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Secondary metabolism occurs after the main growth phase in Streptomyces. A 'transition phase' occurs to remodel global patterns of gene expression at the onset of physiological and developmental differentiation. Many different signals influence this transition phase, integrating, for example, information on nutritional status, growth rate, and stress responses. Several pleiotropic transcription factors that regulate the transition phase have been identified, but aspects of epigenetic control of gene expression are not well understood. This study focused on the characterisation of a novel gene sco2075 in S. coelicolor encoding a protein that combines a histone-like domain with a conserved DksA-like domain, the latter considered a ppGpp cofactor. The protein is important for integrating responses to both oxidative and osmotic stresses. The sco2075- mutant strain is sensitive to oxidative stress at least in part due to reduced induction of the alternative sigma factor sigmaR. SCO2075, similarly to E. coli DksA, may play a possible role in the liberation of core RNA polymerase to bind alternative sigma factors such as sigmaR. In addition DSCO2075 has an altered topological profile of a reporter plasmid under osmotic stress, showing little alteration in negative supercoiling when compared to the significant increase in wildtype. DSCO2075 also has a reduction in aerial hyphae and a possible reduction in actinorhodin production when grown with osmolyte. The histone-like domain of SCO2075 binds DNA non-specifically. SCO2075 expression appears to coincide with diffused FtsZ expression prior to Z-ring formation when SCO2075 appears to become nucleoid associated. Analysis of pre-spore compartment lengths showed SCO2075 is one of several nucleoid associated proteins involved in nucleoid compaction during aerial hyphal erection and sporulation. Absence of sco2075, however, does not affect the production of unigenomic spore chains. Finally, over-expression of SCO2075 suppresses defects in secondary metabolism of a relA mutant affected in ppGpp synthesis. SCO2075 could potentially be a new type of regulator, likely acting as a node to integrate stress and physiological cues by modulating DNA topology/compaction and RNA polymerase activity.

The multidrug resistance plasmid TP228 replicates at low copy number in Escherichia coli. Stable partitioning of this plasmid is mediated by three essential components: a ParA homologue, ParF; a centromere binding protein, ParG; and a centromere site, parH. ParF and ParG jointly assemble on the parH centromere forming the segrosome complex, and thereby direct intracellular plasmid transport. ParG belongs to the ribbon-helix-helix (RHH) class of dimeric DNA binding proteins. ParG specifically binds the parH site and also is a transcriptional repressor of the parFG genes. Previous studies demonstrated that unstructured N-terminal tails in ParG are not important for dimerization. Instead the tails are implicated in assembly of higher order nucleoprotein complexes essential for transcriptional repression and segrosome assembly, and also influence ParF nucleotide hydrolysis and polymerization. In this study we defined the role of residues in the RHH folded domain that are crucial for ParG dimerization and function. To achieve our goal the two α-helices, the intervening loop, and two C-terminal residues were analyzed fully by alanine scanning mutagenesis. Initially, ParG mutants were constructed and assessed for effects on normal plasmid partition activity and on dimerization. In vivo segregation assays and bacterial two-hybrid studies revealed mutation of residues F49 in α-helix 1 and W71 and L72 in α-helix 2 of ParG each resulted in defective plasmid partition activity and impaired dimerization. In vitro chemical cross-linking of purified proteins ParG-F49A, ParG-W71A and ParG-L72A demonstrated predominant monomeric species whereas wild-type ParG formed dimeric species as noted previously. Multiangle light scattering and sedimentation equilibrium analysis of the mutant proteins showed shifts in molar mass towards monomeric species with increased Kd values for dimerization. Protein-DNA interactions studied by gel retardation assays showed impaired interactions of ParG-F49A, ParG-W71A and ParG-L72A with parH. Results of conserved substitutions at position 71 showed that aromatic substitutions of W71 to Y71 or F71 are tolerated and have no apparent effects on ParG mediated plasmid segregation, but the non-aromatic W71L mutation blocked the segregation. However, a ParG double mutant bearing the ‘reversed’ amino acid pair (W71L-A52Y) retained plasmid segregation activity and behaved like wild-type ParG in dimerization assays in vitro and in vivo. Thus, substitution of W71 by tyrosine or phenylalanine does not disturb the monomer-monomer interface interactions that pack α-helix 2 from one monomer against residues of α-helix 1 and α-helix 2 of the partner monomer. Moreover, the permissible amino acid combinations at interacting positions 52 and 71 in ParG show significant flexibility and reveal key roles for these residues in function and dimerization of ParG. Overall, our in vivo and in vitro interaction studies provide novel information about the role of hydrophobic residues F49, W71 and L72 in ParG dimerization and activity. In the longer term, interference with dimerization by ParG and other centromere binding proteins using artificial ligands may provide a novel strategy for destabilization of antibiotic multiresistance plasmids.

AreA, a 96 kDa gene regulatory protein involved in nitrogen metabolite repression in Aspergillus nidulans, is a member of the GATA family of zinc finger DNA binding proteins, and regulates the expression of around 100 genes. This project was designed to examine the domain structure of AreA in this region of the protein, and to characterise the DNA binding domain Limited proteolysis has been employed to identify structural domains in the Cterminal region of AreA, which has been cloned and over-produced in E.coli. A variety of proteases have been used, and each reveals a dominant stable fragment of approximately 17-22 kDa. N-terminal sequencing and mass spectroscopy have been used to identify a number of these fragments. The major product following limited proteolysis by Glu-C is composed of two closely related species, a 164 residue fragment (17,489 Da) and a 157 residue fragment (16,857 Da). Both fragments encompass the Zn-finger motif, and share the same Cterminus, differing at the N-terminus by only 7 amino acids. The DNA sequence coding for the 157 residue fragment (16,857 Da) has been cloned and over-produced as a His-tag fusion protein. Further studies on this domain have shown that this putative domain has a relatively strong DNA binding constant with values in the nanomolar range. Structural analysis using Circular Dichroism, NMR and fluorescence suggests that the domain contains some irregular or unstructured regions. The regions that are structured are likely to be from the zinc-finger region, since DNA binding is maintained.

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No description available.

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