Purification of an SOS repressor from Bacillus subtilis.

We have identified in Bacillus subtilis a DNA-binding protein that is functionally analogous to the Escherichia coli LexA protein. We show that the 23-kDa B. subtilis protein binds specifically to the consensus sequence 5'-GAACN4GTTC-3' located within the putative promoter regions of four distinct B. subtilis DNA damage-inducible genes: dinA, dinB, dinC, and recA. In RecA+ strains, the protein's specific DNA binding activity was abolished following treatment with mitomycin C; the decrease in DNA binding activity after DNA damage had a half-life of about 5 min and was followed by an increase in SOS gene expression. There was no detectable decrease in DNA binding activity in B. subtilis strains deficient in RecA (recA1, recA4) or otherwise deficient in SOS induction (recM13) following mitomycin C treatment. The addition of purified B. subtilis RecA protein, activated by single-stranded DNA and dATP, abolished the specific DNA binding activity in crude extracts of RecA+ strains and strains deficient in SOS induction. We purified the B. subtilis DNA-binding protein more than 4,000-fold, using an affinity resin in which a 199-bp DNA fragment containing the dinC promoter region was coupled to cellulose. We show that B. subtilis RecA inactivates the DNA binding activity of the purified B. subtilis protein in a reaction that requires single-stranded DNA and nucleoside triphosphate. By analogy with E. coli, our results indicate that the DNA-binding protein is the repressor of the B. subtilis SOS DNA repair system.     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

Analyses of binding sequences of the two LexA proteins of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Xanthomonas axonopodis pv. citri (X. axonopodis pv. citri) possesses two lexA genes, designated lexA1 and lexA2. Electrophoretic mobility shift data show that LexA1 binds to both lexA1 and lexA2 promoters, but LexA2 does not bind to the lexA1 promoter, suggesting that LexA1 and LexA2 play different roles in regulating the expression of SOS genes. In this study, we have determined that LexA2 binds to a 14-bp dyad-spacer-dyad palindromic sequence, 5'-TGTACAAATGTACA-3', located at nucleotides -41 to -28 relative to the translation start site of lexA2 of X. axonopodis pv. citri. The two spacer nucleotides in this sequence can be changed from AA to TT without affecting LexA2 binding; all other base deletions or substitutions abolish LexA2 binding. The LexA1 binding sequence in the promoter region of lexA2 is TTAGTACTAAAGTTATAA and is located at -133 to -116, and that in the lexA1 gene is AGTAGTAATACTACT located at nucleotides -19 to -5 relative to the translation start site of lexA1. Any base change in the latter sequence abolishes LexA1 binding.     

Analysis of a cloned colicin Ib gene: complete nucleotide sequence and implications for regulation of expression.

The complete nucleotide sequence of a 2,971 base pair EcoRI fragment carrying the structural gene for colicin Ib has been determined. The length of the gene is 1,881 nucleotides which is predicted to produce a protein of 626 amino acids and of molecular weight 71,364. The structural gene is flanked by likely promoter and terminator signals and in between the promoter and the ribosome binding site is an inverted repeat sequence which resembles other sequences known to bind the LexA protein. Further analysis of the 5' flanking sequences revealed a second region which may act either as a second LexA binding site and/or in the binding of cyclic AMP receptor protein. Comparison of the predicted amino acid sequence of colicin Ib with that of colicins A and E1 reveals localised homology. The implications of these similarities in the proteins and of regulation of the colicin Ib structural gene are discussed.     

Repression of the E coli recA gene requires at least two LexA protein monomers

To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA+. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.     

LexA-binding sequences in Gram-positive and cyanobacteria are closely related

The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.Communicated by R. Devoret     

Properties of BGP1, a poly(dG)-binding protein from chicken erythrocytes.

The chicken beta A-globin gene contains in the neighborhood of its 5' promoter a (dG)-homopolymer sequence 16 base pairs long. The 66 kD protein BGP1 (beta globin protein 1), isolated from chicken erythrocytes, has been shown to bind specifically to this sequence. We describe further purification of BGP1, measure its affinity for the beta A-globin promoter binding site, and analyze its binding properties. The minimal binding sequence is seven dG residues; methylation interference studies show that each of these residues contacts BGP1. Binding competition experiments employing (dG).(dC) oligomers of varying lengths also consistent with (dG)7 as a minimum recognition sequence. All of the data can be explained by a model in which BGP1 binds to any contiguous set of seven (dG) residues, so that the effective constant for binding to (dG)n is proportional to n minus 6. This behavior may be typical of proteins that bind specifically to repeated sequences.     

In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

Both the amino-terminal and the carboxy-terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA-catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non-specific binding to DNA, the purified amino-terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3-fold if the DNA lattice is fully saturated with protein. Further, the amino-terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino-terminal domain harbours a substantial amount of residues in alpha-helical conformation, a prerequisite for DNA recognition via a helix--turn--helix structural motif as proposed for many other regulatory proteins.     

Purification and characterization of YihA,an essential GTP-binding protein from Escherichia coli

YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis. It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics. YihA encodes a putative GTP-binding protein. We have cloned and overexpressed the gene encoding E. coli YihA and initiated biochemical studies as a first step towards understanding its biological function. We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins. It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA. The K(d) values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 microM, respectively. Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence.