Escherichia coli DnaA protein: specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation.

The Escherichia coli dnaA73, dnaA721, and dnaA71 alleles, which encode A213D, R432L, T435K substitutions, respectively, were originally isolated as extragenic suppressors of a temperature-sensitive dnaX mutant. As the A213D substitution resides in a domain that functions in ATP binding and the R432L and T435K substitutions affect residues that recognize the DnaA box motif, they might be expected to reduce ATP and specific DNA binding, respectively. Therefore, a major objective was to quantify the biochemical defects of the mutant DnaAs to understand how the altered proteins suppress the temperature-sensitive phenotype of a dnaX mutant. A second purpose was to address the paradox that mutant proteins with substitutions of amino acids essential for recognition of the DnaA box motifs within the E. coli replication origin (oriC) may well be inactive in initiation, yet chromosomal dnaA mutants expressing DnaA proteins with the R432L and T435K substitutions are viable at temperatures from 30 to 39 degrees C. We show biochemically that mutant DnaAs carrying R432L and T435K substitutions fail to bind to the DnaA box sequence. The A213D mutant is sevenfold reduced in its affinity for ATP compared to wild-type DnaA, and its affinity for the DnaA box sequence is also reduced. However, the reduced activity of the A213D mutant in oriC plasmid replication appears to arise from a defect in DnaA oligomerization. Although the T435K mutant fails to bind to the DnaA box sequence, other results suggest that DnaA oligomerization stabilizes the binding of the mutant DnaA to oriC to support its partial activity in initiation in vitro. These results support a model that suppression of dnaX occurs by reducing the frequency of initiation to a manageable level for the mutant DnaX so that viability is maintained.

Clostridium taeniosporum spore ribbon-like appendage structure, composition and genes.

Clostridium taeniosporum spores have about 12 large, flat, ribbon-like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the 'encasement'. Appendages are about 4.5 microm long, 0.5 microm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair-like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen-like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative sigma(K)-dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.

Suppression of temperature-sensitive chromosome replication of an Escherichia coli dnaX(Ts) mutant by reduction of initiation efficiency.

Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40 degrees C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20 degrees C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40 degrees C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34 degrees C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39 degrees C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39 degrees C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38 degrees C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.