The Genome Sequences of Cellulomonas fimi and “Cellvibrio gilvus” Reveal the Cellulolytic Strategies of Two Facultative Anaerobes,Transfer of “Cellvibrio gilvus” to the Genus Cellulomonas,and Proposal of Cellulomonas gilvus sp. nov

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484^T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic “Cellvibrio gilvus” ATCC 13127^T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that “Cellvibrio gilvus” belongs to the genus Cellulomonas. We thus propose to assign “Cellvibrio gilvus” to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482^T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.     

Growth of Cellulomonas sp. ATCC 21399 on different polysaccharides as sole carbon source Induction of extracellular enzymes

Summary Growth and extracellular enzyme production of Cellulomonas sp. ATCC 21399 on carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel), xylan, galactomannan and starch were compared. The bacteria grew poorly on CMC, whereas high cell densities were obtained on the other substrates. Growth on Avicel resulted in extracellular enzyme activities against CMC, Avicel, xylan, galactomannan and amylose. By contrast, growth on xylan, galactomannan and starch induced only the enzymes neccessary for the degradation of the growth substrate. Extracellular proteinase activity could be measured during growth on all substrates but CMC, and the possibility of proteolytic inactivation of some of the unstable enzymes (i.e. Avicelase and amylase) in discussed.     

Molecular cloning of cellulolytic enzyme genes from Cellulomonas flavigena in E. coli

A genomic bank of Cellulomonas flavigena was constructed in E. coli using the pUC18 vector, and over 14000 clones screened for cellulolytic activity. Three different cellulolytic enzyme genes were cloned, one coding for an endo-β-glucanase (pJS10, CMC activity) and two coding for β-glucosidases, each with a distinct substrate specificity (pJS3, X-glu, and pJS4, X-glu and MUC activities). These three inserts have different restriction patterns to each other and the previously isolated cellulolytic enzyme genes from C. fimi and C. uda.     

Cellulase and hemicellulase production by Cellulomonas flavigena NIAB 441

Summary Cellulomonas flavigena (strain NIAB 441) produced cellulase and hemicellulase activities when grown on Leptochloa fusca L. Kunth (Kallar grass), found to be the best inducer for enzyme production. The enzyme possessed the potential to saccharify bagasse, Kallar grass straw, wheat straw, carboxymethyl cellulose (CMC) and xylan to reducing sugars.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

Synthesis and regulation of D-xylanase from Cellulomonas flavigena wild type and a mutant

The xylanolytic system from Cellulomonas flavigena was enhanced by adding cellulose to the growth medium. The Solka floc:xylan (60:40 w/w) mixture induced xylanase synthesis by more than 3-fold over that induced by growing C. flavigena, wild type and its mutant PN-120 on pure xylan. The hydrolysis pattern of sugar cane bagasse and xylan indicated the presence of debranching endo-;-xylanase activity.     

Mannan-Degrading Enzymes from Cellulomonas fimi

The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.     

Isolation and characterization of a new strain ofCellulomonas flavigena

Summary A cellulolytic bacterium was isolated from leaf litter. Its nutritional characteristics and most of its morphological features closely resemble those of ATCC # 482, which is considered to be the type species ofCellulomonas flavigena (Stackebrandt and Keddie in Bergey's Manual of Systematic Bacteriology). However, when stationary phase cells from cultures using a minimal medium containing an excess carbon and energy source are compared, a very prominent morphological difference is manifest. Phase contrast microscopy and transmission electron microscopy indicate the presence of large swollen polar structures in the type species. The absence of such structures from the new isolate and the production of large amounts of an extracellular polysaccharide indicate that it is a new strain ofCellulomonas flavigena.     

Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399

Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H(3)PO(4)-swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with beta-1-4-D-glucoside bonds. The specific activity of endoglucanase C against H(3)PO(4)-swollen Avicel was low.     

Glucose uptake by Cellulomonas fimi

Glucose uptake by whole-cell suspension of the facultative anaerobe Cellulomonas fimi, which was two-fold higher under aerobic conditions than under N₂ or H₂, was inhibited by inhibitors of electron transport and ATP synthesis and, particularly, by proton and metal ion ionophores. A variety of sugars, including 2-deoxyglucose, did not inhibit glucose uptake but cellobiose was a non-competitive inhibitor. Cells grown on cellobiose medium transported glucose at one half the rate of glucose-grown cells. Cellulomonas fimi has a highly specific active system for glucose transport.     

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.     

<Emphasis Type="Italic">Cellulomonas macrotermitis</Emphasis> sp. nov., a chitinolytic and cellulolytic bacterium isolated from the hindgut of a fungus-growing termite

To investigate the symbiotic roles of the gut microbiota in the fungus-growing termite Macrotermes barneyi, a novel strain with chitinolytic and cellulolytic activity, designated strain an-chi-1^T, was isolated from the hindgut of M. barneyi. Strain an-chi-1^T grows optimally at 28–30 °C, pH 8.0 in PYG medium. On the basis of 16S rRNA gene sequence analysis, this isolate belongs to the genus Cellulomonas with high sequence similarity to Cellulomonas iranensis (99.4%), followed by Cellulomonas flavigena (98.4%), Cellulomonas phragmiteti (97.4%), Cellulomonas oligotrophica (97.2%) and Cellulomonas terrae (97.0%). The DNA–DNA relatedness between an-chi-1^T and the type strains of C. iranensis and C. flavigena DSM20109^T are 35.4% and 23.7%, respectively. The major cellular fatty acids are anteiso-C_15:0 and C_14:0. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylinositol dimannosides and one unidentified phospholipid. The cell-wall sugar is ribose. The peptidoglycan contains glutamic acid, aspartic acid and alanine. The DNA G+C content is 67.3 mol%. Based on its distinctive phenotypic, phylogenetic, and chemotaxonomic characteristics, an-chi-1^T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas macrotermitis sp. nov. is proposed. The type strain is an-chi-1^T (= JCM 31923^T = CICC 24195^T).     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.     

Growth and enzyme production of Cellulomonas sp. ATCC 21399 on microcrystalline cellulose. Effects of increasing concentration of a mineral medium

Summary The kinetics and production of different extracellular enzyme activities were studied during growth of Cellulomonas sp. ATCC 21399 on 2% Avicel with different concentrations of M9 mineral medium. The lag phase and the doubling time increased with increasing ionic strength of the medium. The highest cell density was obtained during growth at 5 x M9 mineral medium and Cellulomonas grew well at this high salinity. The enzyme activities against carboxymethylcellulose and xylan increased with increasing concentration of M9 medium up to 5 x M9. By contrast, activities against microcrystalline cellulose (Avicel), galactomannan and amylose decreased with increasing concentration of M9 medium. The extracellular proteinase activity increased with increasing concentration of M9 medium, and it is possible that the lability of the cellulolytic and amylolytic enzymes may be due to their susceptibility to proteolytic inactivation by the extracellular proteinases.     

Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Fermentation of C14-Labeled Cellobiose by Cellulomonas fimi

Crude cell-free extracts from Cellulomonas fimi contain cellobiose phosphorylase which cleaves cellobiose into glucose and glucose-1-phosphate in the presence of inorganic phosphate. With the aid of this enzyme, two samples of C¹⁴-cellobiose labeled in reducing or non-reducing glucosyl moiety were prepared from uniformly labeled C¹⁴-glucose or C¹⁴-glucose-1-phosphate as substrate, respectively. The labeled preparations have been shown to be radiochemically pure. Analyses of the anaerobic fermentation products from C¹⁴-cellobiose by resting cell suspensions showed that both glucose moieties were fermented almost equivalently. However, relatively small differences in specific activities of the products revealed that significantly larger amounts of formic acid and smaller amounts of acetic acid were produced from the reducing glucose moiety than from the other half of the molecule. Succinic and lactic acids appeared to be produced almost equally from both moieties.     

The curdlan-type exopolysaccharide produced by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> KU forms part of an extracellular glycocalyx involved in cellulose degradation

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.     

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.