Secretome profile of Cellulomonas sp. B6 growing on lignocellulosic substrates.

AIMS: Lignocellulosic biomass deconstruction is a bottleneck for obtaining biofuels and value-added products. Our main goal was to characterize the secretome of a novel isolate, Cellulomonas sp. B6, when grown on residual biomass for the formulation of cost-efficient enzymatic cocktails. METHODS AND RESULTS: We identified 205 potential CAZymes in the genome of Cellulomonas sp. B6, 91 of which were glycoside hydrolases (GH). By secretome analysis of supernatants from cultures in either extruded wheat straw (EWS), grinded sugar cane straw (SCR) or carboxymethylcellulose (CMC), we identified which proteins played a role in lignocellulose deconstruction. Growth on CMC resulted in the secretion of two exoglucanases (GH6 and GH48) and two GH10 xylanases, while growth on SCR or EWS resulted in the identification of a diversity of CAZymes. From the 32 GHs predicted to be secreted, 22 were identified in supernatants from EWS and/or SCR cultures, including endo- and exoglucanases, xylanases, a xyloglucanase, an arabinofuranosidase/ß-xylosidase, a ß-glucosidase and an AA10. Surprisingly, among the xylanases, seven were GH10. CONCLUSIONS: Growth of Cellulomonas sp. B6 on lignocellulosic biomass induced the secretion of a diverse repertoire of CAZymes. SIGNIFICANCE AND IMPACT OF THE STUDY: Cellulomonas sp. B6 could serve as a source of lignocellulose-degrading enzymes applicable to bioprocessing and biotechnological industries.

Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability.

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.

Cellulomonas aerilata sp. nov., isolated from an air sample.

A Gram-positive, aerobic, motile, coccoid or short rod-shaped bacterium, 5420S-23(T), was isolated from an air sample collected in the Republic of Korea. According to phylogenetic analysis based on 16S rRNA gene sequences, strain 5420S-23(T) revealed 97.5, 97.3, 97.3 and 97.2 % similarity, respectively, to Cellulomonas biazotea DSM 20112(T), Cellulomonas cellasea DSM 20118(T), Cellulomonas fimi DSM 20113(T) and Cellulomonas chitinilytica X.bu-b(T). The peptidoglycan type of strain 5420S-23(T) was A4beta, containing l-ornithine-d-glutamic acid. The cell-wall sugars were galactose, glucose and xylose. The major fatty acids were anteiso-C(15 : 0) (49.7 %) and C(16 : 0) (20.0 %). The major menaquinone was MK-9(H(4)) and major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C content was 74 mol%. The results of DNA-DNA hybridization with strains of closely related Cellulomonas species, in combination with chemotaxonomic and physiological data, demonstrated that isolate 5420S-23(T) represents a novel Cellulomonas species, for which the name Cellulomonas aerilata sp. nov. is proposed, with strain 5420S-23(T) (=KACC 20692(T) =DSM 18649(T)) as the type strain.

Cellulomonas chitinilytica sp. nov., a chitinolytic bacterium isolated from cattle-farm compost.

A bacterial strain, designated X.bu-b T, with chitin-, xylan-, cellulose- and starch-degrading activities, was isolated from compost at a cattle farm near Daejeon, Republic of Korea. The strain comprised Gram-positive, aerobic or facultatively anaerobic, non-motile, rod-shaped bacteria. On the basis of an analysis of 16S rRNA gene sequences, the phylogenetic position of X.bu-b T was within the genus Cellulomonas, and the strain exhibited relatively high sequence similarities with respect to Cellulomonas biazotea DSM 20112T (98.1 %), C. cellasea DSM 20118T (98.1 %), C. fimi DSM 20113T (98.0 %), C. terrae DB5T (97.9 %), C. humilata ATCC 25174T (97.7 %), C. xylanilytica XIL11 T (97.5 %), C. uda DSM 20107T (97.4 %), C. gelida DSM 20111 T (97.3 %), C. iranensis OT (97.3 %) and C. flavigena DSM 20109T (97.0 %). The phylogenetic distance from other Cellulomonas species with validly published names was greater than 3 % (i.e. less than 97.0 % sequence similarity). Chemotaxonomic data also supported the classification of strain X.bu-b T within the genus Cellulomonas: L-ornithine was the cell-wall diamino acid, anteiso-C15:0 and anteiso-C17:0 were the major fatty acids, rhamnose, galactose, xylose and ribose were the cell-wall sugars, MK-9(H4) was the predominant menaquinone and diphosphatidylglycerol and phosphatidylglycerol were present in the polar lipids. The G+C content of the genomic DNA was 73.6 mol%. DNA-DNA hybridization experiments showed that the values for DNA-DNA relatedness between strain X.bu-b T and the phylogenetically closest neighbours were below 23 %. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain X.bu-b T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas chitinilytica sp. nov. is proposed. The type strain is X.bu-b T (=KCTC 19133T =DSM 17922T).

Cellulomonas composti sp. nov., a cellulolytic bacterium isolated from cattle farm compost.

A bacterial strain, TR7-06(T), which has cellulase and beta-glucosidase activities, was isolated from compost at a cattle farm near Daejeon, Republic of Korea. It was a Gram-positive, aerobic or facultatively anaerobic, non-motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas, with highest sequence similarity to Cellulomonas uda DSM 20107(T) (98.5 %). Cell wall analysis revealed the presence of type A4beta, L-orn-D-Glu peptidoglycan. The cell-wall sugars detected were mannose and glucose. The predominant menaquinone was MK-9(H(4)); MK-8(H(4)) was detected in smaller quantities. The major fatty acids were anteiso-C(15 : 0), C(16 : 0), C(14 : 0) and C(18 : 0). The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The results of DNA-DNA hybridization and physiological and biochemical tests clearly demonstrated that TR7-06(T) represents a novel species. The combined genotypic and phenotypic data show that strain TR7-06(T) (=KCTC 19030(T)=NBRC 100758(T)) merits description as the type strain of a novel Cellulomonas species, Cellulomonas composti sp. nov.

Cellulomonas terrae sp. nov., a cellulolytic and xylanolytic bacterium isolated from soil.

A bacterial strain (DB5(T)), with polysaccharide-degrading activities, was isolated from garden soil in Daejeon, Republic of Korea. The cells were Gram-positive, aerobic or facultatively anaerobic, non-motile straight rods. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas and that it is most closely related to Cellulomonas xylanilytica LMG 21723(T) and Cellulomonas humilata ATCC 25174(T) (98.0 and 97.9% similarity, respectively). Chemotaxonomic data also supported the classification of strain DB5(T) in the genus Cellulomonas, i.e. L-ornithine as the cell-wall diamino acid, anteiso-C(15:0) and iso-C(15:0) as the major fatty acids, MK-9(H(4)) as the predominant menaquinone and the presence of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol mannosides in the polar lipid profile. The results of DNA-DNA hybridization in combination with chemotaxonomic and physiological data demonstrated that strain DB5(T) (=KCTC 19081(T)=NBRC 100819(T)) should be classified as the type strain of a novel species within the genus Cellulomonas, for which the name Cellulomonas terrae sp. nov. is proposed.

The curdlan-type exopolysaccharide produced by Cellulomonas flavigena 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 (beta-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.