Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10541–10546, September 1997 Biochemistry The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior (signal transductionybacterial chemotaxisyaerotaxis) ANURADHA REBBAPRAGADA*, MARK S. JOHNSON*, GORDON P. HARDING*, ANTHONY J. ZUCCARELLI*†, HANSEL M. FLETCHER*, IGOR B. ZHULIN*, AND BARRY L. TAYLOR*†‡ *Department of Microbiology and Molecular Genetics and †Center for Molecular Biology and Gene Therapy, School of Medicine, Loma Linda University, Loma Linda, CA 92350 Edited by Daniel E. Koshland, Jr., University of California, Berkeley, CA, and approved July 17, 1997 (received for review May 6, 1997) ABSTRACT We identified a protein, Aer, as a signal conformational change in the signaling domain that increases transducer that senses intracellular energy levels rather than the rate of CheA autophosphorylation. The phosphoryl resi- the external environment and that transduces signals for due from CheA is transferred to CheY, which, in its phos- aerotaxis (taxis to oxygen) and other energy-dependent be- phorylated state, binds to a switch on the flagellar motors and havioral responses in Escherichia coli. Domains in Aer are signals a reversal of the direction of rotation of the flagella. similar to the signaling domain in chemotaxis receptors and Evidence that CheA, CheW, and CheY are also part of the the putative oxygen-sensing domain of some transcriptional aerotaxis response (12) led us to propose that the aerotaxis activators. A putative FAD-binding site in the N-terminal transducer would have (i) a C-terminal domain homologous to domain of Aer shares a consensus sequence with the NifL, Bat, the chemoreceptor signaling domain that modulates CheA and Wc-1 signal-transducing proteins that regulate gene autophosphorylation and (ii) a domain that senses oxygen. We expression in response to redox changes, oxygen, and blue have identified a putative aerotaxis transducer in a comput- light, respectively. A double mutant deficient in aer and tsr, erized search of protein databases and confirmed that this which codes for the serine chemoreceptor, was negative for protein, which we named ‘‘Aer,’’ functions as a transducer for aerotaxis, redox taxis, and glycerol taxis, each of which aerotaxis and related responses. We also present evidence that, requires the proton motive force andyor electron transport in addition to Aer, the serine chemoreceptor Tsr functions as system for signaling. We propose that Aer and Tsr sense the an independent transducer for aerotaxis and related responses. proton motive force or cellular redox state and thereby A preliminary report of these findings was presented at the integrate diverse signals that guide E. coli to environments Annual Meeting of the American Society for Biochemistry and where maximal energy is available for growth. Molecular Biology, 1996 (16). In an environment where oxygen is being depleted by growing MATERIALS AND METHODS bacteria, aerotaxis quickly moves Escherichia coli and Salmo- nella typhimurium to a more favorable microenvironment Bacterial Strains and Growth Conditions. The strains and before anoxia develops (1). Aerotaxis, the behavioral response plasmids used are listed in Table 1. Cells were grown in to oxygen, requires a functional electron transport system (2). Luria–Bertani medium containing 1 mM thiamine at 35°C to Oxygen stimulates electron transport through the electron OD600 5 0.45–0.50, unless specified otherwise. transport system, increasing the proton motive force (2, 3). A Construction of Mutants. A 1.2-kb kanamycin cassette postulated aerotaxis-transducing protein responds to the in- excised from pMB2190 (a derivative of pUC4K obtained from crease in electron transportyproton motive force and initiates P. Matsumura, University of Illinois, Chicago) using HincII a signal for the behavioral response to oxygen (3–6). Chemicals was ligated into pGH1 at the SmaI site (nt 349) of aer. The other than oxygen that stimulate electron transport also elicit aer-2::kan construct was excised with XmnI and EheI and an aerotaxis-like behavioral response in bacteria (5, 7, 8). The ligated into the SmaI site of the temperature-sensitive plasmid aerotaxis transducer may mediate other types of bacterial pKO3 (kindly provided by G. M. Church, Harvard University, behavior, such as the newly described taxis to a preferred redox Cambridge, MA). Mutants defective for aer were generated by potential (9) and energy taxis to carbon sources, such as allelic exchange as described by Hamilton et al. (23) and glycerol (10) or proline (11). Each of these behaviors involves modified by Link (24). The pKO3 vector contains a Bacillus modulation of the proton motive force and electron transport subtilis sacB gene that inhibits Escherichia coli growth in the as the initial sensory transduction event (3). presence of sucrose and facilitates the selection of cells that The aerotaxis sensory transduction pathway includes three have lost the vector sequence. Gene replacement was verified proteins that are common to the chemotaxis pathway: the by PCR using Expand Long Taq Polymerase (Boehringer CheA sensor kinase, CheY cognate response regulator, and Mannheim)(25) and Southern blot analysis of the transfor- CheW docking protein (12). The methyl-accepting chemotaxis mants. The absence of fusion products was confirmed by receptors Tsr, Tar, Trg, and Tap (13–15) span the cytoplasmic sequencing the inactivated gene. membrane and have a highly conserved signaling sequence in The aer tsr double mutant BT3311 was generated by P1 thr1 tsr the C-terminal cytoplasmic domain that binds CheA and transduction of a D -7021 fragment from RP5882 into BT3309 (aer-2) recipient cells and by selecting for threonine1 CheW. Repellent binding to a chemotaxis receptor induces a transductants. Colonies that lacked serine taxis on tryptone The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviation: IPTG, isopropylthiogalactoside. accordance with 18 U.S.C. §1734 solely to indicate this fact. A commentary on this article begins on page 10487. © 1997 by The National Academy of Sciences 0027-8424y97y9410541-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: blTaylor@ PNAS is available online at http:yywww.pnas.org. ccmail.llu.edu. 10541 Downloaded by guest on September 30, 2021 10542 Biochemistry: Rebbapragada et al. Proc. Natl. Acad. Sci. USA 94 (1997) Table 1. Strains and plasmids used in this study sequenced by the Blatner group in the E. coli Genome Project and deposited in the GenBank database (accession no. Strainsy U28379). The sequence currently is listed as AIRoECOLI in plasmids Relevant genotype Sourceyreference the SwissProt database (accession no. P50466). Transcription Strains of the ORF506 gene is independent of the flanking ygjG and MM335 wild type (17) yqjI genes. A computerized search revealed that the putative RP437 wild type (18) product of the ORF506 was a 506-residue protein that had a GK100 Dcyo Dcyd (19) predicted C-terminal segment with 96.7% identity to a highly RP5882 D(tsr) 7021 (20) conserved domain of Tsr (Fig. 1C). The N-terminal domain of BT3300 MM335 aer-2::kan This study the ORF506 protein (residues 8–129) shares homology with BT3309 RP437 aer-2::kan This study domains in the NifL protein of Azotobacter vinelandii (28% BT3310 BT3309ypGH1 (Aer11) This study identity, 52% similarity), the Bat protein of Halobacterium BT3360 MM335ypGH1 (Aer11) This study salinarium (22% identity, 54% similarity) and Wc-1, the White BT3311 aer-2::kan Dtsr This study Collar protein of Neurospora crassa (19% identity, 37% sim- BT3313 BT3311ypJL3 (Tsr11) This study ilarity) (Fig. 1A). Both NifL and Bat are known oxygen sensors. Plasmids In response to oxygen, the NifL protein regulates expression of pK03 rep pSC101(ts) sacB G. M. Church* nitrogen fixation genes (33), and the Bat protein regulates q pTrc99A ptac lacI Pharmacia and (21) synthesis of bacteriorhodopsin (34). The Wc-1 protein is a pGH1 pTrc99A aer1 This study central regulator of blue light responses in Neurospora (35). q 1 pJL3 ptac lacI tsr (22) Thus, the ORF506 protein had the expected domains of the *Harvard University, Cambridge, MA. putative aerotaxis-transducing protein (12), and the gene was renamed ‘‘aer’’ (aerotaxis and energy responses) after exper- semisoft agar were further characterized by temporal chemo- imental evidence confirmed its function. taxis assays. Sequence analysis predicted that the topology of Aer in the Overexpression of Aer and Tsr. The expression vector pGH1 membrane is different from the topology of known chemotaxis was constructed by subcloning a 1.5-kb AflIII–SalI PCR frag- receptors that transverse the cytoplasmic membrane (TM1 and ment containing the aer gene into the NcoI and SalI sites of TM2) at either end of a periplasmic ligand-binding domain. pTrc99A and was verified by sequencing. pGH1 was intro- Only one hydrophobic sequence (residues 167–206) was pre- duced into BT3309 cells by electroporation and was induced dicted in Aer. This sequence and these flanking regions are with varying amounts of isopropylthiogalactoside (IPTG). q similar to the TM1 and TM2 transmembrane regions of Tsr The plasmid pJL3 (22) containing the lacI repressor gene (Fig. 1B) but lack the intervening periplasmic