Small RNA‐binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli

Abstract The RNA‐binding protein RapZ cooperates with small RNAs (sRNAs) GlmY and GlmZ to regulate the glmS mRNA in Escherichia coli. Enzyme GlmS synthesizes glucosamine‐6‐phosphate (GlcN6P), initiating cell envelope biosynthesis. GlmZ activates glmS expression by base‐pairing. When GlcN6P is ample, GlmZ is bound by RapZ and degraded through ribonuclease recruitment. Upon GlcN6P depletion, the decoy sRNA GlmY accumulates through a previously unknown mechanism and sequesters RapZ, suppressing GlmZ decay. This circuit ensures GlcN6P homeostasis and thereby envelope integrity. In this work, we identify RapZ as GlcN6P receptor. GlcN6P‐free RapZ stimulates phosphorylation of the two‐component system QseE/QseF by interaction, which in turn activates glmY expression. Elevated GlmY levels sequester RapZ into stable complexes, which prevents GlmZ decay, promoting glmS expression. Binding of GlmY also prevents RapZ from activating QseE/QseF, generating a negative feedback loop limiting the response. When GlcN6P is replenished, GlmY is released from RapZ and rapidly degraded. We reveal a multifunctional sRNA‐binding protein that dynamically engages into higher‐order complexes for metabolite signaling.

. The separated globular domains of RapZ are impaired in binding QseE and QseF and fail to activate glmY expression.
Data information: In (A) and (B), b-galactosidase activities are presented as mean AE SD, n = 3. Source data are available online for this figure.

Muna A Khan et al
The EMBO Journal ª 2020 The Authors The EMBO Journal e103848 | 2020 Figure EV2. Upon GlcN6P depletion, GlmY* still accumulates to a minor extent in DqseF and DqseE mutants and this effect requires rapZ.
Northern blots comparing GlmY levels in MG1655 derivative strains. The wildtype strain Z854, the DqseF mutant Z1081, the DqseE mutant Z1082, and the DqseF DrapZ double mutant Z1083 were assessed under normal growth ("mock") and GlcN6P starvation ("Nva-FMDP") conditions, respectively. Bacteria were grown and analyzed as described for Fig 1A. Total RNAs were subjected to Northern analysis using probes directed against GlmY and 5S rRNA. Total RNA isolated from the various mutants was analyzed on the right halves of the various blots alongside wild-type RNA samples (  Strain Z904 (DglmS; MG1655 derivative) carrying a glmS'-lacZ reporter fusion on the chromosome was grown in LB supplemented with 0.2% GlcN until OD 600 = 0.3. Subsequently, the culture was split, cells were washed, and growth was continued in the presence or absence of 0.2% GlcN. Samples were harvested at hourly intervals and used for determination of b-galactosidase activities (right, bottom) and extraction of total RNA, which was analyzed by Northern blotting using probes specific for GlmY, GlmZ, and 5S rRNA (left). The corresponding growth curves are provided (right, top). The time = 0 h refers to the time of splitting the culture. Data information: b-galactosidase activities and OD 600 values underlying the growth curves are presented as mean AE SD, n = 3. Source data are available online for this figure. A B C Figure EV4. Stabilization of GlmY* upon GlcN6P starvation requires the RNA-binding activity of RapZ.
Strain Z939 (DglmS DrapZ) was used, which harbored a plasmid transcribing either rapZ or rapZ quad from the P Ara promoter. RapZ quad comprises alanine substitutions of four amino acid residues (Lys270, Lys281, Arg282, and Lys283) abrogating RNA-binding activity. The bacteria were grown and analyzed as described for Fig 6. For induction of rapZ expression, 0.2% arabinose was added to the pre-cultures used for inoculation of the test cultures containing or lacking 0.2% GlcN.
A Analysis of strain Z939 harboring plasmid pBGG61 transcribing wild-type rapZ. B Analysis of strain Z939 harboring plasmid pYG30 transcribing rapZ quad . C Semi-logarithmic plots of GlmY* decay for half-life determination.
Data information: In (C), data are presented as mean, n = 2. Source data are available online for this figure.