Jürgen Distler

Streptomycin biosynthesis and its regulation in Streptomycetes

New insights into the gene orders, structures, evolution, and functions of streptomycin (Sm) biosynthetic genes (str) were gained via hybridization studies, determination of nucleotide sequences, and measurement of expression in the str gene clusters of Streptomyces griseus and S. glaucescens. Both str clusters showed considerable divergence in macro and micro structure. Genes putatively involved in pathways leading to the (dihydro-)streptose and N-methyl-L-glucosamine moieties of Sm were identified. Additional regulatory elements, such as gene strS and conserved TTA codons in the N-terminal sections of reading frames, are reported. Evidences for the involvement of physiological state, signal transduction, and activators in the control of Sm production are presented.

Gene cluster for streptomycin biosynthesis in Streptomyces griseus: Analysis of a central region including the major resistance gene

SummaryA central segment of a cluster of biosynthetic genes for the antibiotic streptomycin cloned from Streptomyces griseus was analysed for open reading frames, as well as for transcriptional and translational activity. The nucleotide sequence revealed two significant open reading frames, ORF1 and APH(6), orientated in opposite directions and with a spacer of 885 bp between the start codons. The first, ORF1, had a coding capacity of 38 kDa. One open reading frame, APH(6), was identified as the major resistance gene coding for streptomycin 6-phosphotransferase, a protein of 307 amino acid residues and 33 kDa. Sequence determination of the first 14 N-terminal amino acid residues of the purified APH(6) enzyme protein was in agreement with the proposed primary structure. The possible identity of the presmed gene product of ORF1 with an in vitro translated protein (apparent molecular weight 41 kDa) is discussed. Comparison of the two APH(6) genes from S. griseus and the hydroxystreptomycin-producing S. glaucescens (cf. Vögtli and Hütter 1987) revealed 75% nucleotide sequence homology in the coding region and 74% conservation of the polypeptide sequence. Two protein domains which are highly conserved in other antibiotic and protein phosphotransferases were detected.

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes.

Abstract Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins – together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster – show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the l-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.

Decision phase regulation of streptomycin production in Streptomyces griseus

The streptomycin (Sm) producer, Streptomyces griseus N2-3-11, shows medium-independent biphasic kinetics of the vegetative or exponential growth phase (EGP), reflecting an innate clock-like behaviour of growth and differentiation. The S. griseus growth and development cycle has the following characteristics: (1) after the developmental cycle commences, it cannot be influenced by environmental conditions; (2) the first EGP (decision phase) and its duration seem to be genetically determined, and it is also exhibited in pleiotropic mutants deficient in differentiation and antibiotic production; (3) during this early phase of growth, the decision to produce Sm is established and the fixation of later production and differentiation can only be influenced by effector molecules, e.g. A-factor, during this period; (4) after the onset of the second EGP, the commitment to Sm production cannot be reversed by dilution into fresh medium, nor by effector molecules; (5) the length of time of this effector-insensitive growth phase (second EGP or execution phase) can be extended by dilution into fresh medium; (6) the differentiation cycle of S. griseus is completed on entering stationary phase. The cells of S. griseus then return to a decision-making stage and recover sensitivity to effector molecules. Evidence that this type of phasing is valid for the growth and developmental cycles of all streptomycetes is discussed.

The str gene cluster for the biosynthesis of 5'-hydroxystreptomycin in Streptomyces glaucescens GLA.0 (ETH 22794) : new operons and evidence for pathway-specific regulation by StrR

Two divergently oriented operons,strXU andstrVW, located within the gene cluster for 5′-hydroxystreptomycin (5′-OH-Sm) biosynthesis inStreptomyces glaucescens strain GLA.0 (ETH 22794), were analysed by DNA sequencing and transcription/regulation studies. Three genes,strU andstrVW, are conserved in a similar arrangement but in a different location within thestr/sts gene cluster of the Smproducing strainS. griseus N2-3-11. The four putative products resemble NDP-4-ketohexose 3,5-epimerases (StrX, Mr 20.2 kDa), NAD(P)-dependent oxidoreductases (StrU, 45.6 kDa), and ABC-transporters (StrV, 61.8 kDa; StrW, 63.4 kDa). These genes are apparently involved in the biosynthesis of 5′-OH-Sm because the promoters of both operons are activated in trans by the activator StrR ofS. griseus N2-3-11, when cloned inS. lividans 66 TK23. A sequence motif resembling the consensus sequence GTTCGActG(N)11 CagTcGAAc for binding of StrR was identified within the intergenic region ofstrX andstrV. Specific binding of StrR to this site was demonstrated by gel retardation assays using purified His*Tag-StrR.

A second streptomycin resistance gene from Streptomyces griseus codes for streptomycin-3″-phosphotransferase

Two genes, aphE and orf, coding for putative Mr 29,000 and Mr 31,000, proteins respectively, were identified in the nucleotide sequence of a 2.8 kbp DNA segment cloned from Streptomyces griseus N2-3-11. The aphE gene expressed streptomycin (SM) resistance and a SM phosphorylating enzyme in S. lividans strains. The two genes were found to be in opposite direction and seemed to share a common region of transcription termination. The aphE gene shows significant homology to the aph gene, encoding aminoglycoside 3′-phosphotransferase, APH(3′), from the neomycin-producing S. fradiae. The enzymatic specificity of the aphE gene product was identified to be SM 3″-phosphotransferase, APH(3″). The primary structure of the APH(3″) protein is closely related to the members of the APH(3′) family of enzymes. However, the APH(3″) enzyme did not detectably phosphorylate neomycin or kanamycin. There is only low similarity of the protein to the APH(6) group of SM phosphotransferases. An evolutionary relationship between antibiotic and protein kinases is proposed.

A spectinomycin resistance determinant from the spectinomycin producer Streptomyces flavopersicus

Summary: The spectinomycin (Sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphoryiates Sp and actinamine, the aminocyclitol moiety of Sp.