Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans.

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.     

Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.     

Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.     

Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca.

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 micromol cellobiose.min-1.mL-1, more than 15-fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C. Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.     

Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8

E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a non-catalytic, three-domain protein consisting of a Family 33 CBM, a FNIII domain, followed by a Family 2 CBM, were cloned, expressed, purified, and characterized. Western blots showed that E7 and E8 were induced and secreted when Thermobifida fusca was grown on cellobiose, Solka floc, switchgrass, or alfalfa as well as on beta-1,3 linked glucose molecules such as laminaribiose or pachyman. E8 bound well to alpha- and beta-chitin and bacterial microcrystalline cellulose (BMCC) at all pHs tested. E7 bound strongly to beta-chitin, less well to alpha-chitin and more weakly to BMCC than E8. Filter paper binding assays showed that E7 was 28% bound, E8 was 39% bound, a purified CBM2 binding domain from Cel6B was 88% bound, and only 5% of the Cel5A catalytic domain was bound. A C-terminal 6xHis tag influenced binding of both E7 and E8 to these substrates. Filter paper activity assays showed enhanced activity of T. fusca cellulases when E7 or E8 was present. This effect was observed at very low concentrations of cellulases or at very long times into the reaction and was mainly independent of the type of cellulase and the number of cellulases in the mixture. E8, and to a lesser extent E7, significantly enhanced the activity of Serratia marscescens Chitinase C on beta-chitin.     

Proteomic and physiological experiments to test Thermotoga neapolitana constraint-based model hypotheses of carbon source utilization

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways. In physiological experiments comparing cellotetraose to cellobiose and glucose as growth substrates, product formation yields on cellotetraose, cellobiose, and glucose were similar; however cell yields per mol carbon consumed were higher on cellotetraose than on cellobiose or glucose. Proteomic analysis showed increased expression of several proteins from cells grown on cellotetraose compared with glucose cell cultures, including cellobiose phosphorylase (CTN_0783), endo-1,4-β-glucosidase (CTN_1106), and an ATP-binding protein (CTN_1296). The CTN_1296 gene product should be evaluated further for participation in cellotetraose metabolism and is included as one of two hypothetical gene-protein-reaction associations in the T. neapolitana constraint-based model to reinstate cellotetraose metabolism in model simulations.     

DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.     

Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda

The formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass

Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.     

Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture

When the wild type Cellulomonas flavigena was grown on glycerol, xylose or cellobiose, it produced basal levels of carboxymethyl-cellulase (CMCase), filter-paperase (FPase) and xylanase activities. By comparison, a catabolic derepressed mutant strain of the same organism produced markedly higher levels of these enzymes when grown on the same carbon sources. Sugar-cane bagasse induced both the wild type and the mutant strain to produce three- to eight-time higher levels of FPase and xylanase than was observed with xylose or cellobiose. Continuous culture was used to determine the minimal cellobiose or glucose concentrations that repress the enzyme synthesis in both strains. 2.5 g l(-1) glucose repressed FPase and xylanases from wild type, while 1.6 times more glucose was needed to repress the same activities in the PN-120 strain. In the same way, twofold more cellobiose was needed to reduce by 75% the CMCase and xylanase activities in the mutant compared to the wild type. The FPase in the presence of 4 g l(-1) cellobiose did not change in the same strain. Therefore, its derepressed and feedback resistant characters of PN-120 mutant are evident. On the other hand, isoelectrofocused crude extracts of mutant and wild strains induced by sugar-cane bagasse, did not show differences in protein patterns, however, the Schiffs staining was more intense in the PN-120 than in the wild strain. These results point out that the mutational treatment did not apparently change the extracellular proteins from mutant PN-120 and this could affect their regulation sites, since derepressed and feed-back resistant enzymes may be produced.     

The expansin superfamily

The expansin superfamily of plant proteins is made up of four families, designated α-expansin, β-expansin, expansin-like A and expansin-like B. α-Expansin and β-expansin proteins are known to have cell-wall loosening activity and to be involved in cell expansion and other developmental events during which cell-wall modification occurs. Proteins in these two families bind tightly to the cell wall and their activity is typically assayed by their stimulation of cell-wall extension and stress relaxation; no bona fide enzymatic activity has been detected for these proteins. α-Expansin proteins and some, but not all, β-expansin proteins are implicated as catalysts of 'acid growth', the enlargement of plant cells stimulated by low extracellular pH. A divergent group of β-expansin genes are expressed at high levels in the pollen of grasses but not of other plant groups. They probably function to loosen maternal cell walls during growth of the pollen tube towards the ovary. All expansins consist of two domains; domain 1 is homologous to the catalytic domain of proteins in the glycoside hydrolase family 45 (GH45); expansin domain 2 is homologous to group-2 grass pollen allergens, which are of unknown biological function. Experimental evidence suggests that expansins loosen cell walls via a nonenzymatic mechanism that induces slippage of cellulose microfibrils in the plant cell wall.     

Beta-glucanase and chitinase biosynthesis in a culture of a mycophilic strain of Trichoderma viride

The production of extracellular 1,3-, 1,6-beta-glucanases and chitinase was studied during submerged cultivation of a Trichoderma viride strain 3/78 on various carbon sources: glycerol, glucose, lactose, sucrose, laminaran, starch, pustulan, chitin, and Agaricus bisporus fruit bodies. The synthesis of these enzymes and cellulase was studied also under the conditions of depression at low concentrations (10(-2) and 10(-3)M) of the first five aforementioned carbon sources as well as cellobiose, gentiobiose, N-acetyl-beta-D-glucosamine and 0.1% chitooligosaccharides and A. bisporus cell walls. The experiments were conducted with the washed mycelium of this strain grown for 2 days in a medium with glycerol as a carbon source. The results indicated that 1,3- and 1,6-beta-glucanases of the strain were of the constitutive nature and were repressed by such carbon sources as glycerol and glucose. Chitinase and cellulase were shown to be inducible enzymes. Chitinase was induced by N-acetyl-beta-D-glucosamine, chitooligosaccharides and A. bisporus cell walls as well as by lactose when the fungus was grown on this carbon source. Cellulase biosynthesis was induced by lactose, cellobiose and gentiobiose.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.     

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.