A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230.

A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.     

“Pseudo” γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal γ-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated “pseudo” GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.     

Regulation of jadomycin B production in Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2.

The nucleotide sequence of a region upstream of the type II polyketide synthase genes in the cluster for biosynthesis of the polyketide antibiotic jadomycin B in Streptomyces venezuelae contained an open reading frame encoding a sequence of 196 amino acids that resembeled sequences deduced for a group of repressor proteins. The strongest similarity was to EnvR of Escherichia coli, but the sequence also resembled MtrR, AcrR, TetC, and TcmR, all of which are involved in regulating resistance to antibiotics or toxic hydrophobic substances in the environment. Disruption of the nucleotide sequence of this putative S. venezuelae repressor gene (jadR2), by insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene generated mutants that produced jadomycin B without the stress treatments (exposure to heat shock or to toxic concentrations of ethanol) required for jadomycin B production by the wild type. When cultures of the disruption mutants were ethanol stressed, they overproduced the antibiotic. From these results it was concluded that expression of the jadomycin B biosynthesis genes are negatively regulated by jadR2.     

Engineering a regulatory region of jadomycin gene cluster to improve jadomycin B production in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

Streptomyces venezuelae ISP5230 produces a group of jadomycin congeners with cytotoxic activities. To improve jadomycin fermentation process, a genetic engineering strategy was designed to replace a 3.4-kb regulatory region of jad gene cluster that contains four regulatory genes (3′ end 272 bp of jadW2, jadW3, jadR2, and jadR1) and the native promoter upstream of jadJ (P_J) with the ermEp* promoter sequence so that ermEp* drives the expression of the jadomycin biosynthetic genes from jadJ in the engineered strain. As expected, the mutant strain produced jadomycin B without ethanol treatment, and the yield increased to about twofold that of the stressed wild-type. These results indicated that manipulation of the regulation of a biosynthetic gene cluster is an effective strategy to increase product yield.     

非典型应答调控蛋白活性调控机制

双组分信号转导系统是生物中广泛存在的调控系统,通常由组氨酸激酶和应答调控蛋白(Responseregulator,RR)两个组分构成。典型的RR通过一个磷酸化机制调控活性。非典型应答调控蛋白在细菌中广泛存在,并调控细菌的生长发育、抗生素合成、Fe的转运等多种生理功能。以下主要综述目前研究比较清楚的非典型应答调控蛋白的结构和功能方面的进展,并以链霉菌中杰多霉素生物合成途径中的非典型应答调控蛋白JadR1为例,阐明调控蛋白活性调控的新机制。     

Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

The Mithramycin Gene Cluster of Streptomyces argillaceus Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.     

Identification and characterization of the biosynthetic gene cluster of divergolides from Streptomyces sp. W112

Divergolides are a group of structurally unprecedented ansamacrolactam antibiotics with antibacterial and antitumor activities. A biosynthetic gene cluster predicted to encode the biosynthesis of divergolides was cloned and sequenced from endophytic Streptomyces sp. W112. The gene cluster of divergolides (div) spans a DNA region of 61-kb and consists of 20 open reading frames (ORFs) that encode polyketide synthases (PKSs), enzymes for the synthesis of AHBA and PKS extender units, and post-PKS modifications, proposed regulators, and putative transporters. Disruption of the AHBA synthase gene (divK) completely abolished the production of divergolides proved its involvement in the biosynthesis of divergolides. Bioinformatics analysis suggested that the regulatory gene div8 in div gene cluster might encode a positive regulator for the biosynthesis of divergolides. Constitutive overexpression of div8 improved the production of divergolides E, implying that div gene cluster maybe responsible for the biosynthesis of divergolides. These findings set the stage for fully investigating the biosynthesis of divergolides and rational engineering of new divergolide analogs by genetic modifications, and pave the way to further improve the production of divergolides.     

Gene–gene interactions of drug metabolizing enzymes and transporter protein in the risk of upper aerodigestive tract cancers among Indians

Background: The combined genetic effects of single nucleotide polymorphisms may additively or synergistically contribute to the increased cancer risk. The interactions associated with xenobiotic metabolizing enzymes and transporter protein involved in the biotransformation and transport of xenobiotics could determine the functional outcomes over the independent effects of a single susceptibility gene in the risk of upper aerodigestive tract cancers. Methods: The hospital-based case–control study evaluated CYP1A1 (*2A and *2C), CYP2E1 (*1B, *5B, and *6), GST (M1, T1, and P1) and ABCB1 3435C>T polymorphisms among 408 histopathologically confirmed cases and 220 controls using polymerase chain reaction based methods in an Indian population. Results: The multivariate logistic regression analyses demonstrated potentially high risk gene–gene interactions with the concurrent deletions of the GSTT1 and GSTM1 genes and GSTP1 variant genotypes (OR 5.81; 95% CI 1.01–40.28), the deletions of GSTT1 and GSTM1 genotypes with variant genotypes of CYP1A1*2A (OR 8.21; 95% CI 1.91–49.48), GSTT1 and GSTM1 deficient genotypes along with CYP2E1*1B variant genotypes (OR 6.73; 95% CI 1.32–22.81), the polymorphic genotypes of ABCB1 and deficient GSTT1 (OR 6.08; 95% CI 2.21–16.76) and an enhanced risk with the combined variant genotypes of CYP1A1*2A, GSTT1 and ABCB1 (OR 11.14; 95% CI 2.70–46.02). Conclusion: The findings indicate that the interactions associated with various drug metabolizing enzymes and transporter protein exhibit high risk for UADT cancers than that ascribed to a single susceptible gene. This was particularly established among the polymorphic carriers of CYP1A1*2A, GSTT1 and ABCB1 genes in the population investigated.     

Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome

The tylLM region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains four open reading frames (orfs1^*–4^*). The function of the orf1^* product is not known. The product of orf2^* (tylM2) is the glycosyltransferase that adds mycaminose to the 5-hydroxyl group of tylactone, the polyketide aglycone of tylosin (Ty). A methyltransferase, responsible for 3-N-methylation during mycaminose production, is encoded by orf3^* (tylM1). The product of orf4^* (ccr) is crotonyl-CoA reductase, which converts acetoacetyl-CoA to butyryl-CoA for use as a 4C extender unit during tylactone production.     

Genetic manipulation of pathway regulation for overproduction of angucycline-like antibiotic auricin in <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> CCM 3239

The polyketide gene cluster aur1 is responsible for the production of the antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is low and strictly regulated by two regulators, Aur1P and Aur1R. To improve auricin yield, we genetically manipulated S. aureofaciens CCM 3239 strain to overcome this strict regulation. A regulatory region including aur1R, aur1P, aur1O and the target biosynthetic aur1Ap promoter were replaced by the strong constitutive ermEp* promoter. However, auricin production was decreased in such a genetically manipulated strain. In the second strategy we placed the aur1P gene for auricin pathway-specific activator under the control of the ermEp* promoter. The resulting strain has been shown to produce 2.8-fold higher amount of auricin compared with the WT strain.     

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.