The competence transcription factor of Bacillus subtilis recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix

Abstract

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comG promoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of approximately 65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addAB promoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN5TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK-dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK. The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.

Figure 1

In vitro transcription assays in the absence (−) or presence (+) of ComK. The templates used were plasmid pAN583, plasmid pAN583 containing the comG promoter (pAN-G), and a positive control (pIS109) with transcripts of 142, 184, 455, and 497 nucleotides in length.

Figure 2

DNase I footprinting analysis of ComK binding to the comC, comG, comE, comF, addAB, and comK promoter regions. The left and right halves of each panel represents the footprints of the upper and lower strands, respectively. Footprints obtained in the presence (+) or absence (−) of ComK are flanked by G+A sequence ladders. The protected regions are marked with bars, and the position of the −35 promoter sequence is indicated.

Figure 3

Summary of DNase I footprinting data. Protected bases are marked by solid bars, and hypersensitive sites by arrows. The −35 promoter sequences are underlined.

Figure 4

Gel retardation of permuted DNA fragments containing the comG promoter in the presence of ComK. Restriction enzymes used to release the various fragments from plasmid pBend-G were BglII (lane 1), NheI (lane 2), EcoRV (lane 3), SmaI (lane 4), NruI (lane 5), and BamHI (lane 6). The arrow locates pBend, and retarded and unretarded fragments are marked with + and −, respectively.

Figure 5

Gel retardation assay by use of ³²P-labeled comC promoter fragments incubated with ComK and MBP–ComK mixed in various proportions. (First and penultimate lanes) No MBP–ComK added and no ComK added, respectively. (Last lane) Probe with no protein added.

Figure 6

Hydroxy-radical footprinting analysis of ComK binding to the addAB promoter. Footprints obtained in the presence (+) or absence (−) of ComK are flanked by G+A sequence ladders. The left and right halves represent the footprints of the upper and lower strands, respectively. The protected regions are marked with bars and the position of the −35 promoter sequence is indicated. The origin of the hyperactive bands is unknown, but they are not affected by the presence of ComK and do not interfere with the footprint results. In the sequence, hydroxy-radical-protected residues are marked by ellipses, and DNase I protected residues by bars. Arrows indicate hypersensitive sites in the DNase I footprint.

Figure 7

Helical projections summarizing the ComK footprints on the addAB, comC, comG, comE, comF, and comK promoters. Hydroxy-radical protected nucleotides in addAB are represented by heavy contoured circles, DNase I protections are represented by lightly contoured circles, and hypersensitive sites by diamonds. The −35 promoter sequences are boxed. Separately shown is the ComK–dimer binding recognition pattern, which is outlined on each projection.

Figure 8

Gel retardation of addAB promoter fragments as a function of increasing nucleotide spacing between the AT boxes. The number of base pairs added to the AT box interval of the wild-type addAB promoter (wt) are indicated (+5, +10, +15, and +20, respectively). (A) 0.2 μm ComK was used except for the first lane (−) in which no protein was added; (B) the modified addAB promoter fragments were incubated with 0.4 μm ComK (+).

Figure 9

Gel retardation assay with a ³²P-labeled addAB promoter fragment, a PCR fragment containing the ComK recognition sequence at the end of the cspB gene, and the same fragment from which the ComK recognition sequence was removed (ΔcspB). The ComK (+) concentration was 0.3 μm in all cases.

Figure 10

Location of the hydroxy-radical-protected sugar residues on the addAB promoter backbone. Hydroxy-radical-protected residues are marked by open circles, and the dyad symmetry in the two AT boxes is indicated with arrows. Shaded and black bars in the DNA helix represent adenine and thymine residues, respectively.

Figure 11

Competition of minor groove binding drugs with ComK binding to the addAB promoter. Actinomycin D and distamycin A concentrations were increased in twofold increments from 0.4 to 100 μm, chromomycin A₃ concentrations were increased in twofold increments from 0.04 to 10 μm. Left lanes present ComK-induced retardation of the addAB probe in the absence of drugs.

Figure 12

Summary of the ComK recognition sequence in the promoter regions of all genes known to be ComK controlled. The ComK dimer binding sites (AT boxes) are shaded and the −35 promoter sequences, if known, are printed in boldface type. cspB is the hypothetical ComK binding site located at the end of the cspB gene.