Charles Lee Hershberger

Homology between proteins controlling Streptomyces fradiae tylosin resistance and ATP-binding transport

A tylosin(Ty)-producing strain of Streptomyces fradiae contains at least three genes, tlrA, tlrB, tlrC, specifying resistance to Ty (TyR). The complete nucleotide sequence of the TyR-encoding gene, tlrC, and the transcription start point of the gene were determined. The sequence contains an open reading frame coding for a protein of 548 amino acids (aa) with an Mr of 59129. The TlrC protein was identified by expression of the cloned gene by in vitro coupled transcription and translation in cell-free extracts derived from Streptomyces lividans. The N- and C-terminal halves of TlrC share extensive homology, suggesting that the protein evolved through tandem gene duplication. Each half of the deduced TlrC aa sequence also shows significant homology to numerous eukaryotic and prokaryotic membrane-associated, active-transport protein subunits. The homologous proteins include examples from the systems responsible for efflux of cytotoxic drugs from multidrug-resistant human cells and for export of hemolysin from Escherichia coli. The greatest similarity to TlrC is in regions containing the ATP-binding sites found in these proteins. These results suggest a role for the tlrC gene product as part of a multiple component, ATP-dependent transport system for the active excretion of Ty from the producing organism.

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

SummaryA gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage λ Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

Selective retention of recombinant plasmids coding for human insulin

Plasmids may be lost from Escherichia coli K-12 hosts that are cultured without selection for plasmid retention. This is particularly true for chimeric plasmids that incorporate genes for human insulin into vectors derived from pBR322. The cIts857 gene of bacteriophage lambda was inserted into the bla gene of the human-insulin-coding plasmids, pIA7 delta 4 delta 1, pIB7 delta 4 delta 1 and pHI7 delta 4 delta 1, generating the new plasmids pPR17, pPR18 and pPR19, respectively, which produced the thermosensitive lambda repressor. The cI gene was downstream from the pM and pbla promoters, so that it may have been expressed from either or both promoters. Separate E. coli K-12 RV308 host strains containing the new recombinants were lysogenized with the repressor-defective bacteriophage lambda cI90. Loss of the plasmid from the lysogens causes concomitant loss of the lambda repressor and cell death, because the prophage is induced to enter the lytic growth cycle. The system effectively forces retention of the plasmid in all viable cells in the culture.

Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system

The yeast URA3 gene was used as a reporter to investigate the activities of estrogenic and antiestrogenic compounds in yeast Saccharomyces cerevisiae. The control sequences of the wild type (wt) URA3 promoter were replaced with zero, two, or six copies of estrogen-response elements (ERE). Insertion of two and six copies of ERE rendered the expression of the URA3 gene to be dependent on the presence of the human estrogen receptor (ER) and the hormone 17beta-estradiol (E2). Two versions of the ER genes were constructed: a full-length wild-type ER (ERa-f) and a truncated ER with domains C, D, and E (ERcde). Both forms of the ER were able to activate the ERE-URA3 reporter in a hormone-dependent manner. The growth of yeast transformants were hormone-dependent when the reporter constructs were inserted into chromosomes using yeast integrating vectors (YIp) but not with the 2mu-based episomal (high-copy number, YEp) or centromeric (low-copy number, YCp) vectors. The integrated transformants were employed to investigate the effects of estrogenic and antiestrogenic compounds. The estrogenic compounds, E2, diethylstilbestrol (DES), and estrone (EST), activated expression of the reporter genes at 1 nM concentration, which is the same concentration exhibiting activity in mammalian cells. None of the antiestrogens, at concentrations up to 1 microM, including tamoxifen (TAM), raloxifene (RAL), and ICI 164,384 (ICI) antagonized 1 nM of E2 against either form of the ER. In fact, TAM, RAL, and ICI displayed slight agonistic activity at high concentrations of 300 nM or greater to the ERcde. This system can be used to investigate or clone the missing factor(s) that is responsible for the antagonistic activity of the ER in yeast, and is also suitable for screening for the effectors of the ER.

Metabolic engineering of polyketide biosynthesis.

Three elements came together during the past year to provide the opportunity to generate new polyketides. The first was the availability of cloned genes for several metabolic pathways; the second was a genetically defined host strain able to support the production of polyketides; and the third was the ability to modify specific genes and recombine genes from different pathways using recombinant DNA technology. These tools culminated in the rational design of new molecules and the biosynthesis of large numbers of new molecules using combinatorial biology.

Recombinant DNA Systems for Application to Antibiotic Fermentation in Streptomyces

Publisher Summary This chapter reviews specific selected topics that are critical for successful and efficient application of recombinant DNA to improving productivity and/or generating new antibiotics in Streptomyces. The discovery and subsequent development in the field of restriction endodeoxyribonucleases and their application with other enzymes acting on DNA to recombine DNA molecules in vitro have prompted formation of small, research-oriented companies. The chapter discusses host-controlled restriction. Host-controlled restriction can be a major obstacle to the recovery of recombinant DNA; therefore, most commonly employed cloning systems utilize restrictionless strains as hosts. Several developments have been described that are essential for successful application of recombinant DNA methodologies to antibiotic fermentations in Streptomyces. Studies of host-controlled restriction have identified Streptomyces species without discernible restriction systems. These restrictionless hosts should be particularly advantageous for cloning genes from a variety of Streptomyces and for assembling vectors with general utility. Furthermore, any rational plan for application of recombinant DNA to antibiotic fermentation must include a major commitment to understand and exploit regulation of gene expression. Thereafter, basic biological properties of E. coli plasmids provide guidelines for planning experimental programs to discover characteristics of Streptomyces plasmids. Occurrence of plasmids is common throughout a broad range of Streptomyces species; therefore, virtually unlimited sources of replicons are available for exploitation in construction of recombinant DNA systems. Finally, the genetic markers available for selection and analysis of Streptomyces plasmids have limited the progress toward understanding the systems.

Uses of recombinant DNA for analyses of Streptomyces species.

The advent of recombinant DNA techniques has provided investigators with a new arsenal to attack problems of gene structure, genetic regulation, and genome organization. Already the intellectual rewards have been spectacular in providing solutions to problems that were intractable previously. However, the most important rewards are derived from applications to produce biological products that benefit mankind directly. The first-stage efforts have been mobilized for production of single proteins. September 20, 1982, is a historic date in stage one because Eli Lilly and Company marketed human insulin, which is the first clinically significant product from recombinant DNA that is available to the public. A plethora of additional protein products from recombinant DNA are in various stages of development or research. Undoubtedly, the results will have profound effects on the practice of medicine during the coming decades. Perhaps the next important milestone of pharmaceutical applications will be the production of metabolites such as antibiotics. Essential use of life-saving antibiotics is a cornerstone of modern medicine. Successful development of recombinant DNA methods will provide tools that will help increase the fermentation yields of antibiotics and generate new antibiotic structures that are not available through traditional routes of discovery.’ Full realization of these benefits necessitates developing recombinant DNA systems for Streptomyces (S.) species because two-thirds of all known antibiotics are produced by S. species.2 Researchers, led by David Hopwood and Keith Chater a t the John Innis Institute, have set the tempo for development of recombinant DNA systems in S. species: Hopwood working on plasmid vectors and Chater working on actinophage vectors. Their progress has been described recently and readers are referred to these excellent reviews.”’ Investigators with Stanley Cohen have reported progress in development of plasmid vectors,6.’ Isogai et aZ.* have potential actinophage vectors and Richardson et aL9 have published additional plasmid vectors. Multiple and parallel development of recombinant DNA systems provides the greatest likelihood for success because no one system is likely to work with all S. species or be applicable to all problems. This report describes progress in our program to apply recombinant DNA technology to S. species. Steps are described in the construction of the plasmids pJL197 and pJL198, which should be particularly useful as recombinant DNA cloning vectors. Discovery of amplified DNA in Streptomyces fradiae (S. fradiae) and application of recombinant DNA techniques in the characterization of amplified DNA are described in the latter portion of the report. The features of this high-level DNA amplification are unique to Streptomyces among bacterial species.

INTRACELLULAR SELECTION FOR PLASMIDS THAT CARRY GENES FOR HUMAN INSULIN

Publisher Summary Plasmids may be lost from bacterial hosts that are cultured without selection for plasmid-retention unless the plasmid encodes an active partition function to insure distribution to each progeny at cell division. This is particularly true for certain chimeric plasmids that incorporate foreign genes into vectors derived from Col El-like plasmids. Plasmids coding for chains of human insulin can be lost from virtually the entire population when Escherichia coli (E. coli) K12 strains harboring the plasmids are propagated under nonselective conditions. Alternative selection schemes are necessary to force retention of these recombinant plasmids without continuous exposure to antibiotics. This chapter presents the feasibility of constructing an intracellular conditionally lethal system whereby loss of the plasmid from any progeny cell results in the death of that cell.