Genetic analysis of the iron uptake region of the Vibrio anguillarum plasmid pJM1: molecular cloning of genetic determinants encoding a novel trans activator of siderophore biosynthesis

Abstract

Clones carrying the iron uptake region of the Vibrio anguillarum plasmid pJM1 were subjected to insertion mutagenesis, using transposon Tn3::HoHo1 which carries a promoterless lacZ gene and can thus generate lacZ transcriptional fusions if inserted downstream from an indigenous promoter. Four classes of insertion mutants were obtained based on the level of expression of components of the iron uptake system, and six genetic units were defined according to the phenotype of the mutants. Five of the six genetic units were crucial for biosynthesis of the siderophore anguibactin. Insertions in the remaining genetic unit led to an iron uptake-deficient phenotype and showed either reduced levels of the outer membrane protein OM2 as well as anguibactin activity or a complet shutoff of both OM2 and anguibactin biosyntheses. Analysis of beta-galactosidase production by cells carrying the lacZ fusion derivatives identified iron-regulated and constitutive transcriptional units as well as their orientation in the genetic units. Molecular cloning of pJM1 plasmid DNA noncontiguous to the iron uptake region also identified genetic determinants for a trans-acting factor required for full expression of anguibactin activity. Evidence obtained from bioassays, spectrophotometric measurements, and the lacZ fusion mutants suggested that the trans-acting factor is a novel activator of siderophore biosynthesis at the transcriptional level.

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307.

“…The library was constructed using pVK102 and 8399 chromosomal DNA partially digested with HindIII as described previously (Genti-Raimondi et al, 1991). The Tn3-HoHo1 transposon (Stachel et al, 1985) was used as described previously (Tolmasky et al, 1988) to generate insertional derivatives of recombinant cosmids harbouring A. baumannii 8399 DNA. Recombinant cosmids and Tn3-HoHo1 insertional derivatives were introduced into E. coli DH5α by electroporation.…”

Characterization of the Interaction between Fur and the Iron Transport Promoter of the Virulence Plasmid in Vibrio anguillarum

“…1). Genetic studies utilizing transposon (Tn3::HoHo1) insertion mutagenesis have demonstrated that mutations in fatD, -C, or -B affected FatA expression, suggesting an operon organization of these genes (6,8). A further regulatory linkage between iron transport genes and anguibactin biosynthetic genes has also been proposed since insertion mutations in the iron transport genes led to lowering or complete shutoff of anguibactin biosynthesis (8).…”

“…The positive regulation is mediated by the plasmid-encoded 110-kDa AngR protein whose gene lies immediately downstream of fatA and by trans acting factors encoded in a region located noncontiguous to the iron uptake region in the pJM1 plasmid (5,8,9,10). The negative regulation is afforded by the chromosomally encoded Fur protein (11,12) and by an antisense RNA (RNA␣) (13).…”

Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775

“…Three pJM1-encoded proteins, p40, pOM2, and pAngR, have been shown to be iron regulated and required for iron uptake (3,10,12,29,31), but direct demonstrations that they are also required for virulence of V. anguillarum 775(pJM1) are lacking. This is due in part to the difficulties encountered when attempting to introduce plasmid DNA into V. anguillarum 775(pJM1) in the presence of an apparent pJM1-mediated restriction or exclusion system (6a).…”