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

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. 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 P hospholipids, which are the building blocks of bacterial membranes, are synthesized by a series of enzymes localized in the inner membrane. The biochemistry of the phospholipid synthesis pathway has been well deciphered, especially in the Escherichia coli model bacterium (1). Phospholipid composition is very tightly controlled and is modulated by various parameters, such as growth conditions, growth rate, and stresses. Most of this control is thought to occur at the enzymatic activity level (1). However, transcriptomic data have recently uncovered the possibility that several signaling pathways control phospholipid synthesis through a regulation of enzyme expression levels in E. coli. Indeed, the expression of all the genes for phospholipid

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

“…Some novel pathways identified here that should be of immediate focus are the succinate dehydrogenase complex and the hypothetical gene VK055_3211 and its divergently transcribed regulator, VK055_3212_hdfR [ 51 , 52 ]. Homologues to VK055_3211 and hdfR have been identified in E .…”

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impactKlebsiella pneumoniaefitness

“…Some novel pathways identified here that should be of immediate focus are the succinate dehydrogenase complex and the hypothetical gene VK055_3211 and its divergently transcribed regulator, VK055_3212_hdfR [43, 44]. Homologues to VK055_3211 and hdfR have been identified in E. coli to contribute to the organization of the Ori region during chromosome replication and HdfR has been shown to repress the flagellar master operon ( flhDC ) [43, 45].…”

Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes

“…For genes where expression decreases, this drop may stem from a loss of translational coupling from the upstream gene, while for genes with improved expression, translation of the smORF may impede translation of the downstream gene. It is interesting to note that a mutation (pssR1) that leads to increased expression of pssA mapped to the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by rrnC (64). Further characterization will be required to distinguish smORFs that are simply translated in operons versus those that specifically serve to control the translation of downstream genes and to elucidate the regulatory mechanisms.…”