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THE CHROMOSOMAL REPLICATION INITIATION PROTEIN DnaA, AND THE IDENTIFICATION OF THE CHROMOSOMAL ORIGIN OF REPLICATION FROM SINORHIZOBIUM MELILOTI

Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1 Canada

The organization of the Sinorhizobium meliloti tripartite genome, consisting of one chromosome of 3500kb and two large plasmids (1700kb and 1400kb), has stimulated interest in the mechanisms of replication initiation and replicon segregation during cellular division. Prokaryotic replication initiation has been an intense area of investigation, and has led to the characterization of some essential proteins involved in the process. The DnaA protein has been implicated as one such essential component in the formation of an active complex at the chromosomal origin of replication. The DnaA protein binds to 9 base pair elements in the oriC, leading to the melting of the duplex DNA and the initiation of the replication fork.

The goal of this work was to purify S. meliloti DnaA and identify the origin of replication on the S. meliloti chromosome. The S. meliloti dnaA gene was originally cloned into pBADHisA and S. meliloti DnaA(Hiss) was successfully over-expressed in E. eoli. However, virtually all of the over-expressed protein was represented as insoluble inclusion bodies. Changes in the induction procedure as well as the E. coli expression strain did not result in an increased expression of soluble S. meliloti DnaA(His6). Subsequently, the S. meliloti dnaA gene was cloned as a PCR product in frame into the pGEX5X-l plasmid, which is designed for high level intracellular expression of proteins as fusion to glutathione S-transferase. Over-expression of soluble S. meliloti DnaA as a fusion protein was successful in E. coli. The over-expression of soluble fusion protein was optimized, by inducing protein synthesis once cells had grown to an OD = 0.6 with 0.1 mM IPTG. Cells were then incubated overnight at room temperature.

S. meliloti DnaAGST has been successfully purified. Induced culture (100 mL) was pelleted and resuspended in 10 mL ice-cold STE buffer. Lyozyme was added, incubated on ice for 15 min and just prior to sonication 100 |iL 1M DTT and 1.4 mL 10% Sarkosyl was added. Cells were sonicated and the crude lysate was spun (16,000 rpm) to pellet insoluble material. The effective concentrations of Sarkosyl and Triton X-100 were then brought to 0.7% and 2%, respectively and incubated at room temperature for 45 minutes. The soluble lysate was then incubated with 1 mL of 50% glutathione sepharose in PBS at room temperature for 1 hour with agitation. The binding mixture was then added to a column, washed with PBS and S. meliloti DnaAGST was eluted with 1 mL volumes of elution buffer containing 20 mM GSH. The 25kDa GST domain was effectively cleaved by treatment with Factor Xa to liberate the 57 kDa S. meliloti DnaA.

The origin of replication on the S. meliloti chromosome has not previously been experimentally identified. Analysis of the S. meliloti chromosome sequence revealed a region similar to the oriC locus of the closely related organism Caulobacter crescentus which contains a significant A/T rich region upstream of the hemE gene. A 3 kb fragment containing the entire S. meliloti hemE ORF, the A/T rich region as well as both up and downstream DNA was PCR amplified. To test if the PCR product contained the S. meliloti chromosomal origin of replication it was cloned into pUCP30T, a plasmid unhable to replicate in S. meliloti cells. Conjugal transfer of the recombinant plasmid into S. meliloti showed that the 3 kb S. meliloti hemE region was able to confer autonomous replication to the pUCP30T plasmid. A genetic analysis of the S. meliloti origin of replication is currently underway.

HORIZONTAL TRANSFER OF THE SINORHIZOBIUMMELILOTI pExo MEGAPLASMID

Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1 Canada

Plasmids play one of the strongest roles in prokaryotic evolution, moving from cell to cell, among bacterial strains, related bacterial species and even domains of life. We have phylogenetically analyzed the pExo (pSymB) megaplasmid of the gram-negative soil bacterium Sinorhizobium meliloti and estimate that 60% of the megaplasmid has been acquired through horizontal gene transfer. Previous studies have suggested that horizontal transfer in the rhizobial lineage has occurred only between closely related strains. Our results suggest otherwise; horizontal transfer seems to have taken place between pExo and many distantly related species. A large portion of the horizontally transferred genes appear to be related to Streptomyces coelicolor, Escherichia coli, and Pseudomonas aeruginosa, all of which, like S. meliloti, are found in the soil. Interestingly, the highest number of ORFs related to any one species is a gram-positive species, Streptomyces coelicolor. Analysis of the functional distribution of pExo ORFs reveal that most of the horizontally transferred genes (43%) are involved in cellular processes, which comprise proteins important for the transport of small molecules, DNA uptake, protection responses and nodulation.

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