On the evolution of the tRNA-dependent amidotransferases, GatCAB and GatDE

Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNA^Gln and asparaginyl-tRNA^Asn were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNA^Gln and aspartyl-tRNA^Asn, by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNA^Gln that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNA^Asn, suggesting that tRNA^Asn recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.     

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Archaea make glutaminyl-tRNA (Gln-tRNA^Gln) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA^Gln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA^Gln. Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA^Asn) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228–239.]. Here, we show a similar complex for Gln-tRNA^Gln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA^Gln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K_D = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA^Gln formation, with GatDE reducing the affinity of ND-GluRS for tRNA^Glu by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA^Gln) formation, and has no major effect on the kinetics of tRNA^Gln glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.     

Cyclic peptides identified by phage display are competitive inhibitors of the tRNA-dependent amidotransferase of Helicobacter pylori

In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA^Gln and Asp-tRNA^Asn into Gln-tRNA^Gln and Asn-tRNA^Asn, respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA^Gln, we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylori GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA^Gln, with K_i values of 126 and 392 μM, respectively. The docking models revealed that the Trp residues of these peptides form π-π stacking interactions with Tyr81 of the synthetase active site, as does the 3′-terminal A76 of tRNA, supporting their competitive behavior with respect to Glu-tRNA^Gln in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA^Gln and/or Asn-tRNA^Asn formation.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

Site-Specific N-Glycosylation of Caprine Lysostaphin Restricts its Bacteriolytic Activity Toward Staphylococcus Aureus

Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoid Staphylococcus aureus mastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goat β-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn¹²⁵ but not Asn²³² is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn²³² to Gln²³² substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn¹²⁵ to Gln¹²⁵ substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn¹²⁵ of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus.     

The Predatory Bacterium Bdellovibrio bacteriovorus Aspartyl-tRNA Synthetase Recognizes tRNAAsn as a Substrate

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNA^Asn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNA^Asn formation that GatCAB can then amidate to asparaginyl-tRNA^Asn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNA^Asn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNA^Asn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.     

Trans-kingdom rescue of Gln-tRNAGln synthesis in yeast cytoplasm and mitochondria

Aminoacylation of transfer RNA^Gln (tRNA^Gln) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNA^Gln is generated by direct glutaminylation of tRNA^Gln by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNA^Gln is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNA^Gln amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNA^Gln synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNA^Gln isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNA^Gln isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNA^Gln synthesis can be conferred by a single enzyme.     

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

In most prokaryotes Asn-tRNA^Asn and Gln-tRNA^Gln are formed by amidation of aspartate and glutamate mischarged onto tRNA^Asn and tRNA^Gln, respectively. Coexistence in the organism of mischarged Asp-tRNA^Asn and Glu-tRNA^Gln and the homologous Asn-tRNA^Asn and Gln-tRNA^Gln does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA^Asn intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA^Asn from Asn-tRNA^Asn and Asp-tRNA^Asp by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.     

Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition

In many prokaryotes the biosynthesis of the amide aminoacyl-tRNAs, Gln-tRNA^Gln and Asn-tRNA^Asn, proceeds by an indirect route in which mischarged Glu-tRNA^Gln or Asp-tRNA^Asn is amidated to the correct aminoacyl-tRNA catalyzed by a tRNA-dependent amidotransferase (AdT). Two types of AdTs exist: bacteria, archaea and organelles possess heterotrimeric GatCAB, while heterodimeric GatDE occurs exclusively in archaea. Bacterial GatCAB and GatDE recognize the first base pair of the acceptor stem and the D-loop of their tRNA substrates, while archaeal GatCAB recognizes the tertiary core of the tRNA, but not the first base pair. Here, we present the crystal structure of the full-length Staphylococcus aureus GatCAB. Its GatB tail domain possesses a conserved Lys rich motif that is situated close to the variable loop in a GatCAB:tRNA^Gln docking model. This motif is also conserved in the tail domain of archaeal GatCAB, suggesting this basic region may recognize the tRNA variable loop to discriminate Asp-tRNA^Asn from Asp-tRNA^Asp in archaea. Furthermore, we identified a 3₁₀ turn in GatB that permits the bacterial GatCAB to distinguish a U1–A72 base pair from a G1–C72 pair; the absence of this element in archaeal GatCAB enables the latter enzyme to recognize aminoacyl-tRNAs with G1–C72 base pairs.     

Methanothermobacter thermautotrophicus tRNA Gln confines the amidotransferase GatCAB to asparaginyl-tRNA Asn formation

Many prokaryotes form the amide aminoacyl-tRNAs glutaminyl-tRNA and asparaginyl-tRNA by tRNA-dependent amidation of the mischarged tRNA species, glutamyl-tRNA^Gln or aspartyl-tRNA^Asn. Archaea employ two such amidotransferases, GatCAB and GatDE, while bacteria possess only one, GatCAB. The Methanothermobacter thermautotrophicus GatDE is slightly more efficient using Asn as an amide donor than Gln (k_cat/K_M of 5.4 s⁻¹/mM and 1.2 s⁻¹/mM, respectively). Unlike the bacterial GatCAB enzymes studied to date, the M. thermautotrophicus GatCAB uses Asn almost as well as Gln as an amide donor (k_cat/K_M of 5.7 s⁻¹/mM and 16.7 s⁻¹/mM, respectively). In contrast to the initial characterization of the M. thermautotrophicus GatCAB as being able to form Asn-tRNA^Asn and Gln-tRNA^Gln, our data demonstrate that while the enzyme is able to transamidate Asp-tRNA^Asn (k_cat/K_M of 125 s⁻¹/mM) it is unable to transamidate M. thermautotrophicus Glu-tRNA^Gln. However, M. thermautotrophicus GatCAB is capable of transamidating Glu-tRNA^Gln from H. pylori or B. subtilis, and M. thermautotrophicus Glu-tRNA^Asn. Thus, M. thermautotrophicus encodes two amidotransferases, each with its own activity, GatDE for Gln-tRNA and GatCAB for Asn-tRNA synthesis.     

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA^Asp and tRNA^Asn generating a correctly charged Asp-tRNA^Asp and an erroneous Asp-tRNA^Asn. This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNA^Asp over tRNA^Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNA^Asp and was unable to aspartylate tRNA^Asn. The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.     

Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P₋₁. Although RNase A also contains a P₋₁ subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P₋₁ subsite residues Arg³⁶, Asn³⁹, and Gln⁴⁰ of EDN in its catalytic activity. The individual mutation of Arg³⁶, Asn ³⁹, and Gln⁴⁰ resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P₋₁ subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates. Deepa Sikriwal and Divya Seth contributed equally to this work.     

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA^Asn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA^Asn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNA^Asn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.     

Association of tRNA acceptor identity with phosphate-sugar backbone interactions observed in the crystal structure of the Escherichia coli glutaminyl-tRNA synthetase-tRNA complex

We isolated several mutants with nucleotide substitutions in alanine tRNA (tRNA^Ala) that resulted in glutamine tRNA (tRNA^Gli) acceptor identity in Escherichia coli. These substitutions were in three regions of tRNA structure not previously associated with tRNA^Gln acceptor identity. Only the phosphate-sugar backbone moieties of these nucleotides interact with the enzyme in the previously determined X-ray crystal structure of the complex between tRNA^Gln and glutaminyl-tRNA synthetase. We conclude that these sequence-dependent phosphate-sugar backbone interactions contribute to tRNA^Gln identity, and argue that the interactions help communicate enzyme recognition of the anticodon to the acceptor end of the tRNA and the catalytic center of the enzyme.     

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNA^Asn and tRNA^Gln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA^Asn and Gln-tRNA^Gln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNA^Asn and tRNA^Asp variants, that amidation of Asp acylating tRNA^Asn is promoted by the base pair U₁–A₇₂ whereas the G₁–C₇₂ pair and presence of the supernumerary nucleotide U_20A in the D-loop of tRNA^Asp prevent amidation. We predict, based on comparison of tRNA^Gln and tRNA^Glu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA^Gln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G₄₆ and U₄₇ of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

Overexpression of genes encoding tRNA<Superscript>Tyr</Superscript> and tRNA<Superscript>Gln</Superscript> increases the viability of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains with nonsense mutations in the <Emphasis Type="Italic">SUP45</Emphasis> gene

At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNA^Tyr and four genes encoding wild-type tRNA^Gln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNA^Tyr and tRNA^Gln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed.     

Plasmid Profiles of Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Plasmid profiles were studied in five Acidithiobacillus ferrooxidans strains of various origin cultivated on a medium with Fe²⁺, as well as adapted to such oxidation substrates as S⁰, FeS₂, and sulfide concentrate. The method used revealed plasmids in all A. ferrooxidans strains grown on a medium with Fe²⁺. One plasmid was found in strain TFL-2; two plasmids, in strains TFO, TFBk, and TFV-1; and three plasmids were detected in strain TFN-d. The adaptation of strain TFN-d to sulfide concentrate and the adaptation of strain TFV-1 to S⁰, FeS₂, or sulfide concentrate resulted in a change in the number of plasmids occurring in cells. In cells of strain TFN-d adapted to sulfide concentrate, the number of plasmids decreased from three to two. The number of plasmids in cells of strain TFV-1 adapted to different substrates varied from three to six depending on the energy source present in the medium: three plasmids were found after growth on FeS₂, four after growth on S⁰, and six after growth on sulfide concentrate. The possible role of plasmids in the adaptation of A. ferrooxidans to new energy substrates and in the regulation of the intensity of their oxidation is discussed.     

Specificity of receptors to vasoactive intestinal peptide in guinea pig brain.

The specific binding of VIP to guinea pig brain membranes was tested by 1/ the ability of eight VIP and secretin analogs and fragments to inhibit the binding of ¹²⁵I-VIP and 2/ the capacity of the same peptides to influence basal and VIP-stimulated adenylate cyclase activities. Among all peptides tested, only VIP, secretin, [Val⁵] secretin, and [Gln⁹, Asn¹⁵] secretin (5–27) were able to inhibit ¹²⁵I-VIP binding. The adenylate cyclase activity was stimulated by VIP, secretin and [Val⁵] secretin. [Gln⁹, Asn¹⁵] secretin (5–27) although inactive per se was able to inhibit the VIP-stimulated adenylate cyclase activity competitively.