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.     

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.     

Peptidoglycantyp und Zusammensetzung der Zellwandpolysaccharide vonCellulomonas cartalyticum und einigen coryneformen Organismen

Cellulomonas cartalyticum was found to contain a peptidoglycan type different from that of the other species ofCellulomonas. The diamino acid is lysine instead of ornithine and the interpeptide bridge consists ofd-Asp-d-Ser. The same peptidoglycan type occurs inCorynebacterium manihot, Brevibacterium liticum andArthrobacter luteus. These non cellulolytic organisms are most likely not closely related withCellulomonas cartalyticum, as indicated by the very different G+C content of their DNA, although they formed a narrow cluster includingC. cartalyticum when numeric taxonomical methods were applied.

     

Transformation of protoplasts ofCellulomonas flavigena

Summary Protoplasts ofCellulomonas flavigena (Cm^s) were transformed with plasmid pC194. Transformation frequency was 2.72×10^–3 in MR-1 regeneration medium with 2 g/ml chloramphenicol. Transformation conditions are described.     

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.     

Cyclic AMP regulates the biosynthesis of cellobiohydrolase in <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> growing in sugar cane bagasse

Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.     

Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.     

Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Summary Plasmid-coded -glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The -glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the -glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of -glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK _m values for cellobiose and PNPG indicated that the -glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the -glucosidase fromE. coli pJS3 showed higher affinity for PNPG.     

Biocontrol of tomato late blight with the combination of epiphytic antagonists and rhizobacteria

Control of tomato late blight (LB) in Brazil is heavily based on chemicals. However, reduction in fungicide usage is required in both conventional and organic production systems. Assuming that biological control is an alternative for LB management, 208 epiphytic microorganisms and 23 rhizobacteria (RB) were isolated from conventional and organically grown tomato plants and tested for antagonistic activity against Phytophthora infestans. Based on in vitro inhibition of sporangia germination and detached leaflet bioassays, four EP microorganisms (Aspergillus sp., Cellulomonas flavigena, Candida sp., and Cryptococcus sp.) were selected. These microorganisms were applied either singly or combined on tomato plants treated or not with the RB Bacillus cereus. On control plants, LB progress rate (r), area under disease progress curve, and final disease severity were high. Lowest values of final severity were recorded on plants colonized by B. cereus and treated with C. flavigena, Candida sp. and Cryptococcus sp. There was no reduction on disease severity in plants treated only with RB. Biological control of LB resulted in low values of r and final severity. Integration of biological control with fungicides, cultural practices, and other measures can contribute to manage LB on tomato production systems.     

Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive β-1,4-endoglucanase from Cellulomonas flavigena

A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60°C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.     

Spiromastix saturnispora,a new species from indonesian soil

Spiromastix saturnispora, isolated from a soil sample collected from central Java in Indonesia, is described and illustrated as a new species. The new species was compared with the type ofSpiromastix, S. warcupii, and is similar in having brownish ascomata with a peridium of a loose network of delicate hyphae, peridial appendages which are curved in the manner of a scimitar and never completely coiled, and the absence of an anamorph. The ascospores ofS. saturnispora are characterized as large oblate, 3.2–4.8 × 2.5–3µm, punctate, and with an equatorial rim, which serves to distinguish the species fromS. warcupii and other known species.     

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis>

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a K _m value of 1.95 mg/ml, while Xyl53 had a K _m value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55°C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.     

Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482

The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.     

The generic classification of Fayum anthropoidea

The early anthropoid species initially described asAegyptopithecus zeuxis Simons, 1965, from the Oligocene of Egypt, although retained by many authors in the monotypic genusAegyptopithecus, has been lumped by others into the genusPropliopithecus. Similarly, the species originally described asParapithecus grangeri Simons, 1974, has been ranked by some authors in a monotypic genusSimonsius, while others retain it inParapithecus. Criteria to be considered in resolving these taxonomic debates are (1) the adequacy and consistency of proposed morphological differences between species; (2) analogy with the degree of morphological variation tolerated within extant genera; and (3) nomenclatural conservatism. A philosophy that would require strict monophyletic classification is of insufficient practical value for assessing the validity of Fayum genera. Characters cited as distinguishing vetweenAegyptopithecus andPropliopithecus, and betweenSimonsius andParapithecus, are reviewed and evaluated. The results indicate thatA. zeuxis is generically distinct from species ofPropliopithecus, based on differences in the crown structure and proportions of the molars.Pa. grangeri cannot be shown to differ at the generic level from the type and only known specimen ofPa. fraasi, thus establishing Simonsius as a junior synonym ofParapithecus.     

Systematic implications of the flavonoids and chromosomes ofFlyriella (Compositae—Eupatorieae)

The leaves of five species ofFlyriella were found to contain from one to four glycosides of quercetin and its 4- and 7,4-methyl ethers. These patterns are distinct from those observed for more than seventy species ofBrickellia and support morphological and chromosomal data which indicate thatFlyriella should not be treated as an element ofBrickellia. Alternative treatments are briefly considered.     

Sero-systematics ofCytisus sect.Trianthocytisus (Fabaceae)

The serological reaction of seed proteins provides evidence for a partly new systematic arrangement ofCytisus sect.Trianthocytisus and ofCytisus s.l. Proposed modifications agree with recent advances in morphological taxonomy. Sect.Trianthocytisus includes only two species,C. villosus andC. aeolicus. Its position is central within the genus, and this fact agrees with the proposed retypification ofCytisus (type species:C. villosus).C. emeriflorus, formerly included in the same section, constitutes the monospecific sect.Emeroides, which is intermediate towards the genusLembotropis. This is serologically isolated and includes onlyL. nigricans. It is confirmed thatC. sessilifolius should be removed from the genusCytisus as a monospecific genus:Cytisophyllum Lang which is closely allied toHesperolaburnum and toPodocytisus, the most primitive genera ofGenisteae.     

Non-equivalency of genera inAngiospermae: Evidence from DNA hybridization studies

The problem of taxa equivalency in phylogenetically distant groups can hardly be solved by comparing morphological differences alone. An attempt is made to approach the problem by means of DNA comparisons, e.g., DNA hybridization. Data obtained forCompositae, Umbelliferae andIridaceae indicate that both unique and repetitive DNA sequence comparisons lead to the conclusions that genera within these families are not equivalent, e.g., the differences in the DNA among the species ofIris are much more pronounced than among those ofAchillea; some genera ofUmbelliferae occupy an intermediate position.     

Additions to the genusXenidiocercus (coelomycetes) from Ghana

Two new species of the coelomycete genusXenidiocercus are described and illustrated,X. macrospora on leaves ofMacaranga rowlandii andX. pyriformis on leaves ofM. huraefolia. They differ from the type species in having wider and ellipsoidal or pyriform conidia. A key to species ofXenidiocercus andIdiocercus is provided.     

Three new species and a new combination in the genusTorulomyces from soil

Seven isolates ofTorulomyces from Asian and Australian soil samples were studied in comparison with known taxa of the genus and withMonocillium indicum, the type species ofMonocillium. Three new species,Torulomyces parviverrucosus, T. laevis, andT. ovatus, are described, andT. brunneus is described as a new combination. Conidial characteristics, especially their shape and surface structure, are useful taxonomic criteria for distinguishing species ofTorulomyces. Monocillium is considered to be a distinct genus.     

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the attachment organelle of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. Abbreviations CSS Corn stunt spiroplasma - SEM Scanning electron microscopy - TBS Tris-buffered saline - TEM Transmission electron microscopy