Protein ProQ influences osmotic activation of compatible solute transporter ProP in Escherichia coli K-12

Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Journal of Bacteriology (Impact Factor: 2.81). 04/1999; 181(5):1537-43.
Source: PubMed


ProP is an osmoregulatory compatible solute transporter in Escherichia coli K-12. Mutation proQ220::Tn5 decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter proP transcription or the ProP protein level. Allele proQ220::Tn5 was isolated, and the proQ sequence was determined. Locus proQ is upstream from prc (tsp) at 41.2 centisomes on the genetic map. The proQ220::Tn5 and prc phenotypes were different, however. Gene proQ is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids). The insertion of PCR-amplified proQ into vector pBAD24 produced a plasmid containing the wild-type proQ open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in proQ+ bacteria but not in proQ220::Tn5 bacteria. ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level.

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Available from: Hans Jörg Kunte
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    • "The mechanism behind ProQ regulation of ProP activity, however, remains largely unknown. Disruption of the proQ locus has no effect on the transcription of proP, but the proline uptake activity of ProP is significantly decreased in a ΔproQ strain [11,16]. A post-translational mechanism was initially proposed after ProP protein levels appeared unchanged in a ΔproQ strain [16]; however, a direct physical interaction between ProP and ProQ has not been found. "
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    ABSTRACT: The decrease in proline transport by the proline porter ProP in a ΔproQ strain has been well documented; however, the reason for this phenotype remains undefined. Previous studies have speculated that ProQ facilitates translation of proP mRNA. Here, we demonstrate that ProQ is enriched in the polysome fractions of sucrose gradient separations of E. coli lysates and the 30S fractions of lysates separated under conditions causing ribosomal subunit dissociation. Thus, ProQ is a bona fide ribosome associated protein. Analysis of proQ constructs lacking predicted structural domains implicates the N-terminal domain in ribosome association. Association with the ribosome appears to be mediated by an interaction with the mRNA being translated, as limited treatment of lysates with Micrococcal Nuclease maintains ribosome integrity but disrupts ProQ localization with polysomes. ProQ also fails to robustly bind to mRNA-free 70S ribosomes in vitro. Interestingly, deletion of proP does not disrupt the localization of ProQ with translating ribosomes, and deletion of proP in combination with the proU operon has no effect on ProQ localization. We also demonstrate that ProQ is necessary for robust biofilm formation, and this phenotype is independent of ProP. Binding studies were carried out using tryptophan fluorescence and in vitro transcribed proP mRNAs. proP is transcribed from two differentially regulated promoters, and ProQ interacts with proP mRNA transcribed from both promoters, as well as a control mRNA with similar affinities. In total, these data suggest that ProQ is positioned to function as a novel translational regulator, and its cellular role extends beyond its effects on proline uptake by ProP.
    Full-text · Article · Oct 2013 · PLoS ONE
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    • "These stresses could be changes in turgor pressure caused by osmotic perturbation or even when remodelling the cell wall upon entry into stationary phase or during growth and division (Kung et al., 2010). This genomic conservation, as well as the fact that several prlS homologues are annotated as 'proline sensor' and that proline is a well-known osmoprotectant (Kunte et al., 1999; Racher et al., 1999), are preliminary indications that the TCS PrlS/PrlR could play a role in the Brucella response to changes in osmotic %paper no. mic060863 charlesworth ref: mic060863& "
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    ABSTRACT: Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.
    Full-text · Article · Aug 2012 · Microbiology
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    • "Both the ProU and ProP systems are regulated by the osmolarity of the medium. The proU operon, which encodes the components of the former system, is induced by osmotic stress (Balaji et al., 2005), whereas the latter is regulated by a combination of transcriptional induction (Balaji et al., 2005) and in situ activation (Kunte et al., 1999), resulting in up to 20-fold increase in activity by osmotic stress (Grothe et al., 1986). In addition to ProU and ProP, S. typhimurium LT2 has an additional transport system, OsmU, which can take up glycine betaine in media of high osmolarity (Gutierrez and Csonka, 1989), encoded by the STM1491 to STM1494 genes. "
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    ABSTRACT: In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.
    Full-text · Article · Feb 2012 · DNA and cell biology
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