Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
Correspondence
Jeffrey Green
jeff.green{at}sheffield.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Both NADH dehydrogenases are controlled at the transcriptional level by complex regulatory networks. Expression of the nuoA-N operon responds to oxygen and nitrate availability via the two-component sensorregulators ArcB-A (anaerobic repression) and NarX-L (nitrate activation) and to C4-dicarboxylates via an uncharacterized regulator acting at a far upstream site between -277 and -899 (Bongaerts et al., 1995). The ndh gene is subject to anaerobic repression (Spiro et al., 1989
) by the direct interaction of the oxygen-responsive transcription factor FNR with two sites in the ndh promoter, FNR I and FNR II (Fig. 1
; Green & Guest, 1994
; Meng et al., 1997
). Both FNR sites are required for full repression of ndh expression (Meng et al., 1997
). As well as being subject to FNR-mediated repression, the ndh promoter responds to growth phase and it has been suggested that this response is regulated by the growth-phase-responsive transcription factor Fis (Green et al., 1996
). The ndh promoter region has three Fis-binding sites (Fig. 1
; Green et al., 1996
) and because of the relative affinities of Fis for each site it was suggested that binding to the high-affinity Fis I site might mediate activation, whereas progressive occupation of the lower affinity sites might lead to repression (Fig. 1
; Green et al., 1996
). Here we show that Fis bound at the high-affinity Fis I site has a small positive effect on ndh expression, but that a greater contribution is made by Fis occupying the lower affinity Fis II site. The location of the Fis II site (centred 72 bp upstream of the transcript start) and the observation that Fis-mediated activation of ndh expression is dependent on the C-terminal domain of the RNA polymerase
-subunit, indicates that the ndh promoter is a Class I Fis-activated promoter. Furthermore, it is confirmed that both FNR sites contribute to FNR-mediated repression under anaerobic conditions.
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METHODS |
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Plasmid construction and mutagenesis.
The ndh promoter fragments used were amplified by PCR from pGS418 (Sharrocks et al., 1991) using the EXPAND Hi-fidelity system (Roche). After restriction digestion at unique EcoRI and BamHI sites introduced into the PCR primers, the products were ligated into EcoRI/BamHI-digested pRW50 (Lodge et al., 1992
). Overlap PCR using specific mutagenic primers was used to replace the Fis sites with unrelated DNA sequences (see below). The authenticity of the constructs was confirmed by DNA sequencing.
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RESULTS |
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To investigate the role of Fis in the absence of FNR-mediated repression, -galactosidase activities from the ndh : : lacZ fusions in anaerobic cultures of RK5279 (fnr lac) were measured. The data revealed that deletion of the far upstream Fis I site (fusion N4) reduced ndh expression, suggesting that Fis bound at this site enhances ndh expression (Fig. 2c
). Deletion of the Fis II site (fusion N6) reduced ndh transcription still further, suggesting that Fis also acts positively at this site (Fig. 2c
).
To show that Fis was responsible for the effects observed, -galactosidase activities were measured in a fis lac mutant strain (JRG4656) and in an fnr fis lac mutant strain (JRG4203). These investigations confirmed that under anaerobic conditions both FNR sites contribute to FNR-mediated repression (Fig. 2d
). Moreover, in the absence of Fis and FNR, deletion of the Fis and FNR sites did not significantly alter ndh expression, showing that, as expected, deletion of the upstream Fis-binding sites has no effect on ndh expression in a fis background (Fig. 2e
).
The experiments described above suggested that Fis bound at two upstream sites within the ndh promoter activates ndh expression under anaerobic conditions. The ndh gene is normally expressed under aerobic conditions when FNR is inactive. Therefore, the effects of the ndh promoter deletions were tested under aerobic conditions. To ensure that the bacteria contained no residual active FNR protein, aerobic cultures of RK5279 (fnr lac) and JRG4203 (fnr fis lac) were used. For RK5279 the data show that deletion of the far upstream Fis I site (fusion N4) reduced ndh expression, which was further reduced by deletion of the Fis II site (Fig. 3a). In the absence of Fis, deletion of the Fis sites did not effect ndh expression, which was
5-fold lower than the levels observed in the presence of Fis (Fig. 3b
). Comparison of expression in the fis fnr double mutant in the absence (Fig. 2e
) and presence (Fig. 3b
) of oxygen suggests that at least one other regulatory protein modulates ndh expression. Previous work indicated that IHF, HU and an uncharacterized protein, Arr, interact with ndh promoter DNA (Green et al., 1997
). Thus, one or more of these transcription factors could be responsible for the threefold-lower levels of ndh expression observed under aerobic conditions in the absence of Fis. In conclusion, the experiments under aerobic conditions confirmed that Fis acts as a positive regulator of ndh expression by interaction with two sites located upstream of the basic promoter elements.
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It is well established that the intracellular concentration of Fis in E. coli is growth-rate-responsive (Ishihama, 1999). During early exponential growth Fis is the major nucleoid-associated protein, but during stationary phase it is amongst the least abundant (Ishihama, 1999
). Thus,
-galactosidase activities from the unaltered ndh promoter (Pndh) and the variant with a lesion in Fis II (Pndh+-+) were monitored during aerobic growth to determine the point of the growth cycle at which Fis-mediated activation of ndh expression was most effective. The results are shown in Fig. 5
. For Pndh, expression was greatest during exponential growth, when the intracellular Fis levels are high. As growth proceeded a steady decline in ndh expression was observed (Fig. 5
). In contrast, expression from Pndh+-+, or from Pndh in a fis mutant strain, was more uniform and lower throughout the growth cycle (Fig. 5
). These experiments suggest that Fis activates ndh expression during exponential growth by binding at the Fis II site in the ndh promoter.
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DISCUSSION |
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Fis is a multifunctional nucleoid-associated protein and is the most abundant transcriptional activator in exponential-phase cells (Ishihama, 1999). Fis is a homodimeric protein that binds to DNA through a C-terminal helixturnhelix motif (Osuna et al., 1991
). Because the DNA recognition helices of the Fis dimer are unusually close, a severe bend is induced in the DNA upon Fis binding (Pan et al., 1996
). The bending of target DNA is thought to be important for the function of Fis. To our knowledge this is only the second report showing that Fis can act as a Class I transcription activator, the other example being at rrn P1 promoters (Aiyar et al., 2002
). Class I activators bind to promoter DNA upstream of the -10 and -35 elements. The binding sites are located on the same face of the helix as RNA polymerase. Thus, Class I regulators may be located at, or close to -61, -71, -82 bp, etc., relative to the transcript start (Busby & Ebright, 1994
). At the ndh promoter the major Fis effect is mediated from a site 72 bp upstream of the transcript start. At the rrnB promoter Fis stimulates transcription from a site centred at -71·5 (Aiyar et al., 2002
). Specific protein : protein contacts are established between the C-terminal domain of the
-subunit of RNA polymerase and a small surface-exposed patch of Fis consisting of residues Q68, R71, G72 and Q74 (Aiyar et al., 2002
). These interactions involve only one of the
-C-terminal domains and the downstream subunit of the Fis dimer (Aiyar et al., 2002
). The location of the Fis II-binding site, and the observation that introduction of an RNA polymerase
-subunit lacking the C-terminal domain abolishes Fis-mediated activation, suggests that stimulation of ndh expression by Fis is mediated by identical protein : protein contacts to those established at the rrn promoters.
Investigation of the roles of the Fis I and Fis III sites revealed that the Fis I site has a small positive effect on ndh expression, whereas Fis III site had a small negative effect. Therefore, we are now able to suggest the effects of Fis acting at each of the three binding sites within the ndh promoter. Thus, the question marks in Fig. 1 can be replaced to indicate positive regulation from Fis sites I and II and negative regulation from site III.
Expression of nuoA-N, encoding the proton-translocating NADH dehydrogenase, is also positively regulated by Fis (Wackwitz et al., 1999). It has been suggested that this ensures high ATP yields at the outset of growth (Wackwitz et al., 1999
). This suggestion is consistent with the presence of a low-affinity repressing Fis site (Fis III) at the ndh promoter, which should ensure that Ndh I is used in preference to Ndh II when intracellular Fis levels are highest. As growth proceeds into exponential phase, Fis levels begin to fall. This will release the brake applied to ndh expression by Fis bound at Fis III, allowing Fis-mediated activation of ndh, and also lower Fis-mediated activation of nuoA-N expression. Such a scheme would be consistent with the relative affinities of Fis (estimated from DNase I footprinting reactions) for the nuo promoter (Fis 1,
20 nM; Fis 2,
40 nM; Fis 3,
100 nM; Wackwitz et al., 1999
), which are lower than those for the ndh promoter (Fis I,
0·1 nM; Fis II,
1 nM; Fis III,
10 nM; Green et al., 1996
), and provides a possible explanation for the presence of Fis III in the ndh promoter.
Previous work used site-directed mutagenesis to show that both FNR sites within the ndh promoter are necessary for full anaerobic repression of ndh expression (Meng et al., 1997). The data obtained here from deletion analysis confirms that this is the case. Recent work to investigate regulation of yfiD gene expression, and studies with a series of model promoters with tandem FNR sites, suggested that appropriately spaced tandem FNR dimers act together to repress transcription (Marshall et al., 2001
; Barnard et al., 2003
). Thus, although an FNR dimer located at -50·5 would be expected to interfere with the docking of the
-subunit of RNA polymerase with DNA and thus partially occlude the ndh promoter, the binding of a second upstream FNR dimer would appear to enhance repression, perhaps by stabilizing the FNR : DNA complex, allowing it to effectively compete with the RNA polymerase
-C-terminal domains for this region of the promoter. An
-C-terminal domain that is not bound to either DNA or to a transcriptional activator is thought to inhibit transcription. Moreover, it has been shown here that Fis bound at the Fis II site is a positive regulator of ndh expression. Therefore, by occupying sites located immediately upstream and downstream of the Fis II site, FNR acts as a physical barrier, preventing the formation of the Fis : RNA polymerase contacts necessary for Fis-mediated transcription activation. Thus, it is likely that interactions between the tandem FNR dimers conspire to inhibit ndh transcription at several levels, i.e. by preventing interaction between the
-C-terminal domains of polymerase with DNA and with Fis. This promoter architecture must also prevent the formation of contacts between FNR and RNA polymerase that result in activation of transcription.
In conclusion, it is shown that ndh expression is driven from a Class I Fis-activated promoter and that Fis-mediated activation is inhibited under anaerobic conditions by FNR binding to two sites immediately flanking the region occupied by Fis. Activation of ndh transcription by Fis could account for the enhanced level of ndh expression during periods of rapid growth. This could be necessary to recycle NADH and achieve redox balance independently of energy generation during periods of rapid growth.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 October 2003;
revised 19 November 2003;
accepted 19 November 2003.
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