title: Isolation and Preliminary Characterization of Hydroxamic Acids Formed by Nitrogen-Fixing Azotobacter chroococcum B-8.
abstract: The free-living diazotroph Azotobacter chroococcum B-8 responded to iron-limited growth conditions by forming hydroxamic acids and an 85,000-dalton outer membrane protein. The Fe(III)-binding hydroxamate compounds stimulated the growth of Arthrobacter flavescens JG-9 and gave a positive Csaky reaction for bound hydroxylamines. The hydroxamates were isolated from liquid cultures by benzyl alcohol extraction and purified by size exclusion chromatography and high-performance liquid chromatography. Four high-performance liquid chromatography fractions, designated A, B, C, and D, had the characteristic hydroxamate absorption maximum at 420 to 423 nm, which did not shift over a pH range from 3.0 to 9.0. Cyclic voltammograms of the iron-hydroxamate complexes exhibited reduction potentials of -0.426 to -0.442 V for fractions A, B, and D and of -0.56 V for fraction C versus the normal hydrogen electrode at pH 8.0. Based on mass spectra, nominal molecular weights of 800 and 844 were assigned to ferrated compounds A and B, respectively. Reductive hydrolysis of compounds A and B in 57% hydriodic acid yielded ornithine as detected by gas chromatography-mass spectrometry. All of these physiological and chemical data strongly support the hypothesis that the high-affinity iron-binding compounds isolated from A. chroococcum B-8 are hydroxamic acids and probably function as siderophores for this diazotroph.
authors.affiliation: Departments of Biology and Chemistry, Colby College, Waterville, Maine 04901, and Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588.
authors.forename: F A
authors.initials: FA
authors.lastname: Fekete
authors.forename: R A
authors.initials: RA
authors.lastname: Lanzi
authors.forename: J B
authors.initials: JB
authors.lastname: Beaulieu
authors.forename: D C
authors.initials: DC
authors.lastname: Longcope
authors.forename: A W
authors.initials: AW
authors.lastname: Sulya
authors.forename: R N
authors.initials: RN
authors.lastname: Hayes
authors.forename: G A
authors.initials: GA
authors.lastname: Mabbott