The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing

Abstract

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surfacedisplayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0 Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit $160 Å from the cell surface. The CRE and prolinerich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.

Essential autoproteolysis of bacterial anti-σ factor RsgI for transmembrane signal transduction

“…The C-terminal part of these RsgIs has a divergent carbohydrate-binding module (CBM), which was proposed to sense different types of polysaccharides (27)(28)(29). In Clostridium thermocellum, RsgI1, RsgI2, and RsgI4 contain family 3b CBMs that target cellulose; RsgI3 contains a tandem protective antigen 14 (PA14) motif that recognizes pectin; RsgI5 contains a family 42 CBM that binds to arabinose; RsgI6 contains a family 10 glycosidase hydrolase that targets both xylan and cellulose; RsgI7 and RsgI8 do not contain an obvious CBM, and their functions are currently unknown; and lastly, RsgI9 contains a protease-like domain that is capable of binding cellulose (28)(29)(30). After signal transduction across the cell membrane, the RsgIs release their bound cognate SigIs for initiating transcription of a subset of cellulosomal genes, resulting in the dynamic regulation of cellulosome function, which is one of the major reasons for the high efficiency of anaerobic cellulosome-producing bacteria in lignocellulose degradation (11,(24)(25)(26).…”

Insight into the autoproteolysis mechanism of the RsgI9 anti‐σ factor from Clostridium thermocellum

“…Coupled with prior findings that show RsgI9's C-terminal ectodomain binds cellulose, these results are consistent with it regulating the composition of the cellulosome via a RIP signal transduction mechanism. 22 In this process, polysaccharide binding to the RsgI9 domain would cause the pre-cleaved CRE domain to separate, thereby exposing it to degradation by the RasP intramembrane metalloprotease to release a yet to be identified σ-factor that controls gene transcription. This work builds upon previous studies of the CRE domain by providing insight into the potential mechanism for autoproteolysis.…”

“…[16][17][18][19] In the signaling process for the SigItype σ-factors, each of the membrane-bound anti-σ factors are believed to bind to different types of extracellular polysaccharides. 17,[20][21][22] They then transduce this signal across the membrane, triggering the release of their cognate SigI-type σ-factor from the cytoplasmic membrane. 23 The SigI-type σ-factor then binds to the RNA polymerase complex enabling it to transcribe specific GH encoding genes, as well as genes that encode scaffoldin proteins used to construct the cellulosome.…”

“…These include eight SigI‐type σ‐factors (σ 1 –σ 8 ) whose activities are modulated by at least eight RsgI‐type anti‐σ factors (RsgI1‐8) that are co‐transcribed from the same operon, and σ 24 which is regulated by the Rsi24c anti‐σ factor 16–19 . In the signaling process for the SigI‐type σ‐factors, each of the membrane‐bound anti‐σ factors are believed to bind to different types of extracellular polysaccharides 17,20–22 . They then transduce this signal across the membrane, triggering the release of their cognate SigI‐type σ‐factor from the cytoplasmic membrane 23 .…”

“…Recently we reported the structure of the cellulose‐binding ectodomain from the C. thermocellum RsgI9. It is an “orphan” anti‐σ factor 22 because unlike other RsgI factors, the gene encoding RsgI9 is not located in an operon that also contains a gene encoding a σ‐factor. The ectodomain in RsgI9 adopts an elongated conformation in which a C‐terminal bi‐domain unit is likely projected from the cell surface to engage cellulose, while its conserved CRE domain resides near the membrane to presumably affect signal transduction.…”