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F Malpartida

Publications and source records attributed to F Malpartida.

At least 19 recordsLinked to original sources
PubMed1992

Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin.

A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced. Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis. ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon. The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers. The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis. The ORF6 product does not resemble other known proteins. Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6). The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I).

Acyl Carrier Protein↗
PubMed1992

Heterologous activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.

A DNA fragment of Streptomyces fradiae is able to activate the antibiotic actinorhodin biosynthetic pathway when cloned in Streptomyces lividans. The activator DNA region has been sequenced and its transcription initiation and termination sites accurately mapped in vivo. This DNA encodes a 132 nucleotides long transcript which is apparently responsible for the actinorhodin production phenotype, possibly acting as an antisense RNA. The sequence of the activator gene revealed no homology with any other known Streptomyces coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation.

Amino Acid Sequence↗
PubMed1992

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis.

Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 bp fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1-5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS: a heterodimeric beta-ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a beta-ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORF1-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1991

The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces.

The actII region, flanked by biosynthetic genes in the 25 kb act cluster of S. coelicolor, consists of four open reading frames, including a transcriptional activator for the biosynthetic genes, and genes controlling antibiotic export. A TTA codon (extremely rare in Streptomyces) is present both in actII-ORF2 (encoding a putative transmembrane export protein) and actII-ORF4 (the transcriptional activator gene). Change of the TTA in ORF4 to TTG reverses the normal interruption of actinorhodin synthesis caused by mutation in the pleiotropic regulatory gene bldA (which encodes the cell's tRNA(Leu)(UUA)). We conclude that initiation of actinorhodin synthesis via the actII-ORF4 product, and the final step in production, antibiotic export, are twin targets via which bldA exerts developmental control of actinorhodin production.

Amino Acid Sequence↗
PubMed1991

Transcription of genes involved in the earliest steps of actinorhodin biosynthesis in Streptomyces coelicolor.

A 170bp long BamHI-Sau3A DNA fragment from the actIII-actI intergenic region of the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2) contains two promoters directing transcription in a divergent manner. One of them, the actIII promoter, is responsible for the transcription of the actIII gene and the other controls transcription of the adjacent actI region in the opposite direction. Weak activity of the actIII promoter can be detected in Streptomyces lividans and Bacillus subtilis in the absence but not in the presence of glucose. Neither promoter seems to function in Escherichia coli.

Anthraquinones↗
PubMed1991

Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture.

Three open reading frames (ORFs) in the actII region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2), which are involved in the export of the antibiotic are carried on two divergent transcripts. A monocistronic transcript carries actII-ORF1, encoding a putative repressor protein, and a bicistronic transcript codes for actII-ORF2 and -ORF3, whose products have been postulated to form an antibiotic export complex. The actII-ORF1 and actII-ORF2/3 transcripts each have a single promoter and the promoters for the two transcripts overlap. Both promoters are most active in cultures that have developed to the stage of actinorhodin production. The promoters resemble consensus promoters of the vegetative class in Escherichia coli and Streptomyces. We also demonstrate that these promoters are expressed in E. coli and use this finding to reveal a regulatory role for the repressor, using the xylE reporter gene on promoter-probe shuttle vectors and regulated expression of the actII-ORF1 gene under control of Plac. The actII-ORF2/3 promoter is strongly repressed by the ORF1 product and the ORF1 product also represses its own promoter. The finding that the operator/promoter arrangement, and regulatory interconnection, of an antibiotic export/repressor gene pair in Streptomyces strikingly resemble those for tetracycline resistance in bacteria of clinical importance supports the hypothesis of an evolutionary origin of such genes in an ancestral actinomycete.

Anti-Bacterial Agents↗
PubMed1991

Organisation and functions of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor.

Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and -ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.

Amino Acid Sequence↗
PubMed1990

Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin.

A fragment of DNA carrying the hitherto unisolated members of the cluster of genes (red) for biosynthesis of the red-pigmented antibiotic undecylprodigiosin of Streptomyces coelicolor A3(2) was isolated. This was done by cloning random fragments of S. coelicolor DNA into the closely related Streptomyces lividans 66 and recovering a clone that caused overproduction of undecylprodigiosin. The effect was probably due to the presence of the cloned redD gene, which functions as a positive regulator of the expression of the red cluster, activating the normally poorly expressed red genes of S. lividans. Two fragments from either end of the red cluster were cloned adjacent to each other on a low-copy-number Streptomyces vector. Double crossing-over occurring between these plasmid-borne sequences and the chromosomal copy of the same DNA in S. coelicolor led to isolation of the entire red cluster as a single cloned fragment. Isolation of antibiotic biosynthetic genes by the effects of an activator in a self-cloning experiment, and in vivo reconstitution of a large cluster of genes by homologous recombination, may turn out to be usefully generalizable procedures.

Cloning, Molecular↗
PubMed1989

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1-4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1-4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin-producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.

Amino Acid Sequence↗
PubMed1989

afsB stimulates transcription of the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2) and Streptomyces lividans.

The pleiotropic regulatory gene, afsB, from Streptomyces coelicolor A3(2), possibly encoding a DNA-binding protein, is required for actinorhodin production in this organism. Northern blot hybridization using a DNA fragment covering part of the set of cloned actinorhodin biosynthetic gene cluster (act) as the probe showed lack of the act transcripts in an afsB-negative mutant of S. coelicolor A3(2); the transcripts were restored on introduction of a cloned afsB gene. Introduction of the cloned afsB gene into Streptomyces lividans stimulated transcription of the act genes under conditions in which they are normally silent in this strain, leading to production of actinorhodin in large quantity. These data show that afsB exerts its positive regulatory effect by means of transcriptional stimulation of its target genes.

Anthraquinones↗
PubMed1988

Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor.

The BamHI fragment containing the actIII gene, from the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2), was sequenced. The derived amino acid sequence for the actIII gene shows homology to known oxidoreductases and the actIII product is believed to be responsible for catalysing a beta-keto reductive step during assembly of the Act polyketide chain. High resolution transcript mapping identified the transcription start point at 33 nucleotides upstream of the putative translation start codon. The transcript ends in a large invertedly repeated sequence. In vivo promoter-probe studies suggest that efficient transcription of the actIII gene requires the product of the actII gene.

Alcohol Oxidoreductases↗
PubMed1988

Effect of age on long-term prognosis of patients with myocardial infarction.

We studied 181 patients aged under 65 years and 129 patients over 65 with acute myocardial infarction. There were no major differences in the prevalence of coronary risk factors, angina or previous myocardial infarction. A larger percentage of elderly patients had congestive heart failure (51.4% vs 32.6%, P less than 0.001) and complete heart block (17.1% vs 7.2%, P less than 0.01) during the acute phase. In-hospital mortality was significantly higher in the elderly patients (34% vs 16%, P less than 0.01). Late mortality rates correlated in both groups with the Killip class at the time of infarction and with the occurrence of reinfarction. In the elderly group, it was also associated with complete heart block during the acute phase. Five-year survival was 80% in the older and 72% in the younger patients (P = 0.1). Age did not affect survival of Killip class I patients (85% vs 86%, P = 0.83), but life expectancy was significantly reduced in elderly patients in Killip class greater than II (39% vs 60%, P less than 0.05). In conclusion, elderly patients cannot be considered a homogeneous group of high-risk patients. Clinical variables at the time of infarction can identify low- and high-risk subsets among them. Age constitutes an independent prognostic factor for late mortality when any degree of heart failure is present.

Adult↗
PubMed1987

Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

The Streptomyces hygroscopicus hyg gene encoding a hygromycin B phosphotransferase has been introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli-Saccharomyces cerevisiae shuttle vector YRp7. When this gene was inserted into the BamHI site of pBR322 and then cloned in E. coli phosphorylating activity was not detected, indicating that the hyg gene promoter was not functional in this bacterium. However, when the hyg gene was inserted into either the unique PstI site of pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity was observed. Analysis of the translation products from these constructions by coupled in vitro transcription-translation systems suggested that in all cases transcrition was regulated by a promoter not provided by the inserted hyg gene and that the synthesized polypeptide was identical to that present in S. hygroscopicus.

Base Sequence↗
PubMed1986

Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-terminal amino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3'-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes.

Amino Acid Sequence↗