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Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-β-d-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl β-d-cellobioside (ρ-NPC) and ρ-nitrophenyl β-d-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth.Key points• CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes.• CHU_0961 was identified as a 1,4-β-d-glucan glucohydrolase with unique properties.• CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5′ rapid amplification of cDNA ends (5′RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31 bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter P chu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter P chu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120 kDa in C. hutchinsonii . The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii . Key points • The conserved structure of strong promoter of C. hutchinsonii was elucidated. • Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered. • A new method for induced expression of cellulase in vivo was established. • Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.

Cytophaga hutchinsonii glides rapidly over surfaces and employs a novel collection of cell-associated proteins to digest crystalline cellulose. HimarEm1 transposon mutagenesis was used to isolate a mutant with an insertion in CHU_0170 (sprP) that was partially deficient in gliding motility and was unable to digest filter paper cellulose. SprP is similar in sequence to the Porphyromonas gingivalis type IX secretion system (T9SS) protein PorP that is involved in the secretion of gingipain protease virulence factors and to the Flavobacterium johnsoniae T9SS protein SprF that is needed to deliver components of the gliding motility machinery to the cell surface. We developed an efficient method to construct targeted nonpolar mutations in C. hutchinsonii and deleted sprP. The deletion mutant was defective in gliding and failed to digest cellulose, and complementation with sprP on a plasmid restored both abilities. Sequence analysis predicted that CHU_3105 is secreted by the T9SS, and deletion of sprP resulted in decreased levels of extracellular CHU_3105. The results suggest that SprP may function in protein secretion. The T9SS may be required for motility and cellulose utilization because cell surface proteins predicted to be involved in both processes have C-terminal domains that are thought to target them to this secretion system. The efficient genetic tools now available for C. hutchinsonii should allow a detailed analysis of the cellulolytic, gliding motility, and protein secretion machineries of this common but poorly understood bacterium.

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, employs a novel collection of cell-associated proteins to digest crystalline cellulose. Other Bacteroidetes rely on cell surface proteins related to the starch utilization system (Sus) proteins SusC and SusD to bind oligosaccharides and import them across the outer membrane for further digestion. These bacteria typically produce dozens of SusC-like porins and SusD-like oligosaccharide-binding proteins to facilitate utilization of diverse polysaccharides. C. hutchinsonii specializes in cellulose digestion and its genome has only two susC-like genes and two susD-like genes. Single and multiple gene deletions were constructed to determine if the susC-like and susD-like genes have roles in cellulose utilization. A mutant lacking all susC-like and all susD-like genes digested cellulose and grew on cellulose as well as wild-type cells. Further, recombinantly expressed SusD-like proteins CHU_0547 and CHU_0554 failed to bind cellulose or β-glucan hemicellulosic polysaccharides. The results suggest that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to the cellulolytic bacterium C. hutchinsonii.

Cytophaga hutchinsonii, belonging to Bacteroidetes, is speculated to use a novel cell-contact mode to digest cellulose. In this study, we identified a histone-like protein HU, CHU_2750, in C. hutchinsonii, whose transcription could be induced by crystalline but not amorphous cellulose. We constructed a CHU_2750-deleted mutant and expressed CHU_2750 in Escherichia coli to study the gene’s functions. Our results showed that although the deletion of CHU_2750 was not lethal to C. hutchinsonii, the mutant displayed an abnormal filamentous morphology, loose nucleoid, and obvious defects in the degradation of crystalline cellulose and cell motility. Further study indicated that the mutant displayed significantly decreased cell surface and intracellular endoglucanase activities but with β-glucosidase activities similar to the wild-type strain. Analyses by real-time quantitative PCR revealed that the transcription levels of many genes involved in cellulose degradation and/or cell motility were significantly downregulated in the mutant. In addition, we found that CHU_2750 was important for biofilm formation of C. hutchinsonii. The main extracellular components of the biofilm were analyzed, and the results showed that the mutant yielded significantly less exopolysaccharide but more extracellular DNA and protein than the wild-type strain. Collectively, our findings demonstrated that CHU_2750 is important for cellulose degradation, cell motility, and biofilm formation of C. hutchinsonii by modulating transcription of certain related genes, and it is the first identified transcriptional regulator in these processes of C. hutchinsonii. Our study shed more light on the mechanisms of cellulose degradation, cell motility, and biofilm formation by C. hutchinsonii.

Axenic gametes of the marine green macroalga Ulva mutabilis Foyn (Ria Formosa, locus typicus) exhibit abnormal development into slow-growing callus-like colonies with aberrant cell walls. Under laboratory conditions, it was previously demonstrated that all defects in growth and thallus development can be completely abolished when axenic gametes are inoculated with a combination of two specific bacterial strains originally identified as Roseo-bacter sp. strain MS2 and Cytophaga sp. strain MS6. These bacteria release diffusible morphogenetic compounds (= morphogens), which act similar to cytokinin and auxin. To investigate the ecological relevance of the waterborne bacterial morphogens, seawater samples were collected in the Ria Formosa lagoon (Algarve, Southern Portugal) at 20 sampling sites and tidal pools to assess their morphogenetic effects on the axenic gametes of U. mutabilis. Specifically the survey revealed that sterile-filtered seawater samples can completely recover growth and morphogenesis of U. mutabilis under axenic conditions. Morphogenetic activities of free-living and epiphytic bacteria isolated from the locally very abundant Ulva species (i.e., U. rigida) were screened using a multiwell-based testing system. The most represented genera isolated from U. rigida were Alteromonas, Pseudoalteromonas and Sulfitobacter followed by Psychrobacter and Polaribacter. Several naturally occurring bacterial species could emulate MS2 activity (= induction of cell divisions) regardless of taxonomic affiliation, whereas the MS6 activity (= induction of cell differentiation and cell wall formation) was species-specific and is probably a feature of difficult-to-culture bacteria. Interestingly, isolated bacteroidetes such as Algoriphagus sp. and Polaribacter sp. could individually trigger complete Ulva morphogenesis and thus provide a novel mode of action for bacterial-induced algal development. This study also highlights that the accumulation of algal growth factors in a shallow water body separated from the open ocean by barrier islands might have strong implications to, for example, the wide usage of natural coastal seawater in algal (land based) aquacultures of Ulva.

Abstract The crystalline region of cellulose is the main barrier to the utilization of crystalline cellulose. Cytophaga hutchinsonii actively digests the crystalline region of cellulose by an unknown mechanism. Transposon mutagenesis was done to identify a novel gene locus chu_1557, which is required for efficient disruption of the crystalline region of cellulose, and the absence of CHU_1557 resulted in decreased glucose assimilation efficiency. The defect of the mutant in the disruption of the crystalline region of cellulose was partially retained by additional glucose or pre-culturing the mutant in a low glucose concentration medium which could improve its glucose absorption efficiency. These results suggested that extracellular glucose has important roles in the disruption of crystalline cellulose by C. hutchinsonii. Further study showed that the expression of an outer membrane protein CHU_3732 was downregulated by the absence of CHU_1557 in a low glucose concentration medium. CHU_3732 was involved in uptake of glucose and its expression was induced by a low concentration of glucose. CHU_3732 was predicted to be a porin, so we inferred that it may work as a glucose transport channel in the outer membrane. Based on these results, we deduced that CHU_1557 played a role in the process of glucose assimilation and its disruption affected the expression of other proteins related to glucose transportation such as CHU_3732, and then affected the cell growth in a low glucose concentration medium and disruption of the crystalline region of cellulose.

Cytophaga hutchinsonii is a gliding cellulolytic bacterium that is ubiquitously distributed in soil. The mechanism by which C. hutchinsonii achieves cellulose digestion, however, is still largely unknown. In this study, we obtained a C. hutchinsonii mutant that was defective in utilizing filter paper or Avicel as the sole carbon source by transposon mutagenesis. The interrupted gene locus, CHU_2981, encodes a hypothetical protein with only 130 amino acids. Cell fractionation and western blot detection of CHU_2981 fused with a C-terminal green fluorescence protein (GFP) indicated that CHU_2981 is located in the periplasm. The CHU_2981-disrupted mutant cells exhibited a significant growth defect on Avicel but not on glucose and cellobiose. The absence of CHU_2981 also resulted in a significant defect in colony spreading and individual cell motility compared to wild-type cells. Further analysis demonstrated that the CHU_2981-disrupted mutant cells exhibited a different profile of cellulose-absorbed outer membrane proteins from that of wild-type cells, in which protein varieties and amounts were markedly decreased. Our results showed that CHU_2981, the periplasmic non-cellulolytic protein, plays an important role in both cellulose utilization and cell motility probably by being involved in the appropriate production of outer membrane proteins.

Tenacibaculum maritimum (formerly Flexibacter maritimus) is a filamentous, biofilm-forming member of the Cytophaga-Flavobacterium-Bacteroides group (or Bacteroidetes), which causes the widely distributed marine fish disease tenacibaculosis. A search for N-acylhomoserine lactones (AHLs) quorum-sensing (QS) signals in the culture media of nine representative strains of this species using different biosensor strains revealed the presence of short-type AHL activity in all of them. N-butyryl- l-homoserine lactone (C4-HSL) was identified in T. maritimum NCIMB2154T by LC-MS. A degradation activity for long-acyl AHLs (C10-HSL) was subsequently demonstrated in T. maritimum NCIMB2154T. The acidification of the culture medium after degradation did not allow the recovery of C10-HSL, which indicates a possible acylase-type degradation activity. Even though the physiological processes under the control of AHL-mediated QS in T. maritimum need to be further characterized, this discovery extends the paradigm of AHL-mediated QS signalling beyond the Proteobacteria and reinforces its ecological significance.