames. Comparative analysis of the replication origins of virions and plasmids show them to be conserved, originating from the simplest autocatalytic replicon to highly complex and evolved plasmids, replicating by a rolling circle mechanism. The eventual association of proteins with nucleic acids provided added efficiency and protective advantages for molecular perpetuation. The promiscuous and selfish nature of plasmids is demonstrated by their ability to genetically engineer their host so that the host cell is best able to cope and survive in hostile environments. Survival of the host ensures survival of the plasmid. Sequestering of genes by plasmids occurs when the environmental conditions negatively affect the host. The sequestering mechanism is fundamental and forms the outreach mechanisms to generate and propagate macromolecules of increasing size when necessary for survival. The level of sophistication of plasmids increases with the addition of new genes such as those that allow the host to occupy a specific environment normally inhospitable to the host cell. The vast range of plasmid types which have obtained genes interchangeably reflect the levels of sophistication achieved by these macromolecules. The Ti plasmid in Agrobacterium tumefaciens and the pSym and accessory plasmids in Rhizobium illustrate the level of complexity attained by replicons.

Characterization of flagella genes of Agrobacterium tumefaciens, and the effect of a bald strain on virulence.

Agrobacterium tumefaciens produces flagella that are arranged circumthecally near one end of the bacilliform cell. The flagella are required for motility to facilitate reaching the root surface, and possibly aid in orientating the bacterial cells at various sites for infection. We have identified three flagella genes designated flaA, flaB, and flaC. Mutations in flaA, flaB and flaC result in abberant swimming behaviour. Electron microscopic examination of these mutants revealed the defective flagella. A non-motile, bald mutant strain was generated by deleting all three fla genes. Nucleotide sequencing of flaA, flaB, and flaC showed that they have a potential coding capacity for polypeptides of 307, 321, and 314 amino acid residues, respectively. The predicted amino acid sequences of the A. tumefaciens FlaA and FlaB proteins are similar (66% average identity) to the FlaA and FlaB proteins encoded by flaA and flaB genes, respectively, in Rhizobium meliloti. There was no counterpart FlaC protein reported in R. meliloti, but the A. tumefaciens FlaC is similar in amino acid sequence to the R. meliloti FlaA (59.8% identity) and FlaB (66.7% identity). Distinct from FlaA and FlaB of R. meliloti is the absence of histidine and cysteine residues and their shorter length (by 88 amino acid residues fewer than FlaA and FlaB of R. meliloti). The transcriptional start sites of each fla gene determined by primer extension revealed consensus-sequence boxes representing potential binding sites for sigma 28 RNA polymerase (RNAP) upstream of the transcriptional start of each fla gene. Besides the potential sigma 28-binding site upstream of flaC, also present are additional putative conserved sequences, GC at -11 and GG at -21 from the transcriptional start, that resemble potential binding motifs for sigma 54. Because the sigma 54 promoter is associated with genes regulated by physiological changes in various bacteria, the flaC gene might be similarly regulated in response to A. tumefaciens responding to host plant stimuli. Virulence studies showed that the bald strain was consistently reduced in virulence below that of the parental wild-type strain by at least 38%. The difference is statistically significant and suggests that the flagella may play a role in facilitating virulence.

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens.

The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter. Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli. Because Osa inhibits oncogenicity of A. tumefaciens, and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.

Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa.

The IncW plasmid pSa originally derived from Shigella flexneri completely inhibits the tumor-inducing ability of Agrobacterium tumefaciens when it is resident in this organism. Oncogenic inhibition is mediated through the expression of the osa gene on pSa. This gene is part of a 3.1-kb DNA segment of pSa that contains four open reading frames revealed by sequencing. Specific deletions and TnCAT insertions within this segment localized the oncogenic inhibitory activity to the last open reading frame, orf-4, designated osa (for oncogenic suppression activity). No promoter exists immediately upstream of the coding sequence of osa since TnCAT insertions or deletions into orf-3 caused the loss of oncogenic inhibition. Deletion analysis showed that the promoter of orf-1 is required for osa transcription. The first three orfs have no role in oncogenic inhibition, since osa alone placed under the control of a constitutive Pkm promoter completely inhibited A. tumefaciens oncogenicity. This inhibition of oncogenicity by osa is not limited to a specific host plant but appears to show broad host specificity. Because the osa-encoded product has close homologies to the fiwA-encoded product of the IncP plasmid RP1, osa may be involved in fertility inhibition that would prevent or reduce the formation of stable mating pairs and T-DNA transfer between A. tumefaciens and plants.

Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis.

Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram-negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T-DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity = 50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase-I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase-I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptI operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA-protein complexes.

An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes.

The 9.5 kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84 kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T-DNA transfer process were determined by generating non-polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T-DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection- and tumorigenicity-negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T-DNA and virulence. Furthermore, by using anti-VirB4 serum, the protein product of virB4 was localized to the inner-membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti-VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T-DNA or to assemble the T-DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, respectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T-DNA from A. tumefaciens to plant cells.

The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens.

The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T-DNA processing and undefined virulence functions. Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root-inducing plasmid pRiA4b and to that of the octopine-type plasmid pTiA6NC. However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC. T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively. virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids. Non-polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4. An independently regulated fifth ORF (orf5) is located immediately downstream of 3' end of virD4 and encodes a polypeptide of 97.4 kDa. The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A. tumefaciens. Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence. In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non-polar mutants that were tested for virulence. virD3 and orf5 non-polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants. These data suggest that virD3 and orf5 are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens.

The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala47, upstream of which two additional alanine residues Ala45-Ala46 are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.

The region essential for efficient autonomous replication of pSa in Escherichia coli.

Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.

A gene near the plasmid pSa origin of replication encodes a nuclease.

We have cloned and sequenced a gene (nuc) from the IncW plasmid pSa which shows amino acid sequence similarity to staphylococcal nuclease (EC 3.1.4.7) and to the parB locus of plasmid RP4. The 525 bp open reading frame encodes a 174-amino-acid potential polypeptide of 19.7 kDa. Expression of the gene was confirmed using an in vitro transcription-translation assay which produced a protein of identical size. Nuclease activity was demonstrated using DNA as the substrate in toluidine blue-DNA agar plates. The deduced amino acid sequence revealed a signal sequence, and TnphoA insertion within the open reading frame indicated that a portion of the protein is transported across the bacterial cell membrane.