Molecular detection of streptomycin-producing streptomycetes in Brazilian soils.

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.     

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.     

Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity

DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into BglII-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4.45 and 11.55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1.95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1.5 rather than 2 d, and reached a level over twice that of the original S. griseus strain.     

Cloning and characterization of a gene from Streptomyces griseus coding for a streptomycin-phosphorylating activity

Abstract Six different plasmids expressing streptomycin (SM) resistance and SM phosphotransferase were obtained by cloning genomic DNA from Streptomyces griseus into Streptomyces lividans . The phosphorylating enzymatic activity formed in S. lividans differed in several biochemical properties from the one in S. griseus , though the phosphorylated products were identical.     

Nucleotide sequence of the streptomycinphosphotransferase and amidinotransferase genes from Streptomyces griseus.

Genes for streptomycin phosphotransferase and inosamine-P-amidinotransferase from a streptomycin-producing Streptomyces griseus were cloned on a 3.8kb BamHI-SphI fragment in S. lividans using the multicopy cloning vector pIJ702. The nucleotide sequence of this 3.8kb fragment was determined and the coding sequences for the two genes were identified by comparison with the amino-terminal sequences of the two enzymes purified from S. lividans clones.     

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.     

The nucleotide sequence of a streptomycin streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from a streptomycin producer

The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.     

Coevolution of antibiotic production and counter‐resistance in soil bacteria

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance‐only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Streptomycin-induced expression in Bacillus subtilis of YtnP, a lactonase-homologous protein that inhibits development and streptomycin production in Streptomyces griseus

Bacillus subtilis induces expression of the gene ytnP in the presence of the antimicrobial streptomycin, produced by the Gram-positive bacterium Streptomyces griseus. ytnP encodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium in S. griseus.     

Molecular cloning and expression in Streptomyces lividans of a streptomycin 6-phosphotransferase gene from a streptomycin-producing microorganism

The gene encoding streptomycin 6-kinase involved in the self-resistance of the streptomycin-producing Streptomyces griseus HUT 6037 was cloned in the plasmid vector pIJ703. The resulting plasmid, pSP6, contained 2.5 kb inserts of S. griseus DNA. When streptomycin-susceptible S. lividans 1326 was retransformed with pSP6, all transformants produced streptomycin 6-kinase. Addition of streptomycin to the culture medium of S. lividans carrying pSP6 plasmid brought about a remarkable increase in streptomycin 6-kinase activity in the cell extracts. It is suggested from the results that the production of streptomycin 6-kinase in streptomycin producer was induced by streptomycin accumulated during cultivation.     

Evidence for transfer of antibiotic-resistance genes in soil populations of streptomycetes

Phylogenetic analysis was used to evaluate the hypothesis of gene transfer in streptomycetes, many of which are antibiotic producers. The diversity and possible origins of streptomycin-resistance genes was investigated for a population of Streptomyces strains isolated from a site in Brazil where antibiotic production had previously been implicated. The analysis provides compelling evidence for the transfer of these genes. Examination of other Streptomyces -type strains also reveals a scattered distribution of streptomycin producers with respect to the overall phylogeny. These results suggest that horizontal gene transfer may be an important factor in the evolution of antibiotic genes in streptomycetes.     

Detection and properties of A-factor-binding protein from Streptomyces griseus.

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The "inducing material" virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.     

Streptomycin sensitivity of ribosomes isolated from a streptomycin-producing Streptomyces griseus.

The streptomycin sensitivity of ribosomes derived from a streptomycin-producing Streptomyces griseus was examined in a polyuridylic acid directed 14C-phenylalanine incorporating system. In order to get reproducible results it is essential to use cell-free extracts which do not inactivate streptomycin. This condition can be fulfilled by the combination of washed ribosomes of the streptomycin-producing strain and the 110 000 g supernatant of the streptomycin-nonproducing variant of S. griseus, because the streptomycin-phosphorylating activity can be washed out from ribosomes of younger streptomycin-producing cultures, and the streptomycin-nonproducing S. griseus does not have any streptomycin-inactivating capacity. In this amino acid polymerizing system the ribosomes of the streptomycin-producing strain were as sensitive to streptomycin as the ribosomes of the nonproducing variant or of Escherichia coli.     

The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites

In Streptomyces griseus the expression of at least one streptomycin biosynthetic gene, strB1 , is dependent on the pathway-specific activator protein StrR. We show here that StrR is a DNA-binding protein which specifically interacts with the strB1 promoter fragment. Footprinting experiments demonstrate that the StrR protein binds to an inverted repeat located upstream of the strB1 promoter. Further StrR-binding sites having the consensus sequence GTTCGActG(N)₁₁CagTcGAAc were identified in the str—sts gene clusters of S. griseus and Streptomyces glaucescens by sequence comparison, gel retardation, and footprinting studies. The genetic and biochemical evidence strongly supports the model of the StrR protein activating the expression of streptomycin biosynthetic genes by interacting with multiple binding sites within the str—sts gene clusters of S. griseus and S. glaucescens .     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

The Production of Mannosidostreptomycinase

S ummary : A medium is described which supports the production of high levels of mannosidostreptomycinase (B'ase) activity by Streptomyces griseus. The enzyme can be utilized to convert streptomycin B to streptomycin A in normal fermentations.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.