Isolation and characterization of a catalase-peroxidase gene from the pathogenic fungus,Penicillium marneffei

ABSTRACT We cloned the MP1 gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungusPenicillium marneffei. MP1 is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of Saccharomyces cerevisiae andCandida albicans. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from Escherichia coli to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of P. marneffei. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity.

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Isolation and Characterization of Metabolites fromPseudomonas fluorescens-D7 for Control of Downy Brome (Bromus tectorum)

Weed Science ◽

10.1017/s0043174500076827 ◽

1994 ◽

Vol 42 (3) ◽

pp. 492-501 ◽

Cited By ~ 38

Author(s):

S. Gurusiddaiah ◽

David R. Gealy ◽

Ann C. Kennedy ◽

Alex G. Ogg

Keyword(s):

Cultural Practices ◽

Pathogenic Fungus ◽

Fatty Acid Esters ◽

Gaeumannomyces Graminis ◽

Partial Purification ◽

Downy Brome ◽

Active Fraction ◽

Active Compounds ◽

Isolation And Characterization

Downy brome is a grassy weed that infests millions of acres of winter wheat in the Western United States and costs farmers millions of dollars in lost yields. It can not be controlled consistently using chemicals or cultural practices. Isolates of naturally occurring rhizobacteria have shown potential as biological control agents for downy brome. Centrifuged supernatant from aerobic shake cultures ofPseudomonas fluorescensstrain D7 suppressed germination of seeds and reduced root and shoot growth of downy brome in agar diffusion assays. The present article relates to the isolation and characterization of active compounds from strain D7. The active compounds were denatured and lost activity with commonly used techniques of extraction, concentration, and purification of microbial products. Two unusual techniques for isolation and partial purification of the active compounds from supernatant of strain D7 were developed. With one technique, supernatant was concentrated at 30 to 35 C under vacuum to 10% of the initial volume. Subsequently, the concentrate was chromatographed on sephadex gels to separate the active fraction. With the second technique, cell-free supernatant was frozen, with or without prior concentration, at −12 C for 12 to 24 h. The frozen supernatant was then thawed at 2 to 4 C, resulting in precipitation and crystallization of the active fraction. The active fraction isolated with either method inhibited downy brome. Inhibition was complete at concentrations as low as 1 mg total dry matter L-1agar. Also, the active fraction inhibited the plant pathogenic fungusGaeumannomyces graminisvar.tritici.Chemical analysis of the active fraction complex revealed the presence of chromopeptides and other peptides, fatty acid esters, and a lipopolysaccharide matrix. Separation of any of the components from the complex resulted in nearly complete loss of activity against downy brome.

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