Characterization of a novel, antifungal, chitin-binding protein from Streptomyces tendae Tü901 that interferes with growth polarity.
The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against Paecilomyces variotii in agar diffusion assays. The 384-bp afp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for Streptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of P. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of Aspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.
Bormann
C
C
Mikrobiologie/Biotechnologie, Universität Tübingen, D-72076 Tübingen, Germany. christiane.bormann@uni-tuebingen.de
Baier
D
D
Hörr
I
I
Raps
C
C
Berger
J
J
Jung
G
G
Schwarz
H
H
D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification*; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D048271:Chitosan; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism*
C402702:AFP1 protein, Streptomyces; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004271:DNA, Fungal; D011134:Polysaccharides; D002686:Chitin; D048271:Chitosan; C011952:nikkomycin