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(p)ppGpp modifies RNAP function to confer β-lactam resistance in a peptidoglycan-independent manner

Nature Microbiology volume 9, pages 647–656 (2024)Cite this article

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Abstract

(p)ppGpp is a nucleotide alarmone that controls bacterial response to nutrient deprivation. Since elevated (p)ppGpp levels confer mecillinam resistance and are essential for broad-spectrum β-lactam resistance as mediated by the β-lactam-insensitive transpeptidase YcbB (LdtD), we hypothesized that (p)ppGpp might affect cell wall peptidoglycan metabolism. Here we report that (p)ppGpp-dependent β-lactam resistance does not rely on any modification of peptidoglycan metabolism, as established by analysis of Escherichia coli peptidoglycan structure using high-resolution mass spectrometry. Amino acid substitutions in the β or β’ RNA polymerase (RNAP) subunits, alone or in combination with the CRISPR interference-mediated downregulation of three of seven ribosomal RNA operons, were sufficient for resistance, although β-lactams have no known impact on the RNAP or ribosomes. This implies that modifications of RNAP and ribosome functions are critical to prevent downstream effects of the inactivation of peptidoglycan transpeptidases by β-lactams.

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Fig. 1: Impact of (p)ppGpp and YcbB production on peptidoglycan structure.
Fig. 2: Localization of amino acid substitutions mediating β-lactam and rifampicin resistance in the RNAP holoenzyme.
Fig. 3: Impact of downregulation of rRNA transcription on β-lactam resistance.
Fig. 4: Impact of the downregulation of rrn operons on the relative number of ribosomes per cell.

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Data availability

Source data are provided with this paper. Whole-genome sequencing raw data of RNAP mutants are available at the Sequence Read Archive database (SRA) under accession code PRJNA1044113. Structures used in this study are available on the Protein Data Bank [entry 5VSW (RNAP, DksA and ppGpp complex), 5UAC (RNAP and rifampicin complex) and 7KHB (RNAP and rrnB P1 promoter open complex)]. Proteomic data of the ribosomal tryptic peptides are available at the MassIVE database under accession code MSV000093796 and at the ProteomeXchange database under accession code PXD048334. Source data are provided with this paper.

Code availability

The i2MassChroQ software used to identify and quantify the tryptic peptides is freely available at https://forgemia.inra.fr/pappso/i2masschroq/-/releases (the preferred software version for this work is 1.0.0).

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Acknowledgements

This work was supported by the French National Research Agency ANR ‘RegOPeps’ (grant ANR-19-CE44-0007 to J.-E.H.). H.V. and C.A.-P. are the recipients of doctoral fellowships from Sorbonne-Université (ED 515, Complexité du Vivant). We thank A. Marie for technical assistance in the collection of mass spectra at the Plateau Technique de Spectrométrie de Masse Bio-Organique of the Muséum National d’Histoire Naturelle; E. Maisonneuve for the kind gift of the pool of the ASKA plasmids; D. Bikard for the kind gift of the pFR56 plasmid; and Z. Edoo for proofreading the manuscript.

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  1. These authors contributed equally: Henri Voedts, Constantin Anoyatis-Pelé.

Authors and Affiliations

  1. Centre de Recherche des Cordeliers, Sorbonne Université, INSERM, Université Paris Cité, Paris, France

    Henri Voedts, Constantin Anoyatis-Pelé, Filippo Rusconi, Jean-Emmanuel Hugonnet & Michel Arthur

  2. GQE-Le Moulon/PAPPSO, Université Paris-Saclay, INRAE, CNRS, AgroParisTech, IDEEV, Gif-sur-Yvette, France

    Olivier Langella & Filippo Rusconi

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Contributions

H.V. conceived and designed the project; acquired, analysed and interpreted data; and drafted and revised the article. C.A.-P. conceived and designed the project; acquired, analysed and interpreted data; and revised the article. F.R. and O.L. performed mass spectrometric analysis of ribosome preparations and quantitative mass data processing. J.-E.H. and M.A. conceived and designed the project; analysed and interpreted data; and drafted and revised the article.

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Correspondence to Jean-Emmanuel Hugonnet or Michel Arthur.

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Extended data

Extended Data Fig. 1 Pleiotropic effects of (p)ppGpp in E. coli.

The (p)ppGpp alarmone binds to enzymes involved in multiple metabolic pathways, such as the GuaB inosine monophosphate dehydrogenase, the DnaG DNA primase, and the PPX polyphosphate kinase3. The alarmone also modulates the expression of approximatively 1200 genes by binding to two sites on RNA polymerase (RNAP)4. Modulation of gene expression involves (1) activation of specific promoters involved in amino acid synthesis, (2) recruitment of alternative sigma factors, and (3) downregulation of ribosomal RNA operons2,17.

Extended Data Fig. 2 Reactions catalyzed by PBPs and L,D-transpeptidases.

(a) Peptidoglycan cross-linking by PBPs is a two-step reaction initiated by the activation of the catalytic Ser residue for nucleophilic attack of the carbonyl of D-Ala4 in a pentapeptide donor stem. This first step results in the release of D-Ala5 and formation of an acyl-enzyme. In the second step, the carbonyl of the resulting ester bond undergoes nucleophilic attack by the side-chain amino group of the diaminopimelyl (DAP) residue of an acceptor. This results in the release of the PBP and in the formation of a peptidoglycan dimer (Tetra→Tri or Tetra→Tetra) containing a 4 → 3 cross-link, which connects D-Ala at the 4th position of the donor to DAP at the 3rd position of the acceptor. Tetra→Tri and Tetra→Tetra dimers differ by the presence of a tripeptide or a tetrapeptide in the acceptor position, respectively. D,D-transpeptidase catalytic domains are found in Class A PBPs PBP1a, PBP1b, and PBP1c, which also contain a glycosyltransferase domain for glycan chain elongation. D,D-transpeptidase catalytic domains are also found in monofunctional Class B PBP2 and PBP3. (b) The L,D-transpeptidases YcbB (LdtD) and YnhG (LdtE) also catalyze peptidoglycan cross-linking in a two-step reaction similar to that catalyzed by PBPs. Main differences involve the nature of (i) the nucleophile: a cysteine instead of a serine, (ii) the donor stem: a tetrapeptide instead of a pentapeptide, and (iii) the cross-link: 3 → 3 instead of 4 → 3.

Extended Data Fig. 3 Impact of (p)ppGpp and YcbB production on peptidoglycan structure in strains lacking the chromosomal copy of ycbB.

Relative peak area (%) were computed for muropeptides extracted from strains BW25113 ΔrelA ΔycbB harboring plasmids pHV6 and pHV7 (blue), pHV6 and pKT8(relA’) (orange), pKT2(ycbB) and pHV7 (dashed blue), or pKT2(ycbB) and pKT8(relA’) (dashed orange). Data are means and standard deviations from three biological repeats (n = 3). Tri and Tetra, disaccharide-tripeptide and disaccharide-tetrapeptide monomers, respectively. 3 → 3 and 4 → 3, dimers containing 3 → 3 and 4 → 3 cross-links, respectively.

Source data

Extended Data Fig. 4 Position of the amino acid substitutions in the RNAP β subunit at the proximity of the rifampicin binding site (a) and DNA in the open promoter complex (b).

Cartoon and surface representations of the PDB entry 5UAC42 (a) and 7KHB43 (b) are shown with subunits highlighted in various colors. (p)ppGpp binding sites of RNAP are indicated by red arrows and dashed circles. Substitutions led to resistance to β-lactams only (green), to rifampicin only (red), and to both β-lactams and rifampicin (yellow). The inset in panel (a) shows rifampicin (pink) bound to RNAP (for the sake of simplicity, only the β subunit is shown). The inset in panel (b) shows DNA (grey) of the rrnB P1 open promoter complex. All substitutions are localized in the β subunit except substitutions in positions 333, 1144, 1147, and 1308 of β’ subunit (indicated in parentheses). The figure was generated with PyMOL (v2.3.4).

Extended Data Fig. 5 Role of (p)ppGpp binding to the two RNAP binding sites.

Plating efficiency of BW25113 ΔrelA pKT2(ycbB) pKT8(relA′) and its derivatives obtained by deletion of the rpoZ or dksA genes. Growth was tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) on BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA′, respectively (n = 3).

Extended Data Fig. 6 Role of the Q148L and G449V RpoB substitutions in β-lactam and rifampicin resistance.

Plating efficiency was performed for BW25113 ΔrelA ΔspoT pKT2(ycbB) and its derivatives harboring mutations leading to the G449V or Q148L substitution in the β-subunit of the RNAP (encoded by rpoB). Growth was tested in BHI agar supplemented by the indicated antibiotics and 40 µM IPTG for induction of ycbB. DNA was extracted from the cultures that were used for the phenotypic analysis and whole genome sequencing did not reveal any additional mutation in the genes encoding the RNAP subunits. This control indicates that our analysis is not biased by the accumulation of suppressors of the ΔrelA and ΔspoT mutations. Data are a representative of three independent experiments (n = 3).

Extended Data Fig. 7 Impact of sub-inhibitory concentrations of chloramphenicol on the generation time and expression of ceftriaxone resistance.

(a) Generation time (GT) as a function of supplementation of BHI broth with various concentrations of chloramphenicol. Each point is the median from five biological repeats. (b) Plating efficiency of the RpoB G449V mutant harboring pKT2(ycbB) (positive control) and of the parental BW25113 ΔrelA ΔspoT pKT2(ycbB) strain in the absence (- Cm) or presence (+ Cm) of chloramphenicol at various concentrations. All plates contained 40 µM IPTG for induction of ycbB. Plates contained 8 µg/ml ceftriaxone (+ ceftriaxone) or no drug (- ceftriaxone). Data are a representative of five biological repeats. (c) Plating efficiency of the RpoB G449V mutant harboring pKT2(ycbB) (positive control) and of the parental BW25113 ΔrelA ΔspoT pKT2(ycbB) strain grown at 28 °C. Plates contained 40 µM IPTG for induction of ycbB, 8 µg/ml ceftriaxone (+ ceftriaxone) or no drug (- ceftriaxone). Data are a representative of five biological repeats. The generation time of the parental strain was 38 ± 4 min at 28 °C versus 24 ± 1 min at 37 °C (determined from five biological repeats).

Source data

Extended Data Fig. 8 Lowering rRNA transcription bypasses the requirement of (p)ppGpp for mecillinam resistance.

(a) Sequence alignments of portions of the rrl genes targeted by the sgRNAs. Variable positions exploited to downregulate rrlB, E, and G without affecting rrlA, C, D, and H expression are marked by asterisks. sgRNA sequences shown are the reverse complements (RC) of the sgRNA sequences used to downregulate rrl transcription. See Material and Methods for the design of sgRNAs. (b) Plating efficiency of derivatives of BW25113 ΔrelA ΔspoT harboring plasmids encoding dCas9 under the control of the DAPG-inducible Phlf promoter and four sgRNAs under the control of a constitutive promoter. The sgRNAs targeted seven (7 rrl) or three (3 rrl) of the seven 23 S rRNA genes. The remaining sgRNAs, ctrl1 and ctrl2, do not target any sequence in the E. coli chromosome25. Induction of the dcas9 gene was performed with 25, 50, or 200 µM DAPG. Induction of dcas9 in the presence of the 7 rrl sgRNA prevented growth of BW25113 ΔrelA ΔspoT at DAPG concentrations of 50 and 200 µM. Effective downregulation of all 7 rrl copies is likely to account for the absence of growth. (c) Plating efficiency of the RpoB G449V mutant (positive control), the parental strain BW25113 ΔrelA ΔspoT pKT2(ycbB) (negative control) and its derivatives harboring plasmids encoding dCas9 under the control of the DAPG-inducible Phlf promoter and the four sgRNAs under the control of a constitutive promoter. Growth was tested in the presence of mecillinam at 16 µg/ml (+ mecillinam) or in the absence of the drug (- mecillinam). Induction of the dcas9 gene was performed with 50 µM DAPG. Mecillinam resistance was obtained in the absence of ycbB induction. Induction of the gene encoding dCas9 was associated with a 1.4-fold increase in the generation time (from 28 ± 2 min versus 37 ± 1 min; n = 3).

Extended Data Fig. 9 Expression of ceftriaxone resistance mediated by downregulation of the transcription of rrl genes in the absence of the P376R substitution in the β subunit of the RNAP.

The panel shows the plating efficiency of the BW25113 ΔrelA ΔspoT pKT2(ycbB) pHV136(dcas9; 3 rrl). The parental strain BW25113 ΔrelA ΔspoT was re-sequenced to ensure the absence of mutations in the genes encoding the RNAP subunits. Plasmids pKT2(ycbB) and pHV136(dcas9; 3 rrl) plasmids were introduced in six independent cultures of this strain (clones 1 to 6). The strain appearing in the top row and in Fig. 3 additionally harbored the unexpected P376R substitution in the β subunit of the RNAP. Growth was tested in the presence of 8 µg/mL ceftriaxone (+ ceftriaxone), 16 µg/mL mecillinam (+ mecillinam), or in the absence of the drugs (- β-lactam). Induction of dcas9 was performed with 50 µM DAPG ( + DAPG). BHI agar plates contained 40 µM IPTG for induction of ycbB (n = 6).

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Combined Excel file containing tabs with statistical and raw source data. Source Data Fig. 2 Combined Excel file containing tabs with statistical and raw source data. Source Data Fig. 3 Combined Excel file containing tabs with statistical and raw source data. Source Data Fig. 4 Combined Excel file containing tabs with statistical and raw source data. Source Data Extended Data Fig. 3 Combined Excel file containing tabs with statistical and raw source data. Source Data Extended Data Fig. 7 Combined Excel file containing tabs with statistical and raw source data.

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Voedts, H., Anoyatis-Pelé, C., Langella, O. et al. (p)ppGpp modifies RNAP function to confer β-lactam resistance in a peptidoglycan-independent manner. Nat Microbiol 9, 647–656 (2024). https://doi.org/10.1038/s41564-024-01609-w

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