Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an 'extended −10' motif and a core −10 element

σ³² controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ³² promoters is a long −10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of σ³² and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an 'extended −10' recognition motif that is recognized by K130, a residue universally conserved in β- and γ-proteobacteria. This residue is located in the α-helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α- and δ-/ε-proteobacteria and we found that σ³² from the α-proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ³² is the first Group 3 σ shown to use the 'extended −10' recognition motif.     

Mutational analysis of Escherichia coli σ28 and its target promoters reveals recognition of a composite −10 region, comprised of an 'extended −10' motif and a core −10 element

σ²⁸ controls the expression of flagella-related genes and is the most widely distributed alternative σ factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of σ²⁸ promoters is a long −10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in σ²⁸ participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended −10 motif and is recognized by R91, a residue in Domain 3 of σ²⁸. The downstream CG is the upstream edge of −10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base-specific recognition of the promoter, R91, D81 and D84 are universally conserved in σ²⁸ orthologues. σ²⁸ is the second Group 3 σ shown to use an extended −10 region in promoter recognition, raising the possibility that other Group 3 σs will do so as well.     

A model for cation content of plants based on surface potentials and surface charge densities of plant membranes

The model presented takes into account the interaction between the negatively charged membranes and macromolecules and the cations. A central postulate is that a constant average surface charge density (σ) as well as a constant average surface potential (Ψ) is conserved under different ionic conditions. The model makes it possible to predict the size of σ and Ψ from measurements of Na, K, Mg and Ca content in plant tissues of the same age but grown under two different ionic conditions (e.g. high and low K⁺). Assumptions were made about the relative amounts of free and bound Ca²⁺ and σ and Ψ were calculated from values in the literature. In all cases σ (and Ψ) are predicted to be higher for shoot (−29 to −96 mC m⁻²) than for root membranes (−14 to −27 mC m⁻²). In most cases the predicted σ falls within the range determined experimentally for biological membranes.     

The surface charge density of wheat root membranes

Seedlings of winter wheat ( Triticum aestivum L. cv. Hildur) were grown at 18°C for 7 days in darkness in a complete growth medium in the presence or absence of 1 m M KCl to produce roots with different ion contents (high and low K⁺ respectively). The roots were homogenized, the 3 000 g, 10 000 g, 30 000 g (further fractionated by two phase partitioning) and 100 000 g pellets isolated, and their surface charge densities (σ) determined by the use of 9-aminoacridine fluorescence. The average σ for all membrane fractions weighted for protein content was the same (−18 mC m⁻²) for low and high K⁺ roots. The K⁺, Na⁺, Mg²⁺ and Ca²⁺ content of roots was determined and used to calculate an average σ following the procedure of Bérczi et al. [Physiol. Plant. 61: 529–534 (1984)]. The predicted value (−11 mC m⁻²) does not deviate much from the experimentally determined value. It is concluded as a useful working hypothesis that the average surface charge density is constant and that the ionic content of plant cells is regulated such that the average surface potential is constant.     

New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the σ^E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA , that decreased the σ^E-dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM ( rfaD ) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM ( rfaD ) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn 10 insertions were isolated, which led to an increased activity of the σ^E regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA , fkpA and ompH/skp genes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.     

Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius

The DnrI protein, essential for the biosynthesis of daunorubicin in Streptomyces peucetius , was purified almost to homogeneity from dnrI expression strains of Escherichia coli and S. peucetius through several steps of chromatography. The proteins purified from both organisms had identical chromatographic and electrophoretic behaviour. Purified His-tagged or native DnrI was used to conduct DNA-binding assays by gel mobility-shift analysis, and the results showed no significant difference in the DNA-binding activity of native or His-tagged proteins. DnrI binds specifically to DNA segments containing the intergenic regions separating the putative dnrG–dpsABCD and dpsEF operons, and the dnrC gene and dnrDKPSQ operon. DNase I footprinting assays indicated that the DNA-binding sites for DnrI extended from upstream of the −10 to −35 regions of the dnrG or dpsE promoters to include about 65 bp of the dnrG – dpsE intergenic region and about 80 bp of the dnrC – dnrD intergenic region. Both binding sites contain imperfect inverted repeat sequences of 6–10 bp with a 5'-TCGAG-3' consensus sequence that was present in 4 out of 10 other promoter regions in the cluster of daunorubicin biosynthesis genes.     

Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element

Regulatory elements within the promoter of the pollen-specific NTP303 gene from tobacco were analysed by transient and stable expression analyses. Analysis of precisely targeted mutations showed that the NTP303 promoter is not regulated by any of the previously described pollen-specific cis -regulatory elements. However, two adjacent regions from −103 to −86 bp and from −86 to −59 bp were shown to contain sequences which positively regulated the NTP303 promoter. Both of these regions were capable of driving pollen-specific expression from a heterologous promoter, independent of orientation and in an additive manner. The boundaries of the minimal, functional NTP303 promoter were determined to lie within the region −86 to −51 bp. The sequence AAATGA localized from −94 to −89 bp was identified as a novel cis -acting element, of which the TGA triplet was shown to comprise an active part. This element was shown to be completely conserved in the similarly regulated promoter of the Bp10 gene from Brassica napus encoding a homologue of the NTP303 gene.