Three mutations that alter the DNA sequence specificity of the catabolite gene activator protein (CAP) from AA-TGTGA--T---TCA-ATW to AA-TGTAA--T---TCA-ATW have been isolated. All three mutations affect the same amino acid of CAP, glutamic acid 181. We propose that it is this amino acid of CAP that makes contacts with base pairs 7 and 16 of the symmetrical recognition site.

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin 0. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin 0, by immunoblotting with a specific antiserum against listeriolysin 0. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin 0 would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin 0 (58,000). The protein sequence is highly homologous to those of streptolysin 0 and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin 0 gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

We have determined the nucleotide sequence of the crp gene of Escherichia coli K 12. From a lambda transducing phage, the crp region was subcloned into pBR322. The gene was localized on the cloned fragment by determining the length of deletions which affect its expression. Its nucleotide sequence was established by using the technique of Maxam and Gilbert. The deduced amino-acid sequence is in agreement with the previously published amino acid composition of the protein (1, 2). Analysis of the sequence confirms that the DNA binding domain is located in the C-terminal portion of the protein.

No abstract

Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for APT base pairs at positions 7 and 16 of the DNA recognition site, rather than GC base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related The three-dimensional structures of three proteins that bind to specific DNA sequences have been determined recentlyi.e., the catabolite gene activator protein (CAP) complex with cAMP (1), cro (2), and the amino-terminal fragment of the X repressor (ref. 3; reviewed in ref. 4). Because the structures determined were those of the uncomplexed proteins, efforts to date to elucidate the structures of the protein-DNA complexes have relied on model building (1)(2)(3)(4)(5)(6)(7)(8). The models proposed in the cases of cro and the X repressor and one of four models in the case of CAP exhibit common features. Each model postulates that a pair of 2-fold related ahelices, one from each subunit of the protein, interacts with successive major grooves of right-handed B-type DNA (2, 3, 7). As discussed below, in each instance the a-helix proposed to contact DNA is the second a-helix of a characteristic helix-turn-helix structural motif (9-13). In this paper, we discuss the implications for this analysis of our genetic results (14), which we believe identify a direct contact between an amino acid of a DNA binding protein and the base pair it contacts in the target DNA sequence.

Comparison of the amino acid sequences of 13 procaryotic regulatory proteins, including the products of genes crp (catabolite activator protein; CAP), lacI, galR, lexA, lysR, araC, trpR, and tnpR of Escherichia coli, of genes cI, cII and cro of phage lambda, cro of phage 434, and c2 of phage P22, has revealed two regions of homology. The sites of action of these proteins also share common features in their DNA sequence. Taking into account the models proposed for the lambda repressors, cro and cI, and for CAP, a general type of DNA‐protein interaction is suggested.

The complete nucleotide sequences of the Salmonella typhimurium LT2 supplemented with ampicillin (50 ,ug/ml), chloramphenicol (25 j,g/ml), or tetracycline (12.5 pug/ml).Bacterial strains. For the cloning, the starting stains were S. typhimurium LT2 SL4213 (13) and Shigella flexneri 2B ATCC 12022. Strains carrying crp-20B (strain LU53) (41), crp45 (strain BS680) (9), and A(crp45 cya-06) (strain CA8445) (42)

Expression of the crp gene was studied in vivo by use of a crp-lacZ gene fusion first constructed on a plasmid and then transferred onto the chromosome. Our in vivo data confirm the in vitro findings that crp is negatively autoregulated via the cyclic AMP-catabolite gene activator protein complex. We present evidence that gene crp is repressed by glucose.