Chromosomal tRNA Research Articles

Bacteria can maintain rRNA operons solely on plasmids for hundreds of millions of years

It is generally assumed that all bacteria must have at least one rRNA operon (rrn operon) on the chromosome, but some strains of the genera Aureimonas and Oecophyllibacter carry their sole rrn operon on a plasmid. However, other related strains and species have chromosomal rrn loci, suggesting that the exclusive presence of rrn operons on a plasmid is rare and unlikely to be stably maintained over long evolutionary periods. Here, we report the results of a systematic search for additional bacteria without chromosomal rrn operons. We find that at least four bacterial clades in the phyla Bacteroidota, Spirochaetota, and Pseudomonadota (Proteobacteria) lost chromosomal rrn operons independently. Remarkably, Persicobacteraceae have apparently maintained this peculiar genome organization for hundreds of millions of years. In our study, all the rrn-carrying plasmids in bacteria lacking chromosomal rrn loci possess replication initiator genes of the Rep_3 family. Furthermore, the lack of chromosomal rrn operons is associated with differences in copy numbers of rrn operons, plasmids, and chromosomal tRNA genes. Thus, our findings indicate that the absence of rrn loci in bacterial chromosomes can be stably maintained over long evolutionary periods.

A mouse model for intellectual disability caused by mutations in the X-linked 2′‑O‑methyltransferase Ftsj1 gene

Mutations in the X chromosomal tRNA 2′‑O‑methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.

Analysis and comparative genomics of ICEMh1, a novel integrative and conjugative element (ICE) of Mannheimia haemolytica.

The aim of this study was to identify and analyse the first integrative and conjugative element (ICE) from Mannheimia haemolytica, the major bacterial component of the bovine respiratory disease (BRD) complex. The novel ICEMh1 was discovered in the whole-genome sequence of M. haemolytica 42548 by sequence analysis and comparative genomics. Transfer of ICEMh1 was confirmed by conjugation into Pasteurella multocida recipient cells. ICEMh1 has a size of 92,345 bp and harbours 107 genes. It integrates into a chromosomal tRNA(Leu) copy. Within two resistance gene regions of ∼ 7.4 and 3.3 kb, ICEMh1 harbours five genes, which confer resistance to streptomycin (strA and strB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)] and sulphonamides (sul2). ICEMh1 is related to the recently described ICEPmu1 and both ICEs seem to have evolved from a common ancestor. A region of ICEMh1 that is absent in ICEPmu1 was found in putative ICE regions of other M. haemolytica genomes, suggesting a recombination event between two ICEs. ICEMh1 transfers to P. multocida by conjugation, in which it also uses a tRNA(Leu) as the integration site. PCR assays and susceptibility testing confirmed the presence and activity of the ICEMh1-associated resistance genes in the P. multocida recipient. These findings showed that ICEs, with structurally variable resistance gene regions, are present in BRD-associated Pasteurellaceae, can easily spread across genus borders and enable the acquisition of multidrug resistance via a single horizontal gene transfer event. This poses a threat to efficient antimicrobial chemotherapy of BRD-associated bacterial pathogens.

ICE Slu van, a 94-Kilobase Mosaic Integrative Conjugative Element Conferring Interspecies Transfer of VanB-Type Glycopeptide Resistance, a Novel Bacitracin Resistance Locus, and a Toxin-Antitoxin Stabilization System

A 94-kb integrative conjugative element (ICESluvan) transferable to Enterococcus faecium and Enterococcus faecalis from an animal isolate of Streptococcus lutetiensis consists of a mosaic of genetic fragments from different Gram-positive bacteria. A variant of ICESluvan was confirmed in S. lutetiensis from a patient. A complete Tn5382/Tn1549 with a vanB2 operon is integrated into a streptococcal ICESde3396-like region harboring a putative bacteriophage exclusion system, a putative agglutinin receptor precursor, and key components of a type IV secretion system. Moreover, ICESluvan encodes a putative MobC family mobilization protein and a relaxase and, thus, in total has all genetic components essential for conjugative transfer. A 9-kb element within Tn5382/Tn1549 encodes, among others, putative proteins similar to the TnpX site-specific recombinase in Faecalibacterium and VanZ in Paenibacillus, which may contribute to the detected low-level teicoplanin resistance. Furthermore, ICESluvan encodes a novel bacitracin resistance locus that is associated with reduced susceptibility to bacitracin when transferred to E. faecium. The expression of a streptococcal pezAT toxin-antitoxin-encoding operon of ICESluvan in S. lutetiensis, E. faecium, and E. faecalis was confirmed by reverse transcription (RT)-PCR, indicating an active toxin-antitoxin system which may contribute to stabilizing ICESluvan within new hosts. Junction PCR and DNA sequencing confirmed that ICESluvan excised to form a circular intermediate in S. lutetiensis, E. faecalis, and E. faecium. Transfer between E. faecalis cells was observed in the presence of helper plasmid pIP964. Sequence analysis of the original S. lutetiensis donor and enterococcal transconjugants showed that ICESluvan integrates in a site-specific manner into the C-terminal end of the chromosomal tRNA methyltransferase gene rumA.

In silicocomparison of pKLC102-like genomic islands ofPseudomonas aeruginosa

The genomic island pKLC102 first detected in Pseudomonas aeruginosa clone C strains can cross species barriers and exhibits the highest mobilization rate of a genomic island known to date. Homologous genomic islands of 81-108 kb in size were identified in the completely sequenced P. aeruginosa strains PA7, PA14, 2192, C3719 and PACS2, but not in strains PAO1 and LES. All pKLC102-like genomic islands are integrated in chromosomal tRNA(Lys) genes and share a syntenic set of more than 70 homologous ORFs, part of which are related to DNA replication or mobility genes. The conserved backbone has predilection sites for the uptake of island-specific gene cassettes. A major difference between the islands is the organization of the origin of replication oriV.

Site-specific recombination with the chromosomal tRNA(Leu) gene by the large conjugative Haemophilus resistance plasmid.

Characterization of the sequences involved in recombination of the Haemophilus plasmid p1056 with the Haemophilus influenzae chromosome produced evidence indicating site-specific recombination with chromosomal tRNA(Leu). attP sequences identical to those of p1056 were found in six plasmids of diverse origin, suggesting that a family of Haemophilus plasmids recombines with chromosomal tRNA(Leu).

Functional Evidence for Indirect Recognition of G·U in tRNA Ala by Alanyl-tRNA Synthetase

The structural features of the G.U wobble pair in Escherichia coli alanine transfer RNA (tRNA(Ala)) that are associated with aminoacylation by alanyl-tRNA synthetase (AlaRS) were investigated in vivo for wild-type tRNA(Ala) and mutant tRNAs with G.U substitutions. tRNA(Ala) with G.U, C.A, or G.A gave similar amounts of charged tRNA(Ala) and supported viability of E. coli lacking chromosomal tRNA(Ala) genes. tRNA(Ala) with G.C was inactive. Recognition of G.U by AlaRS thus requires more than the functional groups on G.U in a regular helix and may involve detection of a helical distortion.

In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs.

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.

Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.

The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C. Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.

Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.

The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C. Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.

Isolation and properties of a plasmid which expresses the E. coli Su+7 amber suppressor tRNA gene

The gene of the amber suppressor tRNA derived from tRNATry, Su+7, has been inserted into a col E1-derived vehicle by selecting for its expression. Despite selection for a suppressor phenotype, and the plasmid's stable presence at ca. 180 copies cell during balanced growth, the level mature tRNA maintained by the gene is less than that of the normal haploid tRNATry locus in the bacterial chromosome. Transfer RNA genes, both the plasmid Su+7 gene and chromosomal tRNA's are expressed during inhibition of protein synthesis. During, e.g. chloramphenicol inhibition, Su-7 and Su+7 tRNA can be elevated similarly in the plasmid-containing cell; Su+7 reaches levels of molecules/cell which ordinarily characterize a major tRNA. The recombinant plasmid, but not the cloning vehicle alone, has a more general effect on tRNA levels; accumulation of tRNA from three chromosomal tRNA loci including tRNATry, continues during extensive isoleucine limitation. The plasmid therefore contains a locus which probably alters the relaxed-stringent circuit, whose effect is disseminated to at least 3 widely separated loci.