|Home | About | Journals | Submit | Contact Us | Français|
Many physiological adjustments to nutrient changes involve ppGpp. Recent attempts to deduce ppGpp regulatory effects using proteomics or gene profiling can rigorously identify proteins or transcripts, but the functional significance is often unclear. Using a random screen for synthetic lethals we found a ppGpp-dependent functional pathway that operates through transketolase-B (TktB), and which is “buffered” in wildtype strain by the presence of an isozyme, transketolase-A (TktA). Transketolase activity is required in cells to make erythrose-4-phosphate, a precursor of aromatic amino acids and vitamins. By studying tktB-dependent nutritional requirements as well as measuring activities using PtalA-tktB′-lacZ transcriptional reporter fusion we show positive transcriptional regulation of the talA-tktB operon by ppGpp. Our results show the existence of RpoS-dependent and independent modes of positive regulation by ppGpp. Both routes of activation are magnified by elevating ppGpp levels with a spoT mutation (spoT-R39A) defective in hydrolase but not synthetase activity or with the stringent suppressor mutations rpoB-A532Δ or rpoB-T563P in the absence of ppGpp.
Bacteria have evolved with complex protective global responses to stress. In enteric bacteria, the accumulation of guanosine 5′-diphosphate, 3′-diphosphate and/or guanosine 5′-triphosphate, 3′-diphosphate, collectively referred to as the (p)ppGpp nucleotides (Cashel and Gallant, 1969) is a common response to different sources of nutritional stress. Here we shall use ppGpp as an abbreviation for pppGpp and ppGpp. There are many examples of regulation that involve ppGpp as a function of normal growth and during stress. Phenotypes associated with a complete deficiency of ppGpp (ppGpp0) include multiple amino acid requirements, filamentation, nucleoid partitioning defects, agglutination changes, adhesion and motility defects arising from the absence of fimbriae and flagella respectively and decreased virulence in pathogenic bacteria (Xiao et al., 1991; Cashel et al., 1996; Magnusson et al., 2005, 2007; Breaken et al., 2006).
Random genetic approaches to define ppGpp function have not been frequently used. Instead, mutants of the relA and spoT genes were encountered in different strains of E. coli, mapped, isogenic strains were constructed and phenotypes characterized. Selections based on these phenotypes identified mutations in relB and relC but not the genes involved in the wide range of functions just mentioned (Cashel et al., 1996). One way to identify cellular functions mediated by ppGpp is to isolate spontaneous extragenic suppressors of defects in ppGpp0 cells. Many such mutations have been isolated that reverse the multiple amino acid auxotrophic phenotype of ppGpp0 cells to allow growth on minimal glucose media. So far, such mutations occur exclusively in rpoB, rpoC and rpoD RNA polymerase subunit genes and have been termed “stringent” mutations (Murphy and Cashel, 2003; Zhou and Jin, 1998).
We used a synthetic lethal approach to look for genes involved in ppGpp-dependent functions. Two mutations are synthetic lethal if either in isolation is viable but together cause inviability. Two separate non-lethal mutations that confer a growth defect more severe than either single mutation can be called synthetic growth inhibition (Ooi et al., 2006; Phizicky and Fields, 1995). The interpretation is that synthetic growth inhibition reflects an important genetic interaction, whereas synthetic lethality reflects an essential genetic interaction. Such interactions reveal genes that function in parallel pathways and “buffer” each other biologically or function within the same pathway but independently contribute to the strength of the signal in the pathway. In general, synthetic lethal screens help uncover pathway(s) that are conditionally essential or significantly influence growth.
Here a genetic screen is used to search for pathways that show ppGpp-mediated regulation. We isolated an insertion in tktA that gives synthetic growth defects in a ppGpp0 strain. This led to the identification of tktB as a transcriptional target of ppGpp and evidence that activation of tktB transcription by ppGpp occurs both through the modulation of RpoS levels and independent of RpoS. The physiological relevance of the two modes of regulation is assessed.
The rationale behind the screen is that an unstable plasmid replicon carrying a gene required for growth would be retained through selection during growth conditions that favor plasmid loss through segregation (Phizicky and Fields, 1995). Low copy number plasmid pHR14 is a temperature-sensitive pSC101-replicon with functional spoT and lacIq genes. Replication of pHR14 is stable at 30°C significantly restricted at 38°C and completely abolished at 420. Growth at 38°C without selection causes plasmid loss and dilution of cellular LacI levels. In the ΔlacI (lacZ+)Δ relA251 ΔspoT207 strain CF11722 carrying plasmid pHR14, plasmid loss results in an increase in β-galactosidase expression and appearance of blue colonies in plates containing the chromogenic substrate X-gal. Mutations that limit plasmid loss will give rise to white or pale blue colonies. Among many other possibilities, mutations that render spoT gene functions essential for growth are expected to select against plasmid loss. A similar approach has been used to identify a synthetic lethal mutation in ftsEX mutant (Reddy M, 2007)
CF11722 with pHR14 was subjected to Tn5 transposon mutagenesis and dilutions were plated on LB X-gal plates with trimethoprim to obtain about 200 well separated single colonies on each plate. A pale blue colony was identified after screening 5,000 blue colonies. The transposon insertion in this clone impaired growth when moved into the ppGpp0 strain CF10237 (by P1vir transduction) but not in the wildtype strain CF1648 (See Fig. 1 panel B). Thus, growth inhibition is dependent on ppGpp deficiency and the growth phenotype from the transposon insertion is a severe growth impairment rather than lethality. Sequencing the transposon-chromosome junction localized the insertion to the distal half of the tktA open reading frame and it was designated as tktA::Tp.
In E. coli, tktA and tktB genes encode redundant transketolases that catalyze synthesis of a key metabolic intermediate, D-erythrose-4-phosphate. As shown in Fig. 2, lack of transketolase activity would result in the failure to synthesize erythrose-4-phosphate, a precursor required for biosynthesis of aromatic amino acids, aromatic vitamins like para-amino benzoic acid and pyridoxine (PN), a precursor of pyridoxal phosphate (Fraenkel, 1987; Pittard, 1996; Wallace and Pittard, 1969; Zhao and Winkler, 1994).
We examined LB growth in the presence of tktA and tktB mutations singly and in combination. We chose two tktA alleles, tktA::Tn10, an undefined insertion in tktA (Iida et al., 1993) and the ΔtktA::kan deletion-insertion allele from the Keio collection (National Bioresource project, Japan) as well as two ΔtktB::kan alleles (Iida et al., 1993 and Keio collection). The tktA mutants were slightly slower growing on LB while the tktB mutants showed no growth defect. However when combined, the tktA tktB double mutant shows growth inhibition in LB comparable to that observed in the tktA ppGpp0 strains (Fig. 1B; Table 1A, last column).
Transketolase mutants require aromatic amino acids and pyridoxine (Fraenkel, 1987; Iida et al., 1993; Zhao and Winkler, 1994). The growth requirements of the tktA tktB double mutants are due to the lack of erythrose-4-phosphate. This compound is generated from glyceraldehyde-3-phosphate and fructose-6-posphate by transketolase or from sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate by transaldolase. However, the syntheses of the latter two substrates require transketolase. Therefore, erythrose-4-phosphate is not synthesized in a tktA tktB double mutant (Fig. 2).
Growth on minimal glucose casaminoacids plates with or without tryptophan and pyridoxine is shown in Fig. 1, panels C & D. The growth requirements of tktA ppGpp0 strain differs from that seen in tktA tktB mutant; the former does not show an absolute requirement for pyridoxine; when each of the aromatic amino acids is additionally omitted, both strains fail to show growth (Table 1A). A requirement for pyridoxine as well as aromatic amino acids has been reported in a tktA tktB double mutant (Zhou and Winkler, 1994). Since phenylalanine requirement is also observed in ppGpp0 strain (Xiao et al., 1991), it will not be considered further as a synthetic phenotype of the ppGpp0 tktA mutant. The pyridoxine requirement seen in the tktA tktB double mutant is not observed in ppGpp0 strains with the tktA::Tn10 or the tktA::Tp alleles (Table 1A). This difference could be due to a trace of transketolase activity in the ppGpp0 tktA strain as compared to the complete absence of activity in the tktA tktB mutant (see below). We assume that a small amount of pyridoxine is sufficient to support growth due to the catalytic use of vitamins as opposed to stoichiometric consumption of amino acids. Unlike ppGpp0 tktA mutant the ppGpp0 tktB mutant strain has growth phenotypes identical to the ppGpp0 parental strain (data not shown).
Supplementing LB medium with aromatic amino acids and/or pyridoxine did not improve growth while the addition of 0.2% glucose partially improved growth (data not shown). Colony sizes are equivalent on LB glucose and in minimal glucose with all 20 amino acids and PN when incubated for the same amount of time (data not shown). It is notable that the tktA tktB mutant does not appear to require para-amino benzoic acid and related vitamins under our growth and media conditions. We do not have a good explanation for this phenotype based on our current understanding of the metabolic pathways.
The tktA gene is located at the 66.3 min region of the genome with 6 ORFs (cmtB to yggC) downstream of tktA and oriented in the same direction (Fig. 3A). Therefore tktA could be the first gene of an operon and the synthetic phenotypes of an insertion in tktA might be due to polar effects. In order to ensure that the loss of transketolase activity caused the observed phenotype, we looked for phenotypic rescue by ectopic expression of the transketolase B isozyme (74% amino acid identity with tktA) from an IPTG-inducible promoter in the ppGpp0 tktA mutant. Table 1B shows that IPTG-induced expression of a minimal tktB gene from plasmid pHR30 completely reverses the synthetic growth defect of tktA-ppGpp0 strains while the plasmid vector had no influence on the synthetic growth phenotypes. This verifies that the phenotypes conferred by tktA insertions are a consequence of lowered transketolase catalytic activity. This result implies that ppGpp deficiency could lower transketolase B activity.
There are two genes for ppGpp synthesis in E.coli, namely relA and spoT (Cashel et al., 1996). A relA tktA (spoT +) strain does not exhibit growth impairment on LB, but shows a partial tyrosine requirement (Table 2, rows 1–3). Tyrosine and tryptophan auxotrophy is observed when the entire spoT ORF is deleted (spoT212) in the relA tktA background (Table 2 row 4). We conclude that functional SpoT is sufficient to alleviate synthetic growth phenotypes, especially the growth defect on LB. The converse experiment of deleting spoT in a tktA relA+ strain could not be performed since such a construct is inviable because excess ppGpp inhibits growth (Xiao et al., 1993). However, as described below, RelA-mediated ppGpp synthesis also contributes to the synthetic growth phenotypes.
There are at least two known functions for SpoT protein, namely, ppGpp synthesis and hydrolysis (Xiao et al., 1991). We wanted to understand the SpoT function required to alleviate synthetic growth phenotypes. It is even possible that this function is spoT-dependent but ppGpp-independent, because a number of proteins have been identified that interact with SpoT. We were unable to test this by providing a weak enough source of ppGpp to allow survival of a ΔrelA ΔspoT strain (Table S2, row 3). Examples of proteins that interact with SpoT are acyl-carrier protein (Battesti and Bouveret, 2007); CgtA (Wout et al., 2004; Jiang et al., 2007) and numerous small and large subunit ribosomal proteins (Butland et al., 2005). The balance of SpoT hydrolase and synthetase activities respond to variety of environmental signals (Cashel et al., 1996; Murray and Bremer, 1996) but little is known of the mechanisms coupling SpoT responses to these signals except in the case of fatty acid synthesis (Battesti and Bouveret, 2007).
We constructed a pair of single amino acid substitution alleles of SpoT designed to eliminate either the hydrolase or the synthetase activity of SpoT but otherwise minimally altering the protein. To do this, we exploited predictions from mutants and structures solved for RelSeq, the SpoT homolog from Streptococcus equisimilis (Mechold et.al., 2002; Hogg et al., 2004). The residues chosen to be altered in each of the two catalytic centers were SpoT-R39 to limit hydrolase activity (H−S+) and SpoT-E319 to limit synthetase (H+S−) activity. The residues were selected because their homologs in RelSeq were deduced to display maximum movement during structural changes in their catalytic pocket when ligands bind the opposing catalytic center (Hogg et al., 2004). Growth tests in a relA mutant to characterize spoT-R39A and spoT-E319Q alleles are described in supplementary information (Table S2). The hydrolase mutation (H−S+) slows growth in LB and in minimal media (Table S2) consistent with higher basal levels of ppGpp during growth. The E319Q synthetase mutation in this host entirely eliminates ppGpp synthesis because the mutant fails to grow in minimal media when the relA256 in-frame ORF deletion is present (Xiao et al., 1993).
Substituting the synthetase mutant (H+S−) allele for a complete spoT deletion confers growth requirements in a tktA relA256 background. If this strain is made RelA+, growth is normal on LB and aromatic amino acid requirements are not seen; RelA becomes the source of ppGpp (Table 2, rows 6 & 7). Introduction of the spoT-R39A allele eliminates the growth requirements of the parental tktA ppGpp0 strain (Table 2, row 5) and is not viable in a relA+ background (data not shown). Apparently, transketolase B activity can be down-regulated by a single residue change in the synthetase catalytic center of the 702-residue SpoT protein. This suggests a key role for ppGpp synthetase function and eliminates other putative regulatory functions of SpoT protein that are unaltered in the E319Q allele. The simplest interpretation of the results is that ppGpp regulates transketolase B activity in the tktA relA256 mutant. The extent of tktB activation reflects the cellular capacity to synthesize ppGpp either from RelA or SpoT.
Finding that ppGpp is required for tktB function leads to the need to assess the roles for DksA and RpoS, two proteins whose regulatory functions are coordinated with that of ppGpp in many instances. DksA, a multi-copy suppressor of DnaK (Kang and Craig, 1990), functions at the level of transcription initiation in vitro as a co-factor of ppGpp to mediate both positive and negative regulatory effects on gene expression (Perederina et al. 2004; Paul et al., 2004a, 2004b & 2005). Studying RpoS is relevant because during entry into stationary phase the accumulation of this stationary phase sigma factor is delayed in the absence of ppGpp or dksA; during exponential growth RpoS levels increase upon gratuitous induction of ppGpp (Gentry et al., 1993; Brown et al., 2002). A requirement for ppGpp exists not only at the level of accumulation of RpoS but also for RpoS-dependent gene expression (Kvint and Nystrom, 2000).
Deleting dksA confers several amino acid requirements but these do not include tryptophan or tyrosine (Brown et al., 2002). The same dksA allele when combined with tktA reduces, but does not eliminate growth in the absence of tyrptophan or tyrosine (Table 3, row 2). Apparently the absence of one co-factor (DksA) only partially mimics the absence of the other (ppGpp). The results could be interpreted as independent regulation of tktB expression by dksA or potentiation of ppGpp-mediated regulation by dksA. The latter is supported by the observation that absence of DksA and ppGpp give phenotypes only as severe as those seen in the absence of ppGpp (Table 3, rows 3 & 4).
In an otherwise wildtype host, deleting rpoS does not result in amino acid or vitamin requirements, upon further inactivation of tktA, growth impairment is slight with all amino acids and PN present, similar to a tktA mutant (Table 1, rows 1 & 2; Table 3, rows 5 & 6). However, the rpoS tktA double mutant, unlike each single mutant, shows partial requirements for tryptophan, tyrosine (Table 1, row 2; Table 3, rows 5 & 6) and phenylalanine (data not shown). Adding a relA deletion (rpoS tktA relA) gives strong growth requirements for PN and amino acids (Table 3, rows 7). A similar phenotype is observed in the rpoS tktA ppGpp0 strain, making these strains phenotypically identical to the tktA tktB mutant (Table 1A, row 4; Table 3, rows 7,9). We confirmed that growth requirements in mutant strains arise from reduced levels of TktB by rescuing growth through ectopic tktB expression using plasmid pHR30 (Table 3, rows 6–10). The results indicate independent regulatory roles for ppGpp and rpoS in tktB transcription (see discusson).
As mentioned previously, DksA over-expression using multicopy plasmid can suppress some ppGpp0 phenotypes. Table 4, rows 3 & 4 show that DksA over-expression in the ppGpp0 tktA mutant restores prototrophy for tryptophan and tyrosine and that the suppression requires RpoS. The pyridoxine requirement is overcome by DksA over-expression in a ppGpp0 tktA rpoS mutant (Table 4, compare rows 2 & 4). Therefore, over-expression of DksA can suppress pyridoxine and amino acid requirement in the presence of RpoS and only the pyridoxine requirement in the absence of RpoS.
About 60 spontaneous mutant alleles have been isolated that restore the growth of ppGpp0 strain on minimal glucose and mapped to rpoB, rpoC and rpoD genes. Some of these have been studied extensively in vitro (Cashel et al., 1996; Murphy and Cashel, 2003; Zhou & Jin, 1998; Barker et al., 2001). We chose for this study two well known rpoB alleles that confer rifampicin resistance, rpoB-T563P and rpoB-A532Δ (alias rpoB3370 and rpoB3449 respectively) which mimic ppGpp regulatory behavior in vivo and in vitro (Zhou & Jin, 1998). We first asked if the alleles change RpoS expression pattern in the absence of ppGpp. Figure 4 is an immunoblot using anti-RpoS antibody in ppGpp0 strains with or without the rpoB3449 and rpoB3370 alleles. The presence of the suppressor mutations elevates RpoS protein levels 20-fold over the levels observed in ppGpp0 cells in the log phase of growth. The RpoS level in the rpoB mutant strains are about 5-fold higher than in the wildtype strain in log phase (data not shown).
Table 4 shows that both rpoB alleles completely suppress the growth requirements associated with low transketolase activity in the tktA ppGpp0 host (rows 5 & 8). This is consistent with their ability to induce RpoS accumulation. However, when rpoS is deleted, growth in the absence of PN and tryptophan or tyrosine persists although the suppression is considerably weakened (Table 4, rows 6 & 9). Therefore, the suppression activity of the rpoB alleles is not entirely RpoS-dependent. We thought it was also important to ask if suppression of growth requirements by the rpoB alleles is entirely through the activation of tktB or has an alternate explanation (say, the activation of a cryptic transketolase gene). Table 4, rows 7 & 10 show that suppression requires tktB; these results indicate the rpoB alleles can increase TktB activity independent of RpoS and ppGpp (see below).
The nutritional requirements of the mutant strains indicate regulation of tktB expression by ppGpp, RpoS, DksA and the stringent rpoB mutations. To find out if transcription can account for regulation of growth requirements, reporter activity of tktB-lacZ operon fusions were measured during growth in LB using a tktA+ strain.
Previous studies have identified two closely spaced promoters upstream of talA (P1 and P2 in figure 3B), and one within the talA ORF just upstream of tktB (Lacour and Landini, 2004; Jung et al., 2005). We constructed three transcriptional fusions (Fig. 3B) to look at activity of the promoters. Fusion A measures transcription from P1 P2 promoters upstream of talA, fusion B from the promoter reported in the talA ORF with fusion joints identical to the one described in Jung et al., 2005. Fusion C detects transcription from the entire region upstream of tktB (Fig. 3B) and extends 238 nucleotides upstream of talA.
Table S3 is a survey of reporter activities using the three fusions in wildtype strains for exponential and stationary growth phases. Fusion B is marginally active, whereas Fusions A and C have measurable activities during exponential growth, which are induced 7- and 9- fold respectively in stationary phase. Under our conditions talA and tktB genes seem to comprise an operon. We chose fusion C for the studies reported below.
The reporter activity from fusion C is shown in Fig. 5, measuring activities in log, early stationary and stationary phase for different strains. Transcription is lowered 5–6 fold during all phases of growth in the ppGpp0 strain (panels A & C) whereas the absence of RpoS lowers the tktB activity increasingly during growth from log to stationary phase (7-to 16- fold). In ppGpp0 strain the absence of RpoS lowers activity at least 3- fold further in all growth phases (compared to rpoS mutant) and the activity is virtually absent in the log phase cells (~0.6 Miller units). The results are consistent with an independent regulation of tktB expression by ppGpp and RpoS (see discussion).
The presence of the “stringent” rpoB suppressor mutations T563P and A532Δ (in the ppGpp0 background) results in a large increase in tktB expression (32-fold in rpoBA532Δand 42-fold in rpoBT563P) during exponential growth. When these strains go into stationary phase only a modest additional increase in expression occurs (Fig. 5 panels G and I). For both rpoB mutants the increase in tktB expression is largely RpoS-mediated (panels H and J). However, in the absence of RpoS, they have a 20-fold higher activity in log phase compared to isogenic ppGpp0 strain (compare panel D with H & J).
The activation of tktB transcription by the hydrolase-deficient spoT-R39A allele is consistent with a positive regulatory role for ppGpp. The tktB expression pattern seen when spoT synthetase is not balanced by hydrolase, is strikingly similar to that observed in the rpoB mutant strains: a large increase in exponential phase and a moderate increase thereafter. An 8- fold or 44-fold increase in expression is observed in exponential phase when compared to wildtype or ppGpp0 strains (Fig. 5 compare panels A, C & E). The RpoS-independent tktB expression in the spoT mutant is once again similar to that seen in the rpoB mutant strains (compare panels F, H & J in Fig. 5) underscoring a possibility of similar regulatory mechanisms (see discussion).
A dksA deletion reduces tktB transcriptional activity roughly by half during all phases of growth, compared to wild-type strain; in a ppGpp0 strain the same deletion has no further effect (data not shown). DksA over-expression does not significantly alter tktB transcription in the presence of ppGpp, but restores expression close to wildtype levels in a ppGpp0 strain. This positive effect of DksA on tktB transcription in the ppGpp0 strain is primarily RpoS-dependent but the small RpoS- independent effect is also noted (Fig. 6B). These results are consistent with the growth phenotypes observed during DksA over-expression in the absence of ppGpp and in the presence or absence of RpoS (Table 4, rows 1 to 4).
This work shows that genetic screening for synthetic lethals can be applied to define ppGpp-dependent functions. We explain the synthetic growth defects arising from the inactivation of tktA in a ppGpp0 host strain as due to inactivation of a tktB-dependent redundant pathway. We show that ppGpp regulates tktB transcription through the modulation of RpoS activity and by RpoS-independent positive stringent regulation as well. The contribution of each pathway may depend on growth conditions.
It is unclear how transketolase activity affects growth in LB agar or broth. Supplementing LB with glucose, aromatic amino acids and pryridoxine only marginally improves growth (data not shown). Unlike in LB, in minimal glucose media containing all the supplements, growth of the double mutant is only slightly slower than that of an isogenic wildtype strain (Table 1A). Presence of inhibitors in LB has been suggested previously (Zhou and Winkler, 1994). Alternatively, the unique need for transketolase activity could be specific for growth on carbohydrate-poor complex peptide digest medium like LB and related to its metabolic function at the intersection of gluconeogenesis and pentose phosphate shunt. The synthetic growth defect arising from tktA and ppGpp deficiency or the absence of both transketolase isozymes can be completely reversed by ectopic expression of TktB (Table 1B), indicating that growth defects are due to general transketolase deficiency rather than from a specific function of TktA. It is important to consider this possibility because a genetic selection for mutants with reduced chromosomal negative supercoiling has uncovered a role for TktA and DksA along with H-NS, Fis and SeqA/Pgm in the maintenance of chromosomal superhelicity (Hardy and Cozzarelli, 2005).
The assay for transketolase-B function based on growth requirements indicates that the cellular transketolase-B activity is almost entirely dependent on the presence of ppGpp and RpoS because growth properties of tktA tktB mutant is identical to that of tktA rpoS ppGpp0 mutant. The tktB-lacZ fusion results show that regulation by ppGpp and RpoS is almost entirely at the level of transcription. The data also indicates that there are at least two routes for activation of tktB transcription; one requires ppGpp and RpoS while the other is transcriptional activation by ppGpp independent of RpoS.
The transcriptional fusion C in tktB shows a 16-fold drop in expression in stationary phase in the rpoS mutant, consistent with a microarray study that reported 14-fold RpoS-dependent increase in tktB transcripts in stationary phase (Weber et al., 2005). Previous studies have established that ppGpp0 strains phenocopy RpoS deficiency and gratuitous induction or increase in ppGpp basal levels leads to increased RpoS protein levels (Gentry et al., 1993; Brown et al., 2002). Also, ppGpp facilitates transcription by pre-existing RpoS (Kvint et al., 2000) and its absence diminishes the ability of RpoS to compete against RpoD for the core (Jishage et al., 2002). There is growing evidence from many other studies that lead to the proposal that ppGpp helps alternative sigma factors to compete for RNAP core (Magnusson et al., 2005). Therefore, down-regulation of tktB expression observed in ppGpp0 strain can result from a combination of lowered RpoS protein levels and diminished RpoS function. It is possible that low RpoS levels could be a reason why tktB was not identified in a transcriptional profile of stringent response (Durfee et al., 2008), since the response was elicited in early exponential phase cultures using serine hydroxymate which inhibits protein synthesis. The induction by elevated ppGpp alone in the absence of RpoS was probably below detection levels.
The partial growth defects observed in rpoS tktA mutant indicate low level TktB activity (Table 3). Additional inactivation of relA or the absence of ppGpp eliminates residual activity, because growth requirements of rpoS tktA relA, rpoS tktA ppGpp0 and tktA tktB mutants are identical (Table 3). Similarly, the low lacZ reporter activity in the rpoS mutant is lowered further to barely detectable levels in the absence of ppGpp (Fig. 5). These results are consistent with a role for ppGpp that is independent RpoS in the regulation of tktB expression and suggest a mechanism for feedback regulation in the aromatic amino acid biosynthetic pathway (Fig. 2). Aromatic amino acid starvation activates RelA, leading to ppGpp synthesis, activation of tktB transcription and aromatic amino acid biosynthesis. This leads to disappearance of the signal.
Based on existing models, the transcriptional activation mediated by ppGpp we see could be due to either direct effect of ppGpp and DksA at the promoter (Paul et.al., 2005) or due to an indirect effect resulting from increased availability of free RNAP as a consequence of inhibition of rRNA transcription (Zhou and Jin, 1998; Barker et al., 2001) that facilitates competition by alternative sigma factors for the RNAP core (Jishage et al., 2002; Magnusson et al., 2005; Szalewska-Palasz et al., 2007; Costanzo et al., 2008).
Two transcription start sites were localized upstream of talA and used to deduce promoters P1 and P2 based on a -10 consensus for RpoS dependent promoters (Lacour and Landini, 2004). This microarray study found RpoS-dependence for talA but in contrast to our results and the other microarray study cited earlier (Weber et al., 2005) tktB transcripts were not found to be RpoS-dependent. Interestingly the P1 promoter has a sequence tgctatgcttttt followed by the +1 transcription start site, that is, an extended −10 (underlined) together with AT rich discriminator. This could fuse RpoS-dependence with activation by ppGpp. It is also possible that the two promoters respond differentially, one to RpoS and another to ppGpp perhaps through sigma-70 or an alternative sigma factor.
RpoS-mediated positive and negative regulation of tktB and tktA respectively was reported; it was suggested that isozymes TktA and TktB may play their main roles in exponential and stationary phase respectively (Jung et al., 2005). However, using an identical construction (fusion B, Fig. 3) we are unable to observe activity (Table S2) or observe RpoS regulation (data not shown); the reason for the disparity is not apparent.
Suppression by over-expression of DksA is a feature found for many ppGpp0 phenotypes including the restoration of RpoS induction (Brown et al., 2002 ; Magnusson et al., 2007). Consistent with an effect mediated through RpoS, restoration of tktB expression by DksA over-expression requires functional RpoS (Fig. 6A & B). However a small positive effect can be observed in the absence of RpoS and ppGpp; this is supported by the suppression of vitamin requirement observed in growth assays (Fig. 6B; Table 4). The ability of DksA to substitute for a complete absence of ppGpp argues DksA and ppGpp are not co-factors (like cAMP and CRP).
The spoT-R39A mutation is deduced to change the ppGpp hydrolase-synthetase balance in favor of synthesis and increase ppGpp basal levels (Table S2). The spoT-R39A mutant and the stringent rpoB mutant strains used in this study show essentially identical tktB transcriptional regulation and TktB activity in vivo as deduced from lacZ fusion data and growth requirements of these strains (Tables 2 & 4; Fig. 5). The phenotypic similarities between the strains persist in the absence of RpoS. The results suggest that an increase in intracellular ppGpp level and presumably polymerase conformation changes can have similar functional consequences for positive transcriptional regulation mediated by ppGpp. A previous study with the same polymerase mutants showed they mimic the negative transcriptional regulation mediated by ppGpp (Zhou and Jin, 1998). The rpoB mutants increase RpoS protein levels (Fig. 4) and the same is observed for the spoT-R39A hydrolase mutant (data not shown). Together, the results provide further evidence for the passive regulatory model and extend it for ppGpp-mediated RpoS regulation. The spoT-R39A and stringent rpoB mutants soften the wildtype “stair step” like increase in tktB expression with growth phase. If high ppGpp levels slow the metabolic turnover of RpoS during growth in LB like it does during phosphate starvation through iraP (Bougdour and Gottesman, 2007) similar effects might be expected.
This study shows that tktB is subject to RpoS-mediated regulation through ppGpp, excess DksA in the absence of ppGpp, and “stringent” rpoB mutations. In addition, transcription activation is also seen independent of RpoS (Fig. 7): (i) under conditions that increase cellular ppGpp levels; (ii) by “stringent” rpoB mutations in the absence of ppGpp and (iii) DksA over-expression in the absence of ppGpp. It is possible that in each case transcriptional activation is mediated through a similar conformational change in RNAP. In vitro transcription studies are warranted to find out if the effects are direct or involve additional factors.
Table S1 list the E.coli strain derivatives of MG1655 and plasmids used in this study. Cultures were grown in LB broth in rotary shaker flasks at 37°C. The media used are described by Miller (1972) but modified as follows: LB contains 0.5% NaCl and M9 glucose minimal contains 15 μM thiamine and may be supplemented with either 0.4% casiamino acids, all 20 amino acids (each at 40 μg/ml), 19 amino acids lacking either phenylalanine, tryptophan or tyrosine and pyridoxine (10 μM). Final concentration of antibiotics was: ampicillin (100 μg/ml in LB and 50 μg/ml in minimal media), tetracycline (20 μg/ml), kanamycin (30 μg/ml) and trimethoprim (100 μg/ml). Quantitative estimates of growth at 37°C are based on colony diameters: ± is 0.5 mm or less; + is 1–1.5 mm; + + is 2–2.5 mm; + + + is 3 mm or more. In general, colony sizes are scored on LB plates and on minimal media after incubating for 24 hrs and after 48 hrs respectively.
Plasmid pMA2 is a temperature-sensitive, pSC101 replicon (obtained from the cloning vector collection at NIG, Mishima, Japan). pMA2 was digested with SphI and re-circularized to obtain plasmid pHR13, and was used to clone the spoT gene from plasmid pHX41 (Gentry et al., 1996) as a 2.1 kb SphI fragment yielding pHR14. Plasmid pJK537 has been described in Kang & Craig, 1990. Plasmid pHR30 was constructed using pQE80L (Qiagen vector) for IPTG-inducible TktB expression. A 2041 bp fragment encompassing the entire tktB ORF was PCR-amplified from MG1655 genome using Pfu-polymerase and primers 5′-atgaattccagccacggagt-3′ (upstream) and 5′-ttggatccggcaatcacc atca-3′ (downstream). EcoRI and BamHI sites in the primers (underlined) were used to clone the fragment in the EcoRI/BamHI sites of pQE80L.
Phage P1(vir) transductions were performed by standard procedures (Miller, 1972). When constructing strains with the tktA rpoS, ppGpp0 tktA, and ppGpp0 rpoS tktA genotypes the last allele to be introduced is tktA and transductants were selected and maintained on LB plates containing 0.2% glucose which improved the growth of these strains. Plasmid DNA was extracted using Qiagen kits, transformations and recombinant DNA procedures were performed generally as described in Sambrook et al., 1989. Recombineering was performed as described in Yu et al., 2000, using the linear DNA transformation protocol to engineer the spoT- synthetase and hydrolase mutations.
The talA-tktBprime;::lacZ fusion (fusion-C) was constructed using a 1331 bp fragment retrieved from the chromosome of strain MG1655. This fragment has 238 bp of non-coding region upstream of talA, the entire talA structural gene, the talA- tktB intergenic region and 124 nucleotides of tktB coding sequence; it was recombined into a linear pBR322 derived fragment using a protocol described in Court et al., 2002. The primers for generating the linear fragment for recombination with the chromosomal sequence (italics in the primer sequence) were 5′-cagggtaataatgtgcgccacgttgtgggcaggggaattcgcgt ttcggtgatgacggtg-3′ (bottom strand) and 5′-gggcatggctgatattgccgaagtgctgtggaacggatccga aagggcctcgtgatacgc-3′ (top strand). The chromosomal fragment was then subcloned into the operon fusion vector pRS415 using restriction enzyme EcoRI and BamHI (underlined in the primers) and recombined first into λRS45 and then into the λatt site on the chromosome as described in Simons et al., 1987. The talA′::lacZ fusion (fusion A) was constructed using a 319 bp fragment containing the same 5′ end point as fusion C and 81 bp of the talA coding sequence and obtained by PCR using primers 5′-atgaattccccctgccca caacg-3′ (top strand) and 5′-atggatccgatgataatggcgaatggactc -3′. The ‘talA-tktB’::lacZ fusion (fusion B) contains a 344 bp fragment with 200 bp of the 3′-coding sequence of talA, 19 bp of intergenic sequence and 124 bp of tktB coding sequence and obtained using the primers 5′-atgaattcctgcaggaaaaagtttcgcc -3′ (top strand) and 5′-atggatcctcgttccacagcac ttc-3′ (bottom strand). These fusions were also transferred to the λatt site as described for fusion C.
Random mutagenesis was carried out using the EZ-Tn5™ < DHF R-1> transposon kit from Epicentre (Madison, WI). The protocol for sequencing insertions is published in Epicentre forum (Ducey and Dyer, 2002).
Cultures were grown after a 1:500 dilution of an overnight culture and the lacZ fusions were assayed for β-galactosidase activity as described in Miller, 1972 with activities reported in Miller units.
Lysates were prepared after precipitation with cold trichloroacetic acid using final concentrations of 5% and 10% for exponential and stationary phase cells, respectively. After centrifugation, pellets were washed once with 0.5 ml of ice-cold 80% acetone, air dried and resuspended in SDS-gel loading buffer. Equal quantities of proteins were separated on precast SDS-12% PAGE acrylamide gels (Invitrogen) and transferred to PVDF membranes. The membranes were incubated with anti-RpoS antibody (Neoclone, Madison, WI) at 1:1000 dilution and the blots developed using horseradish peroxide-conjugated goat anti-rabbit antibody by the enhanced chemiluminescence protocol (GE health sciences).
We would like to thank Manjula Reddy for discussions that inspired this study and for sharing plasmid pMA2 and Dick D’Ari making suggestions for the manuscript. This work is supported by the intramural research program of NICHD/NIH.