Omar Orellana

Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates

Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes. They differ in their choice of substrates and use either tRNAGlu or both tRNAGlu and tRNAGln. Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown. A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain. To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. pylori contains one tRNAGlu and one tRNAGln species, whereas A. ferrooxidans possesses two of each. We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities. The H. pylori GluRS1 acylated only tRNAGlu, whereas GluRS2 was specific solely for tRNAGln. The A. ferrooxidans GluRS2 preferentially charged \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{tRNA}}_{{\mathrm{UUG}}}^{{\mathrm{Gln}}}\end{equation*}\end{document}. Conversely, A. ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{tRNA}}_{{\mathrm{CUG}}}^{{\mathrm{Gln}}}\end{equation*}\end{document} species. These three tRNA species have two structural elements in common, the augmented D-helix and a deletion of nucleotide 47. It appears that the discriminating or nondiscriminating natures of different GluRS enzymes have been derived by the coevolution of protein and tRNA structure. The coexistence of the two GluRS enzymes in one organism may lay the groundwork for the acquisition of the canonical glutaminyl-tRNA synthetase by lateral gene transfer from eukaryotes.

Regulation of a glutamyl-tRNA synthetase by the heme status.

Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the C5 pathway. In this route Glu-tRNA is transformed to δ-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase. GluTR is a target of feedback regulation by heme. In Acidithiobacillus ferrooxidans, an acidophilic bacterium that expresses two GluRSs (GluRS1 and GluRS2) with different tRNA specificity, the intracellular heme level varies depending on growth conditions. Under high heme requirement for respiration increased levels of GluRS and GluTR are observed. Strikingly, when intracellular heme is in excess, the cells respond by a dramatic decrease of GluRS activity and the level of GluTR. The recombinant GluRS1 enzyme is inhibited in vitro by hemin, but NADPH restores its activity. These results suggest that GluRS plays a major role in regulating the cellular level of heme.

A dual-specific Glu-tRNAGln and Asp-tRNAAsn amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans

The gatC, gatA and gatB genes encoding the three subunits of glutamyl‐tRNAGln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon‐like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu‐tRNAGln to Gln‐tRNAGln and Asp‐tRNAAsn to Asn‐tRNAAsn. Biochemical analysis revealed that neither glutaminyl‐tRNA synthetase nor asparaginyl‐tRNA synthetase is present in A. ferrooxidans, but that glutamyl‐tRNA synthetase and aspartyl‐tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln‐tRNA and Asn‐tRNA in A. ferrooxidans.

A tRNAGlu that uncouples protein and tetrapyrrole biosynthesis

Glu‐tRNA is either bound to elongation factor Tu to enter protein synthesis or is reduced by glutamyl‐tRNA reductase (GluTR) in the first step of tetrapyrrole biosynthesis in most bacteria, archaea and in chloroplasts. Acidithiobacillus ferrooxidans, a bacterium that synthesizes a vast amount of heme, contains three genes encoding tRNAGlu. All tRNAGlu species are substrates in vitro of GluRS1 from A. ferrooxidans. Glu ‐ tRNA 3 Glu , that fulfills the requirements for protein synthesis, is not substrate of GluTR. Therefore, aminoacylation of tRNA 3 Glu might contribute to ensure protein synthesis upon high heme demand by an uncoupling of protein and heme biosynthesis.

ICEAfe1, an Actively Excising Genetic Element from the Biomining Bacterium Acidithiobacillus ferrooxidans

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation.

Characterization of the two rRNA gene operons present in Thiobacillus ferrooxidans

The organization of rRNA genes from the autotrophic, acidophilic bacterium Thiobacillus ferrooxidans has been examined. Two rRNA operons were found in this microorganism by means of genomic hybridization studies. Recombinant plasmids, pTR‐3 and pTR‐1 that carry a portion of 16/23 S rDNA from one operon and the 5′‐flanking region of the second operon, respectively, were identified and characterized.

A 300 kpb genome segment, including a complete set of tRNA genes, is dispensable for Acidithiobacillus ferrooxidans

The genome sequences from two strains of the acidophilic, autotrophic, chemolithotrophic proteobacterium A. ferrooxidans are available from genome databases. Bioinformatic sequence comparison revealed the existence in one strain of a putative integrative conjugative element (ICE), containing an entire set of clustered tRNA genes. ICE is missing in the other strain, suggesting that this element as well as the tRNA genes cluster is dispensable for the bacterium. Bioinformatic predictions suggest that the tRNA genes cluster might mainly contribute to the translation of ICE encoded genes.

In vivo formation of glutamyl-tRNAGln in Escherichia coli by heterologous glutamyl-tRNA synthetases

Two types of glutamyl‐tRNA synthetase exist: the discriminating enzyme (D‐GluRS) forms only Glu‐tRNAGlu, while the non‐discriminating one (ND‐GluRS) also synthesizes Glu‐tRNAGln, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu‐tRNAGlu, A. ferrooxidans and Helicobacter pylori GluRS2 form Glu‐tRNAGln in E. coli in vivo.

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose‐1,5‐bisphosphate car☐ylase (RuBisCO) were cloned from the obligate autotrophThiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30‐bp intergenic region, the smallest described for the RuBisCO genes. TherbcL andrbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes forrbcL andrbcS found in other species demonstrated that theT. ferrooxidans genes have the closest degree of identity with those ofChromatium vinosum and ofAlvinoconcha hessleri endosymbiont. BothT. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.

Organization of the 16s‐23s intergenic spacer region of the two rRNA operons from thiobacillus ferrooxidans

Abstract The recombinant plasmid pTR‐3 was previously shown to contain part of the 16S and 23S ribosomal RNA genes and the spacer region between the two genes from operon rrnT, from Thiobacillus ferrooxidans. The spacer region was subjected to deletions, using the exonuclease III nested deletions procedure, and the resulting fragments were sequenced. tRNAile‐ and tRNAala‐like sequences were identified near the 3’ end of16S rRNA gene. The spacer DNAs from both rRNA operons were amplified by the polymerase chain reaction (PCR) and the sequences were compared. No differences were observed. A DNA segment identical to putative box A of the antiterminator sequence of Mycoplasma sp. was identified. Comparison between the sequence of the spacer region from strains Torma and A4 showed some minor differences. It implies that only in strain A4 is a recognition site for the Avail restriction enzyme present.