Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

The border sequence of the balhimycin biosynthesis gene cluster from Amycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator

Balhimycin, produced by the actinomycete Amycolatopsis balhimycina DSM5908, is a glycopeptide antibiotic highly similar to vancomycin, the antibiotic of 'last resort' used for the treatment of resistant Gram-positive pathogenic bacteria. Partial sequence of the balhimycin biosynthesis gene cluster was previously reported. In this work, cosmids which overlap the region of the characterized gene cluster were isolated and sequenced. At the 'left' end of the cluster, genes were identified which are involved in balhimycin biosynthesis, transport, resistance and regulation. The 'right' end border is defined by a putative 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (dahp) gene. The proximate gene is similar to a type I polyketide synthase gene of the rifamycin producer Amycolatopsis mediterranei indicating that another biosynthesis gene cluster might be located directly next to the balhimycin gene cluster. The newly identified StrR-like pathway-specific regulator, Bbr, was characterized to be a DNA-binding protein and may have a role in balhimycin biosynthesis. Purified N-terminally His-tagged Bbr shows specific DNA-binding to five promoter regions within the gene cluster. By in silico analysis and by comparison of the DNA sequences binding Bbr, conserved inverted repeat sequences for the Bbr-binding site are proposed. The putative Bbr consensus sequence differs from that published for StrR.     

A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.     

Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene.

The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.     

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.     

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.     

The two-component phoR-phoP system of Streptomyces natalensis: Inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis

The biosynthesis of the antifungal pimaricin in Streptomyces natalensis is very sensitive to phosphate regulation. Concentrations of inorganic phosphate above 1mM drastically reduced pimaricin production. At 10mM phosphate, expression of all the pimaricin biosynthesis (pim) genes including the pathway-specific positive regulator pimR is fully repressed. The phoU-phoR-phoP cluster of S. natalensis encoding two-component Pho system was cloned and sequenced. Binding of the response regulator PhoP to the consensus PHO boxes in the phoU-phoRP intergenic promoter region was observed. A phoP-disrupted mutant and a phoR-phoP deletion mutant were obtained. Production of pimaricin in these two mutants increased up to 80% in complex yeast extract-malt extract (YEME) or NBG media and showed reduced sensitivity to phosphate control. Four of the pim genes, pimS1, pimS4, pimC and pimG showed increased expression in the phoP-disrupted mutant. However, no consensus PHO boxes were found in the promoter regions of any of the pim genes, suggesting that phosphate control of these genes is mediated indirectly by PhoR-PhoP involving modification of pathway-specific regulators.     

Cloning, analysis, and heterologous expression of a polyketide synthase gene cluster of Streptomyces curacoi.

Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed.     

Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30–84

Abstract The DNA sequence of five contiguous open reading frames encoding enzymes for phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30–84 was determined. These open reading frames were named phzF, phzA, phzB, phzC and phzD . Protein PhzF is similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases of solanaceous plants. PhzA is similar to 2,3-dihydro-2,3-dihydroxybenzoate synthase (EntB) of Escherichia coli . PhzB shares similarity with both subunits of anthranilate synthase and the phzB open reading frame complemented an E. coli trpE mutant deficient in anthranilate synthase activity. Although phzC shares little similarity to known genes, its product is responsible for the conversion of phenazine-1-carboxylic acid to 2-hydroxy-phenazine-1-carboxylic acid. PhzD is similar to pyridoxamine phosphate oxidases. These results indicate that phenazine biosynthesis in P. aureofaciens shares similarities with the shikimic acid, enterochelin, and tryptophan biosynthetic pathways.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

Regulation of tyrosine biosynthesis in Escherichia coli K-12: isolation and characterization of operator mutants

Mutant strains of Escherichia coli have been isolated in which the synthesis of two of the enzymes involved in tyrosine biosynthesis, 3-deoxy-d-arabinoheptulosonic acid-7 phosphate synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, is partially constitutive. The mutations involved are closely linked to aroF and tyrA, the structural genes of these enzymes. The gene in which the mutations occur has been designated aroK, and the gene sequence is aroK, aroF, tyrA. In aroK(+)/aroK diploids, the aroK allele only affects the structural genes in the cis position. The mutant allele aroK is not recessive to aroK(+) and aroK/aroK(+) strains exhibit the aroK phenotype of resistance to 4-aminophenylalanine. It is proposed that aroK is an operator locus for an aroF tyrA operon.     

Evolution of the clusters of genes for beta-lactam antibiotics: a model for evolutive combinatorial assembly of new beta-lactams.

beta-Lactam antibiotics are produced by prokaryotic and eukaryotic organisms. The genes for beta-lactam biosynthesis are organized in clusters but the location of the different genes is not identical. Biosynthesis genes are clustered with genes for resistance (bla, pbp) and for the efflux of the antibiotic (cmcT) in prokaryotes. Comparison of proteins reveals much larger differences for primary metabolism enzymes than for beta-lactam biosynthesis enzymes in producing organisms. This suggests a horizontal transfer of the beta-lactam antibiotic biosynthesis genes.