The nucleotide sequence and characterization of the relA gene of Escherichia coli.

The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation. This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein. The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein. A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E. coli chromosome. Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E. coli chromosome.

Nucleotide sequence and promoter mapping of the Escherichia coli Shiga-like toxin operon of bacteriophage H-19B.

We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxin-converting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted A1 fragment of the SLT-1 A subunit. S1 nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene on plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Betley et al. (M. Betley, V. Miller, and J. Mekalanos, Annu. Rev. Microbiol. 40:577-605, 1986) have recently summarized evidence suggesting that the slt operon is under the control of the fur regulatory system. The area of dyad symmetry found in both promoters may represent a regulatory site. A rho-independent terminator sequence was found 230 base pairs downstream from the B cistron stop codon.