P1 plasmid replication: measurement of initiator protein concentration in vivo

Abstract

To study the functions of the mini-Pl replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer deteetable.P1 prophage is a stringently controlled plasmid replicon that is maintained at a copy number approximately equal to the number of Escherichia coli chromosomes (16). The basic plasmid replicon, the minimal region required to maintain the plasmid at its normal copy number, consists of an origin, the gene for the initiator protein RepA, and a control locus. The salient feature of the basic replicon is the presence of two sets of repeated, nearly identical 19-base-pair sequences. A set of five repeats is an essential part of the origin. A separate set of nine repeats constitutes the control locus (3, 4). The purified RepA protein binds to both sets of repeats (1). Extra copies of the repeats decrease the replication frequency, whereas deletion of the control locus increases the frequency (4, 14). Similar results were obtained for mini-F plasmids, whose genetic organization is very similar to that of mini-Pl (25). It has been proposed (25) that the origin and the control repeats compete for RepA, which has been shown to be limiting for replication (13,18), implying that the physiological concentration of the protein is so low that sequestration of a few molecules would have a significant effect on the rate of replication. In other words, the number of repeats and the number of RepA molecules are expected to be comparable in the cell. In this paper we report the measurement of RepA concentration by two approaches. These measurements are consistent with the sequestration hypothesis.The protein is also known to have two other functions. It inhibits the synthesis of the repA transcript and, when overproduced, inhibits replication (6). Knowledge of the protein concentration will thus be helpful in understanding how RepA functions. A preliminary report of some of this work has been presented (5). Protein determination. The protein concentration was determinated by using amido black as described by Schaffner and Weissman (20). All samples were brought to 1% SDS, and BSA was-used as the protein standard. The protein concentration of purified RepA as determined by amino acid analysis was about 30% less than the concentration determined by the amido black method with BSA as standard. The discrepanc...

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

“…X-ray films (AGFA) were exposed and then both the hybridized plasmid bands and the EtBrstained chromosome were quantified using GelDoc (Bio-Rad). The amount of intracellular RepA in P. aeruginosa cells carrying pPSEC plasmids was estimated by Western blotting, as described (47). 200-l culture aliquots were taken at different A 600 values.…”

Location of a P1 plasmid replication inhibitor determinant within the initiator gene

“…However, the mutant plasmid still inhibited lysogeny significantly (pKM102; An in vivo measure of RepA synthesis, albeit indirect, was obtained by assaying the ability of the protein to repress an authentic repA promoter in trans (autorepression). The repA promoter activity was followed by use of gene fusion to lacZ (22). We have shown previously that RepA produced from a wild-type mini-Pl plasmid represses another repA promoter in trans so that its activity is reduced from about 250 Miller units to 1 Miller unit.…”

“…Within the origin repeats is the repA promoter, whose activity remains largely repressed, apparently due to RepA binding. This autorepression sets the basal level of RepA to only about 20 dimers per repA gene (22). Modest increases in RepA concentration by using foreign promoters help to increase plasmid copy number only in the absence of incA.…”

Host range diversification within the IncP-1 plasmid group

“…Poor establishment and poor maintenance of IncP-1c plasmids in alphaproteobacterial hosts can be due to nonoptimal levels of the replication proteins (Swack et al, 1987;Sevastsyanovich et al, 2005), or non-productive interactions between host proteins and either the initiation proteins (Maestro et al, 2002(Maestro et al, , 2003 or the oriV gene (Caspi et al, 2000). The observed difference in the host range between the two IncP-1 subgroups may also be due to a bias of plasmids size, as smaller plasmids can transform and transfer more efficiently than larger plasmids with the same replicon.…”