Chitin degradation by cellulolytic anaerobes and facultative aerobes from soils and sediments.

Species of strictly and facultatively anaerobic cellulolytic bacteria from soils and sediments were examined for the ability to degrade chitin. Of 22 species studied, 16 degraded insoluble chitin. Cellulomonas uda, which was selected for a comparative study of its cellulase and chitinase enzyme systems, produced different enzyme systems for the degradation of cellulose and chitin and different patterns of regulation of production of the two enzyme systems were observed. Moreover, C. uda utilized chitin as a source of nitrogen for the degradation of cellulose. In natural environments, the ability to use chitin as a nitrogen source may confer on cellulolytic microorganisms, such as C. uda, a selective advantage over other cellulolytic microbes.

Clostridium hungatei sp. nov., a mesophilic, N2-fixing cellulolytic bacterium isolated from soil.

Two strains of obligately anaerobic, mesophilic, cellulolytic, N2-fixing, spore-forming bacteria were isolated from soil samples collected at two different locations near Amherst, MA, USA. Single cells of both strains were slightly curved rods that measured between 2 and 6 microm in length and approximately 0.5 microm in diameter. The spores were spherical, terminally located, distended the sporangium and measured 0.8-1.0 microm in diameter. The cells of both isolates (designated strain ADT and strain B3B) stained Gram-negative, but did not have a typical Gram-negative cell wall structure as demonstrated by transmission electron microscope analysis. The cells of both strains were motile with subpolarly inserted flagella and exhibited chemotactic behaviour towards cellobiose and D-glucose. Both strains fermented cellulose, xylan, cellobiose, cellodextrins, D-glucose, D-xylose, D-fructose, D-mannose and gentiobiose. In addition, strain B3B fermented L-arabinose. For both strains, fermentation products from cellulose were acetate, ethanol, H2 and CO2, as well as small amounts of lactate and formate. The G+C content of strain AD was 40 mol% and that of strain B3B was 42 mol%. Based on their morphological, physiological and phylogenetic characteristics, it was concluded that the two isolates are representatives of a novel species of Clostridium. The name Clostridium hungatei is proposed for the new species. The type strain of Clostridium hungatei sp. nov. is strain ADT (= ATCC 700212T).

Ultrastructural diversity of the cellulase complexes of Clostridium papyrosolvens C7.

Transmission electron microscopy was used to investigate the ultrastructural features of diverse cellulase and cellulase-xylanase multiprotein complexes that are components of the cellulase-xylanase system of Clostridium papyrosolvens C7. The multiprotein complexes were separated by anion-exchange chromatography into seven biochemically distinguishable fractions (F1 to F7). Most individual F fractions contained, in relatively large numbers, an ultrastructurally recognizable type of particle that occurred only in smaller numbers, or not at all, in the other F fractions. It is suggested that these ultrastructurally distinct particles represent the biochemically distinct multiprotein complexes that constitute the cellulase-xylanase system of C. papyrosolvens C7. Some of the particles consisted of tightly packed globular components that appeared to be arranged in the shape of a ring with conical structures pointing out along its axis. Other particles had triangular, polyhedral, or star shapes. The major protein fraction (F4) almost exclusively contained particles consisting of loosely aggregated components, many of which appeared to be arranged along filamentous structures. The ultrastructural observations reported here support our previous conclusion that the cellulase-xylanase system of C. papyrosolvens C7 comprises at least seven different high-molecular-weight multiprotein complexes. Furthermore, results of this and earlier studies indicate that the interactions between C. papyrosolvens C7 and cellulose are different from those that have been described for Clostridium thermocellum.

Cellulose degradation in anaerobic environments.

In anaerobic environments rich in decaying plant material, the decomposition of cellulose is brought about by complex communities of interacting microorganisms. Because the substrate, cellulose, is insoluble, bacterial and fungal degradation occurs exocellularly, either in association with the outer cell envelope layer or extracellularly. Products of cellulose hydrolysis are available as carbon and energy sources for other microbes that inhabit environments in which cellulose is biodegraded, and this availability forms the basis of many microbial interactions that occur in these environments. This review discusses interactions among members of cellulose-decomposing microbial communities in various environments. It considers cellulose decomposing communities in soils, sediments, and aquatic environments, as well as those that degrade cellulose in association with animals. These microbial communities contribute significantly to the cycling of carbon on a global scale.

Multicomplex cellulase-xylanase system of Clostridium papyrosolvens C7.

The cellulase system of Clostridium papyrosolvens C7 was fractionated by means of ion-exchange chromatography into at least seven high-molecular-weight multiprotein complexes, each with different enzymatic and structural properties. The molecular weights of the complexes, as determined by gel filtration chromatography, ranged from 500,000 to 660,000, and the isoelectric points ranged from 4.40 to 4.85. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the complexes showed that each complex had a distinct polypeptide composition. Avicelase, carboxymethyl cellulase, and xylanase activity profiles differed from protein complex to protein complex. Three of the complexes hydrolyzed crystalline cellulose (Avicel). Activity zymograms of gels (following electrophoresis under mildly denaturing conditions) revealed different carboxymethyl cellulase-active proteins in all complexes but xylanase-active proteins in only two of the complexes. The xylanase specific activity of these two complexes was more than eightfold higher than that of the unfractionated cellulase preparation. A 125,000-M(r) glycoprotein with no apparent enzyme activity was the only polypeptide present in all seven complexes. Experiments involving recombination of samples eluted from the ion-exchange chromatography column indicated that synergistic interactions occurred in the hydrolysis of crystalline cellulose by the cellulase system. We propose that the C. papyrosolvens enzyme system responsible for the hydrolysis of crystalline cellulose and xylan is a multicomplex system comprising at least seven diverse protein complexes.

Cellulase system of a free-living, mesophilic clostridium (strain C7).

The enzymatic activity responsible for crystalline cellulose degradation (Avicelase activity) by a mesophilic clostridium (strain C7) was present in culture supernatant fluid but was not detected in significant amounts in association with whole cells or in disrupted cells. Cells of the mesophilic clostridium lacked cellulosome clusters on their surface and did not adhere to cellulose fibers. The extracellular cellulase system of the mesophilic clostridium was fractionated by Sephracryl S-300 gel filtration, and the fractions were assayed for Avicelase and carboxymethylcellulase activities. The Avicelase activity coincided with an A280 peak that eluted in the 700,000-Mr region. Nondenaturing polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 700,000-Mr fractions showed that Avicelase was present as a multiprotein aggregate that lost the ability to hydrolyze crystalline cellulose when partially dissociated by sodium dodecyl sulfate treatment. Proteins resulting from the partial dissociation of the aggregate retained carboxymethylcellulase activity. An Avicelase-deficient mutant of strain C7 (strain LS), which was not capable of degrading crystalline cellulose, lacked the Avicelase-active 700,000-Mr peak. The results indicated that an extracellular 700,000-Mr multiprotein complex, consisting of at least 15 proteins, is utilized by the mesophilic clostridium for the hydrolysis of crystalline cellulose. At least six different endo-1,4-beta-glucanases may be part of the cellulase system of strain C7. Sephacryl S-300 column fractions, corresponding to an A280 peak in the 130,000-Mr region, contained carboxymethylcellulase-active proteins that may serve as precursors for the assembly of the Avicelase-active complex by the mesophilic clostridium.

Characterization of the extracellular cellulase from a mesophilic clostridium (strain C7).

An extracellular, 700,000-Mr multiprotein complex that catalyzed the hydrolysis of crystalline cellulose (Avicel) was isolated from cultures of Clostridium sp. strain C7, a mesophile from freshwater sediment. In addition to cellulose (Avicel, ball-milled filter paper), the multiprotein complex hydrolyzed carboxymethylcellulose, cellodextrins, xylan, and xylooligosaccharides. Hydrolysis of cellulose or cellotetraose by the complex yielded cellobiose as the main product. Cellopentaose or cellohexaose was hydrolyzed by the complex to cellotriose or cellotetraose, respectively, in addition to cellobiose. Xylobiose was the main product of xylan hydrolysis, and xylobiose and xylotriose were the major products of xylooligosaccharide hydrolysis. Activity (Avicelase) resulting in hydrolysis of crystalline cellulose required Ca2+ and a reducing agent. The multiprotein complex had temperature optima for Avicelase, carboxymethylcellulase, and xylanase activities at 45, 55, and 55 degrees C, respectively, and pH optima at 5.6 to 5.8, 5.5, and 6.55, respectively. Electron microscopy of the 700,000-Mr enzyme complex revealed particles relatively uniform in size (12 to 15 nm wide) and apparently composed of subunit structures. Elution of strain C7 concentrated culture fluid from Sephacryl S-300 columns yielded an A280 peak in the 130,000-Mr region. Pooled fractions from the 130,000-Mr peak had carboxymethylcellulase activity but lacked Avicelase activity. Except for the inability to hydrolyze cellulose, the 130,000-Mr preparation had a substrate specificity identical to that of the 700,000-Mr protein complex. A comparison by immunoblotting techniques of proteins in the 130,000- and 700,000-Mr preparations, indicated that the two enzyme preparations had cross-reacting antigenic determinants.

Nitrogen fixation by anaerobic cellulolytic bacteria.

Four strains of anaerobic nitrogen-fixing, cellulose-fermenting bacteria were isolated in pure culture from freshwater mud and soil. Nitrogenase activity was demonstrated in these strains and also in several previously described anaerobic cellulolytic bacteria isolated from various natural environments. These are the first anaerobic bacteria known to use cellulose as an energy source for nitrogen fixation. Because cellulose is a plant polysaccharide that abounds in nature, these results raise the possibility that nitrogen-fixing, cellulose-fermenting bacteria may be widespread and thus play a major role in carbon and nitrogen cycling.

Anaerobic cellulolytic bacteria from wetwood of living trees.

Obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from the wetwood of elm and maple trees. The isolation of these bacteria involved inoculation of selective enrichment cultures with increment cores taken from trees showing evidence of wetwood. Cellulolytic bacteria were present in the cores from seven of nine trees sampled, as indicated by the disappearance of cellulose from enrichment cultures. With two exceptions, cellulolytic activity was confined to the darker, wetter, inner section of the cores. Cellulolytic bacteria were also present in the fluid from core holes. The cellulolytic isolates were motile rods that stained gram negative. Endospores were formed by some strains. The physiology of one of the cellulolytic isolates (strain JW2) was studied in detail. Strain JW2 fermented cellobiose, d-glucose, glycerol, l-arabinose, d-xylose, and xylan in addition to cellulose. In a defined medium, p-aminobenzoic acid and biotin were the only exogenous growth factors required by strain JW2 for the fermentation of cellobiose or cellulose. Acetate and ethanol were the major nongaseous end products of cellulose fermentation. The guanine-plus-cytosine content of the DNA of strain JW2 was 33.7 mol%. Cellulolytic bacteria have not previously been reported to occur in wetwood. The isolation of such bacteria indicates that cellulolytic bacteria are inhabitants of wetwood environments and suggests that they may be involved in wetwood development.

Mesophilic cellulolytic clostridia from freshwater environments.

Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit.

Characterization of a halotolerant-psychroloterant bacterium from dry valley Antarctic soil.

The saline soils of the ice free dry valleys of Victoria Land, Antarctica may provide the closest analog on Earth to Martian conditions. We have initiated a study aimed at examining microbial adaptations to the harsh environment of these dry valley soils. In this report we describe the characterization of one bacterium, strain A4a, isolated from Taylor Valley soil. Strain A4a was an obligately aerobic, orange-pigmented, Gram-positive coccus that grew over wide ranges of both temperature (0 degrees C-40 degrees C) and sodium chloride concentration (0-2.0M). The optimal temperature for growth at all NaCl concentrations was 25 degrees C. Phospholipid composition and guanine plus cytosine content of the DNA of the isolate indicate a close relation to the genus Planococcus.

Rifampin as a selective agent for isolation of oral spirochetes.

Spirochetes indigenous to the healthy gingival crevice of the human mouth were isolated directly from colonies in agar medium containing rifampin as a selective agent.

Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down.

Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.