Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface IntroductionStreptomycetes are Gram-positive soil bacteria that colonize moist substrates by forming a branched network of multinucleoid hyphae. At some stage during their life cycle, these bacteria are confronted with a hydrophobic environment. For instance, after a feeding substrate mycelium has been established, hyphae leave the aqueous environment to grow into the hydrophobic air. These aerial hyphae differentiate further by forming chains of uninucleoid cells, which metamorphose into pigmented spores. Spores or hyphae of streptomycetes may also encounter hydrophobic solids such as surfaces of dead or living organisms. When streptomycete hyphae leave their aqueous environment, they change their surface. Hyphae in a moist substrate are hydrophilic, whereas the surfaces of aerial hyphae and spores are hydrophobic.Formation of aerial structures has been best studied in Streptomyces coelicolor (for recent reviews, see Chater, 1998;Kelemen and Buttner, 1998;Wösten and Willey, 2000). Bald (bld) mutants of S. coelicolor were isolated that, when grown on rich medium, are affected in the formation of aerial structures and in the production of a small surface-active peptide called SapB (Willey et al., 1991). Many of these mutants appear to be affected in an extracellular signalling cascade involved in the erection of aerial hyphae (Willey et al., 1993;Nodwell et al., 1996;. Experimental evidence suggests the existence of at least five signalling molecules. It was hypothesized that each signal triggers the synthesis and release of the next signal, ultimately leading to the production and secretion of SapB (Willey et al., 1993;Nodwell et al., 1996). By lowering the water surface tension from 72 to 32 mJ m -2 , SapB enables hyphae to breach the water-air interface to grow into the air (Tillotson et al., 1998).Aerial hyphae and spores of S. coelicolor have several surface layers that make them hydrophobic. One surface layer, called the rodlet layer, has a typical ultrastructure of a mosaic of 8-to 10-nm-wide parallel rods (Wildermuth et al., 1971;Smucker and Pfister, 1978). The nature of the surface layers is not known. SapB is not expected to form one of these layers, as this peptide was localized in the culture medium but could not be detected at the surfaces of aerial structures (Wösten and Willey, 2000). SummaryThe filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growi...

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml ؊1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter؊1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml ؊1 . These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter ؊1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml ؊1 (i.e., 360 mg liter ؊1 ) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter؊1 . In this case, maximal activities were 3,900 and 4,660 nkat ml ؊1 , respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.Filamentous fungi belonging to the homobasidiomycetes offer great potential for industrial and medical applications. They secrete proteins into their culture media with activities or in amounts that are not found in other fungi. For instance, homobasidiomycetes produce various metalloenzymes, such as laccases, which are attractive candidates for a wide variety of applications. These enzymes degrade a large number of recalcitrant pollutants and are a biological and environmentally friendly alternative to the highly contaminating pulping and bleaching treatments of the paper and pulp industries (3, 4). Until now, the expression of basidiomycete metalloenzymes in ascomycete production systems such as Aspergillus ssp. and Trichoderma reesei has had limited success (6). Therefore, basidiomycetes should be developed as hosts for large-scale protein production. The white rot fungus Pycnoporus cinnabarinus is an attractive candidate in this respect. This basidiomycete was selected for its ability to efficiently degrade lignin and to transform lignin-derived compounds such as ferulic acid into vanillin (9,11,22). P. cinnabarinus has a simple ligninolytic system. Neither lignin peroxidase nor manganese peroxidase activity has been detected, but laccase is produced (9). Two laccase genes have been cloned, i.e., lcc3-1 or the allelic form lac1 (10, 23) and lcc3-2 (34). Until now, transformation ...

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PP,-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PP,-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PP,-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P, and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.Actinomycetes are important bacterial producers of secondary metabolites. There is a strong interest in the genetics of secondary-metabolite biosynthesis, with most studies concentrating on these pathways and their control. Many secondary metabolites are initially derived from intermediates of the central pathways of primary metabolism. Little is currently known, however, about the enzymes and regulation of, for instance, glucose metabolism in actinomycetes. This is mostly because of a general lack of physiological studies on primary metabolism in actinomycetes (21). We have initiated such studies with the actinomycete Amycolatopsis methanolica (8), belonging to the family Pseudonocardiaceae (42), which includes many species producing bioactive compounds, e.g., the antibiotics rifamycin and erythromycin. A. methanolica is one of the few methanol-utilizing gram-positive bacteria known (10,12,17). Methanol oxidation via formaldehyde and formate to carbon dioxide results in energy generation (5,17). Carbon assimilation starts by formaldehyde fixation via the ribulose monophosphate (RuMP) cycle (9,17). This cycle involves the specific enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), the glycolytic enzymes 6-phosphofructokinase (PFK) and fructose-1,6-bisphosphate (FBP) aldolase (9), and various enzymes also involved in the related pentose phosphate pathway (Fig. 1) (12).The identity, properties, and regulation of enzymes involved in glucose and methanol metabolism in A. methanolica were examined in this study. MATERUILS AND METHODSMicroorganisms and cultivation. Wild-type A. methanolica NCIB 11946, its maintenance, and the procedures followed for cultivation in batch cultures, harvesting of cells, and growth measurements have been described previously (8)(9)(10)(11)