Histidine ammonia-lyase from Streptomyces griseus.

Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH. The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate. High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate. On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme. Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon. The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida. Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon. Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.     

An endogalactosaminidase from Streptomyces griseus.

An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN-GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN-GalN bonds in this molecule link positions 1 and 4, and are in the alpha-configuration, we are probably dealing with an endo-alpha-(1 leads to 4)-galactosaminidase, bu this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan. The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments greater than or equal to 10(4) daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules.     

Genetic recombination in Streptomyces griseus.

Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described.     

Nucleotide sequence of Streptomyces griseus initiator tRNA.

The primary structure of initiator tRNA from Streptomyces griseus was determined by post-labeling procedures. The nucleotide sequence is pC-G-C-G-G-G-G-U-G-G-A-G-C-A-G-C-U-C-G-G-D-A-G-C-U-C-G-C-U-G-G-G-C-U-C-A-U-A-A-C-C- C-A-G-A-G-G-U-C-G-C-A-G-G-U-psi-C-A-m1A-A-U-C-C-U-G-U-C-C-C-C-G-C-U-A-C-C-A0H. The unique feature of the sequence of this tRNA is that residue 54 is occupied by unmodified U, while ribothymidine is located in that position in most initiator tRNAs from eubacteria.     

Molecular analysis of sporulation in Streptomyces griseus.

Previous evidence suggested that orf1590 from Streptomyces griseus has the potential to encode two polypeptide products from temporally regulated nested open frames (orfs) and that the longer polypeptide may be a DNA-binding protein. We have developed a hypothetical model of the role of orf1590 in sporulation of S. griseus and have begun to test this model by determining the nucleotide sequence of the orf1590 counterpart from Streptomyces coelicolor. The conservation of the helix-turn-helix domain and the two potential translation start codons is consistent with our model. Continued analysis of bald mutants of S. griseus has indicated that several prematurely synthesize sporulation septa and spore walls. One of these nonsporulating strains appears to be a bldA mutant of S. griseus. Complementation analysis suggests that at least three genetic loci are involved in the correct timing of deposition of sporulation septa and wall thickening.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

Tailoring modification of deoxysugars during biosynthesis of the antitumour drug chromomycin A by Streptomyces griseus ssp. griseus

Chromomycin A3 is a member of the aureolic acid group family of antitumour drugs. Three tailoring modification steps occur during its biosynthesis affecting the sugar moieties: two O-acetylations and one O-methylation. The 4-O-methylation in the 4-O-methyl-D-oliose moiety of the disaccharide chain is catalysed by the cmmMIII gene product. Inactivation of this gene generated a chromomycin-non-producing mutant that accumulated three unmethylated derivatives containing all sugars but differing in the acylation pattern. Two of these compounds were shown to be substrates of the methyltransferase as determined by their bioconversion into chromomycin A2 and A3 after feeding these compounds to a Streptomyces albus strain expressing the cmmMIII gene. The same single membrane-bound enzyme, encoded by the cmmA gene, is responsible for both acetyl transfer reactions, which convert a relatively inactive compound into the bioactive chromomycin A3. Insertional inactivation of this gene resulted in a mutant accumulating a dideacetylated chromomycin A3 derivative. This compound, lacking both acetyl groups, was converted in a two-step reaction via the 4E-monoacetylated intermediate into chromomycin A3 when fed to cultures of S. albus expressing the cmmA gene. This acetylation step would occur as the last step in chromomycin biosynthesis, being a very important event for self-protection of the producing organism. It would convert a molecule with low biological activity into an active one, in a reaction catalysed by an enzyme that is predicted to be located in the cell membrane.     

Biosynthesis of N-methyl-L-glucosamine from D-glucose by Streptomyces griseus.

Biosynthesis of N-methyl-L-glucosamine moiety of streptomycin from D-glucose by Streptomyces griseus was studied. A mixture of D-[1-(14) C] glucose and D-[6(-3)H]glucose was given to the culture of S. griseus. The 3H/14C ratio found in N-methyl-L-glucosamine further supports a mechanism that the conversion of D-glucose to L-hexose is carried out without scission of carbon skeleton. When D-[1-14C]glucose and D-[3-3H]glucose were used, the fall of 3H/14C ratio in N-metyl-L-glucosamine showed that the hydrogen atom at C-3 plays a r?le in such a transformation.     

Biosynthesis of streptomycin. dTDP-dihydrostreptose synthase from Streptomyces griseus and dTDP-4-keto-L-rhamnose 3,5-epimerase from S. griseus and Escherichia coli Y10.

dTDP-dihydrostreptose synthase from Streptomyces griseus was purfied about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration on Ultrogel AcA44. The synthase preparation was free of dTDP-4-keto-L-rhamnose 3,5-epimerase (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, EC 5.1.3.13) activity. A new enzyme assay using Escherichia coli Y10 as source for the epimerase and dTDP-glucose 4,6-dehydratase (dTDP-glucose 4,6-hydro-lyase, EC 4.2.1.46) was developed. In the presence of excess epimerase the apparent Km for dTDP-4-keto-6-deoxy-D-glucose was determined to be 25 microM. The molecular weight of epimerase and synthase were determined by their elution volumes from a Sephadex G-100 column to be approx. 67,000 and 32,000, respectively. The pH optimum for the epimerase was between 7.5 and 8.5. The intermediate formation of dTDP-4-keto-L-rhamnose in the epimerase reaction could be shown by detection of 6-deoxy-[3H]talose after NaB3H4 reduction. Results which indicate the existence of dTDP-4-keto-6-rhamnose as a free intermediate in the epimerase reaction are reported.     

Deoxysugar transfer during chromomycin A3 biosynthesis in Streptomyces griseus subsp. griseus: new derivatives with antitumor activity

Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-D-oliose (sugar A) and 4-O-methyl-D-oliose (sugar B), and a trisaccharide of D-olivose (sugar C), D-olivose (sugar D), and 4-O-acetyl-L-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two D-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.