Molecular analysis of a gene encoding a cell-bound esterase from Streptomyces chrysomallus

A gene (estA) encoding a 42-kDa cell-bound esterase, EstA, was found to be located 75 bp upstream of the cyclophilin A gene (cypA) of Streptomyces chrysomallus. Western blot analysis revealed the presence of EstA (42 kDa) in cell extracts of S. chrysomallus X2 and Streptomyces lividans. EstA specifically hydrolyzes short-chain p-nitrophenyl esters. EstA formation starts at the end of growth phase, and its activity level remains constant throughout stationary phase. Expression of estA from the melanin (mel) promoter in plasmid pIJ702 led to a substantial increase of total esterase activity in streptomycetes.     

Molecular cloning of the actinomycin synthetase gene cluster from Streptomyces chrysomallus and functional heterologous expression of the gene encoding actinomycin synthetase II

The actinomycin synthetases ACMS I, II, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism. We have cloned the genes of ACMS I (acmA) and ACMS II (acmB) by hybridization screening of a cosmid library of Streptomyces chrysomallus DNA with synthetic oligonucleotides derived from peptide sequences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and partial sequence analyses indicate that the gene of an additional peptide synthetase, most likely the gene of ACMS III (acmC), is located immediately downstream of acmB in the same orientation. The protein sequence of ACMS II, deduced from acmB, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide synthetases, and an additional epimerization domain. Heterologous expression of acmB from the mel promoter of plasmid PIJ702 in Streptomyces lividans yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme-bound thioesters. It also catalyzes the dipeptide formation of threonyl-L-valine, which is epimerized to threonyl-D-valine. Both of these dipeptides are enzyme bound as thioesters. This catalytic activity is identical to the in vitro activity of ACMS II from S. chrysomallus.     

Synthesis and some properties and antitumor effects of the actinomycin lactam analog, (di(1-L-alpha, beta-diaminopropionic))actinomycin D1

A lactam analog of actinomycin D (AMD) has been synthesized as a potential antitumor chemotherapeutic agent. Both L-threonine residues were replaced by L-alpha,beta-diaminopropionic acid. Starting with Nalpha-benzyloxycarbonyl-Nbeta-tert-butyloxycarbonyl-L-alpha,beta-diaminopropionic acid methyl ester hydrochloride the linear intermediate Nalpha-benzyloxycarbonyl-Nbeta-(tert-butyloxycarbonylsarcosyl-L-N-methylvalyl)-L-alpha,beta-diaminopropionyl-D-valyl-L-proline p-nitrophenyl ester was prepared by conventional methods of peptide synthesis in solution. Selective cleavage of the Nbeta-tert-butyloxycarbonyl group and lactam formation afforded the desired cyclic pentapeptide derivative. The chromophore precursor, Nalpha-(2-nitro-3-benzyloxy-4-methylbenzoyl) substituent, was introduced via its symmetric anhydride. Catalytic reduction followed by ferricyanide-mediated phenoxazinone formation provided the lactam analog, [di(1'-L-alpha,beta-diaminopionic acid)]actinomycin D ([Dpr1]2-AMD). Its binding to natural and synthetic DNA and that of an analogous L-threo-alpha,beta-diaminobutyric acid containing lactam ([Dbu1]2-AMD) compared with the binding of AMD (in which the peptides are in lactone form) was studied by circular dichroic (CD) spectroscopy. The visible and uv CD spectra of free AMD differed from those of the free lactam analogs, indicating that the asymmetric environment of the pentapeptide rings in the region of the chromophore differs in free actinomycin lactone and lactams. In the presence of calf thymus DNA, PM2 DNA, and the synthetic d(A-T)-like copolymers containing 2,6-diaminopurine (DAP), poly[d(DAP-T)], and poly[d(DAP-A-T)], the rotational strengths of the optically active transitions in the visible region of the actinomycins increased, and the CD spectra in the presence of the various DNA duplexes were qualitatively similar. The CD spectra of bound actinomycin lactams resembled the spectrum of bound AMD. This suggests that the lactone and lactam actinomycins acquire a similar environment when bound to DNA. [Dpr1]2-AMD was less cytotoxic than AMD in antibacterial assays but exhibited somewhat higher toxicity in mice than AMD. At optimal dose levels the lactam analog had little or no antitumor activity in three murine tumor systems.     

Instability of artificially circularized chromosomes of Streptomyces lividans

The chromosomes of Streptomyces species are linear molecules, containing long terminal inverted repeats and covalently bound terminal proteins. These chromosomes undergo spontaneous deletions of the terminal sequences at high frequencies and become circularized in several cases examined. Artificial circularization of the Streptomyces lividans chromosome was also achieved by targeted recombination in vivo, in which the terminal inverted repeats of the chromosome were connected by a kanamycin resistance gene (aphII). Under kanamycin selection, the circularized chromosomes harboured tandem amplifications of a 20.2 kb sequence that included the aphII gene flanked by direct repeats and deletions nearby. On release from kanamycin selection, the aphII amplifications and the neighbouring sequences were deleted from the chromosomes, rendering all the cultures kanamycin sensitive. The chloramphenicol resistance gene, which was prone to deletion in wild-type S. lividans, became much more stable in the kanamycin-sensitive derivatives. These results indicate that the telomeres and/or certain terminal sequences may be involved in the structural instability of Streptomyces chromosomes.     

Methodological bases for monitoring the health of the population

Bireplicon plasmids were constructed. The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome. Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance. The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S. lividans. The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S. lividans. No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA. The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants. The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids. As a result actinomycete monoreplicon plasmids were formed. The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map.     

Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements and Escherichia coli mutant defective in the ATPase activity of SecA

The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes. It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins. All the previously described conserved motifs of SecA proteins were conserved in the S. lividans protein. The secA gene of S. lividans restored sensitivity to sodium azide in E. coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein. However, it did not complement the temperature-sensitive mutation in E. coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature. secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

Pool of free amino acids in mycelium and culture fluid of Streptomyces galbus (F) subsp. achromogenes 695, a strain producing actinomycin complex and its variants

Free amino acids in the mycelium and culture fluid of Streptomyces galbus (F) subsp. achromogenes 695 and its active and inactive variants were comparatively studied. It was found that the amino acid pool in the mycelium of the highly productive variant was 14 per cent higher than that of the initial strain and 40 per cent higher than that of the inactive variant. Even so, the highest amounts of the synthesized protein in the three strains were about the same. The free amino acid composition of the mycelium of the active antibiotic-producing strains and the inactive variant was shown to be the same and included all the investigated 16 amino acids. Glutamic acid was the main amino acid. The contents of alanine, serine and valine were comparatively high. The contents of methionine, histidine and phenylalanine were the lowest. It was shown that the quantities of the amino acids or their precursors participating in the construction of the antibiotic molecule were to a higher extent determined by the strain development and growth rate than by the actinomycin biosynthesis. The qualitative and quantitative composition of the amino acid pool in the culture fluid of the active strains was inferior to that in the inactive variant.     

High-frequency transposition of IS1373, the insertion sequence delimiting the amplifiable element AUD2 of Streptomyces lividans

IS1373 is the putative insertion sequence delimiting the amplifiable unit AUD2 of Streptomyces lividans. Two IS1373-derived thiostrepton-resistant transposons, Tn5492 and Tn5494, transposed into multiple sites of the S. lividans chromosome at frequencies as high as 0.4 and 1%, respectively. Hence, IS1373 is a functional insertion sequence and its unique open reading frame, insA, encodes the transposase.     

Structure of cell-wall teichoic acids in Streptomyces roseoflavus var. roseofungini and its Nocardia-like variant

The structure of teichoic acids was studied in the cell walls of Streptomyces roseoflavus var. roseofungini 1128 and its Nocardia-like variant 1-68 differing from the parent strain in the absence of a spore-forming aerial mycelium as well as by the fragmentation of hyphae in the substrate mycelium. The teichoic acids of the both cultures consist of a 1,3-poly(glycerophosphate) chain containing 11-13 glycerolphosphate residues which have glucosamynl units and lysine groups bound through an ester bond. These teichoic acids contain no O-acetyl groups, in contrast to the glyceroteichoic acids of actinomycetes studied earlier. The teichoic acid from the cell wall of the variant has less lysine and glucosamine then the parent strain. The content of teichoic acid in the cell wall of the parent culture is 4.5 times greater than in the wall of the variant.     

Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.     

Chronic immune demyelinating neuropathies

During growth of Streptomyces niveus wild-type in the novobiocin production medium CDM the resistance of mycelia to novobiocin rises from about 25 micrograms/ml to over 200 micrograms/ml. (S. lividans, a novobiocin-sensitive strain, is resistant to approx. 10 micrograms/ml novobiocin.) The initial period of low level resistance extends from the time of inoculation of the culture until approx. 70 h when the culture is still in the growth phase. High level resistance is initiated before the start of novobiocin production and rises rapidly to a maximum level beyond the end of the growth phase. The rise in pH of the unbuffered CDM medium which occurs during S. niveus fermentation was shown not to be the cause of the change in novobiocin resistance. However, mycelia-free CDM from S. niveus cultures expressing high level novobiocin resistance was shown to contain a factor which induced high level novobiocin resistance in germinating S. niveus spores. Kinetic studies revealed that the inducer first appears in the culture medium before the switch to high level resistance begins and reaches its highest concentration before resistance reaches its maximum level.     

Accumulation of intracellular carbon reserves in relation to chloramphenicol biosynthesis by Streptomyces venezuelae

Two chloramphenicol-producing strains of Streptomyces venezuelae accumulated small amounts of polyhydroxybutyrate during exponential growth; the compound disappeared from the mycelium as the cultures entered stationary phase. Depletion of polyhydroxybutyrate coincided with chloramphenicol production but the amount of polymer stored in the mycelium was insufficient to supply the precursor requirement for biosynthesis of the antibiotic. Accumulation of polyhydroxybutyrate in the S. venezuelae strains was appreciably lower than in two other streptomycetes examined. Glycogen and lipids accumulated in the mycelium of S. venezuelae 13s during the stationary phase, after nitrogen depletion; under the culture conditions used, they were the principal storage compounds in S. venezuelae. Trehalose was absent from the mycelium in vegetative cultures grown under nonsporulating conditions but it was abundant in spores obtained from submerged and surface cultures. Glycogen and polyhydroxybutyrate were absent from spores.     

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.     

With regard to "The role of air plethysmography in monitoring results of venous surgery"

By homology to the mgt gene (encoding a macrolide glycosyltransferase) from Streptomyces lividans, a 3.3-kb DNA fragment from the oleandomycin producer, Streptomyces antibioticus, was cloned and sequenced. Analysis of the sequence revealed the presence of the 3' end of a gene (ORF1) and two complete ORFs (ORF2 and oleD), all of them translationally coupled. The deduced product of the sequenced region of ORF1 contained the typical signature of integral membrane proteins responsible for the translocation of substrates across the membrane. The ORF2 product did not show significant similarity with proteins in databases, but contains an N-terminus leader peptide region characteristic of secreted proteins, and a lipid attachment site motif characteristic of membrane lipoproteins synthesized with a precursor signal peptide. The oleD product showed clear similarity with several UDP-glucuronosyl- and UDP-glycosyl-transferases from different origins and particularly with the mgt gene from S. lividans, and might encode a glycosyltransferase activity capable of inactivating macrolides. It is proposed that these three genes could participate in the intracellular glycosylation of oleandomycin and its secretion during antibiotic production.     

A novel beta-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases

A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66, 329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned beta-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical beta-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable beta-glucosidase activity, revealed homology with microbial beta-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of beta-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60 degreesC and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-beta-N-acetylglucosamine were 425.7 microM and 24.8 micromol min-1 mg of protein-1, respectively.