Intergeneric conjugation in Streptomyces peucetius and Streptomyces sp. strain C5: chromosomal integration and expression of recombinant plasmids carrying the chiC gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the phiC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of phiC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.     

Giant linear plasmids of β-lactam antibiotic producing Streptomyces

Abstract A survey of the total cellular DNA from five β-lactam antibiotic-producing Streptomyces spp. by pulsed field gel electrophoresis was conducted to investigate the presence of linear plasmids. Streptomyces clavuligerus NRRL 3585 contained two giant linear plasmids of 120 and 430 kb, in addition to the well-characterized 11.7 kb linear plasmid. Streptomyces griseus NRRL 3851 contained a single giant linear plasmid of 120 kb, and Streptomyces jumonjinensis NRRL 5741 contained two giant linear plasmids (220 and 280 kb), and two smaller linear plasmids. No plasmids were identified in Streptomyces cattleya NRRL 3841 or Streptomyces lipmannii NRRL 3584. Southern hybridization did not reveal any homology shared by these plasmids, and β-lactam antibiotic synthesis gene clusters were located on the chromosome.     

Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis.

The Tn3-like Streptomyces transposon Tn4560 was used to mutagenize Streptomyces avermitilis, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. Tn4560 transposed in this strain from a temperature-sensitive plasmid to the chromosome and from the chromosome to a plasmid with an apparent frequency of about 10(-4) to 10(-3) at both 30 and 39 degrees C. Auxotrophic and antibiotic nonproducing mutations were, however, obtained only with cultures that were kept at 37 or 39 degrees C. About 0.1% of the transposon inserts obtained at 39 degrees C caused auxotrophy or abolished antibiotic production. The sites of insertion into the S. avermitilis chromosome were mapped. Chromosomal DNA fragments containing Tn4560 insertions in antibiotic production genes were cloned onto a Streptomyces plasmid with temperature-sensitive replication and used to transport transposon mutations to other strains, using homologous recombination. This technique was used to construct an avermectin production strain that no longer makes the toxic oligomycin.     

Antibiotic production in a spatially structured environment

Ecological factors influencing the effects of antibiotic production were explored experimentally and theoretically. A spatially structured model was used to model the dynamics of antibiotic-producing and nonproducing bacteria in which growth of the nonproducers was reduced by neighbouring antibiotic producers. Various factors affecting spatial interactions between the bacteria were examined for their impact on antibiotic producers. Spatial clustering had a positive impact on the effect of antibiotic production, as measured by the decline in growth of the nonproducing strain, while increasing the initial density of the nonproducing strain had a negative impact. Experiments examined the growth of antibiotic-producing Streptomyces species and a nonproducing, antibiotic-sensitive strain of Bacillus subtilis that were coinoculated on surface media. There was an effect of the Streptomyces on Bacillus growth in some experiments but not in others. In light of the predictions from the model, unintentional clustering of cells is a more likely explanation for this finding than different initial Bacillus densities. The importance of spatial structure seen in this study is consistent with a terrestrial rather than an aquatic distribution of antibiotic-producing bacteria, and may have implications in the search for novel antibiotics.     

David Hopwood and the emergence of Streptomyces genetics.

Streptomyces spp. are unusual among bacteria in growing as mycelial colonies with sporulating aerial hyphae. They are very important as the source of most of the major antibiotics. Pioneering work by David Hopwood in the 1950s and 1960s established Streptomyces coelicolor A3(2) as the model system for the genus. Since then he has led successive key phases of research on this organism. In the 1970s, plasmids were discovered and characterised, and used both to establish conditions for transformation and in the subsequent development of cloning vectors. Protoplasts were exploited in both transformation and highly efficient cell fusion. In the 1980s, the early cloning of resistance genes from antibiotic-producing strains was followed by the cloning of antibiotic biosynthetic gene clusters, and the development of general methods and probes for the cloning of such clusters from diverse species. Analysis of these gene sets led to wide-ranging inferences about the biosynthesis of the important polyketide class of antibiotics, and to the production of hybrid antibiotics, and then, in the last decade, to more sophisticated combinatorial biosynthesis of designer molecules. In parallel, David Hopwood's work has also provided a crucial platform for studies of the regulation of the morphological and physiological differentiation that is manifested by sporulating antibiotic-producing colonies. Most recently, his involvement in the physical mapping of the entire 8 Mb genome of S. coelicolor A3(2) has culminated in its complete DNA sequencing: a project that should be completed under his management during the year 2000.     

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.     

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Experimental studies on the ecological role of antibiotic production in bacteria

Summary Genetically well-characterized strains of antibiotic-producing soil bacteria (Streptomyces griseus andStreptomyces coelicolor) were used to examine the ecological role of antibiotic production. Streptomycetes were competed against sensitive and resistantBacillus subtilis, another soil bacterium, on surface (agar) culture. The ecological role of antibiotics was examined in three levels of competition. (1) Capacity of antibiotics to allow invasion of producing organisms (B. subtilis established and streptomycetes added later). (2) Capacity of antibiotics to mediate competition between established populations (B. subtilis and streptomycetes co-inoculated). (3) Capacity of antibiotics to prevent invasion by competitors (streptomycetes established andB. subtilis added later). Antibiotic production was found to play a significant role in preventing the invasion of competitors in these experiments. Antibiotic production did not improve the ability of producers to invade a population of sensitive cells nor did it play a strong role in mediating competition between established populations. Antibiotic production also selected for antibiotic-resistant bacteria among invading competitors.     

Effect of the gene for bacterial hemoglobin vhb on the effectiveness of the process of Escherichia coli-Streptomyces interspecies conjugation and production of antibiotics in streptomycetes

The bacterial hemoglobin vhb gene was cloned from sliding bacterium Vitreoscilla sp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhb gene was transferred from Escherichia coli to some Streptomyces strains, producers of antibiotics, by the method of intergeneric conjugation using conjugative-integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhb gene on processes of conjugation and antibiotic production in a number of examined strains was shown.     

Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds

A survey of antibiotic-producing bacteria from the microbial flora attached to seaweeds and the study of their antibiotic capacities were carried out. From 5 species of green and brown marine algae, 224 bacterial strains were isolated and tested for antibiotic production. A total of 38 strains displayed antibiotic activity, withEnteromorpha intestinalis being the source of the highest number of producer strains. All epiphytic bacteria with antibiotic activity were assigned to thePseudomonas-Alteromonas group. Antagonism assays among the isolates demonstrated that each producer strain inhibits the growth of the other producers, as well as of some nonproducer strains also isolated from seaweeds. Likewise, an autoinhibitory effect was observed in all antibiotic-producing strains. Antibacterial spectra of all the strains include activity againstStaphylococcus, Alcaligenes, Pseudomonas, Vibrio, Pasteurella, andAchromobacter. A preliminary characterization of the antibiotic substances produced by these epiphytic bacteria demonstrated that they are low molecular weight compounds, thermolabile, and anionic and are not affected by proteolytic enzymes. The role that these inhibitory substances can play in the natural environment is discussed.     

Studies of protoplast fusion in Streptomyces chrysomallus

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.     

Strain identification in Rhizobium trifolii using DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistances

Abstract Indigenous strains of R. trifolii , originating from different geographical regions, were compared using total DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistance patterns. The results of strain relatedness using 2 of these techniques (DNA restriction and antibiotic resistance patterns) were similar, and indicated that these strains formed a heterogeneous group. A large variation in plasmid DNA profiles was observed among the R. trifolii isolates investigated, indicating heterogeneity of plasmid DNA pools. This variation occurred even in some strains that were shown to be related on the basis of their antibiotic resistance patterns. The application of these techniques to differentiate indigenous R. trifolii strains is discussed in this report.     

Effect of the producer type on the nature of antibiotic fermentation

Fermentation processes in production of bacitracin, a polypeptide antibiotic by Bacillus licheniformis, and oleandomycin, a macrolide antibiotic by Streptomyces antibioticus, were studied comparatively. It was shown that the antibiotic-producing actinomycete was characterized by a prolonged phase of growth retardation. The highest efficiency of the control actions was observed at the beginning of the fermentation. They were aimed at intensifying the substrate usage during the growth phase and activation of cell metabolism. Controlled cultivation of the Bacillus representative was based on its capacity of achieving the maximum growth rate possible under the certain conditions. Therefore, an increase in the quantity of the synthesized antibiotic was due, under such conditions, to inhibition of the culture growth by various means including lower mass exchange intensity.     

Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization.

We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .     

Deletion of scbA enhances antibiotic production in Streptomyces lividans

Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.     

Effect of intercalating dyes on the production of antibiotics by Micromonospora rosaria and Micromonospora purpurea.

The effect of treatment with various intercalating dyes on the ability to produce antibiotics in Micromonospora rosaria and Micromonospora purpurea was studied. Treatment with acriflavine resulted in a high frequency loss of antibiotic productivity in both species. In M. rosaria, the loss of antibiotic-producing ability appeared to be strain-dependent. In M. purpurea, up to 90% of colonies were found to have lost gentamicin-producing ability when protoplasts were used in the test. These antibiotic-nonproducing strains were further studied. The following observations were made: (1) Unlike the producing ability, the resistance to the antibiotics is a very stable character in both species. (2) Protoplast fusion analysis indicates that rosamicin-nonproducing characteristics of MR 217-AF2 and MR 217-AF3 strains induced by the acriflavine treatment is due to chromosomal mutation or rearrangement but not to loss of a plasmid. (3) Gentamicin-nonproducing strains of M. purpurea responded differently to the supplementation of streptamine or DOS in the culture medium. When supplemented with streptamine or DOS, some of these strains regained the ability to produce antibiotic, showing that the biosynthesis of intermediate was affected in these strains.     

Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2)

Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general.     

Streptomyces genes involved in biosynthesis of the peptide antibiotic valinomycin.

We have identified genes from Streptomyces levoris A-9 involved in the biosynthesis of the peptide antibiotic valinomycin. Two segments of chromosomal DNA were recovered from genomic libraries, constructed by using the low-copy-number plasmid pIJ922, by complementation of valinomycin-deficient (vlm) mutants of S. levoris A-9. One set of plasmids restored valinomycin production to only one mutant, that carrying vlm-1, whereas a second set of plasmids restored productivity to seven vlm mutants, those carrying vlm-2 through vlm-8. Additional complementation studies using subcloned restriction enzyme fragments showed that the vlm-1+ gene was contained within a 2.5-kilobase (kb) DNA region, whereas alleles vlm-2+ through vlm-8+ were contained in a 12-kb region, representing at least three genes. Physical mapping experiments based on the isolation of cosmid clones showed that the two vlm loci were 50 to 70 kb apart. Southern hybridization experiments demonstrated that the vlm-2+ gene cluster was highly conserved among other valinomycin-producing Streptomyces strains, whereas the vlm-1+ gene was ubiquitous among Streptomyces species tested. Increasing the copy number of the vlm-2+ gene cluster in S. levoris A-9 by the introduction of low-copy-number recombinant plasmids resulted in a concomitant increase in the level of valinomycin production.