Protein dosage of the lldPRD operon is correlated with RNase E-dependent mRNA processing

Abstract

The ability of Escherichia coli to grow on L-lactate as a sole carbon source depends on the expression of the lldPRD operon. A striking feature of this operon is that the transcriptional regulator (LldR) encoding gene is located between the permease (LldP) and the dehydrogenase (LldD) encoding genes. In this study we report that dosage of the LldP, LldR, and LldD proteins is not modulated on the transcriptional level. Instead, modulation of protein dosage is primarily correlated with RNase E-dependent mRNA processing events that take place within the lldR mRNA, leading to the immediate inactivation of lldR, to differential segmental stabilities of the resulting cleavage products, and to differences in the translation efficiencies of the three cistrons. A model for the processing events controlling the molar quantities of the proteins in the lldPRD operon is presented and discussed.ImportanceAdjustment of gene expression is critical for proper cell function. For the case of polycistronic transcripts, posttranscriptional regulatory mechanisms can be used to fine-tune the expression of individual cistrons. Here, we elucidate how protein dosage of the Escherichia coli lldPRD operon, which presents the paradox of having the gene encoding a regulator protein located between genes that code for a permease and an enzyme, is regulated. Our results demonstrate that the key event in this regulatory mechanism involves the RNase E-dependent cleavage of the primary lldPRD transcript at internal site(s) located within the lldR cistron, resulting in a drastic decrease of intact lldR mRNA, to differential segmental stabilities of the resulting cleavage products, and to differences in the translation efficiencies of the three cistrons.

FIG 1

Genetic structure of the lldPRD operon. Open arrows indicate the positions and directions of the three structural genes. Gene lengths are given in parentheses. Transcriptional elements in the upstream region of the structural genes (such as promoter –35 and –10 sequences), the transcription initiation site, and the ArcA-P and LldR binding sites are indicated. The relative position of the RBS of each gene is shown. In the text boxes, the ribosome binding sites and the start codons are underlined, and the stop codons of the overlapping genes are shown in boldface and labeled with asterisks.

FIG 2

Unequal protein production from the lldPRD operon is not controlled at the transcriptional or translational level. (A) Levels of the LldR–3×FLAG (31.9-kDa) and LldD–3×FLAG (45.4-kDa) proteins in E. coli cells grown aerobically on M9 minimal medium supplemented with 0.2% Casamino Acids and 20 mM l-lactate, as determined by Western blot analyses using anti-Flag antibodies. Cell extracts from strains IFC5032 and IFC5033 were loaded in lanes 1 and 2, respectively. The total protein content loaded in lane 1 is 100 times higher than that loaded in lane 2. The positions of standard protein markers are shown on the left. (B) Stabilities of the LldR–3×FLAG and LldD–3×FLAG proteins after inhibition of protein synthesis with chloramphenicol. (Left) Western blot analysis using anti-Flag antibodies. (Right) Quantitative determination of LldR and LldD from Western blot experiments plotted as a function of time. (C) Probing for possible internal promoters in the lldPRD operon. (Left) Schematic representation of the lldPRD operon and the fragments fused to lacZ to construct strains ECL5002 and IFC5028. (Right) Strains ECL5002 and IFC5028 were grown aerobically on M9 minimal medium supplemented with 0.2% Casamino Acids either in the presence (+) or in the absence (−) of 20 mM l-lactate. At the mid-exponential phase of growth, cells were harvested and assayed for β-galactosidase activity, expressed as a percentage of the activity of strain ECL5002 grown in the presence of the inducer. Averages from four independent experiments are presented, and standard deviations (error bars) are indicated. (D) Translation initiation rates of the three lldPRD genes as determined by lacZ translational fusions. (Left) Schematic representation of the lldPRD operon and the fragments fused to lacZ for strains IFC5029, IFC5030, IFC5031, and IFC5032. The relative position of the RBS of each gene or lacZ fusion is indicated by a triangle. (Right) Strains IFC5029, IFC5030, IFC5031, and IFC5032 were grown aerobically on M9 minimal medium supplemented with 0.2% Casamino Acids and 20 mM l-lactate as the inducer. At the mid-exponential phase of growth, cells were harvested and assayed for β-galactosidase activity, expressed as a percentage of the activity of strain IFC5031. Averages from four independent experiments are presented, and standard deviations (error bars) are indicated. (E) Analysis of the lld transcript by Northern blotting. Strain MC4100 was incubated on M9 minimal medium supplemented with 0.2% Casamino Acids in the presence (+) or absence (−) of 20 mM l-lactate, and total RNA isolated from samples that were harvested during mid-exponential growth was probed for the lld transcript using an lldD-specific DNA probe. A blot representative of results from three independent experiments is shown. The positions of standard RNA markers are shown to the left of the blot. The lower panel shows the ethidium bromide-stained rRNA bands of the corresponding Hybond membrane, used as a loading control.

FIG 3

Analysis of lldPRD mRNA in an rne⁺ and an rne(Ts) strain by Northern blotting. Strains CH1827 (rne⁺) and CH1828 [rne(Ts)] were grown aerobically at 30°C. At an OD₆₀₀ of 0.4, cells were shifted to 43°C, samples were withdrawn at the indicated times, and total RNA was isolated. Shown are autoradiograms of blots with the specific lldP probe (A) and the lldD probe (B). The positions of standard RNA markers are shown on the left. Arrowheads indicate the positions of the lldP, lldD, and lldPRD transcripts. Lower panels show the ethidium bromide-stained rRNA bands of the corresponding Hybond membranes, used as loading controls.

FIG 4

Primer extension analysis of sites of RNase E cleavage into the lldR transcript. (A) Total RNAs from strains CH1827 (rne⁺) and CH1828 [rne(Ts)], grown aerobically in the presence of 20 mM l-lactate at 30°C and 15 min after a shift to a nonpermissive temperature (43°C), were prepared and used for primer extension analysis. A 5′-end-labeled primer specific for the lldR transcript was annealed to total RNA and extended by reverse transcriptase. The resulting cDNA was resolved in an 8% polyacrylamide gel alongside a DNA sequencing ladder. Positions of relevant primer extension products are marked on the right. The relevant portion of the mRNA sequence (corresponding to the complementary sequence of the DNA sequencing ladder) is presented on the left, and the identified 5′ ends are shown in boldface. (B) Schematic representation of the lldPRD operon and RNase E cleavage sites identified by primer extension analysis. Sites of cleavage into the lldR transcript at positions +381 and +389 with respect to the start codon of lldR are shown (▴), and predicted RNase E recognition sequences, which fulfill the proposed RNase E target motif RN↓WUU (where R stands for G or A, W stands for A or U, and N stands for any nucleotide) (30), are marked in boldface. The 5′ end corresponding to the larger primer extension product detected in the RNase E mutant, at position +341 with respect to the lldR start codon, is also indicated (Δ).

FIG 5

Northern blot analysis of lldP and lldD mRNA stabilities. E. coli MC4100 was grown in LB medium supplemented with 20 mM l-lactate at 37°C to the mid-exponential phase prior to the addition of rifampin. Samples were then harvested at the indicated times, and RNA was extracted as described in Materials and Methods. Ten micrograms of total RNA from each time point was used for Northern blot analysis. (Left) Autoradiograms of blots using the specific lldP (top) or lldD (bottom) probe. Lower panels show the ethidium bromide-stained rRNA bands of the corresponding Hybond membranes. (Right) Semilogarithmic plot of lldP and lldD mRNA decay. The calculated half-lives were 6.8 min for lldD and 2.5 min for lldP.

FIG 6

Gene organization of the 301 predicted TFs encoded in the E. coli genome. Open arrows represent genes encoding predicted TFs, and shaded arrows represent other genes. “(n)” stands for a number from 1 to 15. The number of TFs in each organizational group (TFs in monocistronic mRNA units or at the beginning, end, or middle of polycistronic mRNA units) is given on the left, and the number of TFs that are part of a two-component regulatory system (TCS), and share an operon with the cognate histidine kinase, is given in parentheses.