Downloaded from www.microbiologyresearch.org by IP: 54.237.90.162 On: Tue, 12 Jul 2016 22:51:52 Journal of General Microbiology (1989), 135, 2601-2609. Printed in Great Britain 260 1 Oxygen Inhibition of Globin Gene Transcription and Bacterial Haemoglobin Synthesis in Vitreoscilla By KANAK L. DIKSHIT, DOUGLAS SPAULDING, ANNETTE BRAUN AND DALE A. WEBSTER* Department of Biology, Illinois Institute of Technology, Chicago, IL 6061 6, USA (Received 10 April 1989; revised 12 June 1989; accepted 22 June 1989) A soluble dimeric haemoprotein, structurally and functionally similar to plant and animal haemoglobins, is found in the Gram-negative aerobic bacterium Vitreoscilla sp., strain C1. Vitreoscilla haemoglobin (VtHb) increases in concentration when the cells are exposed to hypoxic conditions. The globin part of VtHb is encoded by a single gene (vgb). An RNA transcript, approximately 500 bases long, specific for vgb was detected after Northern hybridization. The relative amount of this mRNA increased in cells grown at low levels of oxygen. Two enzymes important for haemoglobin function are 6-aminolaevulinic acid synthase (ALAS), which is necessary for haem biosynthesis, and NADH-methaemoglobin reductase, which is necessary to keep VtHb in the physiologically functional ferrous state. An increase in ALAS specific activity under hypoxic conditions preceded the increased haem production. Cellular reductase content also increased when the VtHb increased in cells grown under hypoxic conditions. The ratio of cellular reductase activity to VtHb content remained relatively constant in cells grown under a variety of conditions. The data suggest that in Vitreoscilla the transcription of the globin gene and the biosynthesis of two enzymes important for VtHb function are regulated by oxygen. INTRODUCTION Haemoglobins have generally been considered to occur exclusively in eukaryotic organisms. However, Wakabayashi et al. (1986) reported the sequence of a haemoglobin in Vitreoscilla, a Gram-negative bacterium. This haemoglobin (VtHb) is a cytoplasmic, dimeric haem protein that exists primarily in a stable oxygenated form, similar to that of oxymyoglobin (oxyMb) and oxyhaemoglobin (oxyHb) of higher organisms, during aerobic growth and respiration. Alignment of the amino acid sequence of VtHb with other haemoglobins revealed considerable sequence similarity at many critical conserved regions; in particular there was a 24% sequence identity with yellow lupin leghaemoglobin. The biosynthesis of VtHb increases dramatically when oxygen availability is low, and therefore it was suggested that the function of this protein is to ensure an adequate supply of needed oxygen to the respiring membranes of Vitreoscilla, which is an obligate aerobe (Wakabayashi et al., 1986). The taxonomy proposed by Woese et al. (1984) places the genus Vitreoscilla in a subdivision of the purple photosynthetic bacteria; this classification has been supported by studies from several laboratories (Nichols et al., 1986; Reichenbach et al., 1986; Strohl et al., 1986~). The cytochromes in Vitreoscilla have been analysed by Strohl et al. (19866) and in this laboratory (Georgiou & Webster, 1987). The latter study identified b-, c-, and d-type cytochromes in the membrane. The latter two types were present in very small quantities, and some or all of the 6-type cytochromes could be ascribed to the cytochrome o terminal oxidase by its carbon Abbreviations: ALA, 6-aminolaevulinic acid ; ALAS, 8-aminolaevulinic acid synthase ; DO, dissolved oxygen ; metHb, methaemoglobin; oxyHb, oxyhaemoglobin; oxyMb, oxymyoglobin; PYA, peptone/yeast extract/acetate medium; ogb, Vitreoscilla globin gene; VtHb, Vitreoscilla haemoglobin; VtmetHb, Vitreoscilla methaemoglobin. 0001-5525 0 1989 SGM