DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

Abstract

D proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the ؊10 element, and this motif is known to be recognized by region 2.4 and critical for promoter activity.In the course of efforts to define gene expression determinants from the thermophilic bacterium Aeribacillus pallidus AC6 (2, 18), we identified flagellin (Hag) as being among the most highly expressed proteins in this strain. To determine the basis for high-level Hag expression, we isolated and sequenced the hag gene and identified and expressed the protein required for its expression in The resulting peptide sequences displayed high similarity to B. subtilis flagellin (NAQDGISLIQTAEGALTETHAILQR had 96% identity with amino acids [aa] 65 to 89 and LEHTINNL GTSAENLTAAESR had 85% identity with aa 242 to 262). Two degenerate primers (FlaF1 and FlaR1) were used to amplify the flagellin gene (hag) from A. pallidus AC6 chromosomal DNA. An ϳ450-bp product was cloned into pGEM-T Easy (Promega) for DNA sequencing. The remainder of hag and its upstream region were obtained by inverse PCR. The 828-bp hag gene encodes a 275-amino-acid (29.7-kDa) protein having 63% identity with B. subtilis Hag and is preceded by a typical D promoter. To identify sigD, two degenerate primers were used to amplify a PCR fragment which was cloned into the pGEM-T Easy vector system and sequenced. The flanking portions of sigD were obtained by inverse PCR, and the gene was sequenced. The 771-bp sigD gene encodes a 256-amino-acid (28.7-kDa) protein having 67% overall identity with D Bs , with the highest levels of similarity concentrated in conserved regions 2 and 4, known to mediate promoter recognition.A. pallidus AC6 flagellin is expressed from a D -dependent promoter. Transcription of hag initiates from a canonical Ddependent promoter at a G residue 79 bp upstream of the start codon (Fig. 1). Analysis of hag::lacZ fusions integrated into B. subtilis CU1065 and HB4035 (sigD::kan) indicated that activity was D dependent and highest at late logarithmic phase, as previously reported for B. subtilis (19). Optimal promoter activity required an AT-rich region just upstream of the Ϫ35 element (Table 1), which has similarity with the upstream promoter (UP) element previously described for B. subtilis hag (6). Sequence inspection suggests that high-level Hag expression may also benefit from a strong ribosome-binding site and stabilization of the mRNA by a 5Ј hairpin sequence (24). Purification D of A. pallidus and B. subtilis and reconstitution of D RNAP. D proteins from A. pallidus AC6 and B. subtilis were expressed under T7 RNA polymerase (RNAP) control in Escherichia coli BL21(DE3)/pLysS (Novagen) by using pECG1 and pECB1 (Table 2). For purification, inclusion bodies were solubilized with Sarkosyl (1), refolded, and purified using DEAE-Sepharose and heparin-Sepharose chromatography as described previously (9). B. subtilis core RNAP was purified...

The core‐independent promoter‐specific binding of Bacillus subtilis σB

“…The results from an EMSA‐based competition assay and a DNase I footprinting assay showed that B. subtilis σ B possesses core‐independent promoter −35 element binding specificity. The results are different to those reported for B. subtilis σ D . Possible reasons for the discrepancy are discussed.…”

“…We determined the existence of a core‐independent promoter‐specific binding activity of B. subtilis σ B in vitro by using an EMSA‐based competition assay and a footprinting assay. In contrast to the promoter −10 element binding specificity of the B. subtilis alternative σ factor, σ D , and the primary σ factor, σ A , the B. subtilis σ B binds specifically and preferentially to the −35 region of its cognate promoter DNA.…”

SlrA/SinR/SlrR inhibits motility gene expression upstream of a hypersensitive and hysteretic switch at the level of σD in Bacillus subtilis

“…Hypersensitivity often invokes cooperative protein–protein interactions that are difficult to explain in the context of a sigma factor. σ D is unusual among the sigma factors, however, in that it binds to DNA in the absence of core RNA polymerase, and generates supershifted complexes that may indicate more than one protein bound at the promoter (Chen and Helmann, 1995; Bertero et al ., 1999; Sevim et al ., 2011).…”

Mechanism of Bacterial Transcription Initiation: RNA Polymerase - Promoter Binding, Isomerization to Initiation-Competent Open Complexes, and Initiation of RNA Synthesis

“…103 This interaction may lead to the observed autoinhibition of promoter DNA binding by free primary sigma factors. 104 Autoinhibition has not been observed for interaction with the double-stranded promoter DNA of free sigma factors lacking region 1.1 by deletion 105 or naturally, 106,107 or for free σ 70 interacting with the NT strand of promoter DNA. 108 …”