title: Artefactual cleavage of E coli H-NS by OmpT.
abstract: In the bacterium Escherichia coli, H-NS-(H1, H1a) is a heat-stable protein with a molecular mass of 15.5 kDa involved in nucleoid organisation and gene regulation linked to certain signal transduction pathways. We have shown that, following addition of preparations of everted inner membrane vesicles, heat-stable cleavage products of approximately 10 kDa of H-NS are formed in vitro from newly synthesised, radio-labelled H-NS and from purified H-NS. The 15.5 kDa protein and its cleavage products were also recovered from a minicell system. These results raised the possibility that cleavage of H-NS is physiologically significant. However, the cleavage of H-NS observed appears to occur during cell breakage and to depend on the method of protein extraction and the presence of the outer membrane protease, OmpT. Nevertheless, the results indicate that H-NS may contain at least two separate domains with cleavage occurring between these domains at a preferred OmpT site. Failure to take account of H-NS cleavage in sample preparation and analysis can lead to serious underestimation of H-NS levels.
authors.affiliation: Department of Microbiology and Immunology, University of Leicester, UK.
authors.forename: M D
authors.initials: MD
authors.lastname: Goldberg
authors.forename: J R
authors.initials: JR
authors.lastname: Canvin
authors.forename: P
authors.initials: P
authors.lastname: Freestone
authors.forename: C
authors.initials: C
authors.lastname: Andersen
authors.forename: D
authors.initials: D
authors.lastname: Laoudj
authors.forename: P H
authors.initials: PH
authors.lastname: Williams
authors.forename: I B
authors.initials: IB
authors.lastname: Holland
authors.forename: V
authors.initials: V
authors.lastname: Norris
mesh_terms: D016477:Artifacts; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease