Development of a system for cloning a DNA fragment, containing the determinant of resistance to actinomycin for Streptomyces chrysomallus No.2--a producer of actinomycin C]

To study the modes of actinomycin biosynthesis and the mechanism responsible for resistance to the antibiotic producing S. chrysomallus No. 2, the authors undertook an examination and studies into the cloning system for gene(s) of resistance to actinomycin from a S. chrysomallus No. 2 actinomycin C producer and the cloning of a S. chrysomallus No. DNA fragment to the actinomycin-sensitive Streptomyces Sp. 26-115 H-I on the vector plasmid pIJ702. The cloning gave rise to actinomycin-resistant strains. The character of actinomycin resistance is inheritable in a steady fashion.     

Copy number mutants of the broad-host-range Streptomyces plasmid pMG200

pMG200, isolated from the bacteriocin-releasing strain Streptomyces chrysomallus, was further physically mapped. Variants of S. chrysomallus were isolated which inhibited the parental strain. Two types of plasmids, pMG210 and pMG220, were isolated from these variants, with copy numbers of 10-30 and 300, respectively, compared with 1-3 for pMG200. pMG210 is apparently physically identical to pMG200 but presumably differs at a level not detected by simple restriction mapping; pMG220 is deleted for 1.6 kb. Genes for thiostrepton and viomycin resistance were subcloned from pIJ364 on to pMG200 and a fragment containing the gene for nourseothricin resistance was subcloned on to pMG220. In this way nonessential sites were identified.     

Characteristics of defective lysogeny in Streptomyces chrysomallus

When Streptomyces chrysomallus 2703 grows on solid media, lytic zones appear in the form of negative colonies which are not caused by the virulent phage. The material from these colonies and the cultural broth of S. chrysomallus 2703 were examined by electron microscopy. Four different morphological types of particles were revealed, three of which were defective phage particles (tails). Particles of the fourth type had a regular hexagonal shape and a diameter of 200A. The particles prevailed in all of the preparations. Their origin is discussed. S. chrysomallus in considered as a poly- and defective lysogenic culture.     

Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus

The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.     

Isolation of a plasmid from actinomycin C-producing Streptomyces chrysomallus and its characteristics

Until now the identification of plasmids in streptomyces, the producers of actinomycins, has not been reported, although there exist the genetic data on the possible plasmid participation in biosynthesis of these antibiotics. In this paper the data are presented on plasmid identification in two variants of Streptomyces chrysomallus. Plasmids are shown to be identical in both variants differing in productiveness. The restriction map is constructed for this 7000 b. p. plasmid. Plasmid participation in actinomycin biosynthesis and its possible use for molecular cloning in streptomyces are discussed.     

Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.     

Functional expression of the ectoine hydroxylase gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.     

Novel antibacterial proteins from entomocidal crystals of Bacillus thuringiensis ssp. israelensis

Proteins with molecular masses of 36 and 34 kDa (Bti36 and Bti34) were isolated from entomocidal crystals formed by Bacillus thuringiensis ssp. israelensis cells. The samples of Bti36 contained the admixture of a protein with a molecular mass of 33 kDa (Bti33), apparently a product of proteolysis of Bti36. These 3 proteins are significantly different in N-terminal sequences from known delta-endotoxins of B. thuringiensis and show antibacterial activity toward Micrococcus luteus. The combination of Bti36 and Bti33 also suppresses the growth of some other microorganisms including Streptomyces chrysomallus. The effects of the mixture of Bti36 and Bti33 on the M. luteus cell surface and on the surface of S. chrysomallus cells and exospores are similar, but they are different from the effect of endotoxin Cry11A on micrococcal cells.     

Comparative study of the phage sensitivity of actinomycetes and their Nocardia-like variants

The work was aimed at studying the resistance of three streptomycetes (Streptomyces chrysomallus, S. azureus and S. roseoflavus var. roseofungini) and their spontaneous Nocardia-like variants lacking aerial mycelium and spores against nine polyphages isolated mainly from soil. Some Nocardia-like variants were found to differ from their parent cultures in the resistance against certain actinophages. S. chrysomallus VKM Ac-590 and Ac-628 variants lost resistance against the phages. S. azureus VKM Ac-719 and S. roseoflavus var. roseofungini VKM Ac-770 variants became resistant to the phages. The changed phage resistance of the streptomycetes and their Nocardia-like variants was attributed to the disorganised process of adsorption (8 and 7%, respectively, against 70 and 90% for the parent strains).     

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.     

4-Methyl-3-hydroxyanthranilic acid activating enzyme from actinomycin-producing Streptomyces chrysomallus

A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.     

Calcium transport in cells of Streptomyces chrysomallus var. macrotetrolidi-- a producer of macrotetrolide antibiotics]

The dynamics of calcium accumulation by cells in batch cultures and by washed cells was defined with a radiotracer procedure for Streptomyces chrysomallus var. macrotetrolidi producing ionophore macrotetrolide antibiotics. It was shown that macrotetrolides added to the cultivation medium could regulate intracellular contents of calcium by participating in cation transport. Moreover, possible functioning of a Ca-ATPase system for the calcium active transport in the streptomycete cells was demonstrated.     

Analysis of genomic similarity in Streptomycetes belonging to the fluorescent subgroup as a confirmation of their taxonomic revision using a population method

Genomic similarity was analysed in streptomycetes belonging to the fluorescent subgroup: Streptomyces chrysomallus, S. fluorescens, S. galbofluorescens and S. citreofluorescens. The degree of reference S. chrysomalius DNA hybridization with S. fluorescens and S. galbofluorescens DNAs was 75 and 82%, respectively, thus being within the limits of the intraspecial hybridization level. S. citreofluorescens DNA showed a 55% homology with reference S. chrysomallus DNA, which corresponded to the range of interspecies hybridization. These conclusions were confirmed by the results obtained in analysing the thermostability of hybrid duplexes. Therefore, these findings are consistent with the data of revising the species taxonomy of this streptomycetes subgroup which was done using the method of comparative population analysis. The population model proposed by one of the authors can be used to assess the intraspecies level of DNA-DNA hybridization.     

Interactions of Hg(II) ions with DNA as revealed by CD measurements.

The circular dichroism spectra of Hg(II) complexes with native calf thymus DNA, chemically methylated Streptomyces chrysomallus DNA and with Ag(I)-DNA complexes were measured in the region of 220 - 340 nm. As a main result a conversion of the conservative CD spectrum of DNA to a distinct nonconservative type of CD spectrum for the complexes occurs with increasing Hg(II) concentration. The CD spectra of the Hg(II) complexes as well as some additional arguments strongly support the idea, that DNA in the complex undergoes a structural transition to a more condensed state with 4 -like character.     

FK-506-binding proteins from streptomycetes producing immunosuppressive macrolactones of the FK-506 type.

FK-506-binding proteins (FKBPs), which in T cells are supposed to mediate the immunosuppressive effects of the compounds FK-506 and rapamycin, have been isolated from Streptomyces chrysomallus, S. hygroscopicus subsp. ascomyceticus, and S. hygroscopicus. The latter two strains are producers of ascomycin (the ethyl analog of FK-506) and rapamycin, respectively. Like the 12-kDa FKBP in eukaryotic organisms such as humans, bovines, and Saccharomyces cerevisiae, or the FKBPs from gram-positive streptomycetes are peptidyl-prolyl-cis-trans isomerases. Inhibition studies using FK-506, rapamycin, or ascomycin, revealed inhibition of the peptidyl-prolyl cis-trans isomerase activity of the proteins at the nanomolar level, which is in the same range as with eukaryotic FKBPs. The M(r)s of the various FKBPs were 13,500 to 15,000, and they had the same pI of approximately 4.5. The N-terminal sequences of the three FKBPs were nearly identical in the first 20 amino acids. The amino acid sequence deduced from the gene sequence of S. chrysomallus gave a polypeptide of 124 amino acids. The homologies to FKBPs from humans, S. cerevisiae, and Neurospora crassa were 38, 39, and 50% identity in relevant positions, respectively. Significant homology of 38% was also seen with the C-terminal halves of bacterial protein surface antigens like the Mip protein of Legionella pneumophila and the 27-kDa Mip-like protein of Chlamydia trachomatis. In addition, two more open reading frames in Pseudomonas aeruginosa and Neisseria meningitidis of unknown function show regions of homology to the S. chrysomallus FKBP. In contrast to fungi, streptomycetes are resistant to macrolactones. Ascomycin-producing S. hygroscopicus subsp. ascomyceticus excretes the compound almost quantitatively into medium, which indicates that the organism has an efficient self-protection mechanism against its own secondary metabolite.     

Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.     

Effect of heat shock on biosynthesis of antibiotics by three strains of Streptomyces]

Effects of heat shock on the biosynthesis of antibiotics, actinomycin C (in cultures of Streptomyces sp. 26-115 and S. chrysomallus 23209) and antibiotics of the nonactin group (in the culture of S. werraensis 1365) were studied. After heat shock, the formation of antibiotics of the nonactin group and actinomycin C were shown to increase by 30% and 27%, respectively, in comparison to control values. Thus, heat shock stimulates the biosynthesis of antibiotics in all three strains of streptomyces studied.     

噬菌体φHAU3的cos位点及其与寄主相互作用的功能位点attP和attB的定位

ＨＡＵ３是寄主范围很广的放线菌噬菌体。Ｓｏｕｔｈｅｒｎ杂交实验表明,ＨＡＵ３可以整合到吸水链霉菌应城变种１０－２２和变铅青链霉菌６６的突变体ＺＸ１的染色体中,形成溶原,其溶原菌自发释放ＨＡＵ３,不受热激和紫外线照射的诱导。通过比较ＨＡＵ３衍生噬粒ｐＩＪ８３００的ＤＮＡ酶切片段在加热前、后电泳带谱的区别,将ＨＡＵ３的ｃｏｓ位点在ｐＩＪ８３００的图谱上得到了定位。还利用Ｓｏｕｔｈｅｒｎ杂交的方法定位了ＨＡＵ３与宿主形成溶原时附着位点（ａｔｔＰ）,并利用脉冲电泳技术定位了在变铅青链霉菌ＺＸ７和吸水链霉菌应城变种１０－２２中形成溶原的附着位点（ａｔｔＢ）。这些信息均有利于以ＨＡＵ３为基础的载体的发展和优化。     

The effect of heat shock on the growth and mycelial morphology of Streptomyces chrysomallus]

The effect of various conditions of heat shock (1 hour at 35, 38, 40, 42, 45 and 50 degrees C) on the growth and morphological features of Streptomyces chrysomallus, an organism producing actinomycin, was studied. A definite regularity in the mycelium morphological changes at high temperatures was observed. After the shock at 35 and 38 degrees C the biomass volume and morphological features of the streptomycete did not markedly differ from those in the control. The shock at 40 degrees C induced the growth inhibition with decreasing the biomass volume by 50 per cent and appearance of submerged spores. When the shock conditions were more rigid (42, 45 and 50 degrees C) the mycelium growth lacked. It is of interest that the temperature of 42 degrees C induced abundant formation of the spores. With further increasing of the temperature to 45 and 50 degrees C the spore formation was not so abundant. The changes in the growth and development of the streptomycete are discussed in relation to the molecular mechanism of the cell protection from temperature shock.     

Directed synthesis of macrotetrolides

The composition of the macrotetrolide complex was found to be strongly dependent on the conditions of the Streptomyces chrysomallus v. macrotetrolidi cultivation and could be varied by including in the medium 0.2% of organic acids, precursors of macrotetrolides, such as acetic, propionic and succinic. Acetate caused an increase of the nonactin/monactin ratio, and no other homologues were detected. On the contrary, propionate and succinate produced a drop in the nonactin synthesis, which was accompanied by a rise in the amount of the higher homologues. The composition of the macrtetrolide mixture can also be changed by introducing in the cultivation medium specific inhibitors (100-200 micrograms/ml) such as malonate, cobalamin analogue, sulfadimesin. Malonate, an inhibitor of succinate dehydrogenase, increased the biosynthesis of higher ethylated homologues. Inhibition of methylmalonate mutase resulted in an increased yield of the methylated nonactin homologue and in a decreased yield of dinactin. In this case no other homologues were produced. The inhibitor of transmethylation, sulfadimesin, had no effect on the biosynthesis and composition of the macrotetralide mixture.