Isolation and characterization of Bordetella bronchiseptica mutants deficient in siderophore activity

Abstract

Iron acquisition by the gram-negative pathogens Bordetella bronchiseptica and Bordetella pertussis is thought to occur by hydroxamate siderophore-mediated transport as well as an apparently siderophore-independent process by which host transferrins bind to bacterial surface receptors. We constructed B. bronchiseptica mutants deficient in siderophore activity by insertional mutagenesis with miniTn5/LacZl. The mutants could be placed into four distinct complementation groups, as determined from cross-feeding assays which demonstrated restored siderophore synthesis. Mutants deficient in siderophore activity were BRM1, BRM6, and BRM9, exhibiting approximately 36 to 41% of wild-type siderophore levels, and BRM3 and BRM8, which appeared to produce very little or no detectable siderophore. Mutant BRM4 was found to be a leucine auxotroph, while mutants BRM2 and BRM7 could synthesize siderophore only in low-iron medium which was supplemented with various amino acids. Evaluation of all transcriptional fusions revealed an apparent lack of iron-regulated lacZ expression. Genomic regions flanking the transposable element in the siderophore mutants were homologous with B. pertussis chromosomal DNA, while bioassays suggested siderophore cross-feeding between B. pertussis and B. bronchiseptica. These results indicate probable similarity between the siderophore biosynthetic and transport systems of the two species.Complex high-affinity iron transport systems are expressed by microorganisms in response to suboptimal availability of this nutrient. The prototypic transport system involves excretion of low-molecular-weight siderophores for mobilization of iron to the intracellular compartment (30). Recent studies have described a direct contact uptake mechanism in species of Neisseria (24, 38, 43) and Haemophilus (28, 37) which appears to rely solely on the physical interaction of bacterial surface receptors and host iron-binding proteins such as transferrin or lactoferrin.Little is known about iron acquisition in members of the genus Bordetella, which consists of gram-negative respiratory pathogens of a variety of animal species (6,18,31). Bordetella pertussis, the etiologic agent of human whooping cough, and Bordetella bronchiseptica, which primarily infects nonhuman mammals, exhibit a strong affinity for several transferrins (25,33,34). The assimilation of iron from cell-bound transferrin by B. pertussis suggested the existence of a direct-contact iron-sequestering system similar to that used by the Neisseria and Haemophilus species (33). Demonstration that B. pertussis grew well when separated from iron-loaded transferrin by a dialysis membrane (19) suggested siderophore excretion. The assay of Schwyn and Neilands (39) was used to detect siderophore activity in iron-restricted B. pertussis culture supernatants which also yielded positive results with the Csaky assay for hydroxamic acids (11), suggesting that the chelator may be of the hydroxamate siderophore class (19). Iron-stressed B. bronchiseptica and Bordetella parapertussi...

The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin

“…Molecular cloning of mini-Tn5/lacZ1 transposon insertion markers associated with group III B. bronchiseptica siderophore-deficient mutants BRM3 and BRM5, resulting in recombinant plasmids pBRM3 and pBRM5, was accomplished in a previous study (3). B. bronchiseptica genomic sequences flanking the inser- , an approximately 700-bp NotI-SalI fragment of pBRM3 was used to probe a B. pertussis UT25 cosmidbased genomic library by in situ DNA hybridization.…”

“…B. bronchiseptica B013N, a nalidixic acidresistant derivative of strain B013, and the isolation of siderophore biosynthetic mutants BRM3 and BRM5 by mini-Tn5/lacZ1 (16) insertional inactivation have been described previously (3). Virulent-phase B. pertussis UT25 has also been described previously (18).…”

“…Chrome azurol S (CAS) agar plates for scoring of B. bronchiseptica siderophore production were prepared as described by Armstrong and Clements (3). The CAS universal siderophore detection assay (40) was used to monitor siderophore production by Bordetella strains grown in liquid culture by measuring the decrease in A 630 of the CAS dye reaction mixture as reported previously (3). Siderophore activities are expressed relative to uninoculated complete culture medium.…”

“…Conjugal transfer of pCP13 and pRK415 derivatives to Bordetella strains was accomplished by triparental matings with E. coli DH5␣ as the plasmid donor strain and DH5␣ (pRK2013) as the source of mobilization functions. Suspensions of donor, helper, and recipient strains in SS plus 10 mM MgSO 4 were combined at an estimated 1:1:3 cell ratio, spotted onto Luria-Bertani or blood agar plates (for B. bronchiseptica recipients) or Bordet-Gengou agar plates (for B. pertussis recipients), and incubated at 37ЊC for 3 to 5 h. Bacteria were streaked from mating spots directly onto agar plates containing the appropriate selective antibiotics and crude colicin B (3,12). Plates were incubated at 37ЊC for 1 to 4 days, at which time transconjugant colonies were apparent.…”

“…We report the identification of a gene encoding an alcaligin siderophore biosynthetic activity defined by previously reported transposon mutations abrogating alcaligin production in B. bronchiseptica (3) as an ornithine decarboxylase (Odc) gene, indicating that putrescine is an essential precursor for alcaligin siderophore biosynthesis in Bordetella spp.…”