Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta‐ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl‐carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2‐carboxyacyl precursors onto the ACP; a beta‐ketoacyl synthase that, along with the acylated ACP, forms the poly‐beta‐ketoacyl intermediates; a poly‐beta‐ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta‐ketoacyl oxidoreductase that forms beta‐hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.

No abstract

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.

A conidium-producing variant of Streptomyces griseus, strain 45-H, produces a substance, factor C, which is capable of inducing conidium formation in the hyphae of a conidium-non-producing mutant, strain 52-1. Factor C can be determined quantitatively on the basis of this biological effect. The biologically active substance can be purified by ion-exchange chromatography on cellulose phosphate combined with affinity chromatography on DNA-agarose. The purified substance is concentrated at least 1700 times. The molecular weight of factor C, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, is about 34500. On determining the amino acid composition of factor C 60 % of the amino acids were found to be hydrophobic.Cytodifferentiation has been studied in Streptomyces griseus strains [l, 21. From the fermentation liquid of the conidium-producing S. griseus 45-H strain a substance (designated factor C) has been isolated which, if added to the culture medium of the otherwise non-differentiating S. griseus 52-1 strain, has been found to elicit various cytomorphological signs of conidium formation [3,4].In our previous papers [3-61 we have already described the most apparent features of the effect of this endogenous substance which induces differentiation.In the present paper the process of the purification and the chemical properties of factor C are described. MATERIALS AND METHODS Production of Factor CStrain 45-H, a mutant of Streptomyces griseus, was cultivated in 10 1 of filtered soybean medium in a MicroFerm fermentor, model MF-114 (New Brunswick Scientific Co., Inc., U.S.A.), operating at an airflow of 6 l/min at 27 "C. Cultivation was continued for 66-72 h when conidia, or reproductive hyphae, appeared in a large number. Detection of Factor CFactor C can be detected on the basis of its effect inducing cytomorphological changes in the submerged culture of S. griseus 52-1. Its effect was evaluated with a phase-contrast microscope in 72-h submerged cultures of the test strain after glutaraldehyde fixation. The test was regarded positive if formation of reproductive branches could be detected in the test strain which otherwise did not produce conidia. Estimation of the Quantity of Factor CSerial dilution was performed and the amount of factor C found in 1 ml of the highest dilution still definitely positive was taken as unit. Removal of the MyceliaThe pH of the culture filtrate of S. griseus 45-H was adjusted to 6.5 with 2 M HzS04 and after the addition of 0.001 M EDTA the mycelia were centrifuged by a continuous-action rotor at 21 000 x g in an MSE high-speed 18 centrifuge at 4°C. After centrifugation the pH of the culture filtrate was adjusted to 4.5 with 2 M HzS04. Ion-Exchange ChromatographyTwo steps of ion-exchange chromatography were used consecutively.Batchwise Method. 60 g precyclized, equilibrated and fines-free Whatman P-1 cellulose phosphate were added to the 10-1 culture filtrate. Then the mixture was kept in a cold room for 2 h while being stirred continu-