RESUMO
The oppA2 gene encodes an oligopeptide-binding protein similar to the periplasmic substrate-binding proteins of the ABC transport systems. However, oppA2 is an orphan gene, not included in an ABC operon. This gene is located in the clavulanic acid (CA) gene cluster of Streptomyces clavuligerus and is essential for CA production. A transcriptomic study of the oppA2-null mutant S. clavuligerus ΔoppA2::aac showed changes in the expression levels of 233 genes from those in the parental strain. These include genes for ABC transport systems, secreted proteins, peptidases, and proteases. Expression of the clavulanic acid, clavam, and cephamycin C biosynthesis gene clusters was not significantly affected in the oppA2 deletion mutant. The genes for holomycin biosynthesis were upregulated 2-fold on average, and the level of upregulation increased to 43-fold in a double mutant lacking oppA2 and the pSCL4 plasmid. Strains in which oppA2 was mutated secreted into the culture the compound N-acetylglycyl-clavaminic acid (AGCA), a putative intermediate of CA biosynthesis. A culture broth containing AGCA, or AGCA purified by liquid chromatography-mass spectrometry (LC-MS), was added to the cultures of various non-CA-producing mutants. Mutants blocked in the early steps of the pathway restored CA production, whereas mutants altered in late steps did not, establishing that AGCA is a late intermediate of the biosynthetic pathway, which is released from the cells when the oligopeptide-binding protein OppA2 is not available.IMPORTANCE The oppa2 gene encodes an oligopeptide permease essential for the production of clavulanic acid. A transcriptomic analysis of S. clavuligerus ΔoppA2::aac in comparison to the parental strain S. clavuligerus ATCC 27064 is reported. The lack of OppA2 results in different expression of 233 genes, including genes for proteases and genes for transport systems. The expression of the clavulanic acid genes in the oppA2 mutant is not significantly affected, but the genes for holomycin biosynthesis are strongly upregulated, in agreement with the higher holomycin production by this strain. The oppA2-mutant is known to release N-acetylglycyl-clavaminic acid to the broth. Cosynthesis assays using non-clavulanic acid-producing mutants showed that the addition of pure N-acetylglycyl-clavaminic acid to mutants in which clavulanic acid formation was blocked resulted in the recovery of clavulanic acid production, but only in mutants blocked in the early steps of the pathway. This suggests that N-acetylglycyl-clavaminic acid is a previously unknown late intermediate of the clavulanic acid pathway.
Assuntos
Proteínas de Bactérias/genética , Ácido Clavulânico/biossíntese , Proteínas de Membrana Transportadoras/genética , Deleção de Sequência , Streptomyces/enzimologia , Streptomyces/metabolismo , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Ácido Clavulânico/química , Ácidos Clavulânicos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica , Óperon , Streptomyces/genéticaRESUMO
The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.
Assuntos
Vias Biossintéticas/genética , Indóis/metabolismo , Ovomucina/biossíntese , Penicillium/genética , Penicillium/metabolismo , Queijo/microbiologia , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , Evolução Molecular , França , Deleção de Genes , Ordem dos Genes , Genes Fúngicos , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Dados de Sequência Molecular , Família Multigênica , Penicillium/isolamento & purificação , Filogenia , Piperazinas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência , Sintenia , Reino Unido , Estados UnidosRESUMO
A large part (21%) of the wild-type Streptomyces clavuligerus genome is located in a 1.8-Mb megaplasmid that greatly influences secondary metabolites biosynthesis even if the secondary metabolites are chromosomally encoded. The megaplasmid copy number may change depending on the nutritional and environmental conditions. The S. clavuligerus oppA2::aph mutant described by Lorenzana et al. (2004) does not form aerial mycelium, spores, and clavulanic acid, but overproduces holomycin. Transcriptomic studies, polymerase chain reactions (PCR), qPCR, and RT-qPCR analysis showed that S. clavuligerus oppA2::aph has a drastically reduced number of copies (about 25,000-fold lower than the parental strain) of plasmids pSCL1 (10.5 kb), pSCL2 (149.4 kb), and the megaplasmid pSCL4 (1.8 Mb). To clarify the role of the linear plasmids and the function of OppA2 in S. clavuligerus oppA2::aph we constructed oppA2 mutants which contained: (1) a normal copy number of the linear plasmids, (2) completely lack of the linear plasmids, and (3) a parA-parB pSCL4 mutant that resulted in lack of pSCL4. In addition, a strain with a functional oppA2 gene was constructed lacking the megaplasmid pSCL4. The results confirmed that the oppA2 gene is essential for clavulanic acid production, independently of the presence or absence of linear plasmids, but oppA2 has little relevance on differentiation. We demonstrated that the lack of sporulation of S. clavuligerus oppA2::aph is due to the absence of linear plasmids (particularly pSCL4) and the holomycin overproduction is largely due to the lack of pSCL4 and is stimulated by the oppA2 mutation.
Assuntos
Genoma Bacteriano , Plasmídeos , Metabolismo Secundário , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Ácido Clavulânico/metabolismo , Mutação , Deleção de Sequência , Esporos Bacterianos/genética , Streptomyces/genéticaRESUMO
Clusters for clavulanic acid (CA) biosynthesis are present in the actinomycetes Streptomyces flavogriseus ATCC 33331 and Saccharomonospora viridis DSM 43017. These clusters, which are silent, contain blocks of conserved genes in the same order as those of the Streptomyces clavuligerus CA cluster but assembled in a different organization. S. flavogriseus was grown in nine different media, but clavulanic acid production was undetectable using bioassays or by high-performance liquid chromatography analyses. Reverse-transcriptase polymerase chain reaction (RT-PCR) of S. flavogriseus CA biosynthesis genes showed that the regulatory genes ccaR and claR and some biosynthetic genes were expressed whereas expression of cyp, orf12, orf13, and oppA2 was undetectable. The ccaR gene of S. clavuligerus was unable to switch on CA production in S. flavogriseus::[Pfur-ccaR C], but insertion of a cosmid carrying the S. clavuligerus CA cluster (not including the ccaR gene) conferred clavulanic acid production on S. flavogriseus::[SCos-CA] particularly in TBO and YEME media; these results suggests that some of the S. flavogriseus CA genes are inactive. The known heptameric sequences recognized by CcaR in S. clavuligerus are poorly or not conserved in S. flavogriseus. Quantitative RT-PCR analysis of the CA gene clusters of S. clavuligerus and S. flavogriseus showed that the average expression value of the expressed genes in the former strain was in the order of 1.68-fold higher than in the later. The absence of CA production by S. flavogriseus can be traced to the lack of expression of the essential genes cyp, orf12, orf13, orf14, and oppA2. Heterologous expression of S. clavuligerus CA gene cluster in S. flavogriseus::[SCos-CA] was 11- to 14-fold lower than in the parental strain, suggesting that the genetic background of the host strain is important for optimal production of CA in Streptomyces.
Assuntos
Vias Biossintéticas/genética , Ácido Clavulânico/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces/genética , Streptomyces/metabolismo , Perfilação da Expressão Gênica , Família Multigênica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.
Assuntos
DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Transativadores/metabolismo , Sítios de Ligação , Vias Biossintéticas/genética , Cefamicinas/biossíntese , Ácido Clavulânico/biossíntese , Pegada de DNA , Eletroforese em Gel Bidimensional , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Mutagênese Insercional , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Filamentous fungi respond to hundreds of nutritional, chemical and environmental signals that affect expression of primary metabolism and biosynthesis of secondary metabolites. These signals are sensed at the membrane level by G protein coupled receptors (GPCRs). GPCRs contain usually seven transmembrane domains, an external amino terminal fragment that interacts with the ligand, and an internal carboxy terminal end interacting with the intracellular G protein. There is a great variety of GPCRs in filamentous fungi involved in sensing of sugars, amino acids, cellulose, cell-wall components, sex pheromones, oxylipins, calcium ions and other ligands. Mechanisms of signal transduction at the membrane level by GPCRs are discussed, including the internalization and compartmentalisation of these sensor proteins. We have identified and analysed the GPCRs in the genome of Penicillium chrysogenum and compared them with GPCRs of several other filamentous fungi. We have found 66 GPCRs classified into 14 classes, depending on the ligand recognized by these proteins, including most previously proposed classes of GPCRs. We have found 66 putative GPCRs, representatives of twelve of the fourteen previously proposed classes of GPCRs, depending on the ligand recognized by these proteins. A staggering fortytwo putative members of the new GPCR class XIV, the so-called Pth11 sensors of cellulosic material as reported for Neurospora crassa and some other fungi, were identified. Several GPCRs sensing sex pheromones, known in yeast and in several fungi, were also identified in P.â¯chrysogenum, confirming the recent unravelling of the hidden sexual capacity of this species. Other sensing mechanisms do not involve GPCRs, including the two-component systems (HKRR), the HOG signalling system and the PalH mediated pH transduction sensor. GPCR sensor proteins transmit their signals by interacting with intracellular heterotrimeric G proteins, that are well known in several fungi, including P.â¯chrysogenum. These G proteins are inactive in the GDP containing heterotrimeric state, and become active by nucleotide exchange, allowing the separation of the heterotrimeric protein in active Gα and Gßγ dimer subunits. The conversion of GTP in GDP is mediated by the endogenous GTPase activity of the G proteins. Downstream of the ligand interaction, the activated Gα protein and also the Gß/Gγ dimer, transduce the signals through at least three different cascades: adenylate cyclase/cAMP, MAPK kinase, and phospholipase C mediated pathways.
Assuntos
Metabolismo Secundário , Fungos , Receptores Acoplados a Proteínas G , Transdução de SinaisRESUMO
Diploid strains of the yeast Saccharomyces cerevisiae homozygous for a recessive chromosome loss mutation (chl) exhibit a high degree of mitotic instability. Cells become monosomic for chromosome III at a frequency of approximately one percent of all cell divisions. Chromosome loss at this high frequency is also found for chromosome I, and at lesser frequencies for chromosomes VIII and XVI. In contrast, little or no chromosome loss is found for six other linkage groups tested (II, V, VI, VII, XI and XVII). The chl mutation also induces a ten-fold increase in both intergenic and intragenic mitotic recombination on all ten linkage groups tested. The chl mutation does not cause an increase in spontaneous mutations, nor are mutant strains sensitive to UV or irradiation. The effects of chl during meiosis are observed primarily in reduced spore viability. A decrease in chromosome III linkage relationships is also found.
Assuntos
Deleção Cromossômica , Cromossomos Fúngicos , Mitose , Mutação , Saccharomyces cerevisiae/genética , Instabilidade Cromossômica , Mapeamento Cromossômico , Diploide , Genes Fúngicos Tipo Acasalamento , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos da radiação , Esporos FúngicosRESUMO
The expression of genes encoding enzymes involved in antibiotic and other secondary metabolite biosynthesis is down-regulated by easily assimilable phosphate, carbon and nitrogen sources. Phosphate control of antibiotic production appears to act at the transcriptional level by a mechanism similar to that involved in control of phosphatases and other phosphate-regulated enzymes. A phosphate control (PC) sequence, strikingly similar to the phosphate control (pho) boxes of many bacterial genes, has been isolated from the phosphate regulated promoter that controls biosynthesis of the antibiotic candicidin, and characterized. From computer analysis of sequence data, PC sequences appear to be associated with promoter regions of several phosphate-controlled antibiotic biosynthetic genes.
Assuntos
Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/genética , Genes Bacterianos/genética , Fosfatos/fisiologia , Transcrição Gênica , Sequência de Bases , Candicidina/biossíntese , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
The pabS gene of Streptomyces griseus IMRU3570 encodes the enzyme p-aminobenzoic acid synthase, which synthesizes p-aminobenzoic acid (PABA), a precursor of the antibiotic candicidin (Cd). The pabS transcript reached a peak at 12 h of incubation in batch cultures, preceding the formation of PABA synthase and the antibiotic itself. A decay of the pabS transcript was observed with an apparent half-life of 35 min. Inorganic phosphate (Pi; 7.5 mM) reduced the synthesis of the pabS transcript by 90-95%, and consequently the formation of PABA synthase and Cd. Thirty min after addition of 7.5 mM Pi, the cells synthesized only about 15% as much pabS transcript compared to control cultures. However, Pi stimulated two- to threefold total RNA synthesis. The 1.7-kb pabS transcript shown by Northern hybridization was greatly reduced in amount in cells grown in 7.5 mM phosphate. Pi-deregulated mutants, described previously, were impaired in the transcriptional control exerted by Pi. It is concluded that Pi control of PABA synthase and Cd biosynthesis is exerted by repression of formation of the pabS mRNA.
Assuntos
Candicidina/biossíntese , Regulação Bacteriana da Expressão Gênica , Streptomyces griseus/genética , Transaminases/genética , Northern Blotting , Peso Molecular , Fosfatos/fisiologia , RNA Bacteriano/genética , RNA Mensageiro/genética , Fatores de Tempo , Transcrição GênicaRESUMO
The argG gene of Streptomyces clavuligerus (Scl) has been cloned by complementation of argG mutants of Escherichia coli and S. lividans (Sl). The argG nucleotide (nt) sequence showed that it corresponds to a new type of argG different from the corresponding genes of S. coelicolor (Sco) and Sl. It encodes a 43,250-Da protein that showed higher similarity to argininosuccinate synthetases (ASS) from Methanococcus vannielii and Methanosarcina barkeri than to ASS deduced from other Streptomyces argG. No hybridization of the Scl argG was found with the homologous genes of Sl or Sco. The argH gene was located downstream from argG in Scl. The genomic region around argG and argH in Scl was different from the homologous regions in other Streptomyces and is not genetically unstable, unlike in Sco and Sl. Amplification of argG in transformant Scl[pULAR113] results in a 2.3-fold increase in the production of clavulanic acid (CA) in relation to the control strain Scl[pIJ699].
Assuntos
Ácidos Clavulânicos/biossíntese , Streptomyces/genética , Sequência de Aminoácidos , Argininossuccinato Sintase/genética , Sequência de Bases , Ácido Clavulânico , Clonagem Molecular , DNA Bacteriano/metabolismo , Amplificação de Genes , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Streptomyces/enzimologiaRESUMO
Two genes, claR and car, encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb BglII-EcoRI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the cephamycin and clavulanic acid biosynthesis gene cluster. claR encoded a protein of 431 amino acids (deduced Mr 47080), that showed a significant degree of homology with several transcriptional activators of the LysR family. The ClaR protein contained two helix-turn-helix (HTH) motifs in the amino and carboxyl terminal regions. The second gene, car, encoded a protein of 247 amino acids (Mr 26629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus. Amplification of the claR gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas cephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or cephamycin production. Both claR and car are expressed as monocistronic transcripts; the level of transcript declined rapidly after 48h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96h. claR and car were not significantly expressed in mutants disrupted in the ccaR gene, a regulatory gene that controls positively clavulanic acid and cephamycin biosynthesis. These results indicate that clavulanic acid and cephamycin biosynthesis in S. clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.
Assuntos
Oxirredutases do Álcool/genética , Aldeído Redutase/genética , Proteínas de Bactérias/genética , Ácido Clavulânico/biossíntese , Genes Bacterianos/genética , Streptomyces/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência de Bases , Ácido Clavulânico/química , Clonagem Molecular , Códon de Iniciação/genética , Amplificação de Genes/genética , Amplificação de Genes/fisiologia , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Ligação Genética , Dados de Sequência Molecular , Família Multigênica/genética , Mutação/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Reguladoras de Ácido Nucleico/fisiologia , Homologia de Sequência de Aminoácidos , Streptomyces/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
Antifúngicos/biossíntese , Candicidina/biossíntese , Clonagem Molecular , Fosfatos/fisiologia , Regiões Promotoras Genéticas , Sequência de Bases , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Genes , Canamicina Quinase , Dados de Sequência Molecular , Fosfotransferases/genética , Fosfotransferases/metabolismo , Plasmídeos , Mapeamento por Restrição , Streptomyces griseus/genética , Transaminases/genética , Transcrição Gênica , Transformação BacterianaRESUMO
Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC.
Assuntos
Carboxil e Carbamoil Transferases , Cefamicinas/biossíntese , Genes Bacterianos/genética , Nocardia/genética , Streptomyces/genética , Transferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Dados de Sequência Molecular , Nocardia/enzimologia , Fases de Leitura Aberta/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologiaRESUMO
The early steps of cephamycin synthesis by S. clavuligerus are catalyzed sequentially by lysine epsilon-aminotransferase (LAT), delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (cyclase, IPNS). The genes (lat, pcbAB, and pcbC, respectively) are closely linked in the same order as the enzymes act in the biosynthetic pathway and are transcribed in the same direction. Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity. All four mutants are complemented in cephamycin formation by transformation with pNBR1, a plasmid containing a 7.2-kb genomic region of S. clavuligerus in vector pIJ702. The cloned DNA was found to possess no part of the cyclase gene, but instead it contained lat and the 5' upstream part of pcbAB. Doran et al. reported that the 31-bp region between pcbAB and pcbC contains no recognizable promoter or transcription termination sequences. We found that there are 153 bp between the lat ORF and the pcbAB start codon. A potential transcriptional terminator begins 4 to 6 bp downstream of the lat ORF. In the 111-bp segment between the end of the "terminator" and the pcbAB start codon, there are no Streptomyces-like or Escherichia coli-like promoter consensus sequences. However, upstream of the "terminator," that is, in the downstream portion of the lat ORF, are two regions resembling a Streptomyces consensus promoter. Promoter activity in gene fusion constructions was demonstrated in this region. A third potential promoter is upstream of the lat ORF, but only the--10 part is on the cloned DNA. The mechanism by which the cloned DNA (containing lat, the 5' part of pcbAB, and the intervening sequence) influences the expression of the downstream genes encoding ACVS and IPNS, even in strains that possess LAT activity, is an intriguing target of future investigation.
Assuntos
Cefalosporinas/biossíntese , Genes Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Clonagem Molecular , Expressão Gênica , Engenharia Genética , L-Lisina 6-Transaminase , Mutação , Oxirredutases/genética , Peptídeo Sintases/genética , Regiões Promotoras Genéticas , Transaminases/genéticaRESUMO
A bifunctional Streptomyces-E. coli promoter probe vector, pULJA30, has been developed to isolate and characterize nucleotide sequences involved in transcription initiation and regulation. The vector is derived from plasmid pIJ486, carries the pIJ101 replicon and utilizes the promoterless aminoglycoside phosphotransferase (neo) as indicator gene. Important features of the new vector include: wide Streptomyces host range and as high a plasmid copy number as the parental pIJ486, an upstream transcriptional terminator (toop) and a polylinker sequence with unique sites for BamHI and BglII for flexible cloning, fragment re-isolation and direct sequencing of promoter-active inserts. pULJA30 also has an E. coli replicon (from pBR322) and the possibility of selection in Streptomyces and E. coli by using the tsr, neo and bla genes, which makes it very convenient to test the comparative functionality of Streptomyces promoters in E. coli.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Regiões Promotoras Genéticas/genética , Streptomyces/genética , Transcrição Gênica , Sequência de Bases , Clonagem Molecular , Genes Bacterianos , Dados de Sequência Molecular , Plasmídeos , Mapeamento por RestriçãoRESUMO
An expression and purification cassette containing the aminoglycoside phosphotransferase gene (aph) as selective marker has been constructed in the Escherichia coli vector pULHis2. DNA fragments inserted in the cassette can be easily subcloned in pIJ699 to give vectors for overexpression of genes in Streptomyces and purification of proteins by a one-step procedure. The expression system uses the thiostrepton-inducible promoter tipA for expression and a six histidine coding nucleotide sequence that is fused in frame to the foreign gene inserted in the polylinker. The pULHis2-derived expression vector has been used satisfactorily to express and to purify the P7 and P8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7-methoxycephalosporin C.
Assuntos
Proteínas de Bactérias/biossíntese , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Cromatografia de Afinidade , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Histidina/metabolismo , Canamicina Quinase , Metiltransferases/biossíntese , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Dados de Sequência Molecular , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Nocardia/genética , Oligodesoxirribonucleotídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Tioestreptona/farmacologia , Transformação GenéticaRESUMO
Labeled acetate, propionate and p-aminobenzoic acid were efficiently incorporated into candicidin by phosphate-limited resting cells of Streptomyces griseus. The efficiency of incorporation in short-term experiments using phosphate-limited resting cells was similar to that achieved previously in long-term experiments using growing cells. (2-14C)Propionate was more efficiently incorpoated than (1-14C)propionate, (U-14C)propionate, or (U-14C)-acetate. p-Aminobenzoic acid incorporation is linear over a 10-hour period while those of acetate or propionate reach a constant level after approximately 4 hours of incubation. Double-labeled candicidin of high specific activity was prepared by supplementing the resting cell system with (3H)acetate and (14C)p-aminobenzoic acid.
Assuntos
Antifúngicos/biossíntese , Candicidina/biossíntese , Streptomyces griseus/metabolismo , Ácido 4-Aminobenzoico/metabolismo , Acetatos/metabolismo , Cinética , Fosfatos/farmacologia , Propionatos/metabolismo , Fatores de TempoRESUMO
Mutants have been isolated in which phosphate does not inhibit the biosynthesis of candicidin. At high phosphate concentrations, candicidin production by phosphate-deregulated mutants is still inhibited, but to a lesser extent than in the wild type. Some of these mutants are higher candicidin producers than the wild type, not only in phosphate-supplemented medium but also in non-supplemented production medium. The high candicidin production by these mutants is due to (1) a high specific rate of candicidin biosynthesis and (2) an extended production phase. None of the phosphate-deregulated mutants in which uptake of [32P]phosphate was measured was a phosphate-permeability mutant.
Assuntos
Antifúngicos/biossíntese , Candicidina/biossíntese , Fosfatos/farmacologia , Streptomyces griseus/genética , Cinética , Mutação , Fosfatos/metabolismo , Streptomyces griseus/efeitos dos fármacos , Streptomyces griseus/metabolismoRESUMO
The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC-MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains.
Assuntos
Antibacterianos/metabolismo , Cefamicinas/metabolismo , Família Multigênica/genética , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Cefamicinas/farmacologia , Clonagem Molecular , DNA Recombinante , Técnicas de Transferência de Genes , Testes de Sensibilidade MicrobianaRESUMO
Streptomyces clavuligerus ATCC 27064 and S. clavuligerus ΔccaR::tsr cultures were grown in asparagine-starch medium, and samples were taken in the exponential and stationary growth phases. Transcriptomic analysis showed that the expression of 186 genes was altered in the ccaR-deleted mutant. These genes belong to the cephamycin C gene cluster, clavulanic acid gene cluster, clavams, holomycin, differentiation, carbon, nitrogen, amino acids or phosphate metabolism and energy production. All the clavulanic acid biosynthesis genes showed Mc values in the order of -4.23. The blip gene-encoding a ß-lactamase inhibitory protein was also controlled by the cephamycin C-clavulanic acid cluster regulator (Mc -2.54). The expression of the cephamycin C biosynthesis genes was greatly reduced in the mutant (Mc values up to -7.1), while the genes involved in putative ß-lactam resistance were less affected (Mc average -0.88). Genes for holomycin biosynthesis were upregulated. In addition, the lack of clavulanic acid and cephamycin production negatively affected the expression of genes for the clavulanic acid precursor arginine and of miscellaneous genes involved in nitrogen metabolism (amtB, glnB, glnA3, glnA2, glnA1). The transcriptomic results were validated by quantative reverse transcription polymerase chain reaction and luciferase assay of luxAB-coupled promoters. Transcriptomic analysis of the homologous genes of S. coelicolor validated the results obtained for S. clavuligerus primary metabolism genes.