Abstract

DksA is an auxiliary transcription factor that interacts with RNA polymerase and influences gene expression. Depending on the promoter, DksA can be a positive or negative regulator of transcription initiation. Moreover, DksA has a substantial effect on transcription elongation where it prevents the collision of transcription and replication machineries, plays a key role in maintaining transcription elongation when translation and transcription are uncoupled and has been shown to be involved in transcription fidelity. Here, we assessed the role of DksA in transcription fidelity by monitoring stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele, as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization studies. We present data showing that DksA acts to maintain transcription fidelity in vivo and the role of DksA seems to be distinct from that of the GreA and GreB transcription fidelity factors.

INTRODUCTION

All organisms maintain their phenotypic identity by accurate replication of the information encoded in their genes but also by a high fidelity of the processes that generate the products expressed from those genes. Every level of information transfer from DNA to RNA to protein is prone to errors, but not all of these errors are considered to have significant and long-lasting consequences for the organism. Unlike the more permanent effects of genetic mutation and potentially detrimental consequences of errors in protein folding known to cause prion formation, the magnitude and consequences of errors in transcription are just beginning to be explored (1–7). One mRNA error can compromise the cell with the production of many faulty proteins; translation errors however, lead to only one compromised protein among many wild-type proteins produced from the same transcript and are therefore potentially of less consequence, even if translation may be more error-prone than transcription. Indeed, errors in RNA can impart a long-lasting and heritable impact on cellular phenotype when arising in a transcript of a transcription factor involved in a differentiation switch. Specifically, we have shown that alteration of transcription fidelity in vivo promotes heritable phenotypic change in the bistable lac operon system (Figure 1) (1). We showed that error-prone RNA polymerase (RNAP) mutants, as well as loss of the RNAP fidelity factors GreA and GreB, reduce transcriptional fidelity. We also showed that an error-prone message in the transcriptional regulator controlling the differentiation switch promotes transcriptional infidelity (2). Moreover, we showed that both the cis and trans components of the system act synergistically in the control of transcriptional fidelity (2). Thus, the bistability of the lac operon can be used to capture and monitor the consequences of transient transcription errors in living Escherichia coli cells, providing a sensitive tool to study proteins involved in the fidelity of RNA transcription.

Figure 1.

Stochastic switching in the lac bistable gene network. Under maintenance conditions [that concentration of inducer which does not activate transcription of the operon but allows an already induced cell to remain induced (44)], the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer TMG remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence (1,2,4).

Figure 1.

Stochastic switching in the lac bistable gene network. Under maintenance conditions [that concentration of inducer which does not activate transcription of the operon but allows an already induced cell to remain induced (44)], the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer TMG remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence (1,2,4).

Auxiliary transcription factors bind RNAP and can influence gene expression processes without directly interacting with DNA. This family of proteins includes GreA and GreB, Gfh1, Rnk, DksA, DksA2 and TraR (8–14). Each auxiliary factor has been shown, or predicted, to dock to the surface of RNAP and protrude their coiled-coil tip into the secondary channel of the RNAP complex. Among these auxiliary transcription factors, only GreA and GreB, the bacterial homologs of eukaryotic TFIIS transcription fidelity factor (15–17), are known to be required for high fidelity RNA synthesis in vivo and in vitro (1,2,5,18–22). The coiled-coil structures enable these factors to reach close to the active center of the RNAP complex where they promote endonucleolytic cleavage of the nascent transcript. Short pauses, associated with a backtracked RNAP of less than 5 bp and a pause of less than 20 s, are resolved by GreA, which stimulates the cleavage and allows RNAP to proceed with elongation. Longer pauses, which involve the extrusion of the 3′ end of the nascent RNA into the secondary channel, require GreB and its action is necessary to restart RNAP (19). Therefore, GreA and GreB are required to revive backtracked RNAP under different circumstances.

E. coli DksA is a structural homolog of the Gre factors. Although DksA is not related to the Gre proteins on a sequence similarity basis, it interacts with the same area on the surface of RNAP (23–26). DksA was first implicated in transcription initiation, and recently this protein was also unambiguously shown to have a substantial effect on transcription elongation as well where it prevents the collision of transcription and replication machineries (27–29). In addition, DksA has recently been shown to play a key role in maintaining transcription elongation when translation and transcription are uncoupled (30). The structural similarity between the fidelity factors GreA, GreB and DksA, in addition to recent discoveries regarding the role of DksA in transcription elongation in vivo (28) and transcription fidelity (31), encouraged us to investigate the role of DksA in maintaining high RNA quality and epigenetic change in living cells.

Here, we employed a variety of in vivo genetic assays to assess the role of DksA in transcription fidelity including stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization (smFISH) studies. Our results show that DksA acts to maintain transcription fidelity and that the in vivo role of DksA seems to be distinct from that of the GreA and GreB fidelity factors. Our results corroborate a recent in vitro study indicating that DksA increases the fidelity of RNA synthesis by slowing down misincorporation events by RNAP (31) and our overexpression studies complement the in vivo partial phenotypic nonsense suppression that has been observed in ΔdksA cells (31).

MATERIALS AND METHODS

Bacterial strains

Strains are listed in Table 1 and plasmids are listed in Table 2. Unless otherwise stated, all strains used are derivatives of MG1655. Transduction with P1vir and other standard genetic methods were performed as described by Miller (32). To monitor the proportion of cells that are ON or OFF for lac operon expression, we have replaced the lacA gene in the wild-type E. coli MG1655 chromosome with a gfp cassette, so that when the lacZYA::gfp transcript is expressed, β-galactosidase, galactoside permease and green fluorescent protein are produced from the lacZ, lacY and gfp genes, respectively (Figure 1) (1,2).

Bacterial strains

Table 1.
Bacterial strains
Strain Genotype Reference 
CH30 MG1655 laboratory stock 
CH458 lacZYA::gfp-cmR (1
JW0141 ΔdksA::kan (38
CH645 lacZYA::gfp-cmR ΔdksA::kan CH458 x P1 JW0141 
CH568 lacZYA::gfp-cmR ΔgreAFRT ΔgreBFRT (1
CH2193 lacI-A9lacZYA::gfpFRT (2
CH5106 lacI-A9lacZYA::gfpFRT rpoC-F1325L thi3178/Tn10kan CH2193 x P1 CH2300 
CH5110 lacI-A9lacZYA::gfpFRT rpoC-F1325L thi3178/Tn10kan ΔdksA::tet CH5106 x P1 CH2300 
CH543 lacZ-U118 (45
CH580 lacZ-U118 ΔdksA::kan (45
CH2570 lacZ-U118 ΔyjaZ::kan ΔbtuB3191::Tn10 rpoC-E677G (45
CH5035 lacZ-U118 dksA-NN ΔyadD::kan CH543 x P1 CH4247 
CH2417 lacZ-U118 ΔdksA::kan pBA169 CH580 + pBA169 
CH2455 lacZ-U118 ΔdksA::kan pGreB CH580 + pGreB 
CH2418 lacZ-U118 ΔdksA::kan pDksA CH580 + pDksA 
CH2419 lacZ-U118 ΔdksA::kan pDksA-NN CH580 + pDksA-NN 
CH2454 lacZ-U118 ΔdksA::kan pGreA CH580 + pGreA 
CH2455 lacZ-U118 ΔdksA::kan pGreB CH580 + pGreB 
RG8527 ΔdksA::tet rpoC-F1325L thi3178/Tn10kan RL Gourse 
CH2263 lacZYA::gfp-cmR rpoC-F1325L thi3178/Tn10kan CH458 x P1 RG8527 
CH2300 ΔdksA::tet lacZYA::gfp-cmR rpoC-F1325L thi3178/Tn10kan RG8527 x P1 CH2263 
JD1337 dksA-NN JD Wang 
CH4207 dksA-NN pKD46 JD1337 + pKD46 
JW5010 ΔyadD::kan (38
CH4247 dksA-NN ΔyadD::kan CH4207 x P1 JW5010 
CH2714 lacIFRTlacZYA (2
CH5041 lacIFRTlacZYA ΔdksA::tet CH2714 x P1 CH2300 
CH5045 lacIFRTlacZYA ΔyjaZ::kan ΔbtuB3191::Tn10 rpoC-E677G CH2714 x P1 CH2570 
CH5047 lacIFRTlacZYA rpoC-F1325L thi3178/Tn10kan CH2714 x P1 CH2300 
CH5087 lacIFRTlacZYA rpoC-F1325L thi3178/Tn10kan ΔdksA::tet CH5047 x P1 CH2300 
Strain Genotype Reference 
CH30 MG1655 laboratory stock 
CH458 lacZYA::gfp-cmR (1
JW0141 ΔdksA::kan (38
CH645 lacZYA::gfp-cmR ΔdksA::kan CH458 x P1 JW0141 
CH568 lacZYA::gfp-cmR ΔgreAFRT ΔgreBFRT (1
CH2193 lacI-A9lacZYA::gfpFRT (2
CH5106 lacI-A9lacZYA::gfpFRT rpoC-F1325L thi3178/Tn10kan CH2193 x P1 CH2300 
CH5110 lacI-A9lacZYA::gfpFRT rpoC-F1325L thi3178/Tn10kan ΔdksA::tet CH5106 x P1 CH2300 
CH543 lacZ-U118 (45
CH580 lacZ-U118 ΔdksA::kan (45
CH2570 lacZ-U118 ΔyjaZ::kan ΔbtuB3191::Tn10 rpoC-E677G (45
CH5035 lacZ-U118 dksA-NN ΔyadD::kan CH543 x P1 CH4247 
CH2417 lacZ-U118 ΔdksA::kan pBA169 CH580 + pBA169 
CH2455 lacZ-U118 ΔdksA::kan pGreB CH580 + pGreB 
CH2418 lacZ-U118 ΔdksA::kan pDksA CH580 + pDksA 
CH2419 lacZ-U118 ΔdksA::kan pDksA-NN CH580 + pDksA-NN 
CH2454 lacZ-U118 ΔdksA::kan pGreA CH580 + pGreA 
CH2455 lacZ-U118 ΔdksA::kan pGreB CH580 + pGreB 
RG8527 ΔdksA::tet rpoC-F1325L thi3178/Tn10kan RL Gourse 
CH2263 lacZYA::gfp-cmR rpoC-F1325L thi3178/Tn10kan CH458 x P1 RG8527 
CH2300 ΔdksA::tet lacZYA::gfp-cmR rpoC-F1325L thi3178/Tn10kan RG8527 x P1 CH2263 
JD1337 dksA-NN JD Wang 
CH4207 dksA-NN pKD46 JD1337 + pKD46 
JW5010 ΔyadD::kan (38
CH4247 dksA-NN ΔyadD::kan CH4207 x P1 JW5010 
CH2714 lacIFRTlacZYA (2
CH5041 lacIFRTlacZYA ΔdksA::tet CH2714 x P1 CH2300 
CH5045 lacIFRTlacZYA ΔyjaZ::kan ΔbtuB3191::Tn10 rpoC-E677G CH2714 x P1 CH2570 
CH5047 lacIFRTlacZYA rpoC-F1325L thi3178/Tn10kan CH2714 x P1 CH2300 
CH5087 lacIFRTlacZYA rpoC-F1325L thi3178/Tn10kan ΔdksA::tet CH5047 x P1 CH2300 

Plasmids

Table 2.
Plasmids
Plasmid Construct Reference 
pBA169 pTrc99A ΔncoI ApR (34
pCP20 FLP+ λ cI857+ λ pR Repts ApR CmR (37
pDksA pBA169-dksA (35
pDksA-NN pBA169-dksA-NN (28
pGreA pBA169-greA (28
pGreB pBA169-greB (28
Plasmid Construct Reference 
pBA169 pTrc99A ΔncoI ApR (34
pCP20 FLP+ λ cI857+ λ pR Repts ApR CmR (37
pDksA pBA169-dksA (35
pDksA-NN pBA169-dksA-NN (28
pGreA pBA169-greA (28
pGreB pBA169-greB (28

Genetic manipulations

Genes to be overexpressed were cloned into pBA169 or pBR322 vectors by standard cloning and transformation techniques (33–35). Gene replacements were made as reported by Datsenko and Wanner (36,37). Specific mutations were transferred between different genetic backgrounds using P1 phage transduction (32,38).

Growth conditions and media

Bacteria were grown in LB broth and on LB and M9 glucose (0.2%) agar plates. Media were supplemented with the following antibiotics: ampicillin (Amp; 50 μg/ml) or carbenicillin (CB; 50 μg/ml), rifampicin (Rif; 50 μg/ml), tetracycline (Tet; 12.5 μg/ml), chloramphenicol (CM; 12.5 μg/ml) and kanamycin (Kan; 30 μg/ml). Thiamine (10 μg/ml) was added when necessary. Isopropyl-β-D-thiogalactoside (IPTG) was used as an inducer for overexpression of the respective constructs (Table 2). Methyl-β-D-thiogalactoside (TMG) was used in the epigenetic bistable switch assay. 5-Bromo-4-chloro-3-indolyl-β-D-thiogalactoside (Xgal) was used to monitor β-galactosidase levels in colonies on M9 glucose agar plates supplemented with casamino acids.

To demonstrate hysteresis and bistability in lac operon expression in single cells, a bacterial culture grown in minimal A salts (32) plus MgSO4 (1 mM) with succinate (0.2%) and thiamine was diluted 1:5 in fresh medium with (ON culture) or without 1 mM TMG (OFF culture) and shaken at 37°C for 7 h. After this induction period, the two cultures were individually diluted and ∼200 cells were seeded to new tubes containing fresh medium that contained varying amounts of TMG and shaken at 37°C for 42 h. Flow cytometry was used to determine the percentage of cells that were induced for lac operon expression (ON cells).

To determine epigenetic switch frequencies, a bacterial culture grown in minimal succinate media, was diluted and ∼200 cells were seeded to new tubes containing fresh medium, with a maintenance level of TMG, and shaken at 37°C for 42 h, as previously described (1,2,39), and subjected to flow cytometry.

Flow cytometry

The analysis of phenotypic switching was performed as described previously (2), using BD FACSCanto™ II Flow Cytometer (Becton, Dickinson and Company, USA) with Diva™ acquisition software (Becton Dickinson), FloJo™ analysis software (Tree Star, Inc. USA) and Prism™ (GraphPad). All cultures were subjected to arbitrary gating, which encompassed approximately 70% of the most typical events (Supplementary Figure S1).

β-galactosidase activity assay

Cultures were grown overnight at 37°C in LB in the presence of IPTG (0.1 mM) to express the lacZ-U118 allele. Aliquots (0.5 ml) were assayed as described earlier (32), except that samples were centrifuged to pellet cell debris before determination of OD420.

Single-molecule mRNA fluorescent in situ hybridization (smFISH)

The smFISH protocol has been described in detail (40,41). Fluorescently labeled oligonucleotide probe sequences designed against the lacI transcript (purchased from Biosearch Technologies, USA) are described in (39). Bacterial strains for smFISH analysis were grown in minimal A salts with succinate (0.2%) and thiamine at 37°C. The estimation of mRNA number in the cell relies on quantifying localized fluorescence and is not achieved by counting discrete spots. The number of bound probes is measured on the basis of the total fluorescence intensity (photon flux) of the spots, without requiring that individual mRNAs appear as separate spots. By performing a calibration step, the total intensity of spots in the cell can then be converted to the number of target mRNAs (41).

RESULTS

Effect of DksA on epigenetic switching in the lac bistable system

To analyze the impact of DksA on transcription fidelity, we employed a bistable lac system that captures transient errors in mRNA synthesis in the form of a stable and heritable phenotype (1,2). Briefly, the lac operon comprises a bistable switch controlled by the LacI repressor under a specific, maintenance concentration of inducer (TMG). The lacI mRNA is a rare transcript, made approximately once per cell cycle, and transcription errors in lacI can lead to a transient depletion of LacI repressor function that triggers an autocatalytic positive-feedback response that flips the epigenetic switch of the bistable network to the ON state (Figure 1). Previously, we showed that a 5-fold decrease in transcription fidelity due to an RNAP mutation (measured both in vitro and in vivo) (42) leads to a 4- to 6-fold increase in switching frequency in our bistable lac assay (1). Thus, the lac bistable system is sensitive to RNA mistakes. If DksA acts to ensure proper transcription, the absence of DksA should increase the phenotypic switching frequency in our lac bistable assay. In order to circumvent the inability of the ΔdksA strain to grow in the minimal medium required for the phenotypic switching assay, we used a known RNAP suppressor of ΔdksA auxotrophy, β’ F1325L (43). To eliminate the possibility of the suppressor itself affecting our observations on DksA, we assessed whether the rpoC F1325L mutation alone had an effect in the bistable switch assay. The β’ F1325L allele strain shows maintenance of the bistable switch (1,44) at a concentration of 8 μM TMG (Figure 2A and B). Thus, the system remains bistable in the presence of the β’ F1325L allele. Cells with β’ F1325L switch their phenotype from OFF to ON at a frequency of 2% (Figure 2C and D) and when the β’ F1325L allele is combined with deletion of dksA, the phenotypic switch frequency is elevated approximately 7-fold (P = 0.001; Figure 2C and D). Moreover, this frequency fluctuates between the cultures indicating that stochastic events trigger the switching process, similar to that observed for mutation (Figure 2C) (1,2).

Figure 2.

The absence of DksA increases stochastic epigenetic switch frequency in the lac bistable gene network. Loss of DksA confers auxotrophy, and our phenotypic switching assay requires cultivation in minimal media with succinate and thiamine. Therefore, we use a suppressor of the auxotrophy observed in ΔdksA cells, β’ F1325L, as the appropriate control. (A) Representative flow cytometry GFP fluorescence histogram series of β’ F1325L (CH2263) and β’ F1325L ΔdksA (CH2300) cells that were originally ON (green histograms) or OFF (red histograms) were sub-cultured and grown in media containing various concentrations of TMG indicated on the vertical axis (104 cells interrogated). Below 6 μM TMG and above 20 μM TMG, the previous history of the cell (ON or OFF) does not affect the current state of the cell. However, between these TMG concentrations, the system exhibits hysteresis. The shaded area highlights the maintenance concentration of 8 μM TMG for these strains, the TMG concentration at which an ON cell remains ON and an OFF cell remains OFF but has a probability to stochastically switch ON (1, 2). (B) Cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG, as above. Filled symbols denote β’ F1325L cells; open symbols denote β’ F1325L ΔdksA cells. Each value is the average ± SD from four independent cultures. The shaded area highlights the maintenance concentration of 8 μM TMG for these strains. (C) OFF β’ F1325L cells (blue dots), β’ F1325L ΔdksA cells (green dots) were diluted and grown in media containing 8 μM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 30 independent cultures of each strain. Each dot represents the value for an independent culture. Means ± standard error of the mean are indicated. (D) The β’ F1325L ΔdksA epigenetic-switch frequency is significantly increased over the β’ F1325L value (Mann-Whitney Rank Sum Test, P = 0.001). All cultures analyzed carry the β’ F1325L allele.

Figure 2.

The absence of DksA increases stochastic epigenetic switch frequency in the lac bistable gene network. Loss of DksA confers auxotrophy, and our phenotypic switching assay requires cultivation in minimal media with succinate and thiamine. Therefore, we use a suppressor of the auxotrophy observed in ΔdksA cells, β’ F1325L, as the appropriate control. (A) Representative flow cytometry GFP fluorescence histogram series of β’ F1325L (CH2263) and β’ F1325L ΔdksA (CH2300) cells that were originally ON (green histograms) or OFF (red histograms) were sub-cultured and grown in media containing various concentrations of TMG indicated on the vertical axis (104 cells interrogated). Below 6 μM TMG and above 20 μM TMG, the previous history of the cell (ON or OFF) does not affect the current state of the cell. However, between these TMG concentrations, the system exhibits hysteresis. The shaded area highlights the maintenance concentration of 8 μM TMG for these strains, the TMG concentration at which an ON cell remains ON and an OFF cell remains OFF but has a probability to stochastically switch ON (1, 2). (B) Cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG, as above. Filled symbols denote β’ F1325L cells; open symbols denote β’ F1325L ΔdksA cells. Each value is the average ± SD from four independent cultures. The shaded area highlights the maintenance concentration of 8 μM TMG for these strains. (C) OFF β’ F1325L cells (blue dots), β’ F1325L ΔdksA cells (green dots) were diluted and grown in media containing 8 μM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 30 independent cultures of each strain. Each dot represents the value for an independent culture. Means ± standard error of the mean are indicated. (D) The β’ F1325L ΔdksA epigenetic-switch frequency is significantly increased over the β’ F1325L value (Mann-Whitney Rank Sum Test, P = 0.001). All cultures analyzed carry the β’ F1325L allele.

Effect of DksA on the PlacI promoter

To test whether the absence of DksA affects the lacI promoter, we employed a lacI-ZYA operon fusion (2). In this fusion, the Plac promoter sequence between lacI and lacZ has been deleted in-frame so that lacZ is only expressed from the PlacI promoter (Figure 3A). Interestingly, the absence of DksA, or the presence of an RNAP mutation that reduces binding of DksA to RNAP (β’ E677G) (45), significantly affects transcription of this operon fusion. Cells lacking DksA exhibit a very low level of β-galactosidase, indicating a down regulation in PlacI activity (Figure 3B). Importantly however, β’ F1325L fully restores the level of β-galactosidase to the wild-type level in the absence of DksA, showing that β’ F1325L is a valid suppressor of ΔdksA for transcription initiation (43). These data suggest that the increase in epigenetic switching observed in the β’ F1325L ΔdksA strain is not due to a change in PlacI promoter activity. This is important since a recent study showing a role for DksA in transcription fidelity used a plasmid-based lacZ fused to an early T7A1 promoter that was differentially expressed in the presence or absence of DksA in E. coli, and all in vivo results had to be normalized to take differential expression into account (31); no such accounting is needed here (see also Supplemental Figure S2).

Figure 3.

The RNA β’ F1325L polymerase restores PlacI activity to the wild-type level in ΔdksA cells. (A) In this lacI-ZYA operon fusion, the lacI gene encoding the lac repressor is fused with the lac operon genes and therefore the amount of functional lacI mRNA can be measured by assaying the β-galactosidase level in cultures grown in LB (2). (B) Loss of DksA causes a decrease in β-galactosidase level indicating that this transcription factor is required for the initiation of PlacI. The effect of RNAP β’ E677G falls between the wild-type and ΔdksA values indicating incomplete penetrance of the phenotype. Importantly, RNAP β’ F1325L restores PlacI activity to the wild-type level in ΔdksA cells. Average ± standard deviation values are indicated.

Figure 3.

The RNA β’ F1325L polymerase restores PlacI activity to the wild-type level in ΔdksA cells. (A) In this lacI-ZYA operon fusion, the lacI gene encoding the lac repressor is fused with the lac operon genes and therefore the amount of functional lacI mRNA can be measured by assaying the β-galactosidase level in cultures grown in LB (2). (B) Loss of DksA causes a decrease in β-galactosidase level indicating that this transcription factor is required for the initiation of PlacI. The effect of RNAP β’ E677G falls between the wild-type and ΔdksA values indicating incomplete penetrance of the phenotype. Importantly, RNAP β’ F1325L restores PlacI activity to the wild-type level in ΔdksA cells. Average ± standard deviation values are indicated.

Effect of DksA on lacI transcript number in single cells

To confirm that the increase in epigenetic switch frequency observed in the ΔdksA strain is not due to lower levels of lacI mRNA molecules, the number of lacI mRNA in β’ F1325L cells lacking DksA compared to β’ F1325L cells was measured at the single cell level using smFISH (39–41). smFISH was also performed on ΔgreA,B cells for comparison.

Briefly, lacI mRNA was hybridized to a series of fluorescent probes and visualized by fluorescence microscopy (39–41). The total intensity of spots in the cell is then converted to the number of lacI transcripts in the cell (41). Using smFISH it has previously been shown that fully 86% of wild-type lacI cells do not exhibit any lacI mRNA, 11% of cells have one lacI mRNA and 3% have two or more lacI mRNA per cell at any given time (0.18 ± 0.01 transcripts per cell; 2081 cells monitored) (39). All strains analyzed here (Figure 4) exhibit similar lacI mRNA levels, and no strain exhibits lower lacI mRNA levels than the wild-type strain. Compared to the β’ F1325L control, β’ F1325L cells lacking DksA did not show any reduction in lacI mRNA levels: fully 81% of β’ F1325L cells do not exhibit any lacI mRNA and 16% of cells have one lacI mRNA at any given time; similarly for β’ F1325L ΔdksA cells, fully 78% of these cells do not exhibit any lacI mRNA and 18% of cells have one lacI mRNA at any given time (Figure 4). Therefore, the 7-fold increase in lac epigenetic switching observed in the β’ F1325L ΔdksA strain, compared to the β’ F1325L control, is not due to an overall decrease in lacI mRNA (Figure 4). Cells lacking GreA and GreB exhibited the same number of lacI mRNA per cell as the β’ F1325L and β’ F1325L ΔdksA cells: fully 76% of these cells do not exhibit any lacI mRNA and 19% of cells have one lacI mRNA at any given time. Therefore, the greater than 40-fold increase in epigenetic switching observed in ΔgreA,B cells can also not be explained by a decrease in lacI mRNA number (1,2).

Figure 4.

Absence of DksA does not affect the number of lacI transcripts in a cell. Single molecule fluorescence in situ hybridization (smFISH) analysis of lacI mRNA numbers in E. coli cells grown in minimal media with succinate and thiamine was performed (39–41). lacI mRNA was hybridized to a series of fluorescent probes that visualize the transcript. The strains analyzed were β’ F1325L, β’ F1325L ΔdksA and ΔgreAB. The right panels represent microscopy images of the respective strains with lacI transcripts labeled using red fluorescent probes. The left panel shows the distribution of lacI transcripts per cell in the respective strains. Mean and standard error values for absolute lacI mRNA numbers per cell are shown for each strain; n is the number of cells analyzed. For comparison, wild-type E. coli cells exhibit an average of 0.18 lacI transcripts per cell, with fully 86% of cells not containing a lacI transcript at any given time (39).

Figure 4.

Absence of DksA does not affect the number of lacI transcripts in a cell. Single molecule fluorescence in situ hybridization (smFISH) analysis of lacI mRNA numbers in E. coli cells grown in minimal media with succinate and thiamine was performed (39–41). lacI mRNA was hybridized to a series of fluorescent probes that visualize the transcript. The strains analyzed were β’ F1325L, β’ F1325L ΔdksA and ΔgreAB. The right panels represent microscopy images of the respective strains with lacI transcripts labeled using red fluorescent probes. The left panel shows the distribution of lacI transcripts per cell in the respective strains. Mean and standard error values for absolute lacI mRNA numbers per cell are shown for each strain; n is the number of cells analyzed. For comparison, wild-type E. coli cells exhibit an average of 0.18 lacI transcripts per cell, with fully 86% of cells not containing a lacI transcript at any given time (39).

The two complementary methods to assess PlacI activity in the absence of DksA (in the presence of the RNAP β’ F1325L suppressor), the lacI-ZYA operon fusion and lacI mRNA smFISH, provide a consistent result: β’ F1325L ΔdksA cells are not compromised for PlacI activity nor lacI mRNA production compared to wild-type and β’ F1325L cells.

Effect of DksA on mistranscription of a lacZ nonsense mutation

To further study transcription fidelity in vivo, we employed a previously described allele of the gene encoding β-galactosidase, lacZ-U118 (42,46–48), that encodes a nonsense UAA (ochre) codon at amino acid position 18 in β-galactosidase (Figure 5A) (47). This mutation stops translation early thereby unmasking transcription terminator sites in the untranslated mRNA and terminates transcription of the full operon. Partial phenotypic suppression (‘leakiness’) of lacZ-U118 depends principally on transcription errors that correct the ochre mutation at the mRNA level, allowing translation to proceed and yield a full-length transcript, leading to the production of many active β-galactosidase molecules. The leakiness of this non-functional allele can be observed by assessing the level of β-galactosidase either by growing cells on media supplemented with the inducer IPTG and an excess of the lactose analogue Xgal, or more quantitatively by a β-galactosidase assay (32). Wild-type E. coli carrying the lacZ-U118 allele forms white colonies on Xgal plates, and strains with decreased transcription fidelity, such as ack-1 [rpoB P564L (1)] form colonies that are light blue (42). To test whether the absence of DksA has an effect on the leakiness of the nonsense codon in the lacZ-U118 allele, we streaked ΔdksA cells on IPTG Xgal M9 glucose casamino acids plates and observed that the absence of DksA clearly results in darker blue colonies as compared to wild-type (Figure 5B). In addition, we tested an RNAP mutant that has previously been shown to be insensitive to DksA, rpoC E677G (45), as well as a dksA allele that is defective in initiating transcription from DksA-dependent promoters in vitro, DksA-NN (28). We observed that, like the absence of DksA, the presence of the rpoC E677G allele also results in clearly increased β-galactosidase levels, attributed to increased leakiness of the lacZ-U118 allele. The presence of the DksA-NN allele also results in slightly bluer colonies compared to wild-type suggesting that DksA-NN acts during elongation since we have previously shown that DksA-NN has no effect on initiation of the Plac promoter (28). The amount of functional β-galactosidase was then directly measured in the same strains. We have determined that the Plac promoter is unaffected by the presence or absence of DksA which allows a straightforward comparison of β-galactosidase levels in different strains (see Supplemental Figure S2). This is important since a recent study showing a role for DksA in transcription fidelity used a plasmid-based lacZ fused to an early T7A1 promoter that was differentially expressed in the presence or absence of DksA in E. coli, and all in vivo results had to be normalized (by decreasing β-galactosidase levels observed by 2.3-fold) to take differential expression into account (31); no such accounting is needed here. The absence of either DksA, or the lack of sensitivity of RNAP to this transcription factor (rpoC E677G), resulted in a 2-fold increase in β-galactosidase levels (Figure 5C). Again, the presence of the initiation-deficient allele of dksA, DksA-NN, resulted in an intermediate activity of β-galactosidase, indicating only a slight but consistent decrease in transcription fidelity. This result of increased leakiness of a nonsense codon in the absence of DksA is similar to that found in a plasmid-based partial phenotypic suppression assay (31).

Figure 5.

The effect of DksA on transcription fidelity in a partial phenotypic suppression system. (A) The lacZ-U118 polar nonsense allele encodes a truncated non-functional β-galactosidase enzyme. Due to the coupling of transcription and translation, transcription is terminated when the first ribosome cannot match the ochre codon in the mRNA. When RNAP makes an error and phenotypically suppresses the DNA-encoded nonsense mutation, the ribosome does not stall and transcription continues. The wild-type transcription error rate allows for synthesis of a very small amount of β-galactosidase. (B) A wild-type strain carrying the lacZ-U118 allele forms white colonies on M9 IPTG glucose casamino acids Xgal plates. The absence of DksA results in blue colonies, indicating a larger amount of β-galactosidase present (possibly due to an increased transcription error rate). ΔdksA cells can grow on these plates without a suppressor. The RNAP mutant β’ E677G produces blue colonies as well, which agrees with the previously reported insensitivity to DksA (45). A transcription initiation-deficient allele of dksA (DksA-NN) does not mimic the phenotype of complete loss of DksA; therefore, the absence of the active tip could affect DksA-dependent transcription initiation but not necessarily elongation. (C) Quantification of the amount of functional β-galactosidase indicates that ΔdksA and β’ E667G result in a 2-fold increase in lacZ-U118 leakiness, presumably from an elevated transcription error rate. The β-galactosidase activity observed for DksA-NN mutant is intermediate, which indicates partial elongation/fidelity activity of the mutated transcription factor. (D) Overexpression of DksA decreases lacZ-U118 leakiness below the wild-type level suggesting that DksA actively prevents transcription errors in this allele. Overexpression of DksA-NN has a weaker effect but it restores the error rate to the wild-type level. In addition we overexpressed two transcription fidelity factors GreA and GreB and observed only partial suppression by GreA suggesting these fidelity factors have only a mild effect on lacZ-U118 leakiness.

Figure 5.

The effect of DksA on transcription fidelity in a partial phenotypic suppression system. (A) The lacZ-U118 polar nonsense allele encodes a truncated non-functional β-galactosidase enzyme. Due to the coupling of transcription and translation, transcription is terminated when the first ribosome cannot match the ochre codon in the mRNA. When RNAP makes an error and phenotypically suppresses the DNA-encoded nonsense mutation, the ribosome does not stall and transcription continues. The wild-type transcription error rate allows for synthesis of a very small amount of β-galactosidase. (B) A wild-type strain carrying the lacZ-U118 allele forms white colonies on M9 IPTG glucose casamino acids Xgal plates. The absence of DksA results in blue colonies, indicating a larger amount of β-galactosidase present (possibly due to an increased transcription error rate). ΔdksA cells can grow on these plates without a suppressor. The RNAP mutant β’ E677G produces blue colonies as well, which agrees with the previously reported insensitivity to DksA (45). A transcription initiation-deficient allele of dksA (DksA-NN) does not mimic the phenotype of complete loss of DksA; therefore, the absence of the active tip could affect DksA-dependent transcription initiation but not necessarily elongation. (C) Quantification of the amount of functional β-galactosidase indicates that ΔdksA and β’ E667G result in a 2-fold increase in lacZ-U118 leakiness, presumably from an elevated transcription error rate. The β-galactosidase activity observed for DksA-NN mutant is intermediate, which indicates partial elongation/fidelity activity of the mutated transcription factor. (D) Overexpression of DksA decreases lacZ-U118 leakiness below the wild-type level suggesting that DksA actively prevents transcription errors in this allele. Overexpression of DksA-NN has a weaker effect but it restores the error rate to the wild-type level. In addition we overexpressed two transcription fidelity factors GreA and GreB and observed only partial suppression by GreA suggesting these fidelity factors have only a mild effect on lacZ-U118 leakiness.

Moreover, we tested the suppression of the ΔdksA-related increase in lacZ-U118 leakiness by overexpressing auxiliary transcription factors that interact with the secondary channel of RNAP (Figure 5D). We used plasmids carrying DksA, DksA-NN, GreA and GreB (45). Overexpression of DksA has a dramatic effect on the leakiness of the lacZ-U118 allele in ΔdksA cells and nearly completely prevented (below wild-type levels) the production of functional β-galactosidase. Overexpression of the transcription initiation-deficient DksA-NN allele restored leakiness to the wild-type level, indicating that partial elongation activity is maintained for this protein. Overexpression of the transcription factor GreA, but not GreB, modestly decreased the effect on lacZ-U118 leakiness in ΔdksA cells (Figure 5D). These data suggest that the absence of DksA promotes base substitutions that increase the leakiness of the lacZ-U118 ochre mutation and overexpression of DksA reduces base substitution misincorporation. The weaker effect of GreA or GreB overexpression suggest that either the level of these factors is not enough to allow full rescue or that these factors act on a different step of transcription fidelity (see Discussion).

Effect of DksA on transcriptional slippage in vivo

We have previously shown that the combined absence of transcription fidelity factors GreA and GreB results in increased transcriptional slippage as measured by phenotypic switching in our lac bistable system in E. coli that was engineered to have a run of nine adenine residues immediately after the translation start codon (2). Here, we employed the same system to test whether DksA actively prevents RNAP slippage, similarly to GreA and GreB. Specifically, we used our slippage-prone allele of the lac repressor, lacI-A9; this altered gene encodes a fully functional lac repressor, and the nine consecutive adenine residues are ‘slippery’ for RNAP (2,7,49,50) (Figure 6A). Both β’ F1325L lacI-A9 and β’ F1325L ΔdksA lacI-A9 strains exhibit bistability and similar hysteresis patterns, and the maintenance concentration of 15 μM TMG is the same for both strains (Figure 6B).

Figure 6.

DksA does not prevent transcriptional slippage. (A) The lacI-A9 ‘slippery’ sequence is prone to transcriptional slippage (2). (B) A hysteresis graph of strains carrying the β’ F1325L mutation, which is a suppressor of ΔdksA-induced auxotrophy, and the modified lacI-A9 allele, with or without DksA. The method used is that of Figure 2B. (C) The epigenetic switch frequency in presence of the lacI-A9 sequence is identical with or without DksA. (D) The switch phenotype of lacI-A9 is not significantly altered in the presence of DksA. All experiments were done using the lacI-A9 β’ F1325L background.

Figure 6.

DksA does not prevent transcriptional slippage. (A) The lacI-A9 ‘slippery’ sequence is prone to transcriptional slippage (2). (B) A hysteresis graph of strains carrying the β’ F1325L mutation, which is a suppressor of ΔdksA-induced auxotrophy, and the modified lacI-A9 allele, with or without DksA. The method used is that of Figure 2B. (C) The epigenetic switch frequency in presence of the lacI-A9 sequence is identical with or without DksA. (D) The switch phenotype of lacI-A9 is not significantly altered in the presence of DksA. All experiments were done using the lacI-A9 β’ F1325L background.

Analysis of lac bistable switching at the maintenance concentration of TMG for the strains lacI-A9 β’ F1325L with or without DksA revealed no difference in epigenetic switch frequency, indicating that, unlike the GreA,B factors, slippage at the lacI-A9 sequence is independent of DksA (Figure 6B and C). This lack of induction of RNA transcriptional frameshifting supports the idea that DksA specifically influences nucleotide misincorporation (31) as we observed with the leaky behavior of the lacZ-U118 allele in the absence of DksA, and the suppression of leakiness when DksA was overexpressed in ΔdksA cells (Figure 5D).

DISCUSSION

This work provides in vivo evidence that the auxiliary factor DksA is a bona fide transcription fidelity factor and supports the recent finding that DksA reduces misincorporation during transcription in vitro and in an in vivo plasmid T7A1-promoter partial phenotypic nonsense lacZ suppression system (31). Here, we employed a variety of in vivo assays to monitor transcription fidelity in living cells and observed a significant decrease in transcription fidelity when DksA was absent. First, we used the epigenetic lac switch system that is sensitive to transcription errors and found an increase in the frequency of epigenetic switching in the absence of DksA, indicating a decrease in transcription fidelity (Figures 2 and 5)(1,2). This increase in epigenetic switching is not due to a decrease in PlacI activity (Figure 3) or the number of lacI transcripts in ΔdksA cells (Figure 4). Moreover, we show that the rate of misincorporation at the lacZ-U118 locus (that allows for synthesis of fully functional β-galactosidase) is increased in the absence of DksA as compared to the wild-type strain (Figure 5). This assay specifically detects base-pair misincorporation events by RNAP, which indicates that DksA diminishes nucleotide misincorporation into mRNA. Thus, our in vivo results are in agreement with the recent in vitro data showing that DksA/ppGpp decreases misincorporation by RNAP (31) and complement the in vivo partial phenotypic nonsense suppression that has been observed in ΔdksA cells (31).

Mechanistically, the GreA/B factors and DksA are different. Both factors have been shown to bind and compete for the secondary channel of RNAP, but unlike GreA/B, DksA is incapable of promoting endonucleolytic cleavage of the nascent RNA chain positioned in the secondary channel (29). Transcription fidelity factors GreA and GreB assist and stimulate RNAP to cleave nucleotides at the 3′ end of the growing RNA of backtracked RNAP in order to eliminate the mismatch error and restart transcription, and no such activity has been associated with DksA. Therefore, it is most likely that DksA acts at a different step than GreA/B during nucleotide incorporation. In vitro, DksA has been shown to have diverse effects on transcription elongation (29), including inhibition of transcript elongation. Thus, it is possible that DksA might affect fidelity by slowing the rate of transcription elongation, however there are no data supporting that a slowing down of transcription elongation rate affects transcription errors (1,51). In vivo, we show that overexpression of GreA or GreB cannot fully rescue the increased leakiness of the lacZ-U118 in ΔdksA cells, whereas overexpresssion of DksA reduces such lacZ-U118 leakiness below wild-type levels in ΔdksA cells. DksA has been shown to interact with the trigger loop of RNAP (26), a mobile domain involved in nucleotide selection during RNA synthesis and this suggests, as recently proposed (31), that DksA acts on the prevention of errors rather than error correction. Moreover, we show that unlike GreA/B, DksA does not play a role in preventing RNAP slippage at a track of nine adenines, again suggesting that DksA may only affect base substitution misincorporations.

The DksA factor has been shown to play a crucial role in the resolution of conflicts between replication and transcription upon nutritional stress. Recent in vitro data suggest that DksA/ppGpp increases fidelity of transcription by slowing down misincorporation of nucleotides by RNAP (31). Misincorporation is known to cause RNAP backtracking, which may lead to formation of a RNAP roadblock on DNA. Thus, it was proposed that DksA may exert its anti-collision property by preventing formation of backtracked RNAP due to nucleotide misincorporation (31). This explanation may not be the cause of conflict for the following reasons: firstly, the effect of DksA on collision avoidance is ppGpp independent (28), while its role in fidelity in vitro is stimulated by ppGpp (31); secondly, we would like to note that a RNAP infidelity mutant, which decreases fidelity by 6-fold (42), has been shown to suppress the replication collision defect in the absence of DksA (28). Finally, the effect of DksA on resolution of replication/transcription conflicts during starvation is exacerbated by uncoupling transcription and translation (30), without invoking misincorporation of nucleotides by RNAP. The idea that transcription infidelity promotes replication conflict is interesting, but additional experiments will need to be performed in order to elucidate the exact role of DksA in resolving conflicts between replication and transcription, and how DksA affects transcription fidelity.

From our in vivo results, we find that DksA, along with a role in transcription initiation and elongation, also participates in maintaining transcription fidelity in E. coli. It is clear that maintaining transcription fidelity is important to ensure the proper propagation of the phenotypic state of the lac bistable switch and this is likely true for other gene networks. Transcription fidelity should therefore be considered a driving force of epigenetic change.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

We thank Richard Gourse, Carol Gross, Herman Dierick and Priya Sivaramakrishnan for comments on the manuscript. We thank Joel M. Sederstrom of the Cytometry and Cell Sorting Core at Baylor College of Medicine for expert assistance. We thank the three anonymous reviewers for insight and helpful comments.

FUNDING

US National Institutes of Health [R01 GM88653 to C.H., R01 GM082837 to I.G., AI036211, CA125123, and RR024574 to Cytometry and Cell Sorting Core at Baylor College of Medicine]; US National Science Foundation [PHY 1147498, PHY 1430124 and PHY 1427654 to I.G.]; Welch Foundation [Q-1759 to I.G.]. Fund for open access charge: US National Institutes of Health [R01 GM88653].

Conflict of interest statement. None declared.

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