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pecS: a locus controlling pectinase, cellulase and blue pigment production in Erwinia chrysanthemi.

Erwinia chrysanthemi mutants (designated as pecS) displaying derepressed pectate lyase and cellulase synthesis were isolated. In addition, the pecS mutation is responsible for production of an extracellular insoluble blue pigment whose synthesis is cryptic in the wild-type 3937 strain. Transduction analysis indicates that the phenotype is due to a single mutation located near the xyl marker on the strain 3937 chromosome. This mutation was complemented by an R-prime plasmid carrying the xyl and argG genes of E. chrysanthemi, suggesting that the pecS product acts in trans to modulate pectinase, cellulase and blue pigment production. Insertion mutagenesis of the cloned region and recombination of the corresponding mutations in the bacterial chromosome by marker exchange revealed the existence of two divergently transcribed genes, pecS and pecM, that are both involved in the pectate lyase and cellulase regulation. The nucleotide sequences of pecS and pecM were determined. The pecS gene encodes a 166 amino acid polypeptide that shows similarity to the MprA regulatory protein of Escherichia coli whereas the pecM gene encodes a 297 amino acid polypeptide that was shown to be an integral membrane protein. The possible functions of the PecS and PecM proteins derived from the mutant phenotype and sequence analysis are discussed in terms of signal transduction and transcription regulation.

Amino Acid Sequence↗

Pharyngeal edema caused by occupational exposure to cellulase enzyme.

Enzymes can cause allergic asthma and rhinitis. Although patients sometimes complain of symptoms in their throat, we have not been able to find any published report of pharyngeal edema caused by exposure to enzymes. We present the case of a maintenance foreman who worked in a factory producing enzymes, and who had pharyngeal edema, asthma, and rhinitis at work. Prick tests for cellulase enzyme and cellulase RAST were positive. The causal association between his pharyngeal symptoms and cellulase was demonstrated with inhalation provocation tests in an exposure chamber.

Bronchial Provocation Tests↗

Nucleotide sequence of a cellulase gene of Bacillus subtilis.

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.

Amino Acid Sequence↗

Cloning and characterization of a new exo-cellulase gene, cel3, in Irpex lacteus.

A new cellulose-inducible gene (named cel3) was isolated from a strain of the white rot basidiomycete, Irpex lacteus MC-2. The cel3 open reading frame, containing two introns, encodes a polypeptide of 526 amino acids residues with a molecular mass of 55794 Da. Expression of the cel3 gene was induced by various insoluble celluloses and CM-cellulose. Transcription of cel3 was abolished when cells were cultivated in media containing the above cellulosic substrates, but added with glucose, fructose or lactose, while addition of glycerol or mannitol did not affect the cel3 mRNA level. The amino acid sequence of the catalytic domain of the Cel3 protein was homologous to that of fungal exo-type cellulases belonging to family 7 of the glycosyl hydrolases. A phylogenetic study showed that these exo-type cellulases can be clearly separated from family 7 endo-type cellulases.

Amino Acid Sequence↗

Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust.

Transcription of laccase and cellulase genes of Lentinula edodes was examined during growth and development under different temperature and moisture levels on a sawdust-based substrate. RNA was extracted from samples of mycelium and fruit bodies at various stages of development and gene expression was determined by competitive RT-PCR. The level of laccase transcripts was maximal during the mycelial growth stage, and then declined rapidly at the fruiting stage. In contrast, the cellulase transcript level peaked at the veil-break stage during fruit body development. Gene expressions for laccase and cellulase were triggered by microclimatic changes, specifically lower temperature and osmotic pressure.

Cellulase↗

Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression.

Toward a better understanding of the biochemical events that lead to biocontrol of plant pathogenic fungi by Hypocrea/Trichoderma spp., we investigated the importance of carbon catabolite (de)repression and cellulase formation in the antagonization of Pythium ultimum by Hypocrea jecorina (Trichoderma reesei) on agar plates and in planta. Hypocrea jecorina QM9414 could antagonize and overgrow P. ultimum but not Rhizoctonia solani in plate confrontation tests, and provided significant protection of zucchini plants against P. ultimum blight in planta. A carbon catabolite derepressed cre1 mutant of H. jecorina antagonized P. ultimum on plates more actively and increased the survival rates of P. ultimum-inoculated zucchini plants in comparison with strain QM9414. A H. jecorina mutant impaired in cellulase induction could also antagonize P. ultimum on plates and provided the same level of protection of zucchini plants against P. ultimum as strain QM9414 did. We conclude that cellulase formation is dispensable for biocontrol of P. ultimum, whereas carbon catabolite derepression increases the antagonistic ability by apparently acting on other target genes.

Carbon↗

Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically.

The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes beta-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these 'soluble'-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley beta-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound.

Cellulases↗

Factors influencing the production of cellulases by Sporotrichum thermophile.

Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi.

Carbon Dioxide↗

Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188.

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.

Anaerobiosis↗

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.

Animals↗

Substrate-induced production and secretion of cellulases by Clostridium acetobutylicum.

Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of beta-1,3- and beta-1,4-linked beta-D-glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum. Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.

Bacterial Proteins↗

Absence of significant cellulase activity in microbial flora of the female genital tract.

It has been postulated that toxic shock syndrome may be mediated by the hydrolysis of certain tampon fibers by bacteria of the female genital tract, leading to the release of glucose that would in turn serve as a substrate for the multiplication of staphylococci producing the toxic shock marker protein (TSST-1). We sought cellulolytic organisms among microorganisms isolated from the female genital tract throughout the menstrual cycle. A total of 288 aerobic and facultative vaginal isolates from 13 healthy female volunteers, aged 18 to 25, and 57 anaerobes from the same sources were screened for cellulase activity. No evidence of production of glucose or degradation of cellulose was found; hence, none of the strains could be described as cellulolytic. A total of 44 organisms (12.7%) showed weak endoglucanase activity as evidenced by minimal changes in the viscosity of the cellulose substrate, but this activity was not reproducible in all of the strains and was inconstantly observed on repeated examination. Five strains of Staphylococcus aureus isolated from cases of toxic shock syndrome also showed no cellulase activity. Cellulase activity does not appear to be a frequent or regular feature of the microflora of the human female genital tract.

Adolescent↗

Expression of cellulase genes in Rhodobacter capsulatus by use of plasmid expression vectors.

Broad-host-range plasmid vectors were constructed for expression of heterologous genes in the photosynthetic bacterium Rhodobacter capsulatus. These plasmids utilize an RK2-derived replicon for maintenance and conjugative transfer and the R. capsulatus rxcA promoter to obtain transcription of genes within appropriately positioned DNA fragments. The expression vectors were used to obtain synthesis of endoglucanase and exoglucanase in R. capsulatus from cellulase genes present on exogenously derived DNA fragments. The cellulase genes were expressed either by use of their native translation initiation signals or by in-frame fusion with the rxcA B870 beta gene translation initiation signals to form a hybrid protein. The level of cellulase gene expression was found to be modulated in response to the extent of aeration of plasmid host cultures.

Cellulase↗

Sophorose as an inducer of cellulase in Trichoderma viride.

Mandels, Mary (Quartermaster Research and Engineering Center, Natick, Mass.), Fredrick W. Parrish, and Elwyn T. Reese. Sophorose as an inducer of cellulase in Trichoderma viride. J. Bacteriol. 83:400-408. 1962.-The impurity in glucose responsible for cellulase induction in Trichoderma viride QM 6a has been isolated and characterized as sophorose (2-O-beta-d-glucopyranosyl-d-glucose). It is present at 0.0058% in reagent grade glucose. Sophorose is a very powerful inducer of cellulase for Trichoderma viride, being 2500 times as active as cellobiose. Modifications of sophorose, such as reduction or glycoside formation, destroy its inducing ability. The high activity of sophorose as an inducer is specific for T. viride.

Carbohydrate Metabolism↗

Cellulase location in Cellvibrio fulvus.

The location of cellulase in C. fulvus depends on the carbon source for growth and the age of the culture. When cells were grown on glucose or cellobiose all CMC-hydrolyzing enzyme was cell-bound but only part of the activity was located on the cell surface. Treatment of cells with EDTA, lysozyme, and detergents and subsequent fractionation experiments showed that cellulase was also located in the periplasm and bound to a membrane fraction. Growth on cellulose gave cell-free cellulase active against CMC. The enzyme was repressed by glucose but formed at a constant differential rate on cellobiose and amylose. This rate was 8-10 times lower than on cellulose and possible reasons for this are discussed.

Amylose↗