A screen for synthetic-lethal mutations in a ppGpp0 host identifies an insertion in tktA
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
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
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 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.
Figure 1 Growth properties of transketolase mutants. A. Strains on plate: tktA::Tp (CF13942), ppGpp0 i.e., relA256 spoT212 (CF10237), ppGpp0 tktA::Tp (CF13926) and tktA tktB (CF13927); B. LB agar after 18 hours; C. Minimal media with glucose and casamino acids (more ...)
In E. coli
genes encode redundant transketolases that catalyze synthesis of a key metabolic intermediate, D-erythrose-4-phosphate. As shown in , 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
Figure 2 Pathways for biosynthesis of the intermediary metabolite D-erythrose-4-phosphate and amino acids and vitamins derived from it. The enzymes involved at each step are indicated by gene names that encode them; broken arrows represent multiple steps in the (more ...)
A ΔtktB::kan mutation confers synthetic growth defects in the tktA mutant similar to that observed from ppGpp deficiency
We examined LB growth in the presence of tktA
mutations singly and in combination. We chose two 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
strains (; , last column).
Growth deficiency and requirements seen in tktA mutants in the absence of ppGpp arise from lowered transketolase-B activity.
Comparison of growth requirements between tktA ppGpp0 mutant and the tktA tktB double mutant
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 ().
Growth on minimal glucose casaminoacids plates with or without tryptophan and pyridoxine is shown in , panels C & D. The growth requirements of tktA
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 (). 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 (). 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 ppGpp0 – tktA synthetic phenotypes arise from low transketolase activity
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 (). 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. 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.
Figure 3 Schematic representation of the genomic neighborhoods of tktA and tktB genes and the DNA segments in each lacZ operon fusion. A. tktA and proximal ORF’s; B. tktB and proximal ORF’s. Open-reading frames are represented by thick filled arrows; (more ...)
The growth phenotypes of the ppGpp0 tktA mutant strain are alleviated by functional SpoT
There are two genes for ppGpp synthesis in E.coli
, namely relA
(Cashel et al., 1996
). A relA tktA
) strain does not exhibit growth impairment on LB, but shows a partial tyrosine requirement (, rows 1–3). Tyrosine and tryptophan auxotrophy is observed when the entire spoT
ORF is deleted (spoT
212) in the relA tktA
background ( 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.
Growth phenotypes correlate with the loss of cellular ppGpp synthesis.
The growth phenotypes in ppGpp0 tktA mutant strain reflect the overall ppGpp biosynthetic capacity of the cell
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
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−
) and SpoT-E319 to limit synthetase (H+
) 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−
) 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
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 (, rows 6 & 7). Introduction of the spoT-R39A allele eliminates the growth requirements of the parental tktA ppGpp0 strain (, 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.
Independent and synergistic roles of ppGpp: DksA and RpoS modulate tktB-dependent growth requirements
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
). 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
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 (, 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 (, rows 3 & 4).
Independent and synergistic effects of ppGpp, DksA and RpoS in the modulation of tktB-dependent growth requirements.
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 (, rows 1 & 2; , rows 5 & 6). However, the rpoS tktA double mutant, unlike each single mutant, shows partial requirements for tryptophan, tyrosine (, row 2; , rows 5 & 6) and phenylalanine (data not shown). Adding a relA deletion (rpoS tktA relA) gives strong growth requirements for PN and amino acids (, rows 7). A similar phenotype is observed in the rpoS tktA ppGpp0 strain, making these strains phenotypically identical to the tktA tktB mutant (, row 4; , 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 (, 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. , 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 (, 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.
Suppression of growth requirements by multicopy DksA and the stringent rpoB mutations
Suppression of auxotrophic requirements by RNA polymerase mutations
About 60 spontaneous mutant alleles have been isolated that restore the growth of ppGpp0
strain on minimal glucose and mapped to rpoB, rpoC
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 rpoB
3370 and rpoB
3449 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. is an immunoblot using anti-RpoS antibody in ppGpp0
strains with or without the rpoB
3449 and rpoB
3370 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).
Figure 4 Stringent rpoB suppressor mutations increase RpoS protein levels of exponentially growing cells. Extracts were made from LB grown cells taken at different stages of growth. Extracts from cells equivalent to 0.1 A600 were used for immunoblotting with anti-RpoS (more ...)
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 (, 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). , 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).
RpoS and ppGpp activate tktB-lacZ transcriptional fusions
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 ), and one within the talA
ORF just upstream of tktB
(Lacour and Landini, 2004
; Jung et al., 2005
). We constructed three transcriptional fusions () 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
() and extends 238 nucleotides upstream of talA
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
genes seem to comprise an operon. We chose fusion C for the studies reported below.
The reporter activity from fusion C is shown in , 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).
Figure 5 Regulation of tktB transcription – the role of ppGpp, RpoS and stringent rpoB mutations. tktB transcription was monitored during growth in LB with the talA-tktB′::lacZ fusion C. Strains used are, CF14213 and CF14241 (columns A & (more ...)
Stringent rpoB alleles increase tktB-lacZ expression
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 ( 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).
Hydrolase-deficient spoT-R39A allele increases tktB-lacZ expression
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 ( 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 ) underscoring a possibility of similar regulatory mechanisms (see discussion).
Positive regulation of tktB-lacZ expression by DksA
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 (). 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 (, rows 1 to 4).
Figure 6 Effect of over-production of dksA on talA-tktB′::lacZ expression. β-galactosidase specific activities are plotted against A600 during growth in LB in the presence of plasmid pJK537 or the vector control pBR322 in wild-type (CF14213) or (more ...)