Abstract The Escherichia coli nir and nrf operons, which encode alternative nitrite reductases expressed dur- ing anaerobic growth, are subject to catabolite regulation. Transcription from the nir promoter is maximal when bac- teria are grown in rich media such as Lennox broth sup- plemented with glucose. Conversely, expression of the nrf operon is suppressed by rich media, but stimulated during growth in minimal medium with glycerol and fumarate. The role of the catabolite repressor-activator (Cra) protein in catabolite regulation of the nir and nrf promoters was investigated. Transcription from the nir promoter was re- pressed by Cra when cells were grown in minimal me- dium with glycerol and fumarate. Crude protein extracts from a strain overproducing Cra encoded on a multicopy plasmid retarded a nir promoter fragment in a mobility shift assay, confirming that the observed Cra-dependent repression was due to the direct interaction of Cra with the regulatory region of the nir operon. Furthermore, the in- clusion of fructose 1-phosphate, an effector of Cra DNA- binding activity, in the assay decreased the ability of Cra to retard the nir promoter fragment. In contrast, transcrip- tion from the nrf promoter was not regulated by Cra under any of the growth conditions tested. Key words Catabolite regulation · Cra protein · Nitrite reductase regulation · Escherichia coli · Catabolite repression Abbreviations Cra Catabolite repressor-activator protein · nir Genes encoding NADH-dependent nitrite reduction complex · nrf genes encoding the formate- dependent nitrite reduction complex Introduction The generation of ATP is an essential process in all living organisms. Enteric bacteria such as Escherichia coli have evolved numerous pathways for generating energy from various carbon sources under various growth conditions. In addition, complex regulatory mechanisms have also evolved to ensure that ATP synthesis does not exceed the cellular requirements. For example, the addition of glu- cose to the growth medium represses the synthesis of en- zymes involved in the metabolism of alternative carbon sources whilst activating the synthesis of the glucose cata- bolic enzymes, phenomena known as catabolite repres- sion and catabolite activation, respectively. In E. coli, two mechanisms of catabolite regulation have been identified. The more extensively studied sys- tem is dependent on cyclic AMP (cAMP) and its receptor protein, CRP, which activates the transcription of many catabolic operons (Busby and Kolb 1996). In contrast, the Cra protein (c atabolite r epressor-a ctivator, formerly des- ignated the fructose repressor, FruR) mediates catabolite regulation independently of cAMP (Geerse et al. 1986, 1989; Chin et al. 1987, 1989; Ramseier et al. 1993; Saier 1996). In the absence of glucose, Cra binds to the promot- ers of target operons and activates or represses transcrip- tion. However, the binding of fructose 1-phosphate or fructose 1,6-diphosphate, generated from the metabolism of fructose and glucose, respectively, dissociates Cra from the DNA, resulting in catabolite repression (Cra-activated genes) or activation (Cra-repressed genes) (Ramseier et al. 1993, 1995). We have examined the role of the Cra protein in medi- ating catabolite effects at the promoters of two operons encoding alternative nitrite reductases of E. coli that are expressed during anaerobic growth. The nrf (n itrite r educ- tion by f ormate) operon encodes a periplasmic nitrite re- ductase that, in the absence of oxygen, allows E. coli to couple formate oxidation to nitrite reduction, with the concomitant generation of a proton motive force that can subsequently be used to synthesize ATP (Motteram et al. Kerry Tyson · Steve Busby · Jeff Cole Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein Arch Microbiol (1997) 168 : 240–244 © Springer-Verlag 1997 Received: 26 February 1997 / Accepted: 15 May 1997 SHORT COMMUNICATION K. Tyson 1 · S. Busby · J. Cole () School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK Tel. +44-121-414-5440; Fax +44-121-414-3982 e-mail: j.a.cole@bham.ac.uk Present address: 1 Department of Medicine (Level 5), Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK