Simon E S Bailey

Publications of Simon E S Bailey

• Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites.

  Authors: Massimiliano Zampini, Andrew Derome, Simon E S Bailey, Daniela Barillà, Finbarr Hayes

  Journal of bacteriology. 04/2009;

  The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. Additional to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. Additional to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel beta-strands in this fold are presumed to insert into the OF operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The OF locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully-bound operator is cooperatively coated by up to eight dimers. OF was subdivided experimentally into four overlapping 20-bp sites (A-D) each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A-D individually are bound with different affinities by ParG (C > A approximately B >> D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs, in the interbox spacers, as well as in flanking sequences each influence ParG binding. The OF operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.

• The influence of operator site geometry on transcriptional control by the YefM-YoeB toxin-antitoxin complex.

  Authors: Simon E S Bailey, Finbarr Hayes

  Journal of bacteriology. 12/2008;

  YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5' of yefM-yoeB inYefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5' of yefM-yoeB in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The yefM-yoeB operator site arrangement is exemplified in Escherichia coli: a pair of palindromes with core hexamer motifs and a centre-to-centre distance of 12-bp overlap the yefM-yoeB promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained, but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.

• Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis.

  Authors: Jeremy Moore, Simon E S Bailey, Zineb Benmechernene, Christos Tzitzilonis, Natalie J E Griffiths, Mumtaz Virji, Jeremy P Derrick

  The Journal of biological chemistry. 10/2005; 280(36):31489-97.

  The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes ofThe adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 microM and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.