Stress-Induced Block in Dicarboxylate Uptake and Utilization in Salmonella enterica Serovar Typhimurium

Hersch, Steven J.; Radan, Bojana; Ilyas, Bushra; Lavoie, Patrick; Navarre, William Wiley

doi:10.1128/jb.00487-20

“…Wild-type S. Typhimurium displays an extended lag phase and a slow doubling time when grown in media with succinate as the sole carbon source (Hersch et al, 2021). Because deletion of the S. Typhimurium proQ gene is known to modulate expression of hundreds of mRNAs and sRNAs (Smirnov et al, 2016), we investigated whether ProQ modulated the succinate-dependent in vitro growth phenotype.…”

Section: Resultsmentioning

confidence: 99%

“…However, despite the established global activity of ProQ as an RBP, the known phenotypes of Salmonella ΔproQ strains (e.g., impaired motility and reduced invasion of eukaryotic cells (Westermann et al, 2019)) were insufficiently robust for in vivo screens under physiological conditions. Inspired by the reported growth suppression of Salmonella with succinate as sole carbon source (Hersch et al, 2021), we discovered that deletion of the proQ gene generated a gain-of-function phenotype that permitted rapid growth on minimal media containing succinate. Using this strong phenotype for a saturation mutagenesis screen, we have mapped crucial residues for ProQ function in vivo.…”

Section: Introductionmentioning

confidence: 99%

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

Mouali

¹

,

Ponath

²

,

Scharrer

³

et al. 2021

RNA

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The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region that are required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

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“…The first pathway is the modulation of the succinate-dependent growth in vivo , where strains with inactivated ProQ presented a gain-of-function phenotype, with an increased growth rate in the presence of succinate, which when present as a sole carbon source in wild type S . Typhimurium causes an extended lag phase and a slowed doubling time [ 97 , 98 ]. The same phenotype was observed for different ProQ mutants, both in the NTD and CTD.…”

Section: The C-terminal Domain Of Proqmentioning

confidence: 99%

Bacterial RNA chaperones and chaperone-like riboregulators: behind the scenes of RNA-mediated regulation of cellular metabolism

Katsuya-Gaviria

¹

,

Paris

²

,

Dendooven

³

et al. 2022

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In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences. In bacteria, three main proteins – Hfq, ProQ, and CsrA – have been shown to regulate numerous complex processes, including bacterial growth, stress response and virulence. Hfq and ProQ have well-studied activities as global chaperones with pleiotropic impact, while CsrA has a chaperone-like role with more defined riboregulatory function. Here, we describe relevant novel insights into their common features, including RNA binding properties, unstructured domains, and interplay with other proteins important to RNA metabolism.

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“…Succinate has been identified as an important carbon source for Salmonella during colonization of the murine gastrointestinal tract (31), and plays an important role during macrophage infection (32,33). Wild-type S. Typhimurium displays an extended lag phase and a slow doubling time when grown in media with succinate as the sole carbon source (30). Because deletion of the S. Typhimurium proQ gene is known to modulate expression of hundreds of mRNAs and sRNAs (9), we investigated whether ProQ modulated the succinate-dependent in vitro growth phenotype.…”

Section: Proq Suppresses Growth In Succinate-containing Mediamentioning

confidence: 99%

“…However, despite the established global activity of ProQ as an RBP, the known phenotypes of Salmonella ΔproQ strains (e.g., impaired motility and reduced invasion of eukaryotic cells (29)) were insufficiently robust for in vivo screens under physiological conditions. Inspired by the reported growth suppression of Salmonella with succinate as sole carbon source (30), we discovered that deletion of the proQ gene generated a gain-of-function phenotype that permitted rapid growth on minimal media containing succinate. Using this strong phenotype for a saturation mutagenesis screen, we have mapped crucial residues for ProQ function in vivo.…”

Section: Introductionmentioning

confidence: 99%

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

Mouali

¹

,

Ponath

²

,

Scharrer

³

et al. 2021

Preprint

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The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.SIGNIFICANCEProteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.

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Stress-Induced Block in Dicarboxylate Uptake and Utilization in Salmonella enterica Serovar Typhimurium

Cited by 6 publications

References 72 publications

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

Bacterial RNA chaperones and chaperone-like riboregulators: behind the scenes of RNA-mediated regulation of cellular metabolism

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

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