Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants.

The intracellular low-molecular-weight thiols present in five gram-positive Streptomyces species and one Flavobacterium species were analyzed by high-performance liquid chromatography after fluorescence labeling with monobromobimane. Bacteria were chosen to include penicillin and cephalosporin beta-lactam producers and nonproducers. No significant amount of glutathione was found in any of the streptomycetes. Major intracellular thiols in all strains examined were cysteine, coenzyme A, sulfide, thiosulfate, and an unknown thiol designated U17. Those streptomycetes that make beta-lactam antibiotics also produce significant amounts of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a key intermediate in their biosynthesis. In Streptomyces clavuligerus, a potent producer of beta-lactams, the level of ACV was low during the early phase of growth and increased rapidly toward the end of exponential growth, paralleling that of antibiotic production. These and other observations indicate that ACV does not function as a protective thiol in streptomycetes. U17 may have this role since it was the major thiol in all streptomycetes and appeared to occur at levels about 10-fold higher than those of the other thiols measured, including ACV. Purification and amino acid analysis of U17 indicated that it contains cysteine and an unusual amine that is not one of the common amino acids. This thiol is identical to an unknown thiol found previously in Micrococcus roseus and Streptomyces griseus. A high level of ergothioneine was found in Streptomyces lactamdurans, and several unidentified thiols were detected in this and other streptomycetes.     

Arsenical resistance of growth and phosphate control of antibiotic biosynthesis in Streptomyces

Twenty-six wild-type Streptomyces strains tested for resistance to arsenate, arsenite and antimony(III) could be divided into four groups: those resistant only to arsenite (3) or to arsenate (2) and those resistant (8) or sensitive (13) to both heavy metals. All strains were sensitive to antimony. The structural genes for the ars operon of Escherichia coli were subcloned into various Streptomyces plasmid vectors. The expression of the whole ars operon in streptomycetes may be strain-specific and occurred only from low-copy-number plasmids. The arsC gene product could be expressed from high-copy plasmids and conferred arsenate resistance to both E. coli and Streptomyces species. The ars operon expressed in S. lividans and the arsC gene expressed in S. noursei did not render the synthesis of undecylprodigiosin and nourseothricin, respectively, phosphate-resistant. In addition in wild-type strains of Streptomyces phosphate sensitivity of antibiotic biosynthesis did not show strong correlation with resistance of growth to arsenicals.     

Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. Lambda-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage phiC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes

Summary There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning theactI, actIII, actIV, andactVII genes (pANT12) of actinorhodin biosynthesis pathway fromStreptomyces coelicolor in anthracycline producing streptomycetes.Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kbXhoI fragment containing only theactI andactVII loci was necessary for aloesaponarin II biosynthesis byS. galilaeus 31 133. WhenS. galilaeus 31 671 was transformed with theactI, actVII, andactIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-0-methyldesoxyerythrolaccin. WhenS. galilaeus 31671 was transformed with only the intactactIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for theactIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci,dauG, dnrR1, andasaA, on the production of natural and hybrid polyketides were also shown.     

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes

ABSTRACT: BACKGROUND: Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. RESULTS: None of the fifteen Streptomyces isolates inhibited all seven tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of only one of the tested fungi, the mycorrhiza-forming fungus Laccaria bicolor, was stimulated by the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only. CONCLUSIONS: Our results show that the primary characteristic of mycorrhiza associated streptomycetes is to inhibit the growth of fungi and bacteria. In parallel, our study indicates that Streptomyces strains which are not general antagonists may produce previously un-described metabolites.     

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.     

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.     

Evolutionary flux of potentially <Emphasis Type="Italic">bldA</Emphasis>-dependent <Emphasis Type="Italic">Streptomyces</Emphasis> genes containing the rare leucine codon TTA

Previous studies have shown that one of the six leucine codons, UUA, is rare in Streptomyces, and that, while the gene for the UUA-specific tRNA, bldA, can generally be inactivated in diverse streptomycetes without impairing vegetative growth, bldA mutants are typically defective in reproductive aerial growth and in antibiotic production. Here, four complete genome sequences and 143 gene clusters for antibiotic biosynthesis from diverse streptomycetes were analysed in order to evaluate the evolution and function of genes whose possession of TTA codons makes them dependent on bldA. It was deduced that the last common ancestor of the four sequenced genomes, possibly 220 million years ago, already possessed the bldA system, together with perhaps 200 TTA-containing target genes. Some 33 of these genes are retained by the modern descendants, though only three of them retain a TTA in all occurrences. Nearly all of these 33, as well as many of the TTA-containing genes with orthologues in two or three of the four genomes, have the same location on the chromosomes as in their common ancestor. However, the majority of TTA-containing genes (61% overall in the four genomes) are species-specific, and were probably acquired by comparatively recent horizontal gene transfer. Most of these genes are of unknown function, and it is likely that many of them confer specialised ecological benefits. On the other hand, one class of species-specific, functionally recognisable, horizontally acquired genes--the gene clusters for antibiotic production--very often contain TTA codons; and nearly half of them have TTA codons in their pathway-specific regulatory genes.     

Conjugative transfer of a plasmid from Escherichia coli to various strains of the order Actinomycetales

The conjugal transfer of autonomous and integrative plasmids from the donor strain Escherichia coli S17-1 to strains of genera Actinomadura, Arthrobacter, Kitasatoa, Micromonospora, Nocardia, Rhodococcus, Saccharopolyspora, and to 16 strains of the genus Streptomyces was demonstrated. The status of plasmids in recipient strains and the stability of their inheritance were analyzed. Plasmids constructed for strains of the genus Streptomyces were shown to function in a large number of strains belonging to the order Actinomycetales. The well-developed system of Streptomyces vector molecules and cloned genes of antibiotic biosynthesis allows their transfer to those microorganisms for which conventional techniques of plasmid transfer by regenerated protoplast transformation or electroporation have not been developed or are inefficient.     

Evolution and ecology of antibiotic resistance genes

A new perspective on the topic of antibiotic resistance is beginning to emerge based on a broader evolutionary and ecological understanding rather than from the traditional boundaries of clinical research of antibiotic-resistant bacterial pathogens. Phylogenetic insights into the evolution and diversity of several antibiotic resistance genes suggest that at least some of these genes have a long evolutionary history of diversification that began well before the 'antibiotic era'. Besides, there is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in clinically relevant and commensal bacteria. Most likely, the primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens. Dissemination and penetration of antibiotic resistance genes from antibiotic producers were less significant and essentially limited to other high G+C bacteria. Besides direct selection by antibiotics, there is a number of other factors that may contribute to dissemination and maintenance of antibiotic resistance genes in bacterial populations.     

Effect of a Cloned DNA Fragment from Streptomyces chrysomallusNo. 2 on the Metabolism of Certain Streptomyces Strains

The effects on a cloned DNA fragment carrying an actinomycin resistance determinant on physiological processes in strains of streptomycetes with various potencies in producing this antibiotic, their inactive mutants, and the model strain ofStreptomyces lividans66 were studied. This fragment was shown to modulate bacterial resistance to actinomycin and biosynthesis of antibiotics.     

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230.

A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.     

Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains

A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus IMRU 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out of 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains were never before described as polyene producers.