LeuO Expression in Response to Starvation for Branched-chain Amino Acids*

Arundhati MajumderDagger , Ming FangDagger , Kan-Jen Tsai§, Chiharu Ueguchi, Takeshi Mizuno||, and Hai-Young WuDagger **

From the Dagger  Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201, the § School of Medical Technology, Chung Shan Medical and Dental College, Taichung 402, Taiwan, and the  BioScience Center and || Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received for publication, January 31, 2001, and in revised form, March 7, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recently identified role of LeuO in the regulation of transcription has prompted us to search for the specific function(s) of LeuO in bacterial physiology. The cryptic nature of expression of leuO has previously limited such analysis. A conditional leuO expression was found when bacteria enter stationary phase and was shown to be guanosine 3',5'-bispyrophosphate-dependent. Multiple physiological events, including the stringent response, are induced upon the increase of the bacterial stress signal, guanosine 3',5'-bispyrophosphate. In this study, we tested whether LeuO was directly involved in the bacterial stringent response. LeuO was shown to be indispensable for growth resumption following a 2-h growth arrest caused by starvation for branched-chain amino acids in an E. coli K-12 relA1 strain. This result supports a functional role for LeuO in the bacterial stringent response.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The overexpression or underexpression of leuO has been shown to affect a number of bacterial hns- phenotypes (1-3). LeuO may effect these unrelated genes via its transcription regulatory role, based on evidence reported in our recent study (4). By studying the leu-500 activation phenomenon in the Salmonella typhimurium topA- strains (5-10), we have identified a 72-bp1 bacterial gene silencer, AT4, located upstream of leuO. LeuO appeared to negate AT4-mediated gene silencing (4). This provided a molecular basis for the speculated transcription regulatory role of LeuO. However, owing to the cryptic nature of the expression of leuO in rapidly growing bacterial cells under standard growth condition, the physiological impact of the leuO expression was unclear. We have recently found a conditional leuO expression when bacterial cells enter the stationary phase grown in rich media/LB (11). Such a conditional leuO expression was demonstrated to be guanosine 3',5'-bispyrophosphate (ppGpp)-dependent. Since ppGpp is the stress signal in the bacterial stringent response pathway (reviewed in Ref. 12), leuO may be one of those genes that are not essential during exponentially growth of laboratory cultures but important for cell survival in more stressful natural environments.

In order to better understand the physiological impact of leuO expression, we studied the LeuO function in the ppGpp-dependent bacterial stringent response. Bacterial stringent response is caused by a limited availability of amino acids or by exhaustion of the primary carbon source (reviewed in Ref. 12). Hence, the stringent response can be induced by limiting the availability of a certain amino acid when such a strain that is auxotrophic for that amino acid is growing in a chemically defined medium. More interestingly, a stringent response can also be induced in an autotrophic strain due to the toxic effects of some amino acids. For example, an overdose of valine was known to cause isoleucine starvation in the Escherichia coli K-12 strain (13), while supplementation with one-carbon amino acids, serine, methionine, and glycine (SMG) in a chemically defined minimal medium was known to deplete the endogenous branched-chain amino acids (14). The SMG-caused toxic effect can be reversed by supplementing with branched-chain amino acids or the precursors of branched-chain amino acid biosynthesis (15). These observed toxic effects reflect the complex metabolite-mediated feedback controls in the bacterial biosynthetic pathways for amino acids (13).

The relA gene encodes an enzyme responsible for the synthesis of ppGpp, which is crucial in triggering the bacterial stringent response. It was not surprising to find that SMG inhibited the growth of relA mutants (13-15). Under such a branched-chain amino acid-depleted condition, the growth of both the relA wild-type (stringent) strains and the relA mutant (relaxed) strains were dramatically interrupted. Upon the nutrient downshift, the stringent strains resumed normal growth within several hours, whereas the relaxed strains remained inhibited. Since the addition of branched-chain amino acids could efficiently reverse the inhibition (15), it suggested strongly that derepression of the ilv (isoleucine-leucine-valine) operons are impaired in the relA mutants while the derepression can be induced in the relA+ strains within 1-4 h as a stringent response (15). The ilv operons encode acetohydroxy acid synthase isozymes that are responsible for the synthesis of precursors for the biosynthesis of branched-chain amino acids, isoleucine, leucine, and valine (reviewed in Ref. 16). The expression of leuO was shown to be regulated via a promoter relay mechanism (5) that is decisively controlled by the transcription activity of ilvIH, which is one of the three ilv operons in bacteria. We therefore tested whether the specific ilv derepression revealed the potential function of LeuO in response to starvation for branched-chain amino acids. If the expression of leuO is indeed induced upon the derepression of the ilv operon via the promoter relay, then LeuO function may be important for the bacterial stringent response. Clavo's group has demonstrated that increase in cellular ppGpp level is crucial for the expression of the leucine-responsive regulatory protein (Lrp), which is the positive regulator for the expression of ilvIH (17, 18). This result provides a reasonable explanation for why the biosynthesis of ppGpp is required for the expression of leuO (11). However, it was unclear whether the level of leuO expression correlated with the cellular ppGpp level.

To address these questions, we took advantage of the E. coli relA1 strains that are "relaxed" but retain a residual "stringent response capability" owing to a low level ppGpp synthetic capacity (19). The relA1 mutation is due to an insertion of an IS2 element between the codons 85 and 86 of the relA gene (20). Owing to promoter activity originating from the IS2 insert, the mutant gene retains a low level of ppGpp synthetic activity. The residual stringent response capability of the relA1 strain may be important for a "slow onset" derepression of ilv operons that allows detection of transient leuO induction upon the ilv transcription activity. The ilv derepression is expected to be efficient in a relA+ stringent strain, as are other stringent responses due to the "rapid onset" of cellular ppGpp in the relA+ strain. These phenotypes are in contrast to that of a completely relaxed strain, such as a relA/spoT double mutant, which will not cause the derepression of ilv due to the absence of ppGpp and will not survive the starvation of branched-chain amino acids either (21).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacteria-- Bacteria used in this study were derived from an E. coli K-12 strain, MC4100 (22). The genotypes are listed in Table I. TO2 is a Delta leuO strain due to the replacement of the BstXI-ApaI fragment of the putative leuO coding region with a DNA fragment carrying the camr gene (3). MF1 was prepared by introducing the leuO::cam mutation into MC4100 (recipient strain) by P1 transduction using TO2 as a donor strain. Hence, MC4100 and MF1 was a leuO+ and leuO- isogenic pair. Disruption of chromosomal leuO in MF1 was confirmed by the restriction enzyme cleavage pattern and the size of the DNA product generated by polymerase chain reaction (PCR) using primers flanking the camr insert. Western blotting (Fig. 4G) further confirmed the LeuO deficiency of MF1.

                              
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Table I
Bacterial strains used in this study

RH147 and RH12079 were gifts from Dr. Hengge-Aronis (Freie University Berlin, Germany). The isogenic pair of relA+ and relA1 strains was constructed by P1 transduction of relA+ or relA1 allele linked fuc-3072::Tn10 into MC4100 (23). RH12079 is essentially MC4100, and RH147 is the relA+ derivative of MC4100.

Bacteria were grown aerobically at 37 °C in a synthetic (chemically defined) medium base, SSA, which is a minimal salt buffer (K2HPO4 (Sigma), 10.5; KH2PO4 (Sigma), 4.5; (NH4)2SO4 (Sigma), 1.0; sodium citrate dihydrate (Sigma), 0.97 (each component is in grams per liter of deionized water)) plus 4 µg/ml thiamin, 0.2% glucose, 20 µg/ml guanine, 40 µg/ml adenine, and 50 µg/ml MgSO4. The no-aa SSA medium is the SSA medium base without any amino acid supplement. The following amino acids were supplemented to the SSA medium base for the various SSA media as described under "Results." 20 µg/ml each of L-histidine, L-lysine, L-tyrosine, L-tryptophan, L-threonine, L-cysteine, L-methionine, L-serine, L-alanine, and L-aspartic acid, and 40 µg/ml each of L-phenylalanine, L-glutamic acid, L-proline, L-arginine, L-valine, L-isoleucine, L-leucine, L-asparagine, L-glutamine, and L-glycine were used when necessary. Ampicillin (50 µg/ml) was added to cultures as needed.

Due to the possible selection of RNA polymerase mutants in the synthetic media with various amino acid supplements (15), fresh cultures inoculated from the frozen stocks were prepared for each experiment. Prolonged exposure to the selection pressure was prevented. As a precaution, at the end of each experiment, the relA1 strains were checked for the characteristics of the relA1 mutation using both the serine hydroxamate inhibition assay (24), and the susceptibility assay for the toxic effect mediated by SMG, or by an excessive amount of valine or leucine as described previously (14).

Plasmid-- pWU204 was made by a two-step cloning procedure. First, a 1291-bp HindIII-NdeI fragment containing galK coding region was deleted from pWU804 (10) to generate pWU804S. Second, a 1919-bp fragment containing E. coli chromosomal sequence from the ilvIH promoter (+53 of the ilvIH operon; Ref. 25) to the leu promoter (+64 of the leu operon; Ref. 26) was generated by PCR. The sequence of the 1.9-kilobase pair DNA has been determined and deposited to the GenBankTM data base (NCBI accession no. AF106955). BamHI and AccI sites were introduced into the 5' and 3' end of the PCR product, respectively, by incorporating mismatch base pairs in the primers. The BamHI/AccI-digested PCR product was cloned into pWU804S for replacing the 1.9-kilobase pair S. typhimurium sequence flanked by BamHI and AccI sites and resulted in pWU204. The parental plasmid, pWU800, from which pWU804 was originally derived (9) was used as the vector control.

Western Blot Analysis-- Bacterial cells were harvested and prepared for total protein lysates. Cell densities of the harvested bacterial cultures were determined by the viable cell counts using LB agar plates. The protein concentration of lysate was determined using BCA protein assay kit (Pierce). Based on the obtained information, lysate prepared from 5 × 106 viable cells contains ~25 µg of total protein. A fixed amount (25 µg) total protein from each lysate was electrophoresed on a SDS-PAGE and transferred onto a nitrocellulose (NC) paper for the subsequent immunoblotting. Hence, the detached LeuO level was normalized to both total cellular protein content and viable cell number.

A LeuO-specific antiserum was raised by injecting the purified overexpressed S. typhimurium His-tagged LeuO into a rabbit. The affinity-purified IgG (1.4 mg/ml) from the antiserum was used at a dilution factor of 1:5000 as the primary antibody to detect the cellular LeuO protein. The secondary antibody was anti-rabbit IgG conjugated to alkaline phosphatase. The blot was developed by ECF using ECF Western blotting kit (Amersham Pharmacia Biotech). The chemifluorescent signal was detected and quantified by a Storm 840 imaging system (Molecular Dynamics).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A relA1 Mutation-dependent 2-h Growth Arrest Was Found during the Exponential Growth of MC4100 in the 17-aa SSA Medium-- An E. coli K-12 relA1 strain, MC4100, was used for testing the possible LeuO function in the bacterial stringent response upon starvation for branched-chain amino acids. The branched-chain amino acid starvation was achieved by growing MC4100 in a chemically defined SSA medium base supplemented with all amino acids except the three branched-chain amino acids: isoleucine, leucine, and valine (the 17-aa SSA medium). The relA1 strains were shown to grow in such a medium normally without the SMG-mediated toxic effect due to the reverting power of threonine in the medium (15). However, the combination of the absence of the exogenous branched-chain amino acids, the presence of SMG-mediated branched-chain amino acid depletion, and the exhaustion of intracellular pools of branched-chain amino acids due to rapid protein synthesis is expected to cause a temporary branched-chain amino acid shortage (starvation) when cells are exponentially dividing in the log phase. The relA1 strain should be susceptible to such amino acid starvation and thus to the derepression of ilv operons. The promoter relay mechanism (5, 6) predicts that the expression of leuO should be detectable upon ilv derepression. In contrast to the rapid onset stringent response in the relA+ strains, the process is expected to be slow due to the low ppGpp level in the relA1 strains. This slow response is expected to cause a distinct growth "phenotype" compared with the growth of the relA+ strains in the 17-aa SSA medium. This rationale provided the base of the study and was tested experimentally.

Indeed, a 2-h growth arrest during the log phase was observed when MC4100 was grown in the 17-aa SSA medium (Fig. 1). The growth resumed after the 2-h arrest, suggesting that some major physiological events must have occurred during that period. This was strikingly consistent with our prediction that the branched-chain amino acids are most likely depleted in the rapidly dividing cells under exponential growth in this medium. Presumably due to the low ppGpp level in the relA1 strain, a 2-h period was necessary to allow the derepression of ilv operons and to finally overcome the temporary starvation for branched-chain amino acids during the exponential growth. Hence, this growth arrest should be specific to the relA1 genetic background.


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Fig. 1.   A 2-h growth arrest during the log phase of MC4100 growth in the 17-aa SSA medium. Optical density was plotted against postinoculation time. The two arrows mark the start and the end points of the 2-h growth arrest period. The reported growth curve was one of the seven experiments that all reproduced the 2-h growth arrest.

The specificity was tested using an isogenic relA+ and relA1 strain pair, RH147 versus RH12079, constructed by Dr. Hengge-Aronis's group (23). This E. coli pair is appropriate for testing the phenotype caused by the relA1 mutation since they were constructed by introducing either the wild-type relA+ or relA1 into MC4100. A phenotype caused by the relA1 mutant (RH12079) should be eliminated in RH147 due to the restoration of the wild-type relA without a possible interference from any second site suppresser. In the 17-aa SSA medium, the growth curve of the relA1 mutant, RH12079, again showed a 2-h growth arrest in the log phase (Fig. 2A, unfilled circles; time points 1-4). The 2-h arrest was not seen in the growth curve of RH147 in which wild-type relA was restored (Fig. 2A, filled circles). This observation provided strong evidence that the 2-h growth arrest was indeed dependent on the relA1 genetic background in MC4100 or RH12079.


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Fig. 2.   LeuO expression correlates with the relA1 genetic background-dependent 2-h growth arrest. The isogenic relA+ and relA1 pair, RH147 and RH12079, were grown in the 17-aa SSA medium. Panel A, optical density was plotted against postinoculation time. The difference between the growth curves of RH147 and RH12079 was reproducible in three independent experiments. At the marked time points (1-5), cells were harvested. Total protein extracts were prepared from the harvested cells and an equal amount (25 µg) total protein from each protein extract was loaded onto a SDS-PAGE for the subsequent immunoblotting assay, shown in panel B. The lane numbers correspond to the time points marked on the growth curves in panel A. Panel C, the LeuO band was quantified and plotted against the lane number.

The 2-h Growth Arrest Was Caused by the Starvation for Branched-chain Amino Acids-- The addition of the three branched-chain amino acids in the amino acid supplement of the growth medium abolished the 2-h growth arrest (Fig. 3, unfilled triangles). This result argued that the 2-h growth arrest is most likely due to the starvation for branched-chain amino acids. Both the well known SMG-mediated depletion of branched-chain amino acids and the fast exhaustion of branched-chain amino acids due to rapid protein synthesis may contribute to temporary amino acid starvation during exponential growth. Indeed, by omitting all amino acids from the supplement (and thus in the absence of the SMG-mediated toxic effect), the relA1 strain grew normally without the 2-h growth arrest (Fig. 3, unfilled circles). It was therefore possible that the 2-h growth arrest in the log phase is due to starvation for branched-chain amino acids, which is partially caused by the SMG-mediated depletion. As expected, a short (approximate 4 h) lag phase was observed in the chemically defined rich (20-aa) medium, whereas a 14-h lag phase was required for the growth of the same bacterial strain in the 17-aa SSA and no-aa SSA media. The branched-chain amino acid starvation-specific 2-h growth arrest in the relA1 strain was used to study the potential physiological function of LeuO in bacterial stringent response.


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Fig. 3.   The 2-h growth arrest is due to the branched-chain amino acid starvation during the exponential growth. RH12079 was grown in the SSA medium base with various amino acid supplements as indicated. Optical densities of the various RH12079 cultures were plotted against postinoculation time. The arrow indicates the 2-h growth arrest. The reported growth curve was one of the four repeated experiments that all reproduced the demonstrated difference.

A Transient Increase in LeuO Expression Correlated with the relA1 Genetic Background-dependent 2-h Growth Arrest-- Based on the possibility that ilv was derepressed upon the starvation for branched-chain amino acids (15), we hypothesized that leuO expression may be associated with the relA1 genetic background-specific growth arrest. To address this issue, the cellular LeuO level was immunologically detected during the growth of the isogenic relA+ and relA1 strains (Fig. 2B). Western results clearly showed that the level of LeuO was elevated during the growth slow-down in the relA1 strain, whereas a constant low LeuO level was detected in the isogenic relA+ strain (time points 1-4 in Fig. 2B). Quantitation of the Western blot indicated an ~5-fold increase of LeuO at the first two time points (Fig. 2C). The low but constant LeuO level in the relA+ strain was likely due to the rapid onset of stringent response when growing in the chemically defined medium (limited nutrients), since LeuO was almost undetectable during the log phase when a wild-type E. coli K-12 strain was grown in a nutrient-rich broth, LB medium (11).

LeuO Is Essential for the Resumption of the Growth of relA1 Strain after the 2-h Growth Arrest-- The transient LeuO peak in the relA1 strain, MC4100, was confirmed in a more detailed Western analysis (Fig. 4E). The cellular LeuO level was almost undetectable (Fig. 4E, lane 1), gradually increased (Fig. 4E, lane 2), peaked during the 2-h growth arrest (Fig. 4E, lanes 3 and 4), and was reduced when cells resumed their growth (Fig. 4E, lane 5). The LeuO peak showed an exact correlation with the 2-h growth arrest in MC4100 grown in the 17-aa SSA medium (Fig. 4, A and E). This correlation suggested that, rather than just a consequence of the growth slow-down, LeuO may play an important role during the 2-h growth arrest. If so, the growth resumption after the 2-h growth arrest may be dependent on the expression of leuO. This possibility was examined by knocking out the leuO in MC4100. The leuO::cam in TO2 (the donor strain) was introduced to MC4100 (the recipient strain) using P1 transduction to form an isogenic knock-out strain, MF1. Immunoblotting confirmed that LeuO was absent from MF1, whereas it was present in the parental leuO+ strain, MC4100 (Fig. 4, compare F and G), when expression reached maximum during the growth arrest at A600 = 1.2 (Fig. 4E).


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Fig. 4.   LeuO is functionally important for the resumption of growth after the 2-h growth arrest. Panel A, the growth curves of MC4100 (filled circles) and MF1 (unfilled circles) cultures in the 17-aa SSA medium. Panel B, the growth curves of MC4100 (filled circles) and MF1 (unfilled circles) cultures in the 20-aa SSA medium. Panel C, the growth curve of MF1 harboring either pWU204 (filled triangles) or pWU800 (unfilled triangles) were superimposed with the growth curve of MC4100 (gray filled circles) and MF1 (gray unfilled circles). All four strains were grown in the 17-aa SSA medium. The reported growth curves were reproducible in four independent experiments. Panel D, aliquots of MC4100 cultures in 17-aa SSA medium were harvested and total protein extracts prepared for a SDS-PAGE. Equal amount (25 µg) of total protein for each sample was loaded. Both the A600 readings and the elapsed incubation time of the MC4100 culture at the harvest are shown above each sample in the Coomassie Blue-stained gel (panel D). The gel was transferred on an NC paper for immunological detection of LeuO protein (panel E). Total protein extracts were also prepared from MC4100 and MF1 cultures in the 17-aa SSA medium 17 h after inoculation when LeuO expression was induced in MC4100 (refer to panel E, lane 4). Various amounts (from high to low, lanes 1-5) of the total protein extracts of MC4100 and MF1 were subject to electrophoresis (panel F) and transferred to an NC paper and subsequently immunoblotted with LeuO-specific antibody (panel G). Lane M shows the high range protein molecular size standards (Life Technologies, Inc.).

Comparing the growth curves of the isogenic leuO- and leuO+ pair in the 17-aa SSA (Fig. 4A), both MF1 and MC4100 grew exponentially without a significant difference until the growth slowed down at A600 = 1.2. Although MC4100 resumed exponential growth 2 h later and entered the stationary phase at A600 = 2.1 (filled circles in Fig. 4A), the leuO knock-out strain, MF1, failed to resume growth and entered directly into a resting state (optical density remained constant) at A600 = 1.2 (open circles in Fig. 4A). This result confirmed an essential role of LeuO for the growth resumption of the relA1 strain after the 2-h growth arrest.

The growth resumption of MF1 after the 2-h growth arrest was largely reversed by harboring pWU204 but not the control vector, pWU800 (compare the filled and unfilled triangles in Fig. 4C). pWU204 contains the wild-type leuO and its upstream and downstream regulatory regions. Otherwise it is identical to the vector, pWU800. The growth restoration by the exogenous LeuO provided in trans clearly indicated that the growth blockade was indeed due to the deficiency of leuO expression in MF1. Therefore, it is clear that the transient LeuO increase during the 2-h growth arrest (Fig. 4E) is responsible for the subsequent growth resumption of MC4100 in the 17-aa SSA medium. In the absence of the growth stress (branched-chain amino acid starvation), both the leuO+ and leuO- relA1 strains grew normally in the chemically defined rich (20-aa SSA) medium (Fig. 4B). It is, therefore, clear that leuO expression is a specific cellular response upon the growth stress caused by the branched-chain amino acid starvation.

LeuO-dependent Growth Resumption (Optical Density Increase) Is Due to Cell Division Subsequent to the 2-h Growth Arrest-- So far, the observed LeuO effects on cell growth have been based on the measurement of optical density. The difference in optical density readings could, however, be due to some nonspecific causes such as alteration of light scattering properties of the bacterial cells. In order to address the LeuO-dependent effect on the cell growth more directly, viable cell counts were carried out (Fig. 5B) during the growth of MC4100 and MF1 cells in 17-aa SSA medium (Fig. 5A). The result clearly indicated that the growth of leuO-deficient cell, MF1, was arrested but that the arrested cells remained viable up to 24 h after the inoculation (unfilled circles in Fig. 5B). More importantly, the relA1 MC4100 resumed its cell division (cell number doubled) after the transient leuO expression during the 2-h growth arrest (filled circles in Fig. 5B, compare the 18-20-h period and the 20-22-h period). The cell density during the arrested period was ~5.8 × 108 cells/ml of culture (filled arrow in Fig. 5B). At the end of the stationary phase (24 h after inoculation), the cell density of the same culture had reached 4.8 × 109 cells/ml of culture. The 8-fold increase in cell density should be the result of at least three rounds of cell division. Hence, the transient leuO expression during the 2-h growth arrest period is likely to be crucial for triggering the subsequent cell divisions.


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Fig. 5.   Viable cell counts during the growth of MC4100 and MF1 in 17-aa SSA medium. During the growth of MC4100 and MF1 in the 17-aa SSA medium (panel A), viable cells were counted by plating diluted cultures on LB agar plates at various time points. The obtained colony formation units were plotted against postinoculation time (panel B). The two arrows mark the time points when LeuO level in MC4100 peaked (Fig. 4E, lane 3 (unfilled arrow) and lane 4 (filled arrow)).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results can best be explained by the promoter relay mechanism (5, 6) whereby ilvIH transcription activity (ilv derepression) triggers the activation of the intermediate leuO and subsequently enhances the transcriptional activity of the leuABCD operon. Upon the turn on of ilvIH, a 3-4-fold enhancement of the transcription activity of leuABCD was documented (5). Increased leuABCD transcriptional activity may provide additional leucine biosynthesis for cells to overcome the temporary but severe branched-chain amino acid starvation during the exponential growth in the testing condition. This explanation is also consistent with the observed transient LeuO peak during the 2-h growth arrest, since leucine deprivation is required for the activation of ilvIH by Lrp (18, 25). Once the cellular leucine level increased due to the 3-4-fold enhancement of leuABCD activity, ilvIH activity was repressed and so was the expression of leuO.

The stress signal, ppGpp, may be required for activation of ilvIH via the increase of cellular Lrp level (17) and subsequent activation of leuO expression. However, this event does not apparently correlate with the cellular ppGpp level. As the 2-h growth arrest proceeds (Fig. 2, B and C) in the relA1 strain, the LeuO levels begin to decrease slowly. Thus, LeuO levels change inversely to the presumed slow rise in ppGpp levels in the relA1 strain. More importantly, constant low LeuO levels were detected (Fig. 2, B and C) in the relA+ strain, where cellular ppGpp level is expected to be high under the experimental condition. Since such a background LeuO level during exponential growth was not observed when relA+ strains were grown in rich broth/LB (11), the low but constant LeuO level in the relA+ strain was likely due to the rapid onset of the stringent response when bacteria were growing in the chemically defined medium. The well known leucine-mediated feedback control on the expression of ilvIH (18, 25) may provide the explanation for the observation of the low but constant LeuO level in the relA+ strain. The rapid onset of cellular ppGpp level in the relA+ strains is expected to efficiently activate the ilvIH-leuO-leuABCD regulatory cassette via increasing the cellular level of Lrp, the positive transcription regulator for the expression of ilvIH (17), and result in the increase of cellular leucine level. Then the increased cellular leucine negatively regulates the regulatory cassette by repressing the transcriptional activity of ilvIH since leucine is known to weaken the binding of Lrp with its binding sites located upstream of ilvIH (18, 25). The equilibrium of these two counteracting regulatory activities is likely to be responsible for the low but constant LeuO expression in the relA+ strains. At the equilibrium, the changes of cellular LeuO levels were too transient to be detected. If so, the failure of detecting LeuO level changes does not suggest the absence of the similar stress-responsive event in the relA+ strains.

This argument is reasonable since the enzyme activities required for the stringent response should be readily turned on (derepressed) in a stringent strain due to the rapid onset of cellular ppGpp accumulation, whereas the enzyme activities remain repressed in the relaxed relA1 strain due to the slow onset of cellular ppGpp accumulation. This rationale also explains why the relaxed relA1 strain was in a physiological impasse (the 2-h growth arrest) from which escape by derepression of the repressed genes (the ilv operons and hence leuO) was necessary for the resumption of growth. Presumably due to the already derepressed leuO and ilv operons in the stringent relA+ strain, the RH147 surpassed the temporary branched-chain amino acid starvation without a clear delay (arrest) during the log phase growth (Fig. 2A). In contrast, the ilv operons (and hence the leuO gene) remain repressed in relA1 strain prior to the 2-h growth arrest (Fig. 4). The LeuO level was almost undetectable (Fig. 4E, lane 1) and gradually increased (Fig. 4E, lane 2) and peaked during the 2-h growth lag (Fig. 4E, lanes 3 and 4).

Apparently, ppGpp is required for triggering the event including the leuO expression. Subsequently, a ppGpp level-independent regulatory mechanism must determine the transient leuO expression. The leucine-mediated feedback control mechanism discussed above is almost certain to be one of such ppGpp-independent regulatory mechanisms. In addition, a possible positive feedback mechanism effect directly on the expression of leuO has been suggested in our recent study (4). It appears that leuO expression is regulated in a complex manner. Additional studies are required to fully understand the auto-regulation of leuO expression.

Although our results report on a very specific condition for LeuO expression in a specific strain (relA1 strain), there is, however, a clear-cut leuO- phenotype (cell growth is interrupted at A600 = 1.2 in the absence of the functional leuO gene). Although this particular experimental condition will provide an excellent opportunity for evaluating the more complete physiological functions of LeuO in the relA1 strain, we believe that LeuO is likely to be also important for normal bacterial physiology in all strains. This is a typical difficulty of identifying the functions of genes that are not essential for exponentially growing cells. Lack of phenotypes under standard laboratory testing conditions does not, however, mean the lack of function physiologically. In fact, due to cryptic phenotypes in laboratory conditions, the regulation and functions of many genes have been overlooked. We believe that LeuO is one of those underappreciated genes in bacteria.

The present study suggested a role of LeuO in the cell physiology, which is essential for the growth (cell division) resumption after the 2-h growth arrest in relA1 strain (Figs. 4C and 5B). What could be the physiological function of LeuO? Apparently, LeuO does not cause the growth inhibition (the 2-h growth arrest) since the leuO- relA1 strain, MF1, also stopped growing under the testing condition (Fig. 4A). Instead, a regulatory role in transcription has been documented for LeuO (4). A direct and global transcription regulatory function for LeuO is currently being considered and studied in the laboratory. The conditional leuO expression identified in the study provides the initial step toward elucidating the physiological function of LeuO in the interesting but largely unknown bacterial stringent response (reviewed in Ref. 12).

    ACKNOWLEDGEMENT

We are in debt to Dr. Heggna-Aronis for providing crucial bacterial strains and to Drs. Ray Mattingly and Ellen Tisdale for their critical readings of the manuscript. We also appreciate comments from the anonymous reviewers who reviewed the earlier submissions of this work.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM53617 (to H.-Y. W.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF106955.

** To whom correspondence should be addressed: Dept. of Pharmacology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1584; Fax: 313-577-6739; E-mail: haiwu@med.wayne.edu.

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M100945200

    ABBREVIATIONS

The abbreviations used are: bp, base pair(s); Lrp, leucine-responsive regulatory protein; ppGpp, guanosine 3',5'-bispyrophosphate; aa, amino acid(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; NC, nitrocellulose; SMG, serine, methionine, and glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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