Antibiotic Thiostrepton Research Articles

Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates sigma(B) through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of sigma(B) from an anti-sigma(B) factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the sigma(B) stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of sigma(B). B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of sigma(B). Neither the energy-responsive activation of sigma(B) nor stress-dependent chaperone gene induction (a sigma(B)-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of sigma(B) are shown to be active in the RplK(-) strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of sigma(B) and suggest that the ribosomes may themselves be the sensors for stress in this system.

Anion recognition by Thiostrepton

A bicyclic polypeptide antibiotic thiostrepton forms both 1:1 and 1:2 complexes with anions (as tetrabutylammonium salts) in organic solvents with K 2 > K 1 for F − and K 2 ≪ K 1 for all other anions studied. Relative stabilities of 1:1 complexes in DMSO are AcO − ∼ F − ≫ Cl −, Br −, HSO 4 −, H 2PO 4 −, but in CHCl 3 they follow a different order: Cl −∼HSO 4 − >F −∼AcO −>Br −>H 2PO 4 −. The binding mode of anions to thiostrepton is discussed on the basis of solvent effects on the complexation selectivity.

A Corynebacterium glutamicum mutant with a defined deletion within the rplK gene is impaired in (p)ppGpp accumulation upon amino acid starvation.

The rplK gene of Corynebacterium glutamicum ATCC13032 comprises 438 nucleotides and encodes a protein of 145 amino acids with a molecular mass of 15.3 kDa. The amino acid sequence revealed extensive similarities to the large ribosomal subunit protein L11 from several Gram-positive and Gram-negative bacteria. The C. glutamicum rplK gene is located downstream of secE, representing part of the protein export apparatus, and of nusG, encoding a transcription antiterminator protein. The rplK gene is followed by an ORF homologous to rplA encoding the 50S ribosomal protein L1. Northern analysis revealed that transcription of the rplK-rplA cluster resulted in two different transcripts of 1.5 and 0.6 kb. The 1.5 kb transcript corresponds to the entire rplK-rplA cluster and the short transcript originates from the rplK gene. A C. glutamicum rplK mutant strain carrying a 12 bp in-frame deletion within rplK, which resulted in the loss of the tetrapeptide Pro-Ala-Leu-Gly in the L11 protein, was constructed. The mutant failed to accumulate (p)ppGpp in response to amino acid starvation and exhibited an increased tolerance to the antibiotic thiostrepton. Evidently, the C. glutamicum rplK gene is required for (p)ppGpp accumulation upon nutritional starvation.

Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance technique.

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.

Replacement of L7/L12.L10 Protein Complex in Escherichia coli Ribosomes with the Eukaryotic Counterpart Changes the Specificity of Elongation Factor Binding

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.

Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome.

The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the alpha-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA or of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.

Thiostrepton binds to malarial plastid rRNA

Binding of the thiazolyl peptide antibiotic thiostrepton to the GTPase domain of 23S rRNA involves a few crucial nucleotides, notably A1067 ( E. coli). Small RNA transcripts were prepared corresponding to the GTPase domain of the plastid 23S rRNA and the two forms of cytosolic 28S rRNAs found in the human malaria parasite Plasmodium falciparum, as well as the plastid form of rRNA of the AIDS-related pathogen Toxoplasma gondii. Binding affinities of the wild type and mutated RNA sequences were as predicted; the malarial plastid sequence had by far the highest affinity, whereas that from toxoplasma did not bind thiostrepton.

General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid.

Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation procedure is described for both organisms. The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S. acidocaldarius. A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S. acidocaldarius. Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection. Growth inhibition studies indicate that both P. furiosus and S. acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol. Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S. acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P. furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA). These mutated genes are being developed as selective markers. Moreover, two beta-galactosidase genes from P. furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95 degrees C with O-nitrophenyl beta-D-galactopyranoside as substrate.

Cooperative interactions of RNA and thiostrepton antibiotic with two domains of ribosomal protein L11.

Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit ribosomal RNA; both the protein and its RNA target have been highly conserved. The antibiotic thiostrepton recognizes the same RNA domain, and binds to the ribosome cooperatively with L11. Experiments presented here show that RNA recognition and thiostrepton cooperativity can be attributed to C- and N-terminal domains of L11, respectively. Under trypsin digestion conditions that degrade Bacillus stearothermophilus L11 to small fragments, the target RNA protects the C-terminal 77 residues from digestion, and thiostrepton and RNA in combination protect the entire protein. A 76-residue C-terminal fragment of L11 was overexpressed and shown to fold into a stable structure binding ribosomal RNA with essentially the same properties as full-length L11. An L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements with the C-terminal fragment detected no cooperativity with thiostrepton. L11 function is thus more complex than simple interaction with ribosomal RNA; we suggest that thiostrepton mimics some ribosomal component or factor that normally interacts with the L11 N-terminal domain.

Recognition determinants for proteins and antibiotics within 23S rRNA.

Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecular genetic and biochemical probing techniques, we have concentrated on regions of the rRNA that are connected with specific functions. These are located in different domains within the 23S rRNA and include the ribosomal GTPase-associated center in domain II, which contains the binding sites for r-proteins L10.(L12)4 and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations here and by modification of A2058 by ErmE methyltransferase. ErmE recognizes a conserved motif displayed in the primary and secondary structure of the peptidyl transferase loop. Within domain VI of rRNA, the alpha-sarcin stem-loop is associated with elongation factor binding and is the target site for ribotoxins including the N-glycosidase ribosome-inactivating proteins ricin and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains are constrained by tetiary interactions, including a pseudoknot in domain II and long-range base pairings in the center of the molecule that bring domains II and V closer together. The phenotypic effects of mutations in these regions have been investigated by expressing 23S rRNA from plasmids. Allele-specific priming sites have been introduced close to these structures in the rRNA to enable us to study the molecular events there.

Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA.

To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3' domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within the 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UGA, through suppression of UAG and UGA, but not UAA, to suppression of all three termination codons. The two helices in 16S rRNA have previously been associated both physically and functionally with the decoding center of the ribosome. The 23S region is part of the binding site for the large subunit protein L11 and the antibiotic thiostrepton, both of which have been shown to affect peptide chain termination. Finally, we have demonstrated that the 23S mutant A1093, which suppresses trpA UGA mutations very efficiently, is lethal at temperatures above 36 degrees C (when highly expressed). This lethality is overcome by secondary 23S rRNA mutations in domain V. Our results suggest that specific regions of 16S and 23S rRNA are involved in peptide chain termination, that the lethality of A1093 is caused by high-level UGA suppression, and that intramolecular interaction between domains II and V of 23S rRNA may play a role in peptide chain termination at the UGA stop codon.

A Base Substitution within the GTPase-associated Domain of Mammalian 28 S Ribosomal RNA Causes High Thiostrepton Accessibility

A molecular basis for the insensitivity of eukaryotic ribosomes to the antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long fragments covering the GTPase domain of 23/28 S rRNA. Filter binding assay showed no detectable binding of the rat RNA to thiostrepton, but the binding capacity was markedly increased by base substitution of G1878 to A at the position corresponding to 1067 of Escherichia coli 23 S rRNA. The association constant (K alpha) for the rat A 1878 mutant was 0.60 x 10(6) M-1, which was comparable with that of the E. coli RNA (K alpha = 1.1 x 10(6) M-1). This suggests that the eukaryotic G 1878 participates in the resistance for thiostrepton. On the other hand, the RNA fragments of the two species had a similar binding capacity for E. coli ribosomal protein L11 and its mammalian homologue L12. Gel electrophoresis under a high ionic condition, however, revealed a difference between the two proteins. E. coli L11 formed stable complexes with both the E. coli RNA and the rat A 1878 mutant RNA in the presence of thiostrepton, while rat L12 failed to exhibit such complex formation. This suggests that the eukaryotic L12 protein may also be an element giving the resistance for thiostrepton. These results are discussed in terms of preserved three-dimensional conformation of the RNA backbone between prokaryotes and higher eukaryotes.

Thermodynamics of RNA unfolding: Stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion

RNAs with interesting secondary and tertiary structures tend to melt in several broad and overlapping transitions over a wide temperature range, and it has been consequently difficult to resolve the thermodynamics of individual unfolding steps. In the case that a ligand selectively binds a single folded state of the RNA, it is possible to obtain reliable thermodynamic parameters for both RNA unfolding and RNA-ligand binding simply from the hyperchromicity of RNA denaturation. The analysis procedure involves fitting a three-dimensional surface to absorbance data collected as a function of both temperature and ligand concentration. Analysis of the unfolding of a fragment of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1109) is presented; both an antibiotic (thiostrepton) and ammonium ion specifically stabilize a tertiary structure within this RNA. A consistent set of thermodynamic parameters (delta H and tm) for the first two sequentially linked unfolding transitions is obtained from the experiments, and the binding constants obtained for the two ligands are consistent with other independent measurements. The approach is applicable to a variety of RNAs that specifically bind proteins, antibiotics, ions or other ligands.

The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A.

The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escherichia coli 23S rRNAs with single mutations at positions 1067 or 1095 were expressed in vivo. Mutant ribosomes are functional in protein synthesis, although those with transversion mutations function less effectively. Antibiotics were bound under conditions where wild-type and mutant ribosomes compete in the same reaction for drug molecules; binding was analysed by allele-specific footprinting. Transversion mutations at 1067 reduce thiostrepton binding more than 1000-fold. The 1067G substitution gives a more modest decrease in thiostrepton binding. The changes at 1095 slightly, but significantly, lower the affinity of ribosomes for thiostrepton, again with the G mutation having the smallest effect. Micrococcin binding to ribosomes is reduced to a far greater extent than thiostrepton by all the 1067 and 1095 mutations. Extrapolating these results to growing cells, mutation of nucleotide 1067A confers resistance towards micrococcin and thiostrepton, while substitutions at 1095A confer micrococcin resistance, and increase tolerance towards thiostrepton. These data support an rRNA tertiary structure model in which 1067A and 1095A lie in close proximity, and are key components in the drug binding site. None of the mutations alters either the higher order rRNA structure or the binding of r-proteins. We therefore conclude that thiostrepton and micrococcin interact directly with 1067A and 1095A.

Ribosomal Proteins L11 and L10.(L12) 4 and the Antibiotic Thiostrepton Interact with Overlapping Regions of the 23 S rRNA Backbone in the Ribosomal GTPase Centre

The Escherichia coli ribosomal protein (r-protein) L11 and its binding site on 23 S ribosomal RNA (rRNA) are associated with ribosomal hydrolysis of guanosine 5′-triphosphate (GTP). We have used hydroxyl radical footprinting to map the contacts between L11 and the backbone riboses in 23 S rRNA, and to investigate how this interaction is influenced by other ribosomal components. Complexes were characterized in both naked 23 S rRNA and ribosomes from an E. coli L11-minus strain, before and after reconstitution with L11. The protein protects 17 riboses between positions 1058 and 1085 in the naked 23 S rRNA. Within the ribosome, L11 also interacts with this rRNA region, although the protection effects are subtly different and extend to nucleotide 1098. The pentameric r-protein complex L10.(L12) 4 binds to an adjacent site on the rRNA, protecting riboses at positions 1043, 1046 to 1049, 1053 to 1055 and increasing the accessibility of position 1068. The overlap in the positions affected by r-proteins L11 and L10.(L12) 4, and the increase in protection between positions 1078 and 1084 when they are bound at the same time, reflect the mutually cooperative nature of their interaction with the rRNA. The data support a model for the tertiary configuration of the rRNA region, in which two stem-loop structures fold so that the loops lie in close proximity, with the main ribose interactions of L11 within the minor groove of one of the stems. The conformation of the rRNA-L11 interaction is modulated by L10.(L12) 4 and other proteins within the ribosome. The antibiotics thiostrepton and micrococcin inhibit the catalytic functions of this region by slotting in between the accessible loops and interacting with nucleotides there.

Specific ammonium ion requirement for functional ribosomal RNA tertiary structure.

In compactly folded RNAs, coordination or hydrogen bonding of cations in specific sites is a potentially important aspect of the tertiary structure. NH4+ specifically stabilizes the tertiary structure of a conserved, 58-nt fragment of the large subunit ribosomal RNA, as judged in two ways: a melting transition associated with tertiary interactions is sharpened and stabilized more effectively by NH4+ than by any alkali metal cation, and the affinity of the RNA fragment for ribosomal protein L11 or the antibiotic thiostrepton is approximately 10-fold stronger when measured in NH4+ than in Na+. The dependence of the melting temperature on NH4+ concentration shows that a single bound ion is responsible for these effects. The requirement of different ribosome functions for NH4+ suggests that other such sites exist in ribosomal RNAs.

Biosynthesis of the modified peptide antibiotic thiostrepton in Streptomyces azureus and Streptomyces laurentii

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBiosynthesis of the modified peptide antibiotic thiostrepton in Streptomyces azureus and Streptomyces laurentiiUrsula Mocek, Zhaopie Zeng, David O'Hagan, Pei Zhou, Lai Duen G. Fan, John M. Beale, and Heinz G. FlossCite this: J. Am. Chem. Soc. 1993, 115, 18, 7992–8001Publication Date (Print):September 1, 1993Publication History Published online1 May 2002Published inissue 1 September 1993https://doi.org/10.1021/ja00071a009RIGHTS & PERMISSIONSArticle Views551Altmetric-Citations85LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans.

Although the antibiotic thiostrepton is best known as an inhibitor of protein synthesis, it also, at extremely low concentrations (< 10(-9) M), induces the expression of a regulon of unknown function in certain Streptomyces species. Here, we report the purification of a Streptomyces lividans thiostrepton-induced transcriptional activator protein, TipAL, whose N-terminus is similar to a family of eubacterial regulatory proteins represented by MerR. TipAL was first purified from induced cultures of S.lividans as a factor which bound to and activated transcription from its own promoter. The tipAL gene was overexpressed in Escherichia coli and TipAL protein purified in a single step using a thiostrepton affinity column. Thiostrepton enhanced binding of TipAL to the promoter and catalysed specific transcription in vitro. TipAS, a second gene product of the same open reading frame consisting of the C-terminal domain of TipAL, is apparently translated using its own in-frame initiation site. Since it is produced in large molar excess relative to TipAL after induction and also binds thiostrepton, it may competitively modulate transcriptional activation.

Transformation of an insect symbiont and expression of a foreign gene in the Chagas' disease vector Rhodnius prolixus.

A shuttle plasmid was developed that is capable of replicating both in Escherichia coli and in Rhodococcus rhodnii, a bacterial symbiont of the Chagas' disease vector Rhodnius prolixus. We have been able to transform R. rhodnii with this plasmid, infect aposymbiotic R. prolixus with the transformed symbionts, select with the antibiotic thiostrepton, and re-isolate genetically altered symbionts from the insects following successive molts. Symbiotic bacteria are potentially valuable as vehicles for the stable introduction of foreign genes into insects with the goal of eventually altering the ability of the insect to transmit a pathogenic agent.