Bordetella pertussis fur gene restores iron repressibility of siderophore and protein expression to deregulated Bordetella bronchiseptica mutants

Abstract

We report the isolation and preliminary phenotypic characterization of manganese-resistant Bordetella bronchiseptica mutants with respect to deregulation of siderophore and iron-regulated protein expression. The fur gene of Bordetella pertussis was cloned by genetic complementation of this deregulated phenotype and confirmed as fur by nucleotide sequence analysis.In the host, iron-binding proteins withhold iron from disease-causing microorganisms (34), suppressing their proliferation. Hence, the ability of a microorganism to overcome this host defense mechanism can be considered a virulence trait. Furthermore, expression of a number of bacterial products contributing to infectious disease pathology is elevated in response to iron limitation (7,23,29).Mechanisms of iron acquisition by members of the genus Bordetella are not well understood. All Bordetella species with the exception of Bordetella avium have been shown to excrete putative hydroxamate-type siderophores in response to iron starvation (12), and there is evidence in B. pertussis and B. bronchiseptica of a cell-associated siderophore-independent iron uptake system allowing direct iron removal from the host iron-binding proteins transferrin and lactoferrin (22,24).In characterized bacterial systems, iron-regulated gene expression is governed principally by the DNA-binding repressor protein Fur (3). Fur homologs have been reported for at least 13 species of gram-negative bacteria, including Escherichia coli (13,25), Salmonella typhimurium (9), Yersinia pestis (27, 28), Vibrio anguillarum ( By analogy to other bacterial species, we hypothesized the involvement of a Fur homolog in the regulation of iron acquisition systems in Bordetella species. Manganese-resistant mutants of B. bronchiseptica B013N (2) were selected on LuriaBertani agar plates containing 10 mM MnCl 2 essentially as described by Hantke (14) for the selection of fur mutants of E. coli K-12. This procedure yielded three manganese-resistant mutants, designated B013N Mn r 4, B013N Mn r 16, and B013N Mn r 23, that appeared to be completely defective in their ability to repress siderophore expression. Mutants were cultured in parallel under both high-iron and low-iron conditions (2) and tested for constitutive production of siderophore activity by both the chrome azurol S universal siderophore assay (26) and the Csaky assay for hydroxamate class siderophores (8). In addition, iron-regulated protein expression in cell samples taken from the same cultures was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (19), as well as immunoblotting (32) of duplicate gels using murine antiserum raised to the 92-kDa iron-regulated outer membrane protein IR92 (1) of B. bronchiseptica B013N. As shown in Fig. 1A, iron-regulated protein expression by the wild-type parent strain B013N was derepressed by iron starvation whereas the mutants expressed iron-regulated proteins constitutively. Deregulated expression of IR92 by the mutants was confirmed by immunoblot analysis as shown in ...

Isolation and analysis of a fur mutant of Neisseria gonorrhoeae

“…Manganese selection (31) has been used in several other species to isolate fur mutants (15,31,39,53,63,67). Although it was possible to isolate Mn-resistant isolates under conditions of excess inorganic iron on GCB plates (complex medium), none of the isolates screened showed any defect in iron regulation as measured by their phenotype on GCB-BCIP medium (data not shown).…”

“…Mn selection is a relatively specific method for isolating fur mutations in many species of bacteria (15,31,39,53,63,67). Although the mechanism of this selection is poorly understood (31), we based our modification of this technique on the premise that the selection works through the ability of Mn to substitute for ferrous iron, causing Fur to bind its cognate binding sequence (4).…”

“…The fur locus was first described for Salmonella typhimurium (26) but has been best characterized for Escherichia coli (42,59). Recently, fur genes from Neisseria gonorrhoeae (6), Neisseria meningitidis (66), Vibrio cholerae (40), Vibrio vulnificus (41), Vibrio anguillarum (67), Pseudomonas aeruginosa (53), Pseudomonas putida (71), Bordetella pertussis (5,15), Haemophilus ducreyi (8), Yersinia pestis (62), Legionella pneumophila (35), Campylobacter jejuni (78), and Klebsiella pneumoniae (73) have been cloned and sequenced.…”

“…Unsuccessful attempts to isolate insertional null mutations have been reported for P. aeruginosa (53), P. putida (71), V. anguillarum (67), H. ducreyi (8), N. meningitidis (66), and N. gonorrhoeae (6). For P. aeruginosa (53) and V. anguillarum (67), as well as for a number of other species, point mutations have been successfully isolated by manganese selection (15,31,39,63). We used a variation of this manganese selection system to isolate a point mutation in N. gonorrhoeae fur and have begun to define the extent of the Fur regulon.…”

Iron stress and genetic response in cyanobacteria : fur genes from Synechococcus PCC 7942 and Anabaena PCC 7120

“…Fur homologues have been found in several other, ¡Gram negative bacteria, including some important human pathogens like Pseudomonas (Prince et al 1993), Vibrio (Litwin et al 1992), Neisseria (Thomas & Sparling 1994), Yersinia (Staggs & Perry 1991), Helicobacter pylori (Bereswill et al 1998), Bordetella (Brickman & Armstrong 1995), Campylobacter (Wooldridge et al 1994), Acinetobacter baumannii (Daniel et al 1999), Legionella (Hinkey & Cianciotto 1994) or Haemophilus (Carson et al 1996). However, Fur has not been deeply investigated in cyanobacteria, having only been described in the unicellular cyanobacteria Synechocystis PCC 6803 (Kaneko et al 1996) and Synechococcus PCC 7942 (Ghassemian & Straus 1996), which are unable to fix nitrogen.…”