Comparative analysis of FimB and FimE recombinase activity.
FimB and FimE are site-specific recombinases, part of the lambda integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four 'half sites'. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.
Holden
Nicola
N
Centre for Infectious Diseases, Royal (Dick) School of Veterinary Medicine, Chancellor's Building, University of Edinburgh, Edinburgh EH16 4SB, UK.
Blomfield
Ian C
IC
Biomedical Research Group, Department of Biosciences, University of Kent, Kent CT2 7NJ, UK.
Uhlin
Bernt-Eric
BE
Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden.
Totsika
Makrina
M
Centre for Infectious Diseases, Royal (Dick) School of Veterinary Medicine, Chancellor's Building, University of Edinburgh, Edinburgh EH16 4SB, UK.
Kulasekara
Don Hemantha
DH
Department of Genome Sciences, University of Washington, 1705 NE Pacific, Seattle, WA 98195, USA.
Gally
David L
DL
Centre for Infectious Diseases, Royal (Dick) School of Veterinary Medicine, Chancellor's Building, University of Edinburgh, Edinburgh EH16 4SB, UK.
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D045522:Recombinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D045522:Recombinases; D014157:Transcription Factors; C116245:atpD protein, E coli; C081956:fimB protein, E coli; C081955:fimE protein, E coli; D019426:Integrases