TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA

Abstract

ABSTRACT Siderophore nutrition tests with Caulobacter crescentus strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. C. crescentus did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [ 59 Fe]apoferrichrome and 59 Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [ K d ], 1 nM; Michaelis-Menten constant [ K M ], 0.1 nM; V max , 19 pMol/10 9 cells/min) were similar to those of Escherichia coli FhuA. Transport properties for 59 Fe-citrate were similar to those of E. coli FecA ( K M , 5.3 nM; V max , 29 pMol/10 9 cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028 , 00138 , 02277 , and 03023 as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to Escherichia coli FepA and BtuB. A Δ 02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for h eme/hemoglobin u tilization). IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus , identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by C. crescentus , its heme/hemoglobin transporter (HutA).