The glycopeptide teicoplanin is used for the treatment of serious infections caused by Gram-positive pathogens. The tcp gene cluster, devoted to teicoplanin biosynthesis in the actinomycete Actinoplanes teichomyceticus, was isolated and characterized. From sequence analysis, the tcp cluster spans approximately 73 kb and includes 39 ORFs participating in teicoplanin biosynthesis, regulation, resistance and export. Of these, 34 ORFs find a match in at least one of the five glycopeptide gene clusters previously characterized. Putative roles could be assigned for most of the tcp genes. The two glycosyltransferases responsible for attaching amino sugars to amino acids 4 and 6 of the teicoplanin aglycon were overexpressed in Escherichia coli and characterized. They both recognize N-acetylglucosamine as the substrate. tGtfA can add a sugar residue in the presence or absence of N-acetylglucosamine at amino acid 4, while tGtfB can only glycosylate the teicoplanin aglycon.

؉ -dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD ؉ -dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed. Methanol dehydrogenase (MDH)1 of Bacillus methanolicus belongs to family III of NAD(P)-dependent alcohol dehydrogenases (ADHs) (2, 3), distinct from the zinc-containing medium chain dehydrogenases/reductases (family I) and the zinc-lacking short chain 32 ADHs (family II) (4, 5). The initial members of family III all were iron-dependent ADHs. In time, with an increasing number of member proteins characterized, it became clear that not all members were iron-dependent. Where investigated, other metals like zinc and magnesium also were found instead of iron (5). B. methanolicus MDH contains one Zn 2ϩ and one or two Mg 2ϩ ions/subunit (3). Identification of members of family III ADHs increasingly became based on overall sequence similarity. Three unique, conserved amino acid sequence motifs have been defined for this family, aiding in ADH classification (2, 6) ( Table I). Over 100 fully sequenced members of family III ADHs are now found in data bases. Many of these are putative proteins, with no biochemical data available.The genes encoding MDH of B. methanolicus, methanol:pnitroso-N,NЈ-dimethylaniline oxidoreductase (MNO) of Amycolatopsis methanolica, MNO of Mycobacterium gastri MB19, ADH of Desulfovibrio gigas, and ADH of Desulfovibrio HDv enzymes of B. methanolicus, A. methanolica, D. gigas, and Desulfovibrio HDv have been cloned and characterized by us (2).2 Classification of the M. gastri enzyme was based on Nterminal amino acid sequence analysis (Fig. 1A). Characterization of the five purified enzymes revealed that each of the proteins possesses a decameric quaternary structure (7-10). The first three are nicotinoproteins, containing a tightly but noncovalently bound NAD(P)(H)/subunit (8, 11). It is unknown whether other members of family III are nicotinoproteins as well. The bound NAD(P)(H) species of MDH and A. methanolica MNO act as cofactors; they become reduced when the enzymes oxidize primary alcohols to the respective aldehydes (8,11). B. methanolicus MDH requires a second, exogenous NAD ϩ for methanol oxidation, serving as a coenzyme and resulting in reoxidation of the NADH cofactor (11). These two NAD(H) molecules are not exchanged during the reaction (11). In vitro, the relatively low coenzyme NAD ϩ -dependent MDH activity is strongly stimulated by a M r 50,000 activator protein from the same organism, resulting in a 40-fold increase in the MDH turnover rate (11,12).Activator protein-mediated activation of MDH is characterized by hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) and converts the Ping-Pong type of reaction mechanism of MDH to a ternary complex mechanism, implyin...

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Accessory genetic elements, such as plasmids and integrative elements, are widespread amongst actinomycetes, but little is known about their functions and mode of replication. The conjugative element pMEA300 from Amycolatopsis methanolica is present mostly in an integrated state at a single specific site in the chromosome, but it can also replicate autonomously. Complete nucleotide sequencing, in combination with deletion studies, has revealed that orfB of pMEA300 is essential for autonomous replication in its host. In this study, it was shown that purified OrfB protein binds specifically to the 39 end of its own coding sequence. Within this short sequence, a putative hairpin structure is located, which contains several direct and inverted repeats, and a nucleotide stretch that resembles the nicking site of the pC194 family of rolling circle replicating plasmids. Additional binding studies revealed that OrfB binds to an 8 bp inverted repeat that occurs three times within the hairpin structure. The data presented show that OrfB is the replication initiator (Rep) protein of pMEA300, and is therefore termed RepAM. Surprisingly, RepAM lacks significant sequence similarity with known prokaryotic Rep proteins, but it is highly similar to a number of yet uncharacterized ORFs that are located on integrative and conjugative elements of other actinomycetes. It is concluded that RepAM and its homologues are members of a novel class of Rep proteins. INTRODUCTIONActinomycetes are Gram-positive mycelium-forming bacteria that play an important role in mineralization processes, and are abundant producers of antibiotics and other secondary metabolites. Accessory genetic elements, including circular and linear plasmids, and integrative and conjugative elements, are widespread in these organisms. Many elements encode a wide variety of known functions, such as antibiotic resistance, or proteins for unique catabolic pathways (Jacoby & Shapiro, 1977). However, to date, only a single phenotype has been found to be associated with some of the actinomycete plasmids and integrative elements: when plasmid-carrying donor cells are grown on plates together with an excess of plasmid-lacking recipient cells, inhibition zones are formed after plasmid transfer, and these are known as pocks (Bibb et al., 1977).The small circular actinomycete plasmids and integrative elements for which the replication mechanism has been identified have all been found to replicate via the rolling circle replication (RCR) mechanism (Hagege et al., 1993). RCR plasmids are divided into five families: pT181, pC194, pMV158 and pSN2 (del Solar et al., 1998), and a recently described family of RCR replicons of Corynebacterium spp. (Nesvera et al., 1997;Osborn et al., 2000). Actinomycete RCR plasmids and integrative elements belong either to the pC194 plasmid family (Hagege et al., 1993;Muth et al., 1995;Servin-Gonzalez, 1993;Suzuki et al., 1997), which is widely distributed in Gram-positive and Gram-negative bacteria, or to the fifth family (RCR replicons of Corynebacter...

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