1 Department of Biochemistry and Microbiology, Division of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA
2 Department of Biology, Georgia State University, Atlanta, GA 30303, USA
3 School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA
Correspondence
Igor B. Zhulin
igor.zhulin{at}biology.gatech.edu
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ABSTRACT |
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INTRODUCTION |
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E. coli also responds chemotactically to physico-chemical parameters that affect the redox status of the electron transport system (ETS) and proton motive force (PMF). These behavioural responses are collectively known as energy taxis (Taylor & Zhulin, 1998; Taylor et al., 1999
; Alexandre & Zhulin, 2001
). In energy taxis, cells respond not to a chemical per se, but to changes in the ETS caused by the chemical, i.e. in a metabolism-dependent manner. Stimuli for energy taxis include terminal electron acceptors (oxygen, nitrate, etc.) and redox-active chemicals that interfere with electron transport (Taylor et al., 1979
; Shioi et al., 1988
; Grishanin et al., 1991
; Bespalov et al., 1996
; Zhulin et al., 1996
, 1997
; Alexandre et al., 2000
).
In energy taxis of E. coli, changes in the ETS are detected by membrane-bound chemoreceptors Aer and Tsr (Bibikov et al., 1997; Rebbapragada et al., 1997
). Aer is anchored in the membrane; however, both its sensing and signalling domains are cytoplasmic. It contains a sensory PAS (found in period clock protein, aryl hydrocarbon receptor and single-minded protein) domain (Taylor & Zhulin, 1999
) that binds an FAD cofactor potentially capable of changing its redox potential to reflect the status of the ETS (Bibikov et al., 1997
, 2000
; Rebbapragada et al., 1997
; Repik et al., 2000
). Because Aer is able to sense and signal changes in the redox potential (Bespalov et al., 1996
; Rebbapragada et al., 1997
), oxidation and reduction of FAD within the PAS domain is proposed to produce the signal (Taylor et al., 1999
; Repik et al., 2000
). Aer is not only responsible for taxis to terminal electron acceptors and redox taxis, but was also proposed to mediate positive taxis to glycerol and succinate (Rebbapragada et al., 1997
). Both substrates are effective donors of reducing equivalents for the ETS, and it was established in a direct experiment that positive taxis to glycerol in E. coli results from its oxidation (Zhulin et al., 1997
). Tsr is a classical ligand-binding transmembrane receptor for serine, but it is also known to govern energy taxis (Rebbapragada et al., 1997
). Because Tsr lacks any oxygen- or redox-responsive prosthetic group, it was proposed to sense changes in the PMF by an as yet unknown mechanism. Earlier suggestions that pH-sensing capabilities of Tsr may contribute to PMF sensing (Taylor et al., 1999
; Levit & Stock, 1999
) are not supported by findings that other transmembrane chemoreceptors in E. coli are also capable of pH-sensing (Yamamoto et al., 1990
; Umemura et al., 2002
), whereas only Tsr acts as an energy taxis transducer (Rebbapragada et al., 1997
). If Aer and Tsr respond to changes in electron transport and/or the PMF, then even in the absence of specialized chemoreceptors, Aer or Tsr should govern cell migration along the gradients of effectively oxidizable substrates.
Here we present experimental evidence that Aer or Tsr when present as a sole chemoreceptor in E. coli mediates metabolism-dependent responses to various oxidizable substrates including some of those that are detected by other chemoreceptors via the ligand-binding metabolism-independent mechanism.
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METHODS |
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Temporal gradient assay for chemotaxis.
Cells were transformed and selected as described above in semi-soft plates, then grown in LB broth at 30 °C and harvested at OD600 0·4. A temporal gradient assay for chemotaxis (Spudich & Koshland, 1975) was performed using a video microscope essentially as described previously (Bespalov et al., 1996
). When necessary, the tumbling frequency of individual cells was analysed using a real-time Hobson Bactracker motion analysis system (Hobson Tracking Systems).
Gel electrophoresis and Western analysis.
Cells were grown to OD600 0·4 at 37 °C and induced with 0200 µM IPTG for 2 h. Cells were then centrifuged and resuspended in SDS sample buffer (2 % SDS, 1 % 2-mercaptoethanol, 15 % glycerol and 62 mM Tris base, pH 6·8), boiled for 2 min and applied to SDS-10 % polyacrylamide gels. The resolved proteins were electroblotted to nitrocellulose membranes and probed. Aer was detected using an anti-N-terminal Aer polyclonal antibody (a gift from M. S. Johnson, Loma Linda University School of Medicine, CA, USA) and Tsr was detected using an anti-Tsr polyclonal antibody (a gift from J. S. Parkinson, University of Utah, Salt Lake City, UT, USA). Expression of both Aer and Tsr from the plasmids was detected even without addition of IPTG, whereas due to the low copy numbers no Aer was detected in wild-type cells.
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RESULTS |
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Redox-responsive co-factor of Aer is required for taxis to oxidizable substrates
To verify whether Aer-governed motility along gradients of oxidizable substrates was due to its redox-sensing capabilities (i.e. occurred via the energy taxis mechanism), we tested an Aer mutant [BT3388(pAVR26), aer D68C] unable to bind the redox-responsive FAD cofactor (Repik et al., 2000). The results of these experiments are shown in Table 2
. Behavioural assays were carried out on semi-soft swarm plates supplemented with IPTG to induce expression of the mutant Aer protein, if necessary. Protein expression was verified by SDS-PAGE (data not shown). The mutant was unable to show chemotaxis to any substrate tested over a range of IPTG concentrations from 0 to 200 µM. The mutant also lost the negative response to 2,4-benzoquinone in a temporal gradient assay.
Tsr governs chemotaxis in gradients of effective growth substrates
Tsr was expressed in BT3388 by transforming the cells with a plasmid (pJL3) harbouring the tsr gene under control of the trc lactose inducible promoter (Rebbapragada et al., 1997) and chemotaxis was assayed similarly to that for strains expressing Aer. Protein expression was verified by Western blotting using anti-Tsr antiserum supplied by J. S. Parkinson. We found that Tsr-carrying cells formed chemotactic rings in gradients of the sugars ribose, galactose, maltose, glucose, fructose, mannose, sorbitol and glycerol, and to a significantly lesser extent in gradients of proline and alanine. No chemotactic rings were formed by the Tsr-carrying strain in gradients of succinate, malate, glutamate and aspartate (Table 2
).
Tsr preferentially directed taxis in gradients of sugars that are the most efficient growth substrates for E. coli. To evaluate further this apparent relationship between energy efficiency and chemotaxis, we measured doubling times of the wild-type cells grown on different substrates in minimal medium. We found a correlation between the efficiency of the chemical as a stimulus for Tsr-mediated chemotaxis and as a growth substrate. The best chemoeffectors for Tsr, such as fructose, glucose and maltose, were also the best growth substrates. Chemicals that did not elicit a chemotactic response, such as succinate and arabinose, were poor or were not carbon substrates for growth (Table 3). For the Aer-mediated response, no correlation was found between the efficiency of a chemical as an attractant and a growth substrate.
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DISCUSSION |
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We found that Tsr efficiently governs chemotaxis in gradients of sugars, which are the best growth substrates for E. coli, and demonstrated a correlation between the efficiency of a chemical as a growth substrate and an attractant. Such a correlation is indicative of energy taxis (Alexandre et al., 2000). The substrates that caused strong responses for Aer- and Tsr-mediated behaviours were different. This difference is consistent with the hypothesis that Aer and Tsr mediate energy taxis by different mechanisms, redox and PMF sensing, respectively (Taylor et al., 1999
). If PMF sensing is indeed a mechanism by which Tsr governs energy taxis, then it remains to be seen why other chemotaxis transducers that are sensitive to internal pH (Yamamoto et al., 1990
; Umemura et al., 2002
) are not energy taxis sensors. One of the possible explanations is that different amino acid residues of transducers are involved in pH taxis versus energy taxis signalling. For example, the residues that are involved in pH sensing were found in the cytoplasmic HAMP domain (Umemura et al., 2002
), whereas residues that might control the transducer mobility in the membrane in response to changes in the electrochemical proton gradient are likely to be located in the membrane/cytoplasm interface (i.e. between the transmembrane helix and the HAMP domain), as suggested for other transmembrane proteins (Schuenemann et al., 1999
).
The energy taxis receptors Aer and Tsr were found to be insensitive to substrates at low concentrations compared to the specialized chemotaxis receptors Tar and Trg. Aer and Tsr are capable of navigating E. coli cells in gradients of ribose, galactose and maltose, but only at concentrations above 1 mM, whereas Tar and Trg sense these sugars at 10 µM concentrations (in a spatial gradient assay). This low sensitivity is typical of the energy taxis responses (Zhulin et al., 1997; Alexandre et al., 2000
) and likely reflects the necessity for a high substrate concentration in order to support electron flow via the ETS and generation of the PMF.
Taken together, our results demonstrate that energy taxis receptors of E. coli even in the absence of highly sensitive specialized chemoreceptors enable the cells to navigate towards a preferred concentration of carbon and energy sources. Energy taxis is a dominant type of behaviour in some species (Alexandre et al., 2000) and is proposed to be widespread in bacteria (Taylor et al., 1999
; Alexandre & Zhulin, 2001
). By widening the repertoire of chemicals that are detected by energy taxis receptors, this study further supports this hypothesis.
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ACKNOWLEDGEMENTS |
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This work was supported, in part, by the National Institutes of Health grant GM29481 (to B. L. T.) and by the start-up funds from Georgia State University (to G. A.) and Georgia Institute of Technology (to I. B. Z.).
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Received 17 February 2003;
revised 2 May 2003;
accepted 5 May 2003.