Two extracellular enzymes (MsP1 and MsP2) capable of efficient beta-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of approximately 150 and approximately 120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of approximately 23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).

The generic position of a gram-positive, facultatively methylotrophic actinomycete known as Nocardiu sp. strain 239 was determined by comparing reverse transcriptase sequences of 16s rRNA. The assignment of the organism to the genus Amycolatopsis was strongly supported by chemotaxonomic and morphological data. A comparison with the type strains of validly described Amycolatopsis species showed that the organism formed the nucleus of a new species. The name proposed for this new species is Amycolatopsis methanolica. The organism has been deposited in the National Collection of Industrial Bacteria as NCIB 11946.Methanol-utilizing bacteria that assimilate formaldehyde via the ribulose monophosphate cycle (1, 42) are potential vehicles for fermentative overproduction of aromatic amino acids (7,38,40). The precursors of the shikimate pathway in these strains, namely, erythrose-4-phosphate and phosphoenolpyruvate, are intermediate and end products, respectively, of the ribulose monophosphate cycle. Gram-negative ribulose monophosphate cycle bacteria are not amenable to the extensive physiological and genetical manipulations needed for strain development in view of their obligate methylotrophic nature (26). There is, however, evidence that gram-positive, facultatively methylotrophic bacteria (6, 8) are suitable for strain improvement studies (4, 35).The single gram-positive ribulose monophosphate cycle actinomycete already described was initially labeled Streptomyces sp. strain 239 (29-31) and then Nocardia sp. strain 239 (24). Stable mutants have been isolated from this metabolically versatile organism, which has been grown under diverse conditions in batch and continuous cultures (4, 24). Regulation of aromatic amino acid biosynthesis in the strain has been studied in detail (9, and examination of the systematic deregulation of these control systems and the development of a transformation system are under way. Preliminary chemosy stematic studies included in this report showed that Nocardia sp. strain 239 has chemical properties consistent with its assignment of the family Pseudonocardiaceae (ll), which encompasses the genera Actinopolyspora, Amycolatopsis, Faenia, Pseudonocardia, Saccharomonospora, Saccharopolyspora, and in all probability, Amycolata (T. Bowen, E . Stackebrandt, M. Dorsch, and M. Embley, J. Gen. Microbiol., in press). In the present investigation, Nocardia sp. strain 239 was further characterized and designated the type strain of Amycolatopsis methanolica sp.nov . MATERIALS AND METHODSTest strains and cultivation conditions. Nocardia sp. strain 239 (strain LMD 80.32

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A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.

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