Carbohydrate Utilization in Bacteria: Making the Most Out of Sugars with the Help of Small Regulatory RNAs

Manifold roles of sRNAs in regulation of carbohydrate metabolism in bacteria. The figure summarizes the major roles of sRNAs (depicted in red) in regulation of carbohydrate metabolism in bacteria. (A) Regulation of uptake and utilization of particular carbohydrates by sRNAs in various species. In Enterobacteriaceae, the trans-encoded sRNA SgrS counteracts phosphosugar stress through repression of glucose transporters and activation of the sugar phosphatase YigL. sRNA ChiX downregulates the chitosugar-specific porin ChiP, setting the threshold concentration for induction of degrading enzymes. Further examples include regulation of host glycan and mannitol uptake by cis-encoded sRNAs in Bacteroides and Vibrio species, respectively. (B) Role of sRNAs in CCR. In Enterobacteriaceae and Vibrionaceae, the sRNA Spot 42 represses genes for utilization of secondary carbon sources. Spot 42 is repressed by cAMP-CRP and therefore only active in the presence of preferred sugars generating low cAMP levels. In Pseudomonas, translation of mRNAs for utilization of secondary carbon sources is repressed by Hfq. In the absence of preferred substrates, the CbrA/CbrB TCS activates expression of the decoy sRNA CrcZ, titrating Hfq from target transcripts. (C) sRNAs coordinate carbohydrate metabolism with carbohydrate, oxygen, and iron availability. The RNA-binding protein CsrA activates glycolysis and represses gluconeogenesis by binding to corresponding RNAs. CsrA activity is counteracted through sequestration by sRNAs CsrB/CsrC, whose levels are regulated by signals from metabolism. In the absence of oxygen, sRNAs such as FnrS in E. coli and RoxS in B. subtilis redirect metabolism from oxidative phosphorylation to anaerobic respiration or fermentation. Upon iron starvation, sRNA RyhB represses TCA cycle enzymes to save iron for essential processes. (D) Example of an anabolic pathway regulated by sRNAs. In Enterobacteriaceae, two homologous sRNAs regulate the key enzyme GlmS to achieve homeostasis of glucosamine-6-phosphate (glucosamine-6-P), an essential precursor for cell envelope synthesis.

The transcriptional regulator cAMP-CRP and sRNA Spot 42 cooperate to trigger CCR in Enterobacteriaceae. (A) CRP and Spot 42 participate in coherent feedforward loops to prevent utilization of the indicated secondary carbon sources when the preferred carbon source glucose is present. In addition to cAMP-CRP, Spot 42 is regulated by base-pairing with the sponge RNA PspH. (B) The validated Spot 42 regulon to date. Target genes that are also positively controlled by cAMP-CRP at the level of transcription are boxed. Microarray analysis of Spot 42 pulse expression ( 75 ) and improved software prediction algorithms ( 32 , 76 ) fostered the identification of most targets. Additional targets were identified by human inference or by a CLIP-seq approach mapping Hfq binding sites on a global scale ( 74 , 82 , 173 , 181 ). Several of these targets were recovered by RIL-seq ( 77 ).

Posttranscriptional regulation of central carbon metabolic pathways in E. coli. Effects of sRNAs (depicted in red) and of the RNA-binding protein CsrA (blue) on synthesis of enzymes involved in glycolysis, gluconeogenesis, and the TCA cycle. A green asterisk and bold letters indicate direct regulation by CsrA ( 106 ). Anabolic pathways directing synthesis of glycogen, UDP-sugars, and the biofilm compound PGA are also shown.

Model of the interconnection of the CsrA system with central carbon metabolism. Decoy sRNAs CsrB and CsrC regulate CsrA activity by sequestering the protein from its target mRNAs. CsrA indirectly activates csrB/csrC transcription, creating a negative feedback loop. In fast-growing cells, when CsrB/C levels are low, CsrA activates glycolytic genes and represses the TCA cycle, gluconeogenesis, and glycogen synthesis. Metabolism of glycolytic carbon sources causes accumulation of FBP, which activates pyruvate kinase, thereby reducing the PEP/pyruvate ratio. Intake of PTS substrates and a low PEP/pyruvate ratio trigger dephosphorylation of EIIAGlc, leading to activation of CsrD, which triggers degradation of CsrB/CsrC by RNase E. Upon accumulation of short carboxylic acids (R-COOH) as metabolic end products, expression of csrB/csrC is induced by the BarA/UvrY TCS. Deceleration of glycolytic activity elevates the PEP/pyruvate level and increases EIIAGlc phosphorylation, leading to stabilization of CsrB/CsrC and titration of CsrA. EIIAGlc∼P stimulates adenylate cyclase CyaA, which converts ATP to cAMP. The cAMP-CRP complex inhibits transcription of csrB/csrC. Involvement of EIIAGlc in regulation of CsrB/CsrC synthesis as well as decay allows integration of further cues.

Role of RNase E adaptor protein RapZ in feedback regulation of GlmS synthesis in E. coli. When GlcN6P is plentiful in the cell, RapZ prevents glmS upregulation by targeting its activating sRNA GlmZ to cleavage by RNase E. Within the tripartite complex formed, the sRNA is envisioned to be sandwiched between the tetrameric RapZ protein and the N-terminal domain (NTD) of RNase E, which also forms a tetramer ( 168 ). Processing results in functional inactivation of GlmZ and subsequent decline in GlmS levels. Conversely, under GlcN6P depletion, RapZ is predominantly sequestered in complexes with the homologous sRNA GlmY, whose levels increase under this condition. Consequently, GlmZ remains in its active full-length form and stimulates glmS expression. Higher levels of GlmS replenish GlcN6P levels in the cell. Whether RapZ has an active role in sensing GlcN6P via direct binding of the metabolite within its C-terminal domain (CTD) is currently under investigation. The unusual tetrameric structure of RapZ is schematically depicted in the box in the upper left. Each monomer is represented by one color and consists of two globular domains, NTD and CTD, connected via flexible linkers. Three distinct surfaces involved in self-interaction can be discerned: CTD-CTD, NTD-NTD, as well as CTD-NTD ( 168 ).