DpiA binding to the replication origin of Escherichia coli plasmids and chromosomes destabilizes plasmid inheritance and induces the bacterial SOS response

Abstract

The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size-both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB-both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids-reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

FIG. 1.

DNA sequences and gel shifts with DpiA protein. (A) The sequence of the repeat regions of the cit operator-promoter from K. pneumoniae and E. coli as well as the A+T-rich repeats from the origins of the plasmids pSC101 and P1 and the E. coli chromosome, oriC, are shown. (B) Binding of DpiA protein and the mutant proteins to the origin regions of pSC101 and P1 are shown. Lanes 1 to 5 contain 0.6 ng of pSC101 origin region/lane, and lanes 6 to 10 contain 0.6 ng of P1 origin region/lane. Five micrograms of protein was added to each reaction mixture: lanes 1 and 6, no protein added; lanes 2 and 7, 5 μl of control GST extract; lanes 3 and 8, 5 μg of DpiA12D-L protein; lanes 4 and 9, 5 μg of wild-type DpiA protein; lanes 5 and 10, 5 μg of DpiA57D-E protein. (C) Binding of DpiA protein and the mutant proteins to pSC101 origin fragment. All lanes have 0.6 ng of the pSC101 origin fragment. The amounts of protein vary to show the binding specificities of various DpiA proteins relative to that of the wild-type protein. Lane 1, no protein; lane 2, 5 μl of GST control extract; lanes 3 to 5, 10, 20, and 30 μg, respectively, of DpiA12D-L protein; lanes 6 to 8, 7.5, 12.5, and 20 μg of wild-type DpiA protein; lanes 9 to 12, 2.5, 5, 12.5, and 17.5 μg of DpiA57D-E protein. (D) Binding of DpiA protein to pSC101 origin fragment and oriC. Lanes 1 and 2, 0.2 ng of pSC101 origin fragment; lanes 3 to 5, 0.2 ng of oriC fragment; lanes 1 and 3 have no protein added, lane 2 has 5 μg, lanes 4 has 10 μg, and lane 5 has 20 μg of wild-type DpiA protein added. Lane 4 shows 50% less labeled DNA, and lane 5 shows 90% less labeled DNA, than lane 3 by PhosphorImager analysis; however, no shifted band is visible.

FIG. 2.

DNA footprint of DpiA on the A+T-rich region of the pSC101 origin. The sequence of the pSC101 origin is known (49), and the sequencing tracts are shown for orientation. The sequence between the known binding sites for DnaA and IHF is shown. The italicized region is the two 13-mers, and the bold region is the area protected by the DpiA protein in the footprint. The footprint and protein purification of DpiA was performed as described in Materials and Methods. The amount of wild-type DpiA added is shown in micrograms; all lanes were treated with DNase I for 1 min.

FIG. 3.

Overexpression of DpiA increases the average DNA content and size of cells. Wild-type strain 1088 (in panels A and B and top row of table) and 1088pHI1429, twofold overproducer of DpiA, (in panels C and D and second row of table) were grown in CAA plus glucose. Flow cytometry data of exponential growing cells (A and C) and cells treated with rifampin and cefalexin (B and D) are shown. The average amount of DNA/cell (calculated from panels A and C) and the average mass/cell are relative numbers. The average origin number/cell is calculated from panels B and D.