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J Biol Chem, Vol. 273, Issue 45, 29929-29934, November 6, 1998

Suppression of leu-500 Mutation in topA⁺ Salmonella typhimurium Strains
THE PROMOTER RELAY AT WORK^*

Ming Fang and Hai-Young Wu

From the Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201

	ABSTRACT

Top Abstract Introduction Procedures Results Discussion References

Suppression of leu-500 mutation in Salmonella typhimurium topA strains has been one of the most fascinating examples for the DNA supercoiling effect on transcription initiation control. Previous studies have indicated possible involvement of transcription-driven DNA supercoiling in the activation of the leu-500 promoter in topA strains. Our recent studies have shown that ilvIH transcription activity located 1.9 kilobase pairs upstream is the initial supercoiling signal for leu-500 activation via a promoter relay mechanism. In the present communication, we show that the ilvIH transcription activity-initiated promoter relay can result in leu-500 activation in topA⁺ strains. In addition, suppression of the chromosomal leu-500 mutation correlates with the transcription activities of ilvIH and leuO rather than the TopA level in the topA⁺ strain. It appears that the leu-500 suppression in a topA strain is due to the constant ilvIH transcription activity.

	INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

The leu-500 mutation, originally isolated from 5-bromouracil-treated Salmonella typhimurium LT2, is an A to G transition at the Pribnow box of the promoter of the leucine operon. This mutation significantly reduces the promoter activity and results in leucine auxotrophy (1). The second mutation in an unlinked suppressor gene (supX), located in the cysteine B and tryptophan region of the S. typhimurium chromosome, was subsequently shown to suppress the leu-500 mutation (2, 3). The supX was later identified to be the mutation in topA (4), the structural gene for topoisomerase I (TopA) which relaxes negative DNA supercoils (5). The mutation in topA affects the overall DNA superhelicity which is primarily maintained by the counter enzymatic activities of TopA and gyrase in bacteria (6-8) (reviewed in Refs. 9 and 10). The hypernegative DNA supercoiling in the topA mutants was interpreted to be responsible for the restoration of transcriptional initiation from the mutant leu-500 promoter (4). However, the suppression of the leu-500 mutation correlated only with the absence of TopA but not with the degree of overall negative superhelicity in S. typhimurium (11). When the minimal leu-500 promoter (80 to +87 of the operon) was subcloned in an extrachromosomal DNA, the plasmid-borne leu-500 promoter failed to be activated in the topA mutant (11). These findings have challenged the overall supercoiling explanation and raised the possibility that in addition to the topA genetic background, some cis-factors must be required for leu-500 activation. Transcription-driven supercoiling has been shown to be one of the possible cis-factors which are required for activating the leu-500 promoter. However, the transcription-driven supercoiling effect is usually short-ranged (<250 bp)¹ (12-14). We have recently shown that the short-range supercoiling effect can be extended up to 400 bp when a stronger promoter, ptac, is used to drive the transcription-induced supercoiling.² When searching for any upstream transcription activity responsible for leu-500 activation in the chromosomal context, we have subsequently demonstrated that ilvIH promoter activity located 1.9 kb upstream is responsible for activating the leu-500 promoter in a S. typhimurium topA strain, CH582 (15). Further characterization has revealed that the long range (1.9 kb) interaction between the ilvIH and leu-500 promoters is mediated by a promoter relay mechanism whereby transcription activity of the ilvIH promoter activates the leuO promoter located within the 1.9-kb intervening sequence, and both the leuO promoter activity and the LeuO protein are required for subsequent leu-500 activation (16).

Thus far both the absence of TopA and the promoter relay initiated by ilvIH transcription activity are shown to be involved in leu-500 activation in the topA strain. Mechanistically, it is important to understand how these two factors play roles in activating the leu-500 promoter. Our previous study showed that the mutation in either ilvIH or leuO promoter abolished leu-500 activation in a topA strain (15, 16), suggesting that the absence of TopA (the topA genetic background) itself is insufficient to activate the leu-500 promoter. In the present study, we examined the effect of the promoter relay alone on leu-500 activation in topA⁺ strains. Strikingly, we found that the ilvIH activity-initiated promoter relay can result in leu-500 activation in topA⁺ strains, while the absence of TopA enhances the promoter relay-mediated leu-500 activation by two-fold. Furthermore, suppression of chromosomal leu-500 mutation in a topA⁺ strain correlated only with the expression of ilvIH and leuO genes but not with the cellular TopA level. The leu-500 activation in a topA strain also correlated with constant ilvIH transcription activity as determined by Northern analysis. It appears that ilvIH was turned on which resulted in the suppression of leu-500 mutation in topA strains. We therefore conclude that the ilvIH transcription activity-initiated promoter relay mechanism plays a decisive role while TopA plays a negative regulatory role in leu-500 activation.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Plasmid Constructs-- The 3476-bp NotI-NotI fragment containing the -galactosidase gene (lacZ) isolated from pSV (CLONTECH) was used to replace the 1291-bp HindIII-NdeI fragment of pWU804, pWU805, pWU807, and pWU804M (16) to generate plasmids, pWU804LZ, pWU805LZ, pWU807LZ, and pWU804MLZ (plasmid maps in Figs. 1 and 3). The lacZ coding region was, therefore, transcriptionally fused with the leu-500 promoter to report the promoter activity in these plasmids.

Bacterial Strains-- S. typhimurium LT2 derivatives: CH582, a topA2726 leu-500 ara9 and its isogenic parental strain, CH601, leu-500 ara9 (8) were provided by Dr. David Lilley. CH601 was derived from PM596 as described previously (8). PM596, an araB9,leu-500 strain, was derived from CV468, an araB9,gal205 S. typhimurium LT2 strain (3). Both PM596 and CV468 were provided by Dr. Joseph Calvo. Unless stated otherwise, the strains were grown aerobically at 32 °C in the synthetic medium SSA without the leucine supplement (17). 40 µg/ml leucine was supplemented when necessary. Plasmids were transformed into bacteria by electroporation.

RNA Isolation and Northern Analysis-- Total RNA was isolated from cell cultures as described previously (15). RNA concentration was measured by the absorbance at 260 nm. 100 µg of total RNA per sample were fractionated in the denaturing agarose gel as described previously (18). The fractionated RNA was then transferred and immobilized to a nitrocellulose membrane (Schleicher & Schuell, BA85). The size and amount of ilvIH or leuO mRNA was determined by hybridizing the membrane with the specific ³²P-labeled DNA probe at 42 °C overnight. The DNA probe for detecting ilvIH mRNA was the 279-bp AccI-AccI fragment isolated from the ilvI coding region. A synthetic 28-bp oligomer consisting of the DNA sequence downstream of the leuO transcription start site was used for detecting leuO mRNA. Nick translation was used to label the ilvIH probe. T4 kinase was used to end-label the leuO probe with [-³²P]ATP. The hybridized nitrocellulose membrane was washed with high stringency buffer (15 mM NaCl, 0.5 mM NaH₂PO₄, 0.5 mM Na₂HPO₄, and 0.1% SDS) twice at 50 °C for 10 min each time. The hybridization results were visualized using autoradiography.

TopA Immunoblotting-- Cells were washed and resuspended in Tris-Cl pH7.5, 1 mM EDTA, and 1 mM dithiothreitol. The resuspended cells were then sonicated on ice for 1 min in four 15-s pulses with 30 s of cooling time on ice between pulses. The protein concentration of each sample was determined by BCA assay (Pierce). 25 µg of total protein from each sample was loaded for 8% SDS-PAGE. Two identical gels were prepared. One of the gels was stained with Coomassie Blue to verify protein loading. The other gel was electroblotted to the nitrocellulose membrane. Escherichia coli DNA topoisomerase I antibody (obtained from Dr. Rolf Menzel and Dr. Haiyan Qi) was the primary antibody. A secondary peroxidase-linked anti-rabbit IgG antibody was used for ECL detection (Amersham Life Science Ltd). Due to the high sequence homology of topA between E. coli and S. typhimurium, the rabbit antibody raised against E. coli TopA antibody is equally efficient in immunologically detecting S. typhimurium TopA as previously stated (19).³

-Galactosidase Assay-- -Galactosidase assays were performed as described by Miller (20) with slight modifications. At each time point, the growth of the bacterial cultures were stopped by placing aliquots of the culture in an iced H₂O bath for a minimum of 20 min before proceeding with the assay. Cells (4.5 ml) were made permeable by mixing (10-s vortex) them with 0.5 ml of 10× Z buffer (formula as described in Miller (20)), 175 µl of chloroform, and 100 µl of 0.1% SDS. After incubating at 28 °C for 5 min, the permeabilized cells were incubated with 1 ml of 8 mg/ml o-nitrophenyl--D-galactoside at 28 °C. The -galactosidase kinetics was determined by measuring the activity at several time points during the incubation. At each time point, 1 ml of sample was transferred into a tube containing 416 µl of 1 M Na₂CO₃ stop solution. After centrifugation at 14,000 × g for 10 min, the supernatant was used to measure A₄₂₀ and A₅₅₀. The Miller units of the -galactosidase activity were calculated using the formula in Miller's procedure (20). The -galactosidase activity was normalized according to the plasmid copy number as determined by Southern analysis. All reported -galactosidase activities are the averages of data from three separate experiments. The standard deviation is included as the error bar in the graphs.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Activation of the Plasmid-borne leu-500 Promoter in a topA⁺ Strain-- Using a plasmid model, we have demonstrated the long range interaction between the ilvIH and leu-500 promoters in CH582 (15) to explain the original observation of the suppression of leu-500 mutation in the S. typhimurium topA strain (2, 3). The fact that leu-500 activation is mediated by the upstream promoter activity has led to the suggestion that topA genetic background may not have a direct effect on leu-500 activation. In other words, the ilvIH activity-dependent leu-500 activation may also occur in topA⁺ strains as long as the upstream ilvIH promoter is active. The plasmid pWU804LZ derived from the previously established pWU800 series (15) was used to test this possibility in both topA⁺ and topA strains, CV468 and CH582, respectively. The pWU804LZ-harboring cells were grown in SSA(Leu) medium since the absence of leucine in the medium is required for activating the ilvIH promoter (21-24). The -galactosidase assay results showed that the plasmid-borne leu-500 promoter was active in CV468 when cells grew to mid-log phase, and leu-500 promoter activity was 2-fold higher in CH582 than that in CV468 (Fig. 1). This result indicated that, in the presence of active ilvIH transcription, the leu-500 mutation can also be suppressed in a topA⁺ strain. The enhanced activation in the topA strain suggests that TopA plays a negative regulatory role in leu-500 activation. To further determine that leu-500 activation is mediated by the upstream ilvIH transcription activity, we measured the plasmid-borne leu-500 promoter activity in pWU805LZ, where the ilvIH promoter was deleted, in both topA⁺ and topA strains. In good agreement with the previously shown primer extension results (16), the deletion of ilvIH promoter abolished leu-500 activation in CH582 (Fig. 1), indicating that the -galactosidase level from the lacZ reporter provided a reliable indicator of the leu-500 promoter activity. As shown in the topA strain, CH582, deletion of the ilvIH promoter also eliminated leu-500 activation in CV468 (Fig. 1), suggesting that it is the ilvIH transcription activity rather than the topA genetic background that plays a decisive role in leu-500 activation.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   leu-500 activation in a S. typhimurium topA+strain. The pWU804LZ contains the lacZ reporter gene which is transcriptionally fused with the leu-500 promoter; otherwise it is identical to pWU804 (15, 16). Plasmid pWU805LZ is identical to pWU804LZ except that the ilvIH promoter is absent. The -galactosidase activities of pWU804LZ- or pWU805LZ-harboring CV468 (lanes 1 and 2, respectively) and CH582 (lanes 3 and 4, respectively) were assayed at OD650 of 0.8.

The Suppression of Chromosomal leu-500 Mutation in a topA⁺ S. typhimurium-- The activation of the plasmid-borne leu-500 promoter in a topA⁺ strain has prompted us to hypothesize that the chromosomal leu-500 promoter can also be activated in topA⁺ strains as long as the ilvIH expresses under certain growth conditions. This hypothesis was tested by monitoring the growth of CH601 in SSA(Leu) medium. CH601, the parental strain of CH582, is considered to be leucine auxotrophic, because it carries the leu-500 mutation and the wild type topA on chromosome (8, 11). The growth of CH601 in leucine-free medium (leucine prototrophy) is a good indicator of leu-500 activation in the topA⁺ strain. Strikingly, we observed that CH601 grew in SSA(Leu) after an unusually prolonged lag phase (16 h after 1:250 inoculation or 19 h after 1:500 inoculation) (Fig. 2). In contrast, with the leucine supplement, CH601 started to grow 6 h post 1:500 inoculation (Fig. 2). The fact that grown cells repeated the prolonged lag phase in many runs of reinoculation has ruled out the possibility that mutants were selected during the period of prolonged lag phase. The growth curve shown in Fig. 2 is the result of the second inoculation. This striking result prompted us to test the growth of another S. typhimurium leu-500 strain, PM596, which was the parental strain of CH601 (8). Similar leucine-independent growth after a prolonged lag phase was observed (data not shown). These S. typhimurium leu-500 strains did not grow on SSA(Leu) plates but grew in SSA(Leu) liquid medium, indicating that some physiological changes must occur during the prolonged lag phase in the liquid medium. Since the plasmid-borne leu-500 activation is mediated by the ilvIH transcription activity-initiated promoter relay in CV468 (Fig. 1), we tested whether the suppression of leu-500 mutation in the topA⁺ strain, CH601, is also due to the ilvIH transcription activity.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The growth of the topA+ strain, CH601, in the leucine-free medium. Growth of CH601 in either SSA(+Leu) or SSA(Leu) medium was monitored after 1:250 dilution of the overnight culture at 32 °C by measuring OD650 at the indicated time.

The leu-500 Activation in CH601 Is Mediated by ilvIH Promoter Activity-initiated Promoter Relay-- Again, using the reporter plasmid, pWU804LZ, we were able to detect the plasmid-borne leu-500 activation in the presence of ilvIH promoter activity when pWU804LZ-harboring CH601 were grown in SSA(Leu) medium. The level of leu-500 activity is comparable with that in CV468 and 50% less than that in CH582 (Fig. 3A). The ilvIH promoter activity is crucial for leu-500 activation, since mutation or deletion of the ilvIH promoter in pWU807LZ or pWU805LZ significantly diminished leu-500 activation in these reporter plasmids (Fig. 3B, compare columns 1 and 2, or columns 1 and 3). We have recently demonstrated that expression of the intermediate leuO gene relays the ilvIH transcription activity to the leu-500 promoters in CH582 (16). To examine whether the promoter relay is also the case in CH601, the reporter plasmid pWU804MLZ, which carries the mutant leuO promoter, was tested. The -galactosidase activity was shown to be significantly lowered due to the mutation of the leuO promoter (Fig. 3B, column 4), suggesting that the plasmid-borne leu-500 activation in CH601 is also mediated by the ilvIH promoter activity-initiated promoter relay mechanism.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Promoter relay-mediated leu-500 activation in CH601. The -galactosidase activities of pWU804LZ-harboring CV468, CH582, and CH601 were assayed at OD650 of 0.8 as shown in panel A. The relevant regions in pWU804LZ, pWU805LZ, pWU807LZ, and pWU804MLZ are illustrated. The ilvIH promoter is deleted in pWU805LZ and is mutated (X) in pWU807LZ. The leuO promoter is mutated (X) in pWU804MLZ. Otherwise, these plasmids are identical. The -galactosidase activities of CH601 carrying one of the above plasmids were assayed at OD650 of 0.8. and shown in panel B, columns 1, 2, 3, and 4, respectively.

The above results led us to speculate that the suppression of leu-500 mutation on the chromosome is likely due to the promoter relay as well. If so, the ilvIH and leuO transcription activities should correlate with leu-500 activation in the prolonged lag phase of CH601 in SSA(Leu) medium. To investigate this possibility, Northern analysis was performed to monitor the ilvIH and leuO mRNAs at different time points during the prolonged lag phase. CH601 grew in SSA(+Leu) medium until the OD₆₅₀ reached approximately 0.6. After spinning down and washing with SSA(Leu) medium, the cells were continued to be incubated aerobically in SSA(Leu) medium. CH601 cells resumed growth 16 h after the medium change (the growth curve in Fig. 4). Our Northern blot results revealed that both ilvIH and leuO genes were active just before cells resumed growing (15 h after the medium change), but were silent at early lag phase (6 h after medium change) (Fig. 4, A and B). Therefore, the ilvIH transcription activity may serve as the supercoiling signal for chromosomal leu-500 activation via activating the leuO gene in CH601. It appears that ilvIH transcription is highly regulated in CH601 in response to nutrient (leucine) starvation.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Expression of chromosomal ilvIH and leuO correlates with activation of the chromosomal leu-500 promoter in CH601. Total RNA was isolated from aliquots of CH601 grown in SSA(Leu) at (1) 6 h and (2) 15 h post medium change and fractionated on a 1% agarose gel. The ethidium bromide-stained RNA gel is shown in panel C. The Northern blot detecting ilvIH or leuO mRNAs is shown in panels A and B, respectively.

The TopA Level Remains Constant While ilvIH Expression Is Highly Regulated in CH601-- So far, our data have indicated that the promoter relay is responsible for leu-500 activation regardless of topA genetic background. Previous studies, however, have shown that leu-500 activation was dependent on the absence of TopA, although leu-500 activation did not correlate with the overall DNA superhelicity (11). TopA must play some kind of a role in leu-500 activation. TopA may indirectly affect leu-500 activation via either (a) regulating ilvIH transcription activity or (b) suppressing the negative supercoiling signals generated by ilvIH and leuO transcription processes. In order to examine the first possibility, we monitored the TopA level and ilvIH transcription activity simultaneously during the growth of CH601 in SSA(Leu) medium. CH601 were grown in SSA(+Leu) medium to OD₆₅₀ of 0.6, and the growth resumed in SSA(Leu) medium as described above (Fig. 4). Cells were harvested at three time points after the medium change (Fig. 5 growth curve, points 1, 2, and 3). The harvested cells from each time point was divided into two parts for monitoring the TopA protein level using immunoblotting (Fig. 5A) and the ilvIH mRNA using Northern blotting (Fig. 5C). While the Northern blot data indicated that ilvIH expression was highly regulated, it was active near the end of the prolonged lag phase and silent once cells resumed exponential growth (Fig. 5C), the immunoblot indicated that TopA level remained constant across the three time points during the CH601 cell growth in SSA(Leu) medium (Fig. 5A). This result ruled out the possible effect of TopA levels on ilvIH expression.

View larger version (55K): [in this window] [in a new window]   Fig. 5.   ilvIH expression is highly regulated in CH601 while the cellular TopA level remains constant. Total bacterial protein lysates or RNA were prepared from aliquots of CH601 grown in SSA(Leu) at (1) 6 h, (2) 15 h, and (3) 20 h post medium change. The Coomassie Blue-stained protein gel and the TopA (approximate 75 kDa) Western blot results are shown in panels B and A, respectively. The ethidium bromide stained RNA gel and the ilvIH mRNA Northern blot results are shown in panels D and C, respectively. The lanes 1, 2, and 3 in all panels correspond to the above time points.

If TopA does not affect leu-500 activation via regulating ilvIH expression (illustrated in Fig. 6), the remaining possibility is that the promoter relay is affected negatively by TopA on ilvIH and/or leuO transcription-generated negative supercoiling signals (illustrated in Fig. 6). Hence, TopA affects leu-500 activation indirectly only when ilvIH and leuO promoters are active in the promoter relay. When TopA is absent, the supercoiling signals generated from the ilvIH and leuO promoters are expected to be maximal and activation of the leu-500 promoter is thus facilitated.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   The roles of TopA in promoter relay-mediated leu-500 activation. See text.

Since the ilvIH transcription activity plays an indispensable role in leu-500 activation, we predicted that ilvIH must be active in CH582 during normal growth conditions. This prediction was confirmed by the Northern blot data that ilvIH was expressed constantly during the log phase of CH582 growth (Fig. 7). This may explain why the suppression of the leu-500 mutation was originally observed in the topA strains including CH582 (2, 3, 11).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   The constant ilvIH expression in CH582. The total RNA was isolated at two time points (1 and 2) during the exponential growth of CH582. ilvIH mRNA detected by Northern blot and total RNA stained with ethidium bromide are shown in panels A and B, respectively. Lanes 1 and 2 in both panels correspond to the samples of the two time points.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

We have demonstrated using both plasmid and chromosome systems that in the presence of the 1.9-kb chromosomal fragment upstream of the leuABCD operon, ilvIH activity-initiated leu-500 activation is independent of topA genetic background, since CH601 (the topA⁺ strain) can overcome its leucine auxotrophy and grow in leucine-free medium while the cellular TopA level remains constant. However, previous studies have shown that activation of the plasmid-borne leu-500 promoter is absolutely dependent on the absence of TopA (topA genetic background) when the leu-500 promoter is flanked by various plasmid sequences (12-14, 25). These contrasted results suggest that, owing to the difference in DNA sequence context where the leu-500 promoter is located, the mechanisms underlying these two types of leu-500 activation are different despite the involvement of transcription-driven DNA supercoiling in both cases. The topA-dependent leu-500 activation by an adjacent promoter activity was demonstrated using a different, non-native sequence flanked by a pair of divergent promoters (12-14). It appears that upstream transcription-driven negative supercoiling propagates along the intervening sequence and directly activates the leu-500 promoter. Lilley's group also demonstrated that the coupling of tetA-mediated transcription and translation induced an increase in overall negative supercoiling and activated the leu-500 promoter in a closed circular DNA (25). TopA relaxes the negative supercoiling, therefore the absence of TopA is required for the direct effect of transcription-driven supercoiling on leu-500 activation. When the leu-500 promoter is associated with the 1.9-kb upstream natural sequence, the long range interaction between the ilvIH and leu-500 promoters is somewhat more complex than the direct effect of DNA supercoiling. Although ilvIH transcription-induced supercoiling initiates the long range promoter interaction, other protein factors and DNA elements within the intervening sequence relay the promoter activity over a long (1.9 kb) distance. We have recently shown that ilvIH first activates the leuO gene located within the intervening sequence, and both the leuO promoter activity and the LeuO protein are required for subsequent leu-500 activation (16). Although the distance between the two divergent leuO and leu-500 promoters is within the 400-bp limit for the direct transcription-driven supercoiling effect, the leuO promoter activity alone was insufficient to activate the leu-500 promoter (16), suggesting that the direct supercoiling effect does not account for the suppression of leu-500 mutation in the chromosomal context. The evolutionarily conserved AT-rich sequence (26) between the two divergent leuO and leu-500 promoters may repress the direct supercoiling effect, and the LeuO protein can derepress and activate the leu-500 promoter in the presence of the cis-acting leuO promoter activity.² It appears that ilvIH transcription-induced DNA supercoiling is relayed via the 1.9-kb intervening sequence involving protein factors and DNA elements in a very efficient manner, so that TopA negatively regulates but does not efficiently abolish the supercoiling signal which may be constrained by the protein factors and DNA elements during the relaying process.

Since ilvIH expression is crucial in initiating the promoter relay, it is important to know what regulates ilvIH expression. The Northern blot data indicated that ilvIH transcription is under tight regulation (Fig. 5C). The ilvIH promoter activity is normally silent until cells are under nutrient (leucine) starvation such as the prolonged lag phase of CH601 in the SSA(Leu) medium. We have ruled out the possibility that ilvIH expression is affected by the cellular TopA level (Fig. 5). It has been shown that ilvIH promoter activity is under the positive control of Lrp (leucine-responsive regulatory protein), which is a global transcription regulator whose cellular level is up-regulated by cellular guanosine 3',5'-bispyrophosphate (ppGpp) in response to nutrient limitation (27, 28). Combined with the previous studies, these results have suggested that ilvIH transcription activity-initiated promoter relay is a transcription regulatory mechanism elicited in response to stress. We are currently conducting experiments to test the roles of promoter relay in this stress response pathway.

Unlike CH601, where ilvIH expression is cryptic during exponential growth, ilvIH is constantly expressed in CH582 (Fig. 7). We observed that the growth rate of CH582 is two times slower than that of CH601. The slow growth rate is shown to up-regulate lrp gene expression (28), which presumably activates the ilvIH promoter constantly in CH582. The constant ilvIH transcription activity is likely to be the reason why suppression of the leu-500 mutation occurs constantly in the topA strains.

Co-detection of the normally silent leuO gene expression with ilvIH indicated that LeuO protein may play a role in such a stress response in bacteria. Besides affecting wild type leuABCD promoter (16), random screening studies have recently shown that LeuO overexpression affects three other unrelated genes at distinct chromosomal locations (29-31), suggesting its global regulatory role in gene expression regulation under stress. The exact physiological implication of leuO expression under the control of the promoter relay at this chromosomal location is very interesting but remains unclear. We may have just begun to unravel the importance of global transcription regulatory functions of LeuO protein through understanding the mysterious leu-500 activation phenomenon in topA mutant which was first reported more than 30 years ago (2).

	ACKNOWLEDGEMENTS

We thank Dr. Amit Banerjee and Terry Barrette for critical reading and comment on the manuscript. We are grateful to Drs. Rolf Menzel and Haiyan Qi for providing E. coli TopA antibody; Drs. David Lilley and Joseph Calvo for providing S. typhimurium strains.

	FOOTNOTES

^* This work was funded by National Institutes of Health Grant GM53617.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 313-577-1584; Fax: 313-577-6739; E-mail: haiwu{at}med.wayne.edu.

The abbreviations used are: bp, base pair(s); kb, kilobase pair(s).

² M. Fang and H.-Y. Wu, unpublished data.

³ J. C. Wang and L. F. Liu, unpublished data.

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