Outer membrane proteins related to SusC and SusD are not required for Cytophaga hutchinsonii cellulose utilization

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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%

“…Bacterial adhesion to cellulose was measured, as previously described (27), by mixing cells with Avicel PH-101, sedimenting the cellulose particles and attached bacteria by centrifugation at 100 ϫ g for 1 min, and measuring the optical density of the supernatants (see Supplemental Methods in the supplemental material for details).…”

Section: Methodsmentioning

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

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“…A cellulose binding experiment was carried out as described by Zhu et al (45). Protein was incubated with 10% (wt/vol) Avicel (or 1% [wt/vol] RAC) in a final volume of 400 l that contained 1 mg/ml protein and 5% Avicel (or 0.5% RAC).…”

Section: Primermentioning

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.

“…C. hutchinsonii has two susC-like genes and two susD-like genes (3). Recent results, however, have revealed that the susC-like and susD-like genes are not required for efficient cellulose utilization or for growth of C. hutchinsonii on crystalline cellulose (17), indicating that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to C. hutchinsonii cellulose utilization. C. hutchinsonii may thus adopt an alternative mode of utilization in which the cell surface enzymes of C. hutchinsonii digest cellulose extracellularly to cellobiose and glucose, eliminating the need for SusC-like and SusD-like proteins to import longer oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

“…For C. hutchinsonii, previous results indicated that, besides cellulose, glucose and cellooligosaccharides can also be rapidly assimilated, and such reducing sugars do not accumulate in the culture supernatant when C. hutchinsonii is cultivated with cellulose as the sole carbon source (4,7). Although two susC-like genes, each arranged in tandem with a susD-like gene, could be identified in the C. hutchinsonii genome, functional characterization revealed that the two sus-like pairs are not required for C. hutchinsonii cellulose utilization (17).…”

mentioning

confidence: 99%

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

Zhou

Wang

Yang

et al. 2016

Self Cite

bCytophaga hutchinsonii specializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential for C. hutchinsonii cellulose utilization. Disruption of CHU_1276 in C. hutchinsonii resulted in complete deficiency in cellulose degradation, as well as compromised assimilation of cellobiose or glucose at a low concentration. Further analysis showed that CHU_1276 was an outer membrane protein that could be induced by cellulose and low concentrations of glucose. Transcriptional profiling revealed that CHU_1276 exerted a profound effect on the genome-wide response to both glucose and Avicel and that the mutant lacking CHU_1276 displayed expression profiles very different from those of the wild-type strain under different culture conditions. Specifically, comparison of their transcriptional responses to cellulose led to the identification of a gene set potentially regulated by CHU_1276. These results suggest that CHU_1276 plays an essential role in cellulose utilization, probably by coordinating the extracellular hydrolysis of cellulose substrate with the intracellular uptake of the hydrolysis product in C. hutchinsonii. Cytophaga hutchinsonii is a common cellulolytic soil bacterium that belongs to the phylum Bacteroidetes (1-4). A unique strategy is thought to be used by C. hutchinsonii to digest crystalline cellulose and to utilize filter paper as the sole carbon source. The mechanism, however, is largely unknown. Most other wellstudied cellulolytic microorganisms apply one of two strategies, the extracellular-free-cellulase system or the cell surface-anchored multiprotein cellulosome, to achieve the efficient degradation of cellulose (5). C. hutchinsonii has been assumed to use a third but poorly understood strategy involving cell surface cellulases that produce cellulo-oligosaccharides that are directly transported into the periplasm for further digestion (6-8). Analysis of the genomic sequences of C. hutchinsonii revealed that all the annotated endoglucanases lack recognizable cellulose binding modules (CBMs), and no obvious homologs of cellobiohydrolases are present (3). Recently, endoglucanases that are processive and may act as functional equivalents of exocellulases, and cellulose binding proteins (CBP) on the outer membrane (OM), have been identified in C. hutchinsonii (9-11), though their exact roles in cellulose digestion remain to be determined.Active import of oligosaccharides released by cell surface enzymes across the outer membrane has been proven to be crucial in the efficient utilization of polysaccharide substrates by many members of the phylum Bacteroidetes (12). This strategy has been well illustrated for starch utilization by the intestinal anaerobe Bacteroides thetaiotaomicron and involves a series of starch utilization system (Sus) proteins, specifically the cell surface lipoprotein SusD and the porin SusC, responsible for the binding and transport of oligosaccharides (13-16). For C. hutchinsonii, previo...