The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

Abstract

In wild‐type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two‐ to threefold relative to wild type. Here, we show that the DnaA protein concentration was two‐ to threefold lower in the dnaA204 mutant compared with the wild‐type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild‐type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half‐life of the protein. Our results suggest that the DnaA204 protein has essentially wild‐type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA–DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.

The Escherichia coli datA site promotes proper regulation of cell division

“…The cells were washed in 0.02 M Tris buffered saline, pH 7.5, and resuspended in the same buffer. The DNA was stained for 30 min in 1.5 mg Hoechst 33258 ml 21 (Sigma) in the same buffer (Torheim et al, 2000). The samples were analysed on an LSR II flow cytometer (BD Biosciences) as mentioned previously (Morigen et al, 2009).…”

Replication patterns and organization of replication forks in Vibrio cholerae

“…A 450/50 nm bandpass filter was used to collect Hoechst 33258 fluorescence. The peaks of the FITC-negative standard were positioned at fixed channels using the photomultiplier tube (PMT) voltage, allowing the DNA content of the FITC-positive sample cells to be accurately calculated (Torheim et al, 2000).…”

“…Cell extract for non-specific absorption of antibody was prepared from a DseqA strain, EBO193 (Torheim et al, 2000). Cells from 1000 ml of exponentially growing culture were harvested, washed once in 10 ml 50 mM KH 2 PO 4 /K 2 HPO 4 , pH 7.4, and resuspended in 1.5 ml of the same buffer.…”

Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

“…Transcription of the dnaA gene is autoregulated, and ATP-DnaA represses about twofold more tightly dnaA transcription than ADP-DnaA in vitro (Speck et al 1999). The cellular DnaA molecules are present at a level of 1000-2000 molecules per cell and they are very stable (Torheim et al 2000;Shimuta et al 2004). Thus, fluctuations in dnaA transcription should not necessarily largely affect the total cellular DnaA levels.…”