Glutamine synthetase mRNA releases sRNA from its 3´UTRto regulate carbon/nitrogen metabolic balance in Enterobacteriaceae
- University of Tsukuba, Japan
- Keio University, Japan
- Chiba University, Japan
- Kyushu University, Japan
- Tokyo Institute of Technology, Japan
Abstract
Glutamine synthetase (GS) is the key enzyme of nitrogen assimilation induced under nitrogen limiting conditions. The carbon skeleton of glutamate and glutamine, 2-oxoglutarate, is supplied from the TCA cycle, but how this metabolic flow is controlled in response to nitrogen availability remains unknown. We show that the expression of the E1o component of 2-oxoglutarate dehydrogenase, SucA, is repressed under nitrogen limitation in Salmonella enterica and E coli. The repression is exerted at the post-transcriptional level by an Hfq-dependent sRNA GlnZ generated from the 3´UTR of the GS-encoding glnA mRNA. Enterobacterial GlnZ variants contain a conserved seed sequence and primarily regulate sucA through base-pairing far upstream of the translation initiation region. During growth on glutamine as the nitrogen source, the glnA 3´UTR deletion mutants expressed SucA at higher levels than the S. enterica and E. coli wild-type strains, respectively. In E. coli, the transcriptional regulator Nac also participates in the repression of sucA. Lastly, this study clarifies that the release of GlnZ from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA. Thus the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.
Data availability
The RNA-seq data have been deposited in DDBJ DRA under accession number DRA012682.
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Regulatory network analysis of mRNA 3'UTRs in SalmonellaDDBJ Sequence Read Archive, DRA012682.
Article and author information
Author details
Masatoshi Miyakoshi
Funding
Japan Society for the Promotion of Science (JP19H03464)
- Masatoshi Miyakoshi
Japan Society for the Promotion of Science (JP19KK0406)
- Masatoshi Miyakoshi
Japan Society for the Promotion of Science (JP21K19063)
- Masatoshi Miyakoshi
Japan Society for the Promotion of Science (JP22H02236)
- Kan Tanaka
Japan Society for the Promotion of Science (JP16H06279)
- Hiroki Takahashi
- Tetsuya Hayashi
Waksman Foundation of Japan
- Masatoshi Miyakoshi
Takeda Medical Research Foundation
- Masatoshi Miyakoshi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Alexander J. Westermann, Helmholtz Centre for Infection Research, Germany
Version history
- Preprint posted: July 25, 2022 (view preprint)
- Received: August 3, 2022
- Accepted: November 27, 2022
- Accepted Manuscript published: November 28, 2022 (version 1)
- Accepted Manuscript updated: November 29, 2022 (version 2)
- Version of Record published: December 8, 2022 (version 3)
Copyright
© 2022, Miyakoshi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Glutamine synthetase mRNA releases sRNA from its 3´UTRto regulate carbon/nitrogen metabolic balance in Enterobacteriaceae
eLife 11:e82411.
https://doi.org/10.7554/eLife.82411
Further reading
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- Medicine
- Microbiology and Infectious Disease
Background:
End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines.
Methods:
The immune response to the COVID-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and 7 d after the second dose (V2D7) using anti-spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified 6 mo after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response.
Results:
Transcriptomic analyses demonstrated differing time courses of immune responses, with prolonged myeloid cell activity in HD at 1 wk after the first vaccination dose. HD also demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p<0.05). Anti-spike IgG remained elevated above baseline at 6 mo in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development.
Conclusions:
Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance HD subjects comparable to healthy controls and identify transcriptomic and clinical predictors of anti-spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of ESRD.
Funding:
F30HD102093, F30HL151182, T32HL144909, R01HL138628. This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003.
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- Microbiology and Infectious Disease
In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to finetune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.
