Molecular cloning and expression of the nag1 gene (N-acetyl-beta-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1.
A 72-kDa N-acetyl-beta-D-glucosaminidase was purified from the mycoparasitic fungus Trichoderma harzianum P1; antibodies were raised against it, and aa-sequences were obtained. The antibody reacted with a single 72-kDa protein band in culture filtrates of T. harzianum grown on chitin, and was subsequently used to clone the corresponding nag1 gene from a lambdagt11 cDNA expression library. It was interrupted by two short introns and encoded a protein of 580 amino acids. The deduced protein sequence contained aa-sequence areas of high similarity to N-acetyl-glucosaminidases from other eukaryotes such as Candida albicans, and invertebrate and vertebrate animal tissues. The highest similarity was observed with the corresponding gene from the silkworm. The aa-sequence of a tryptic fragment of purified N-acetyl-beta-D-glucosaminidase from T. harzianum corresponded to a deduced aa sequence from a portion of the cloned gene, thus verifying that the protein is encoded by nag 1. Southern analysis showed that nag 1 is present as a single-copy gene in T. harzianum. Expression of nag1-mRNA was strongly induced upon growth on chitin, N-acetyl-glucosamine and the cell walls of Botrytis cinerea used as a carbon source. The appearance of the corresponding N-acetyl-beta-D-glucosaminidase protein, as determined by Western analysis, paralleled the pattern of nag 1 expression, thereby suggesting that its formation is regulated at the level of transcription.
Peterbauer
C K
CK
Institut für Biochemische Technologie und Mikrobiologie, Abteilung für Mikrobielle Biochemie, TU Wien, Getreidemarkt 9/1725, A-1060 Wien, Austria.
Lorito
M
M
Hayes
C K
CK
Harman
G E
GE
Kubicek
C P
CP
D000118:Acetylglucosaminidase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002244:Carbon / Q000378:metabolism; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D002686:Chitin; D002244:Carbon; D000118:Acetylglucosaminidase