Pantoea stewartii subsp. stewartii produces an endoglucanase that is required for full virulence in sweet corn

Pantoea stewartii subsp. stewartii, a xylem-dwelling bacterium, is the causal agent of Stewart's wilt and blight of sweet corn. The goal of this study was to characterize the only gene in the P. stewartii subsp. stewartii genome predicted to encode an endoglucanase (EGase); this gene was designated engY. Culture supernatants from P. stewartii subsp. stewartii and Escherichia coli expressing recombinant EngY protein possessed both EGase and xylanase activities. Deletion of engY abolished EGase and xylanase activity, demonstrating that EngY appears to be the major EGase or xylanase produced by P. stewartii subsp. stewartii. Most importantly, our results show that EngY contributes to movement in the xylem and disease severity during the wilting phase of Stewart's wilt but is not required for water-soaked lesion formation.     

Metazoan cellulase genes from termites: intron/exon structures and sites of expression.

Endogenous endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) cDNAs were cloned from representatives of the termite families Termitidae and Rhinotermitidae. These EGases are all composed of 448 amino acids and belong to glycosyl hydrolase family 9 (GHF9), sharing high levels of identity (40-52%) with selected bacterial, mycetozoan and plant EGases. Like most plant EGases, they consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. Using a PCR-based strategy, the entire sequence of the coding region of NtEG, a gene putatively encoding an EGase from Nasutitermes takasagoensis (Termitidae), was determined. NtEG consists of 10 exons interrupted by 9 introns and contains typical eukaryotic promoter elements. Genomic fragments of EGase genes from Reticulitermes speratus (Rhinotermitidae) were also sequenced. In situ hybridization of N. takasagoensis guts with an antisense NtEG RNA probe demonstrated that expression occurs in the midgut, which contrasts to EGase expression being detected only in the salivary glands of R. speratus. NtEG, when expressed in Escherichia coli, was shown to have in vitro activity against carboxymethylcellulose.     

Cloning and Characterizing the Thermophilic and Detergent Stable Cellulase CelMytB from Saccharophagus sp. Myt-1

We previously isolated and reported a second species of the Saccharophagus genus, Saccharophagus sp. strain Myt-1. In the present study, a cellulase gene (celMytB) from the genomic DNA of Myt-1 was cloned and characterized. The DNA sequence fragment contained an open reading frame of 1,893 bp that encoded a protein of 631 amino acids with an estimated molecular mass of 66.8 kDa. The deduced protein, CelMytB, had a catalytic domain that contained a conserved signature sequence (VIYEIYNEPL) of glycosyl hydrolase family 5 and a CBM6 cellulose binding module. CelMytB showed optimal activity at 55 °C and pH 6.5, which is similar to the optimal temperature and pH profile of cel5H, an endoglucanase from the closely related S. degradans 2-40. However, the cellulase (degradation of soluble cellulose) and avicelase (degradation of crystalline cellulose) activities of CelMytB were about 3-fold and 100-fold higher, respectively, than the equivalent activities of cel5H. Moreover, CelMytB could degrade xylan. From the zymogram results, we speculated that the catalytic domain of CelMytB had high activity even without the cellulose binding module. The presence of some detergents stimulated the cellulase activity of CelMytB.     

A rice family 9 glycoside hydrolase isozyme with broad substrate specificity for hemicelluloses in type II cell walls

An auxin analog, 2,4-D, stimulates the activity of endo-1,4-beta-glucanase (EGase) in rice (Oryza sativa L.). The auxin-induced activity from three protein fractions was purified to homogeneity from primary root tissues (based on SDS-PAGE and isoelectric focusing after Coomassie brilliant blue staining). Amino acid sequencing indicated that the 20 N-terminal amino acid sequence of the three proteins was identical, suggesting that these proteins may be cognates of one EGase gene. An internal amino acid sequence of the the rice EGase (LVGGYYDAGDNVK) revealed that this enzyme belongs to glycosyl hydrolase family 9 (GHF9). The major isoform of this rice GHF9 [molecular weight based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): 51,216, isoelectric point (pI): 5.5] specifically hydrolyzed 1,4-beta-glycosyl linkages of carboxymethyl (CM)-cellulose, phosphoric acid-swollen cellulose, 1,3-1,4-beta-glucan, arabinoxylan, xylan, glucomannan, cellooligosaccharides [with a degree of polymerization (DP) >3] and 1,4-beta-xylohexaose, indicating a broader substrate range compared with those of other characterized GHF9 enzymes or EGases from higher plants. Hydrolytic products of two major hemicellulosic polysaccharides in type II cell walls treated with the purified enzyme were profiled using high-performance anion exchange chromatography (HPAEC). The results suggested that endolytic attack by rice EGase is not restricted to either the cellulose-like domain of 1,3-1,4-beta-glucan or the unsubstituted 1,4-beta-xylosyl backbone of arabinoxylan, but results in the release of smaller oligosaccharides (DP <6) from graminaceous hemicelluloses. The comparatively broader substrate range of this EGase with respect to beta-1,4-glycan backbones (glucose and xylose) may partly reflect different roles of gramineous and non-gramineous GHF9 enzymes.     

Cloning and characterization of a new functional human aldehyde dehydrogenase gene

We cloned a new functional ALDH gene (ALDHx) from a human genomic library in cosmid pWE-15 by screening with a 29-nucleotide probe partially matched to a conserved region of the ALDH1 and ALDH2 genes. The new ALDHx gene does not contain introns in the coding sequence for 517 amino acid residues. The degree of resemblance between the deduced amino acid sequences of the new ALDHx gene and the ALDH2 gene is 72.5% (alignment of 517 amino acid residues), while that between the ALDHx and the ALDH1 gene is 64.6% (alignment of 500 amino acid residues). The amino acid residues (Cys-162, Cys-302, Glu-268, Glu-487, Gly-223, Gly-225, Gly-229, Gly-245 and Gly-250), which exist in both ALDH1 and ALDH2 isozymes and have been implicated in functional and structural importance, are also preserved in the deduced sequence of the new ALDHx gene. Northern blot hybridization with ALDHx probe revealed the existence of a unique mRNA band (3.0 kilobases) in the human liver and testis tissues. Using the new ALDHx probe, we cloned the cDNA of the gene from a human testis cDNA library in lambda gt11 vector. The nucleotide sequence of the cDNA differs from that of the genomic sequence at three nucleotide positions resulting in the exchange of 2 deduced amino acid residues. These positions are polymorphic as further demonstrated by the PCR amplification of the targeted region followed by nucleotide sequence analysis of the genomic DNA from eight unrelated individuals. Alignment of the genomic and cDNA sequence indicates that although the ALDHx gene appears to have no intron in its coding sequence, an intron of 2.6 kilobases is found to interrupt the 5'-untranslated (5'-UT) sequence. Primary extension and S1 mapping analysis indicate the existence of at least two 5'-UT exons. The new ALDHx gene was assigned to chromosome 9 by Southern blot hybridization of DNA samples from a panel of rodent-human hybrid cell lines.     

Increased production of xylanase by expression of a truncated version of the xyn11A gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

High-Temperature Perturbation of Starch Synthesis Is Attributable to Inhibition of ADP-Glucose Pyrophosphorylase by Decreased Levels of Glycerate-3-Phosphate in Growing Potato Tubers

A cDNA (Cel1) encoding an endo-1,4-β-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria × ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. × ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.