A 3′ UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria

Abstract

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3′ untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3′ UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA–RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblots and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ impacts growth of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

Glutamine synthetase mRNA releases sRNA from its 3′UTR to regulate carbon/nitrogen metabolic balance in Enterobacteriaceae

“…We detected GlnZ sRNAs corresponding to the glnA 3′UTRs in size and their processed species, which were induced when Gln was used as the sole nitrogen source ( Figure 2C ). This result is consistent with the study by Walling et al, 2022 , but not the previous report by Jia et al, 2021 . In support, SroC was also induced under the nitrogen limiting condition in the two pathogenic E. coli strains devoid of IS 5 insertion upstream of gltI .…”

“…Using the pXG30-sf constructs of glnHP and deoBD intergenic regions, we verified that the expression of glnP and deoD was repressed by S. enterica GlnZ1 and GlnZ2 through the base-pairing mechanism ( Figure 3C and D ). It is noteworthy that the glnP target region is located just upstream of the SD sequence and is conserved in E. coli as verified by the others ( Walling et al, 2022 ). However, Salmonella GlnZ1 binds to the deoD mRNA at the first three codons, which are synonymously mutated in E. coli .…”

“…Since E. coli GlnZ is classified as a class I sRNA which binds to proximal and rim faces of RNA chaperone Hfq ( Kavita et al, 2022 ), Hfq is implicated in the stability of GlnZ. Using the probe that hybridizes the 3′ end region of K-12 GlnZ, we detected a few bands of GlnZ, denoted as GlnZ 213 , GlnZ 194 , and GlnZ 174 by the others ( Walling et al, 2022 ). In the hfq mutant, the glnA mRNA was induced under the nitrogen limiting conditions, but we detected only faint fragments of GlnZ 174 ( Figure 5A ).…”

“…It has recently been reported that GlnZ of E. coli K-12 is also a substrate of RNase III in vitro as it contains a long stem-loop structure ( Walling et al, 2022 ). We tested whether the processing of glnA mRNA is modified in the rnc14 mutant using the pBAD expression system.…”

“…Likewise, the glnA mRNA contains a Rho-independent terminator with a moderate strength located upstream of glnLp ( Ueno-Nishio et al, 1984 ), which can be a ligand of RNA chaperone Hfq ( Chen et al, 2019 ). Indeed, an sRNA has been detected in the 3′UTR of glnA mRNA in E. coli , renamed as GlnZ ( Kawano et al, 2005 ; Walling et al, 2022 ), and also in the same locus of S. enterica ( Sittka et al, 2009 ). Therefore, GlnZ is regarded as a RpoN-dependent sRNA in charge of the non-coding arm of the NtrC regulon.…”