Expression and secretion of interferon-alpha 1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene.

A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.

Effects of polyene macrolide antibiotics on normal and protoplast type L-form cells of Escherichia coli W1655F+.

The action of the polyene macrolide antibiotics mycotrienin, pimaricin, lucensomycin, tetramycin, rimocidin, nystatin, filipin, lagosin, pentaene antibiotic 2814P, flavomycoin, flavofungin, hexaene antibiotic 5001P, and candicidin, including perhydro derivatives of them, on wall-less stable protoplast type L-form and normal rod form cells of E. coli W1655F+ was studied. No inhibition of the normal rod form cells was detected. In contrast to these results the growth of the L-form cells was inhibited by all of the substances tested, with the exception of pimaricin. Further experiments have shown that the differences in sensitivity of normal and L-form cells cannot be explained by differences in sterol content, the target site of polyene antibiotics in sensitive eukaryotic cells. According to our results it is obvious that the cell wall of the normal cells functions as a penetration barrier to polyene antibiotics.

beta-1.3.-1.4-Glucanase in spore-forming microorganisms. VI. Genetic instability of beta-glucanase production in a high-producer strain of Bacillus amyloliquefaciens grown in a chemostat.

A Bacillus amyloliquefaciens strain high-producing for beta-1.3-1.4-glucanase has gradually lost the ability to produce this enzyme during long-time continuous cultivation, independent of the culture conditions. Mutant strains isolated after long-term cultivation exhibited changed behaviour concerning extracellular enzyme formation and sporulation. By agarose gel electrophoresis of alkaline DNA extracts isolated form original and mutant strains we demonstrate that the observed pleiotropic phenomena are not caused by the loss of a complete plasmid present in the original strain. From extracts of both the original and mutant strains plasmid DNAs with approximately the same molecular weight of about 35 Mdal were isolated.

Gene expression after transformation of Streptomyces lividans protoplasts with plasmid pIJ2 DNA.

A new method has been developed for the selection of antibiotic-resistant clones after transformation of Streptomyces protoplasts with plasmid DNA. This method is based on establishing a spatial concentration gradient for the antibiotic, the resistance to which is encoded by the transforming plasmid. By this method, the resistance development of regenerating protoplasts can be followed. The results suggest that antibiotic resistance is inducible. In addition, we were able to show that resident plasmids incompatible with the incoming ones are eliminated when this direct selection principle is used. Moreover, this method, which may facilitate the application of gene technology in Streptomyces, works even though the transformation procedure gives variable results.

[Effect of polyene macrolide antibiotics on normal and cell wall deficient Escherichia coli W1655F+ cells]. / Wirkung von Polyen-Makrolidantibiotica auf normale und zellwandlose Zellen von Escherichia coli W1655F+.

The polyene macrolide antibiotics are active against yeast, fungi, and other eukaryotic cells, but are with a few exceptions inactive against bacteria. The resistance of bacteria against these compounds is usually explained by the absence of sterols in their cells, the target sites of polyene antibiotics. However, in our experiments with mycotrienin, nystatin, tetramycin, lucenosmycin, rimocidin, filipin, lagosin, flavofungin, flavomycoin, antibiotic 2814P, antibiotic 5001P, and candicidin it was demonstrated that bacteria may be susceptible to polyene antibiotics, too, if the wall-less stable protoplast type L-form of E. coli W1655F+ is used. The measured growth inhibition concentrations were comparable with those of typical antibacterial antibiotics. Our experiments have shown that the normal rod form and the wall-less L-form of E. coli W1655F+ contain traces of sterols in nearly the same concentration range. This means that the selective sensitivity of the L-form cannot be explained by higher sterol content of these cells in comparison to the resistant normal rod form cells. We assume that the bacterial cell wall is responsible for the resistance of the normal rod form by masking internal target sites.

Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat.

The maintenance and genetic stability of the vector plasmids pBR322 and pBR325 in two genetically different Escherichia coli hosts were studied during chemostat cultivation with glucose and ammonium chloride limitation and at two different dilution rates. The plasmid pBR322 was stably maintained under all growth conditions tested. However pBR325 segregated from both hosts preferentially during glucose limitation and at low dilution rate. In addition to this general segregation process a separate loss of tetracycline resistance was observed. The remaining plasmid conferred resistance to ampicillin and chloramphenicol only, without any remarkable alteration of its molecular weight. Cultivation conditions in the chemostat were found that allowed the stable genetic inheritance of both plasmids in the hosts studied.

Genetic segregation in a high-yielding streptomycin-producing strain of Streptomyces griseus.

The streptomycin-producing Streptomyces griseus HP spontaneously segregated non-reverting derivatives with altered phenotypes. Clones characterized by increased spore formation and decreased streptomycin production were found. Two other types of derivatives were defective in aerial mycelium and streptomycin formation as well, but differed in the capacity to synthesize a yellow pigment. These derivatives were examined with respect to further properties. The stability of S. griseus HP was investigated in relation to conditions of continuous culture. Both at 26 and 30 degrees C, under glycerol and NH4Cl limitation a rapid segregation and enrichment of streptomycin-non-producing derivatives occurred. At 34 degrees C and glycerol limitation segregation began only after about 35 generations of continuous culture. In NH4Cl-limited chemostats the original strain was stable during 80 generations. In the course of the continuous culture experiments it was shown that the onset of genetic segregation within mycelia can be detected before it becomes obvious in colonies grown from the mycelia. This was achieved by fractionation of the mycelia by protoplast formation and subsequent plating on regeneration medium allowing colony growth and differentiation.