A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene

Summary An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins.     

Fine-structure mapping and complementation analysis of the Escherichia coli cysB gene.

Sixty-two point mutations were isolated in Escherichia coli by means of transduction with mutagenized phage P1. Twenty-two deletions extending into cysB but able to recombine with at least some of the point mutations were isolated on a transmissible E. coli plasmid. Mapping of the point mutations against the deletions divided the former into 16 deletion groups. Nine merodiploids were constructed in which the chromosome carried one of the three point mutations most distal to the trp operon and in which a plasmid carried one of the three point mutations most proximal to the trp operon. All of these showed a Cys-phenotype. It follows that mutations at the two extreme ends of the region belong to the same complementation group.     

Identification of the dnaA and dnaN gene products of Escherichia coli

Summary A specialized transducing lambda phage carrying the dnaN and dnaA genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 kd protein, and the dnaA gene product as a weakly basic 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the other hand, do not affect the synthesis of the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.Abbreviations kd kilodaltons - SDS sodium dodecyl sulfate     

Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli

Summary Extragenic suppressor mutations for dnaA(Ts) mutations mapping in the rpoB gene (-subunit of RNA polymerase) were isolated by selection of spontaneous rifampicin resistant mutants and screening for temperature resistance. Six rpoB mutations were analysed for suppression of 12 different dnaA(Ts) mutations. The analysis showed that all dnaA(Ts) mutations could be suppressed by some rpoB mutation. All six rpoB mutations showed allele specificity when tested for suppression of 12 dnaA (Ts) mutant strains. The allele specificity was found to correlate with the map position of the dnaA (Ts) alleles.     

Dominance of dnaA+ to dnaA in Escherichia coli.

The dominance of dnaA+ to the dnaA508 mutation was complete and was unaffected by the presence of a copy of the chromosomal replication origin on the episome. These results prove that the dnaA gene of Escherichia coli produces a diffusible product.     

Rifampicin-induced replication of the plasmid pBR322 in Escherichia coli strains carrying dnaA mutations

The replication pattern of the plasmid pBR322 was examined in the dnaA mutants of Escherichia coli. The rate of pBR322 DNA synthesis is markedly decreased after dnaA cells are shifted to the restrictive temperature of 42 degrees C. However, addition of rifampicin (RIF) to cultures of dnaA strains incubated at 42 degrees C after a lag of 90 min results in a burst of pBR322 synthesis. This RIF-induced pBR322 replication remains dependent on DNA polymerase I activity. Efficient plasmid pBR322 replication is observed at 42 degrees C in the double mutant dnaA46cos bearing an intragenic suppressor of dnaA46. Though replication of pBR322 in dnaA46cos growing at 42 degrees C is initially sensitive to RIF plasmid synthesis is restored after 90 min incubation in the presence of the drug. RIF-induced replication of the plasmid pBR327, lacking the rriB site implicated in RIF-resistant synthesis of the L strand of ColE1-like plasmids (Nomura and Ray 1981; Zipursky and Marians 1981), was observed also in dnaA46 at 42 degrees C.     

DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations.

Summary In this article we describe some in vivo properties of a coldsensitive ribosomal mutant from Escherichia coli. The mutation affects the rplV gene which is the structural gene of ribosomal protein L22.Our work shows that at 22°C, the biosynthesis of both ribosomal subunits and the maturation processing of 16S and 23S ribosomal RNA are impaired. Integration of our results in a general model of in vivo ribosomal assembly in E. coli is presented.