An Outer Membrane Protein Involved in the Uptake of Glucose Is Essential for Cytophaga hutchinsonii Cellulose Utilization

The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane. IMPORTANCEThe bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels. C ytophaga hutchinsonii is a common cellulolytic soil bacterium that belongs to the phylum Bacteroidetes (1). Cells of C. hutchinsonii digest crystalline cellulose and grow with filter paper as the sole source of carbon and energy. C. hutchinsonii specializes in cellulose digestion and is known to grow only on cellulose or cellulose digestion products (1, 2). Direct contact of cells with their insoluble s...

Section: Discussionmentioning

confidence: 99%

Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

Zhu

Han

Hefferon

et al. 2016

“…C. hutchinsonii appears to use a contact-dependent digestion strategy to utilize cellulose (23); however, details of the mechanism are still a mystery. Recently, several proteins have been found that play important roles in cellulose degradation by C. hutchinsonii, such as the outer membrane proteins CHU_1276 (24) and CHU_1277 (22), the periplasmic protein CHU_2981 (25), and the protein CHU_0170 (26), which is a component of the type IX secretion system. Zhu et al found that two periplasmic endoglucanases were required for use of crystalline cellulose as a sole carbon and energy source (20).…”

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confidence: 99%

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

Wang

Zhao

Bai

et al. 2017

Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii. A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN 3 . Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220. Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases.IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii.

“…Specifically, a significant number of genes which had been involved in glycan biosynthesis and carbohydrate metabolism were discovered to be downregulated within the category of metabolism (62/148). Given that the integrity of the outer membrane protein profile has been found to be critical for cellulose utilization by C. hutchinsonii and given that outer membrane deficiency is often seen in mutant strains which have defects in cellulose utilization (20)(21)(22)(23)27), we observed that a further nine genes which encode putative outer membrane proteins that had unclassified functions were independently grouped within the regulon (see Fig. 7 and Data Set S3).…”

Section: Figmentioning

confidence: 97%

“…Among others, outer membrane proteins which might directly contact cellulose substrate or its digested hydrolysate are considered to play key roles in the utilization of cellulose by C. hutchinsonii (20). Specifically, mutations of genes predicted to encode hypothetical outer membrane proteins, including the genes CHU_1276, CHU_1277, CHU_1719, and CHU_3220, have been reported to lead to defective cellulose assimilation (21)(22)(23)(24). Moreover, a selection of other genes that are involved in the cellulose utilization of C. hutchinsonii have been identified by either random transposon mutagenesis or targeted mutagenesis.…”

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confidence: 99%

“…We have previously analyzed the expression profiles of C. hutchinsonii in response to different carbon sources. It was observed that the significant up-or downregulation of a large number of genes, including the majority of annotated endoglucanases and the CHU_1276 gene cluster, were strongly dependent on carbon sources that are present in the growth media (22). The CHU_1276 mutant strain displayed expression profiles which seemingly differed from those of the WT, not only on Avicel but also on glucose (29).…”

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confidence: 99%

See 1 more Smart Citation

An Extracytoplasmic Function Sigma Factor Controls Cellulose Utilization by Regulating the Expression of an Outer Membrane Protein in Cytophaga hutchinsonii

Wang

Zhang

Zhou

et al. 2019

Self Cite

The common soil cellulolytic bacterium known as Cytophaga hutchinsonii makes use of a unique but poorly understood strategy in order to utilize cellulose. While several genes have been identified as being an active part of the utilization of cellulose, the mechanism(s) by which C. hutchinsonii both (i) senses its environment and (ii) regulates the expression of those genes are not as yet known. In this study, we identified and characterized the gene CHU_3097 encoding an extracytoplasmic function (ECF) σ factor (σcel1), the disruption of which compromised C. hutchinsonii cellulose assimilation to a large degree. The σcel1 and its putative partner anti-σcel1, encoded by the CHU_3096 gene found immediately downstream from CHU_3097, copurified in vitro. The σcel1 was discovered to be associated with inner membrane when cells were cultured on glucose and yet was partially released from the membrane in response to cellulose. This release was found to occur on glucose when the anti-σcel1 was absent. Transcriptome analyses found a σcel1-regulated, cellulose-responsive gene regulon, within which an outer membrane protein encoding the gene CHU_1276, essential for cellulose utilization, was discovered to be significantly downregulated by CHU_3097 disruption. The expression of CHU_1276 almost fully restored cellulose utilization to the CHU_3097 mutant, demonstrating that CHU_1276 represents a critical regulatory target of σcel1. In this way, our study provided insights into the role of an ECF σ factor in coordinating the cellulolytic response of C. hutchinsonii. IMPORTANCE The common cellulolytic bacterium Cytophaga hutchinsonii uses a unique but poorly understood strategy in order to make use of cellulose. Throughout the process of cellulosic biomass breakdown, outer membrane proteins are thought to play key roles; this is evidenced by CHU_1276, which is required for the utilization of cellulose. However, the regulatory mechanism of its expression is not yet known. We found and characterized an extracytoplasmic function σ factor that is involved in coordinating the cellulolytic response of C. hutchinsonii by directly regulating the expression of CHU_1276. This study makes a contribution to our understanding of the regulatory mechanism used by C. hutchinsonii in order to adjust its genetic programs and so deal with novel environmental cues.