ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 293, No. 2, March, pp. 241-245, 1992 The Bacterial Hemoglobin from vitreoscilla Can Support the Aerobic Growth of Escherichia co/I’ Lacking Terminal Oxidases’ Rajendra P. Dikshit: Kanak L. Dikshit,3 Yixiang Liu, and Dale A. Webster Department of Biology, Illinois Institute of Technology, Chicago, Illinois 60616 Received July 25, 1991, and in revised form October 29, 1991 Two Escherichia coli mutants that lack both cyto- chrome o and d terminal oxidases are able to grow with glucose as the carbon source but not with the aerobic substrates succinate or lactate. One of these, GVlOl, is a deletion mutant of cytochrome o and a point mutation of cytochrome d. The other, GKlOO, is a total deletion mutant of all the genes for both cytochromes. When these mutants were transformed with a plasmid containing the gene for the bacterial hemoglobin from Vitreoscilla, they were capable of growth in the presence of succinate or lactate and showed aerobic respiration in the presence of these substrates, unlike the parent strains. Cells transformed with a plasmid containing the gene for the hemoglobin hut lacking the native promoter did not ex- press the hemoglobin and did not respire. Membrane vesicles prepared from the cells consumed oxygen in the presence of succinate. This succinate-supported respi- ration decreased with successive washings of the vesicles but was restored by adding E. coli cytosol containing the hemoglobin or by adding the hemoglobin purified from Vitreoscilla. This respiration was inhibited by cyanide. 0 1992 Academic. Press, Inc. The aerobic bacterium, Vitreoscilla, has two unusual respiratory characteristics. First, it possessesa hemoglo- i Supported by US PHS NIH Grants GM27085 and 2 SO7 RR07027. The authors are indebted to Professor Robert Gennis for providing us with the cytochrome deficient mutants of E. coli and to both him and Professors Jonathan Wittenberg and Benjamin Stark for their helpful suggestions. Each author contributed approximately equally to this re- search. ’ Current address: Department of Microbiology, Panjab University, Chandigarh-160014, India. 3 Current address: Molecular Biology Section, Institute of Microbial Technology, Sector 39, Chandigarh-160014, India. ooo3-9&x/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. bin (VtHb)4 which is currently the only known bacterial hemoglobin. VtHb is a homodimeric protein having about 25% sequence homology with leghemoglobin (1). Its pu- tative function is to capture oxygen and feed it to the terminal oxidases under oxygen limiting conditions. The evidence for this is (i) the cellular content of VtHb in- creases under hypoxic conditions (2), (ii) it exists in the oxygenated form during aerobic respiration (3,4) (iii) the rate constant for oxygen association (k,,) is relatively normal, whereas the rate constant for oxygen dissociation (kifJ is unusually large (5, 6), and (iv) about 40% of the protein is in the periplasmic space (7). This localization would make it well situated to transfer oxygen to the ter- minal oxidases under hypoxic conditions by the mecha- nism of facilitated diffusion (8). The gene (ugb) for VtHb has been cloned in Escherichia co&, where it is strongly expressed (4, 9), and in this organism it also is in the physiologically active oxy form during aerobic respiration (4). Further, E. coli strains carrying this gene grow better under microaerobic conditions (10,ll). The second unusual characteristic of Vitreoscih res- piration is that its respiratory chain is Na+ motive rather than H+ motive (12). When the purified cytochrome o terminal oxidase from Vitreoscih was incorporated into synthetic proteoliposomes it pumped Na+ from one side of the membrane to the other on the addition of substrate (13). This is in contrast to the E. coli cytochrome o, which is a H+ pump (14). We attempted to clone the Vitreoscilla cytochrome o using a Vitreoscilh DNA library to allow more detailed analyses of its structure and function. Two terminal oxidase-deficient mutants of E. coli were used * Abbreviations used: Amps, ampicillin resistant; CmR, chloramphen- icol resistant; cyd, gene coding for cytochrome d; cyo, gene coding for cytochrome o; KmR, kanamycin resistant; lacZ, gene coding for 6-ga- lactosidase; LB, Luria broth; ret-, recombination minus; StrR, strep- tomycin resistant; TcR, tetracycline resistant; Tris, tris(hydroxy- methyllaminomethane; ugb, gene coding for Vitreoscilla globin; VtI-Ib, Vitreoscilla hemoglobin; wt, wild type. 241