TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA

Abstract

Siderophore nutrition tests with <i>Caulobacter crescentus</i> 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. <i>C. crescentus</i> 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 [⁵⁹Fe]apoferrichrome and ⁵⁹Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [<i>K_d</i> ], 1 nM; Michaelis-Menten constant [<i>K_M</i> ], 0.1 nM; <i>V</i>_max, 19 pMol/10⁹ cells/min) were similar to those of <i>Escherichia coli</i> FhuA. Transport properties for ⁵⁹Fe-citrate were similar to those of <i>E. coli</i> FecA (<i>K_M</i> , 5.3 nM; <i>V</i>_max, 29 pMol/10⁹ cells/min). Bioinformatic analyses implicated Fur-regulated loci <i>00028</i>, <i>00138</i>, <i>02277</i>, and <i>03023</i> 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 <i>Escherichia coli</i> FepA and BtuB. A Δ<i>02277</i> mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the <i>02277</i> structural gene as <i>hutA</i> (for <u>h</u>eme/hemoglobin <u>u</u>tilization).<b>IMPORTANCE</b> The physiological roles of the 62 putative TBDT of <i>C. crescentus</i> are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of <i>C. crescentus</i>, 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 <i>C. crescentus</i>, its heme/hemoglobin transporter (HutA).