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. 2017 Jun 27;199(14):e00202-17.
doi: 10.1128/JB.00202-17. Print 2017 Jul 15.

The Long Hunt for pssR-Looking for a Phospholipid Synthesis Transcriptional Regulator, Finding the Ribosome

J Bartoli  1 , L My  1 , Lucid Belmudes  2 , Yohann Couté  2 , J P Viala  1 , E Bouveret  3
Affiliations

The Long Hunt for pssR-Looking for a Phospholipid Synthesis Transcriptional Regulator, Finding the Ribosome

J Bartoli et al. J Bacteriol. .

Abstract

The phospholipid (PL) composition of bacterial membranes varies as a function of growth rate and in response to changes in the environment. While growth adaptation can be explained by biochemical feedback in the PL synthesis pathway, recent transcriptome studies have revealed that the expression of PL synthesis genes can also be tuned in response to various stresses. We previously showed that the BasRS two-component pathway controls the expression of the diacylglycerol kinase gene, dgkA, in Escherichia coli (A. Wahl, L. My, R. Dumoulin, J. N. Sturgis, and E. Bouveret, Mol Microbiol, 80:1260-1275, 2011, https://doi.org/10.1111/j.1365-2958.2011.07641.x). In this study, we set up a strategy to identify the mutation responsible for the upregulation of pssA observed in the historical pssR1 mutant and supposedly corresponding to a transcriptional repressor (C. P. Sparrow and J. Raetz, J Biol Chem, 258:9963-9967, 1983). pssA encodes phosphatidylserine synthase, the first step of phosphatidylethanolamine synthesis. We showed that this mutation corresponded to a single nucleotide change in the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by the rrnC operon. We further demonstrated that this mutation enhanced the translation of pssA Though this effect appeared to be restricted to PssA among phospholipid synthesis enzymes, it was not specific, as evidenced by a global effect on the production of unrelated proteins.IMPORTANCE Bacteria adjust the phospholipid composition of their membranes to the changing environment. In addition to enzymatic regulation, stress response regulators control specific steps of the phospholipid synthesis pathway. We wanted to identify a potential regulator controlling the expression of the phosphatidylserine synthase gene. We showed that it was not the previously suggested hdfR gene and instead that a mutation in the anti-Shine-Dalgarno sequence of 16S RNA was responsible for an increase in pssA translation. This example underlines the fact that gene expression can be modulated by means other than specific regulatory processes.

Keywords: Escherichia coli; hdfR; maoP; phosphatidylserine synthase; phospholipid synthesis; pssA; pssR; ribosomal mutations; rrnC; yifE.

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Figures

FIG 1
FIG 1
Upregulation of pssA in the pssR1 mutant. (A) Strains AC1 (pssR+), AC5 (pssR1), AC1 ΔhdfR::Chlr (EB356), AC5 ΔhdfR::Chlr (EB886), MG ΔhdfR pssR+ (EB1011), and MG ΔhdfR pssR1 (EB430) (Table 2) were transformed with the pssA-GFP translational fusion and the corresponding control plasmids (pEB1853 and pEB1900), and expression of pssA-GFP was measured as described in Materials and Methods. Data are the means from four replicates and the error bars show the standard deviations. (B) AC1 and AC5 strains producing tagged phospholipid enzymes, EB160 and EB161 (PssA-SPA), EB1052 and EB1053 (PlsB-SPA), EB1050 and EB1051 (PlsC-SPA), and EB1054 and EB1055 (PgsA-TAP) (Table 2), were grown in LB at 37°C and stopped in exponential growth phase. Whole-cell extracts were separated by 10% SDS-PAGE and the SPA-tagged or TAP-tagged proteins were analyzed by Western blotting using anti-Flag or PAP antibodies, respectively. (C) The experiment shown in panel B was repeated with three or four replicates for each strain, and the Western blots were quantified on a Li-Cor imager. The error bars show the standard deviations.
FIG 2
FIG 2
hdfR is not pssR. (A) Wild-type MG1655 was transformed by the pBAD24, pBAD-hdfR, or pBAD-hdfR(G237S) plasmid. Cells were grown in LB at 37°C and expression was induced with 0.5% arabinose for 2 h. Whole-cell extracts were separated by 10% SDS-PAGE, and proteins were stained with Coomassie blue. Strains MG1655 and EB814 (ΔhdfR) were transformed simultaneously with the fusion pTrad-pssA (pEB1611) (B) or pUA-yifE (C) and the pBAD-hdfR or pBAD-hdfR(G237S) plasmid (Table 3). Cultures were performed in LB supplemented with ampicillin, kanamycin, and 0.5% arabinose, and expression was monitored as described in Materials and Methods. Data are the means from 4 replicates and the error bars show the standard deviations.
FIG 3
FIG 3
The rrsC(C1538T) mutation is responsible for the increase in pssA expression in AC5. (A) rrnC operon. The mutations present in AC5 but not in AC1 are indicated in red. (B) AC1 and AC5 strains with portions of the rrnC operon deleted (strains EB350, EB349, EB1062, EB352, EB351, and EB1063) (Table 2) were transformed with the pssA-GFP translational fusion (plasmid pEB1853), and expression was monitored as described in Materials and Methods. Data are the means from 4 replicates and the error bars show the standard deviations. (C) Strains AC1 and AC5 were transformed with the pssA-GFP reporter fusion (plasmid pEB1853) and the indicated plasmids [cont, pEB0354; prrnC, pEB1858; prrnC(C1538T), pEB1866]. Expression was monitored as described in Materials and Methods. Data are the means from 4 replicates and the error bars show the standard deviations.
FIG 4
FIG 4
The rrsC(C1538T) mutation affects pssA translation initiation. (A) The 5′ UTR sequence of pssA and wild-type (wt) or mutated (mutC1538U) anti-SD sequences of 16S RNA are shown. Mutations are indicated in red. TSS, transcription start site. The different constructions of GFP fusions with portions of the pssA gene are shown below. (B and C) Strains AC1 and AC5 were transformed with the indicated reporter fusions (plasmids pEB1853, pEB1864, and pEB1878 for panel B and plasmids pEB1611, pEB1856, and pEB1895 for panel C). Expression was monitored as described in Materials and Methods. Data are the means from 4 replicates and the error bars show the standard deviations.
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