A putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance.

Mutations in rrn encoding ribosomal RNA (rRNA) and rRNA modification often confer resistance to ribosome-targeting antibiotics by altering the site of their interaction with the small (30S) and large (50S) subunits of the bacterial ribosome. The highly conserved central loop of domain V of 23S rRNA (nucleotides 2042-2628 in Escherichia coli; the exact position varies by species) of the 50S subunit, which is implicated in peptidyl transferase activity, is known to be important in macrolide interactions and resistance. In this study, we identified an A2302T mutation in the rrnA-23S rRNA gene and an A2281G mutation in the rrnC-23S rRNA gene that were responsible for resistance to erythromycin in the model actinomycete Streptomyces coelicolor A3(2) and its close relative Streptomyces lividans 66, respectively. Interestingly, genetic and phenotypic characterization of the erythromycin-resistant mutants indicated a possibility that under coexistence of the 23S rRNA mutation and mutations in other genes, S. coelicolor A3(2) and S. lividans 66 can produce abundant amounts of the pigmented antibiotics actinorhodin and undecylprodigiosin depending on the combinations of mutations. Herein, we report the unique phenomenon occurring by unexpected characteristics of the 23S rRNA mutations that can affect the emergence of additional mutations probably with an upswing in spontaneous mutations and enrichment in their variations in Streptomyces strains. Further, we discuss a putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance.

Three putative DNA replication/repair elements encoding genes confer self-resistance to distamycin in Streptomyces netropsis.

Distamycin (DST) is a well-characterized DNA minor groove binder with antivirus activity and antitumor potency. Two separate gene clusters (a 28-kb cluster and a 7-kb cluster) have recently been identified to coordinately encode the biosynthetic machinery of DST in Streptomyces netropsis. Here we report a gene cassette, which is linked to the aforementioned smaller dst gene cluster and plays an important role in the self-resistance to DST in S. netropsis. This cassette consists of three uncharacterized genes that might be implicated in DNA replication/repair. Knockout of the cassette led to the decrease in the production of DST, while heterologous expression of part of the cassette in S. lividans made it become resistant to both DST and mitomycin C, another DNA-binding agent. More interestingly, homologs of these three genes were found in genomes of other actinomyces that produce DNA-binding antibiotics, suggesting that a novel common mechanism in addition to pumping may enable these strains to resist the cytotoxic metabolites they produced.

The metabolic switch can be activated in a recombinant strain of Streptomyces lividans by a low oxygen transfer rate in shake flasks.

BACKGROUND: In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures. RESULTS: Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF. CONCLUSION: Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.

Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans.

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.

Regioselective acylation of congeners of 3-amino-1H-pyrazolo[3,4-b]quinolines, their activity on bacterial serine/threonine protein kinases and in vitro antibacterial (including antimycobacterial) activity.

It was found by virtual screening that 3-amino-1H-pyrazolo[3,4-b]quinolines could have wide protein kinase inhibitory activity. Amides of titled amines and thioureas were synthesized regioselectively. 3-Amino-7-methoxy-1H-pyrazolo[3,4-b]quinoline demonstrated in vitro significant inhibitory activity on bacterial serine/threonine protein kinases (inhibition of resistance to kanamycin in Streptomyces lividans regulated by protein kinases). The studies of Structure Activity Relationship (SAR) showed that the substitution of the NH2 group and 1-NH of pyrazole ring or aromatic ring at the position 6 decreased or removed inhibitory activity.

Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans.

Thiazolyl peptides are bacterial secondary metabolites that potently inhibit protein synthesis in Gram-positive bacteria and malarial parasites. Recently, our laboratory and others reported that this class of trithiazolyl pyridine-containing natural products is derived from ribosomally synthesized preproteins that undergo a cascade of posttranslational modifications to produce architecturally complex macrocyclic scaffolds. Here, we report the gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host Streptomyces lividans. GE37468 harbors an unusual ß-methyl-δ-hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding. Isotope feeding and gene knockout were employed in the engineered S. lividans strain to identify the P450 monooxygenase GetJ as the enzyme involved in posttranslational transformation of isoleucine 8 to ß-methyl-δ-hydroxy-proline through a predicted tandem double hydroxylation/cyclization mechanism. Loss of Ile8 oxygenative cyclization or mutation of Ile8 to alanine via preprotein gene replacement resulted in a 4-fold and 2-fold drop in antibiotic activity, respectively. This report of genetic manipulation of a 29-member thiazolyl peptide sets the stage for further genetic examination of structure activity relationships in the EF-Tu targeting class of thiazolyl peptides.

Acyl depsipeptide (ADEP) resistance in Streptomyces.

ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959-SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.

Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression.

Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of Escherichia coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein over-production (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from Streptomyces lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria.

Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12.

Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5alpha the strain retained the plasmid even after 100 generations of unselected growth but produced a low level of the anti-tumour antibiotic violacein. Markedly higher levels of violacein synthesis were obtained from E. coli K12 DH5alpha pUC18-vioABCDE and Sphingomonas sp. JMP4092 pPSX-vioABCDE. Unfortunately, both strains were extremely unstable regardless of presence or absence of antibiotic selection to retain the plasmid. The current study was undertaken to determine if strains of E. coli K12 could be isolated which stably over produce violacein. When a range of E. coli K12 strains were transformed with pPSX-vioABCDE, most produced small amounts of violacein. However, a small number of related strains of E. coli K12 JM101, JM105 and JM109 not only over-produced violacein, but also maintained the high stability. In addition, E. coli K12 JM109 strongly expressed an alpha amylase gene (amyA) from Streptomyces lividans indicating that the S. lividans amyA promoter is highly active in E. coli K12 JM109. In another set of experiments, a violacein overproduction mutation (opv-1) of the plasmid pPSX-vioABCDE was isolated which enabled E. coli K12 DH5alpha to overproduce violacein while retaining high stability. The plasmid pPSX-vioABCDEopv-1 possesses a single base pair deletion in the promoter region of the violacein operon. By combining the over producing strain E. coli K12 JM109 and the over producing plasmid pPSX-vioABCDEopv-1, a stable hyper producing strain (E. coli K12 JM109 pPSX-vioABCDEopv-1) was constructed. Finally, two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis studies.

[Ca2+ -dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)].

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca2+ ions. The inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca2+ -dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent STPK reduced the Ca2+ -induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.

Characterization and large-scale production of recombinant Streptoverticillium platensis transglutaminase.

Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was approximately 33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.

Molecular mechanism of the sea anemone toxin ShK recognizing the Kv1.3 channel explored by docking and molecular dynamic simulations.

Computational methods are employed to simulate the interaction of the sea anemone toxin ShK in complex with the voltage-gated potassium channel Kv1.3 from mice. All of the available 20 structures of ShK in the Protein Data Bank were considered for improving the performance of the rigid protein docking of ZDOCK. The traditional and novel binding modes were obtained among a large number of predicted complexes by using clustering analysis, screening with expert knowledge, energy minimization, and molecular dynamic simulations. The quality and validity of the resulting complexes were further evaluated to identify a favorable complex structure by 500 ps molecular dynamic simulations and the change of binding free energies with a computational alanine scanning technique. The novel and reasonable ShK-Kv1.3 complex structure was found to be different from the traditional model by using the Lys22 residue to block the channel pore. From the resulting structure of the ShK-Kv1.3 complex, ShK mainly associates the channel outer vestibule with its second helical segment. Structural analysis first revealed that the Lys22 residue side chain of the ShK peptide just hangs between C and D chains of the Kv1.3 channel instead of physically blocking the channel pore. The obvious loss of the ShK Ser20Ala and Tyr23Ala mutant binding ability to the Kv1.3 channel is caused by the conformational change. The five hydrogen bonds between Arg24 in ShK and H404(A) and D402(D) in Kv1.3 make Arg24 the most crucial for its binding to the Kv1.3 channel. Besides the detailed interaction between ShK and Kv1.3 at the atom level, the significant conformational change induced by the interaction between the ShK peptide and the Kv1.3 channel, accompanied by the gradual decrease of binding free energies, strongly implies that the binding of the ShK peptide toward the Kv1.3 channel is a dynamic process of conformational rearrangement and energy stabilization. All of these can accelerate the development of ShK structure-based immunosuppressants.

A multidrug efflux system is involved in colony growth in Streptomyces lividans.

Multidrug resistance (MDR) genes are abundant in Streptomyces genomes, and yet these bacteria are generally drug sensitive under routine laboratory conditions, indicating low or no expression of these genes. Drug-resistant mutations have been isolated that lie in regulatory genes adjacent to the MDR genes, suggesting that resistance arises by derepression. This study identified a divergently oriented pair consisting of a TetR-family regulator (ebrS) and a major facilitator-family MDR pump (ebrC) gene in Streptomyces lividans, which is widely conserved in Streptomyces species. EbrS represses transcription of ebrC as well as its own transcription. Deletion of ebrS causes overexpression of ebrC, resulting in elevated resistance to many drugs. The ebrS and ebrC promoters were used in a reporter system to test inducibility by various chemicals. Among the 15 compounds (including five EbrC target drugs) tested, none induced ebrC transcription. On the other hand, the ebrS promoter was induced by rifampicin and high concentrations of calcium and magnesium. Deletion of ebrS-ebrC did not change rifampicin sensitivity, indicating that the EbrC pump is not involved in rifampicin efflux. Moreover, deletion of ebrC caused retardation of colony growth on selected media, and the defect could be suppressed by supplementation with high concentrations of Ca(2+), Mg(2+), Na(+) or K(+). Based on these results, it is proposed that the primary biological role of most MDR systems in Streptomyces species is not removal of extrinsic drugs, but rather export of specific toxic compounds endogenously synthesized during growth.

Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans.

The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Deltappk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.