A weight matrix for binding recognition by the redox‐response regulator ArcA‐P of <i>Escherichia coli</i> (1999)

Sir, In Escherichia coli, gene expression in response to redox conditions is partially mediated by the Arc signal transduction system, which comprises the membrane-associated ArcB sensor kinase and its cognate response regulator ArcA. Under anoxic conditions, ArcB undergoes autophosphorylation and then catalyses the transphosphorylation of ArcA. Phospho-ArcA (ArcA-P) subsequently represses certain target operons (e.g. icd, lld [lct ], sdh and sodA) or activates others (e.g. cyd and pfl ). To date, some 30 operons, most of which are involved in respiratory metabolism, are known to be regulated by ArcA-P [Lynch and Lin, 1996. In Regulation of Gene Expression in E. coli. Lynch, A.S. and Lin, E.C.C. (eds). Austin, TX: Landes, pp. 361–376]. Initially, the sequence 5′-TATTTaa-3′ (the lower case letters being less well-conserved nucleotides) was proposed as a putative signature for recognition by ArcA-P, based on DNase I protection experiments at the pfl promoter region (Drapal and Sawers, 1995, Mol Microbiol16: 597–607). A homology search that included ArcA-P protected promoter regions of cyd, pfl, gltA, lld, sdh, sodA and entire promoter regions of 16 additional operons, whose expression is under ArcA control, led to the suggestion of 5′-nGTTAATTAn-3′ (n being A or T) as the ArcA-P binding consensus (Lynch and Lin, 1996, J Bacteriol178: 6238–6249). This consensus proved useful for locating the ArcA-P binding site at two targets that are not involved in respiratory metabolism: the tra operon of resistance plasmid R1 (Strohmaier et al., 1998, J Mol Biol277: 309–316) and the psi site for Xer-based recombination in plasmid pSC101 (Colloms et al., 1998, Mol Microbiol28: 521–530). Support for ArcA-P binding at the consensus was obtained recently by footprinting and site-directed mutagenesis at the aldA promoter region (Pellicer et al., 1999, Mol Gen Genet261: 170–176). Nonetheless, this ArcA-P binding consensus was criticized for failing to single out truly functional sites within the promoter region of the sdh operon (Shen and Gunsalus, 1997, Mol Microbiol26: 223–236). The palindromic character of the consensus and its high AT content can make the identification of functional ArcA-P binding sites difficult in AT-rich promoter regions. To refine the recognition sequence, we searched for a common motif within ArcA-P footprinted promoter sites using Alignace (Roth et al., 1998, Nature Biotechnol16: 566–571), which uses the Gibbs sampler algorithm (Lawrence et al., 1993, Science262: 208–214). The operons included in the analysis were: aldA (Pellicer et al., Mol Gen Genet261: 170–176), cyd, lld (Lynch and Lin, 1996, J Bacteriol178: 6238–6249);glcC, glcD (Pellicer et al., J Biol Chem274: 1745–1752); gltA, sdh (Lynch and Lin, 1996, J Bacteriol178: 6238–6249; Shen and Gunsalus, 1997, Mol Microbiol26: 223–236); icd (Chao et al., 1997, J Bacteriol179: 4299–4304), lpdA (Cunningham et al., 1998, FEMS Microbiol Lett169: 403–408), pfl (Drapal and Sawers, 1995, Mol Microbiol16: 597–607); sodA (Tardat and Touati, 1993, Mol Microbiol9: 53–63) and traY (Strohmaier et al., 1998, J Mol Biol277: 309–316). The program Alignace identified a highly conserved 15 bp stretch that was shared by all of the above promoters (−3 to −17 from the transcriptional start site of aldA ; −308 to −322, −286 to −300 and −75 to −89 from promoter 1; −73 to −87 from promoter 2 of cyd ; −19 to −33 of glcC ; +5 to +19 of icd ; −7 to −21 of lld ; −11 to −25 of lpdA ; −184 to −198 of pfl ; −28 to −42 and −19 to −33 of sdh ; −11 to −25 of sodA ; and −45 to −59 of traY ). By determining the base frequency at each position, the 15 bp sequence was converted into a weight matrix (Fig. 1). The sequence of most conserved bases, 5′-GTTAATTAAATGTTA-3′, strongly resembles the previously proposed consensus. However, the first nucleotide of the Lynch–Lin consensus (5′-A/T) turned out to be poorly conserved and therefore is not included in the present motif. On the other hand, the motif is extended by five residues at the 3′ end. . Sequence logo (Schneider and Stephens, 1990, Nucleic Acids Res 18: 6097–6100) for the ArcA-P recognition motif in E. coli. The sequence conservation, measured in bits, is shown as the height of a stack of letters at each base position. The relative frequency of each base is given by its relative height. The total sequence conservation is 15.18 ± 0.55 bits. The weight matrix was then used to search the E. coli genome for potential ArcA-P binding sites, using the scoring function of Berg and von Hippel (J Mol Biol193: 723–750). The search turned up 16 sequences (14 of which are in a non-coding region; two are in the C-terminal region of the previous operon) that scored better than the mean of the scores of the footprinted sites, and 1334 sequences that scored within two standard deviations below the mean of the input set. Of the latter, 46% are in non-coding regions. Spacing analysis of intergenic regions within 600 bp upstream of a start codon revealed nine clusters of four or more ArcA-P recognition boxes and 17 clusters of three ArcA-P boxes. Sixty-seven pairs of recognition sites were found within 600 bp upstream of the start codon of 96 operons (29 operons share a pair with a divergently transcribed operon): in 22 of these, the pairs are overlapping; in 15, the pairs are separated by less than 100 bp, whereas in the 30 others, they are separated by 100–500 bp. There are 294 single recognition sites in intergenic regions spanning 600 bp from the start codon and, among these, 260 sites lie within 200 bp from the start codon. The list of all sites will be available on the web (http://arep.med. harvard.edu/ecoli_matrices). The new matrix is significantly improved in its ability to detect potential ArcA-P binding sites, as a weight matrix constructed directly from the Lynch–Lin alignment gave 46 943 intergenic hits upon screening the genome. The weight matrix also seems to be more effective in identifying functional binding sites within the ArcA-P protected regions. In the case of the sdh promoter, three ArcA-P protected segments were found in previous footprinting experiments (Lynch and Lin, 1996, J Bacteriol178: 6238–6249; Shen and Gunsalus, 1997, Mol Microbiol26: 223–236). However, only one, covering the −35 to −10 region, was shown to be functional by genetic analysis (Shen and Gunsalus, 1997, Mol Microbiol26: 223–236). Using the matrix criterion, this −35 to −10 region contains two ArcA-P boxes, whereas the other two footprinted sites contain none. In the pfl promoter region, four distinct ArcA-P protected segments were found (Drapal and Sawers, 1995, Mol Microbiol16: 597–607). Using the matrix criterion, two overlapping ArcA-P boxes were identified only in the region that has highest in vitro affinity for ArcA-P. The matrix method was then used to identify potential ArcA-P target operons of known function. Promoters from genes of known function containing multiple ArcA-P boxes are presented in Table 1. It is noteworthy that the role of several of these targets is not directly connected with respiratory metabolism. Functional verification of ArcA-P control of such operons may enhance our physiological insight into the ArcA modulon and networking. The matrix may also be helpful in predicting a direct or indirect role for ArcA-P in the regulation of operons whose expression is redox dependent or is altered by arc mutations (for references, see Lynch and Lin, 1996, J Bacteriol178: 6238–6249). Among these operons, the promoters of fadB, glpD, cyo, nuo, mdh and pdhR contain one or more ArcA-P boxes, as defined by the matrix. In contrast, betT has a single recognition site that lies within the coding sequence, whereas arcA, betI and dad do not have an ArcA-P box at all. In summary, matrix-based genomic screening for regulator binding sites (Robison et al., 1998, J Mol Biol284: 241–254) seems to have afforded a much improved way of identifying functional ArcA-P binding sites in the E. coli chromosome. Further analyses of the disparate functions of the identified target operons — some of which belong to regulons controlled by specific regulators — may reveal the selective advantage of their organization into the Arc modulon. On the other hand, further studies of the degree of conservation at different positions in the ArcA-P box may increase our understanding of contextual protein–DNA interactions, especially when multiple proteins act in concert at a particular target site. P.D.W. is a postdoctoral D. Collen fellow of the Belgian American Educational Foundation. A.M.M. is a predoctoral fellow of the Howard Hughes Medical Institute. Public Health Service grants GM40993 and GM39693 from the National Institute of General Medical Sciences supported this work.