Gene clusters for β-lactam antibiotics and control of their expression: why have clusters evolved, and from where did they originate?

While β-lactam compounds were discovered in filamentous fungi, actinomycetes and gram-negative bacteria are also known to produce different types of β-lactams. All β-lactam compounds contain a four-membered β-lactam ring. The structure of their second ring allows these compounds to be classified into penicillins, cephalosporins, clavams, carbapenens or monobactams. Most β-lactams inhibits bacterial cell wall biosynthesis but others behave as β-lactamase inhibitors (e.g., clavulanic acid) and even as antifungal agents (e.g., some clavams). Due to the nature of the second ring in β-lactam molecules, the precursors and biosynthetic pathways of clavams, carbapenems and monobactams differ from those of penicillins and cephalosporins. These last two groups, including cephamycins and cephabacins, are formed from three precursor amino acids that are linked into the α-aminoadipyl-L-cysteinyl-D-valine tripeptide. The first two steps of their biosynthetic pathways are common. The intermediates of these pathways, the characteristics of the enzymes involved, the lack of introns in the genes and bioinformatic analysis suggest that all of them should have evolved from an ancestral gene cluster of bacterial origin, which was surely transferred horizontally in the soil from producer to non-producer microorganisms. The receptor strains acquired fragments of the original bacterial cluster and occasionally inserted new genes into the clusters, which once modified, acquired new functions and gave rise to the final compounds that we know. When the order of genes in the Streptomyces genome is analyzed, the antibiotic gene clusters are highlighted as gene islands in the genome. Nonetheless, the assemblage of the ancestral β-lactam gene cluster remains a matter of speculation. [Int Microbiol 2006; 9(1):9-19

Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine

[EN] The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrationsSIThis work was supported by grant BIO2000-272 and a fellowship (to A. de la Fuente) from the Spanish Ministry of Science and Technology (Madrid, Spain). We thank Rosario Pérez-Redondo for her help with RNA experiments

Characterization of a two-gene operon epeRA involved in multidrug resistance in Streptomyces clavuligerus

[EN] Two genes, epeR and epeA, are located downstream of argH in the Streptomyces clavuligerus genome. EpeR belongs to the TetR family of transcriptional regulators. It is homologous to PqrA of Streptomyces coelicolor (74.3% identity) and to NfxB of Pseudomonas aeruginosa (30.9% identity). EpeA encodes a protein with 14 transmembrane spanning domains (TMS) of the major facilitator superfamily. It shares 68.9% identity to PqrB of S. coelicolor and 46.5% identity to LfrA, conferring resistance to fluoroquinolones in Mycobacterium smegmatis. Disruption of epeR results in a S. clavuligerus epeR::aph mutant which shows increased resistance to ethidium bromide and proflavine (16- and 32-fold higher than the wild type). Taking into consideration the sensitivity to drugs of different transformants carrying functional copies of either epeR or epeA, it might be concluded that both genes appear to be co-transcribed, with epeR encoding a regulatory protein which controls the expression of epeASIThis work was supported by CICYT grant BIO2003-3274 and GEN2003-20245. We thank Matthew Smith (University of Nottingham) and María Alvarez (University of León, Spain) for critical reading of the manuscript

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus

[EN] The putative regulatory CcaR protein, which is encoded in the β-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter regionSIThis work was supported by the Spanish Ministry of Science and Technology (FD97-1419-CO2-O2). I. Santamarta received a fellowship from the University of León. We are grateful to A. de la Fuente, F. J. Enguita, and C. de Torre for their interest and helpful discussions, to A. Jiménez for revising the manuscript, and to M. Mediavilla for technical assistance

Transcriptional studies on a Streptomyces clavuligerus oppA2 deletion mutant: N-Acetylglycyl-Clavaminic acid is an intermediate of clavulanic acid biosynthesis

[EN] 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 availableSIThis work was supported by grant BIO2013-34723 from the Spanish Ministry of Economy and Competitiveness. R. Álvarez-Álvarez and Y. Martínez-Burgo received a PFU fellowship from the Spanish Ministry of Science and Innovatio

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite produced by Streptomyces clavuligerus

Background: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chal cone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. Results: A compound produced by Streptomyces clavuligerus has been identified by LC–MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, narin genin production increases in cultures supplemented with phenylalanine or tyrosine. Conclusion: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A ten tative pathway for naringenin biosynthesis has been proposed

Characterization and expression of the arginine biosynthesis gene cluster of Streptomyces clavuligerus

Revista continuada por Microbial Physiology[EN] A cluster of genes argCJBDRGH containing most of the arginine biosynthesis genes has been found in Streptomyces clavuligerus after sequencing a 8.3 kb DNA region containing overlapping sequences of two DNA fragments known to contain arginine biosynthesis genes. Subcloning, complementation of E. coli arginine auxotrophic strains and enzymatic assays confirmed the identity of each gene. S1 nuclease mapping studies and Northern hybridization analysis revealed the formation of two large transcripts corresponding to argCJBDR and argGH. The amount of each of these mRNAs is 10 to 44 times higher in a S. clavuligerus argR-disrupted mutant than in the wild type confirming the existence of an ArgR-mediated control of arginine biosynthesis gene expression. A low level constitutive monocistronic transcript of argR was observed in S. clavuligerus cells. Most of the argGH transcript initiating at an adenine 29 nt upstream of the argG initiation codon appears to stop at a termination stem and loop structure present downstream of the argG geneSIThis work was supported by grants from the CICYT (Madrid) Bio96-0827 and by Antibióticos SA, León. We thank H. Kieser (Norwich, U.K.) for providing the S. coelicolor cosmid library. Álvaro de la Fuente and Rosario Pérez-Redondo received fellowships from the PFPI (Madrid) and the University of León, respectively

ArgR of Streptomyces coelicolor is a versatile regulator

[EN] ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSASIThis work was supported by Grants from the Spanish Comisión Interministerial de Ciencia y Tecnología GEN2003-20245, BIO2009-09820, and by the European Project LSHM-CT-2004-005224. AB received a fellowship from the Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Streptomyces tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources

[EN] In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. tsukubaensis and the model species Streptomyces coelicolor. A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus productionSIThis study was funded by the Government of Spain (grant number PIM2010-EEI00677). María Ordóñez-Robles received a FPU grant from the Government of Spain (grant number AP2009-4508)

ArgR of Streptomyces coelicolor is a pleiotropic transcriptional regulator: effect on the transcriptome, antibiotic production, and differentiation in liquid cultures

[EN] ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotypeSIGrant BIO2013-34723 from the Spanish Ministry of Science and Innovation to PL. Work in the AM's laboratory was funded by the European Research Council (ERC Starting Grant; Strp-differentiation 280304) and by the Spanish Ministry of Economy and Competitiveness (Grant BIO2015-65709-R)