Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport

Abstract

The Acinetobacter baumannii 8399 clinical isolate secretes dihydroxybenzoic acid (DHBA) and a high-affinity catechol siderophore, which is different from other bacterial iron chelators already characterized. Complementation assays with enterobactin-deficient Escherichia coli strains led to the isolation of a cosmid clone containing A. baumannii 8399 genes required for the biosynthesis and activation of DHBA. Accordingly, the cloned fragment harbours a dhbACEB polycistronic operon encoding predicted proteins highly similar to several bacterial proteins required for DHBA biosynthesis from chorismic acid. Genes encoding deduced proteins related to the E. coli Fes and the Bacillus subtilis DhbF proteins, and a putative Yersinia pestis phosphopantetheinyl transferase, all of them involved in the assembly and utilization of catechol siderophores in other bacteria, were found next to the dhbACEB locus. This A. baumannii 8399 gene cluster also contained the om73, p45 and p114 predicted genes encoding proteins potentially involved in transport of ferric siderophore complexes. The deduced products of the p114 and p45 genes are putative membrane proteins that belong to the RND and MFS efflux pump proteins, respectively. Interestingly, P45 is highly related to the E. coli P43 (EntS) protein that participates in the secretion of enterobactin. Although P114 is similar to other bacterial efflux pump proteins involved in antibiotic resistance, its genetic arrangement within this A. baumannii 8399 locus is different from that described in other bacteria. The product of om73 is a Fur-and iron-regulated surface-exposed outer-membrane protein. These characteristics together with the presence of a predicted TonB box and its high similarity to other siderophore receptors indicate that OM73 plays such a role in A. baumannii 8399. The 184 nt om73-p114 intergenic region contains promoter elements that could drive the expression of these divergently transcribed genes, all of which are in close proximity to almost perfect Fur boxes. This arrangement explains the iron-and Fur-regulated expression of om73, and provides strong evidence for a similar regulation for the expression of p114. INTRODUCTIONBacteria survive and multiply under iron-limiting conditions, such as those found in natural and medical environments, by expressing active systems that gather this micronutrient, which is essential for most micro-organisms with some exceptions such as lactobacilli. Some systems involve the secretion of low-molecular-mass ferric-binding compounds called siderophores, which can be classified into different categories based on their chemical structure (Neilands, 1981(Neilands, , 1995. Many of these high-affinity ironchelating molecules contain catecholate groups that are part of the iron-binding site. Enterobactin, the prototype catechol siderophore produced by Escherichia coli, is a cyclic trimer of 2,3-dihydroxybenzoyl-L-serine (O'Brien & Gibson, 1970;Walsh et al., 1990). In contrast, siderophores such as vibriobactin and anguibactin, produced by...

Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

“…The production and secretion of acinetobactin by the 2010 SecA mutant (Fig. 4) also indicate that this mutant produces the BarA and BarB cytoplasmic membrane-associated proteins, which are predicted to be involved in acinetobactin secretion (11,36). Taken together, these results indicate that, with the exception of BauA, the acinetobactin secretion and transport proteins are translocated by a SecA-independent mechanism, which most likely involves the signal recognition particle system because of their membrane location (59).…”

“…5). Considering the protein composition and location of the acinetobactin ABC transporter (11,36), it is possible to speculate that the 2010 derivative also produces the BauC and BauD cytoplasmic-associated acinetobactin transport proteins. The production and secretion of acinetobactin by the 2010 SecA mutant (Fig.…”

“…The column was washed with equilibration buffer, and recombinant BauB was eluted with elution buffer (50 mM NaH 2 PO 4 , 0.3 M NaCl, 250 mM imidazole, pH 8.0). The lysate-insoluble fraction containing most of the recombinant BauE protein was solubilized in equilibration buffer (0.1 M NaH 2 PO 4 , 8 M urea, pH 8.0) and purified as described previously (36). Affinity chromatography of BauB and BauE preparations yielded purified proteins as demonstrated by SDS-PAGE analyses, which showed only a single 14.7-kDa or 20.9-kDa protein band, respectively.…”

“…All overexpressing vectors coding for His-tagged proteins were transformed into E. coli One Shot BL21 Star (DE3) competent cells (Life Technologies) for protein overproduction after induction with isopropyl ␤-D-1-thiogalactopyranoside (IPTG). Recombinant His-tagged SecA was purified from the insoluble fraction of cell lysates prepared with the B-PER protein extraction reagent (Thermo Fisher Scientific) using affinity chromatography over nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) under denaturing conditions as described before (36). The purity of the 110-kDa isolated protein was confirmed by SDS-PAGE (37).…”

“…For the detection of the BauA, BauB, and BauE acinetobactin transport proteins, bacteria cultured in LB broth under iron-rich and iron-chelated conditions were used to prepare whole-cell lysates as well as inner and outer membrane fractions as described before (36,37). After SDS-PAGE size fractionation on 12.5% polyacrylamide gels (37), proteins were blotted to nitrocellulose (39) and then incubated overnight with the appropriate antibodies at 4°C.…”