The alanyl-tRNA synthetase AARS1 moonlights as a lactyltransferase to promote YAP signaling in gastric cancer.

Lactylation has been recently identified as a new type of posttranslational modification occurring widely on lysine residues of both histone and nonhistone proteins. The acetyltransferase p300 is thought to mediate protein lactylation, yet the cellular concentration of the proposed lactyl-donor, lactyl-coenzyme A, is about 1,000 times lower than that of acetyl-CoA, raising the question of whether p300 is a genuine lactyltransferase. Here, we report that alanyl-tRNA synthetase 1 (AARS1) moonlights as a bona fide lactyltransferase that directly uses lactate and ATP to catalyze protein lactylation. Among the candidate substrates, we focused on the Hippo pathway, which has a well-established role in tumorigenesis. Specifically, AARS1 was found to sense intracellular lactate and translocate into the nucleus to lactylate and activate the YAP-TEAD complex; and AARS1 itself was identified as a Hippo target gene that forms a positive-feedback loop with YAP-TEAD to promote gastric cancer (GC) cell proliferation. Consistently, the expression of AARS1 was found to be upregulated in GC, and elevated AARS1 expression was found to be associated with poor prognosis for patients with GC. Collectively, this work found AARS1 with lactyltransferase activity in vitro and in vivo and revealed how the metabolite lactate is translated into a signal of cell proliferation.

AARS2 as a novel biomarker for prognosis and its molecular characterization in pan-cancer.

INTRODUCTION: The mitochondrial alanyl-tRNA synthetase 2 (AARS2) as one of aminoacyl-tRNA synthases (ARSs) performs amino acid transportation and involves protein synthesis. However, its role in cancer remains largely unexplored. METHODS: In this study, more than 10,000 samples were enrolled to explore genomic alterations, biological function, prognosis, and clinical treatment based on AARS2 across pan-cancer. The molecular characterization of AARS2 was confirmed in hepatocellular carcinoma (HCC) using proteomics analysis, quantitative real-time PCR, western blotting, immunohistochemical staining, and cell experiments. RESULTS: For genomic landscape, the AARS2 was dramatically upregulated in multiple cancers, which might be mainly caused by copy number alteration rather than mutation and methylation. The abnormal expression of AARS2 was prominently associated with activity of cancer pathways and performed oncogenic roles in most cancers. Systematic experiments in vitro substantiated the elevated expression of AARS2, and the deficiency of it inhibited cell proliferation and cell migration in HCC. Meanwhile, our findings suggested that AARS2 could serve as a novel promising and stable biomarker for assessing prognosis and immunotherapy. Moreover, a variety of therapeutic drugs and targeted pathways were proposed for cancer treatment, which might enhance clinical efficacy. CONCLUSION: The AARS2 could serve as a new oncogenic gene that promotes cell proliferation and migration in HCC. The comprehensive investigations increased the understanding of AARS2 across human cancers and generated beginning insights of AARS2 in genomic landscape, molecular biological function, prognosis, and clinical treatment.

Distal hereditary neuropathy associated with a novel mutation in alanyl-aminoacyl-tRNA synthetase.

To report a new genetic cause of distal hereditary motor neuropathy (dHMN), which is likely associated with worsening during pregnancy. We collected the clinical data of a patient with severe weakness of the lower limbs induced by repeated pregnancy and performed relevant experimental examinations, including neuromuscular electrophysiological examination, neuromuscular biopsy, and genetic testing. The patient reported weakness of the right lower extremity after delivery of the first child. Initially, the right foot was weak during lifting, and symptoms gradually progressed to weakness when landing on the toe during walking. She then developed weakness of the right lower extremity and thinning of the right leg. After an interval of 2.5 years, after delivery of the second child, her left lower extremity developed asthenia, with the same symptoms as previously reported for the right lower extremity. Subsequently, weakness of both lower extremities became progressively worse, and she developed difficulty sitting up, getting out of bed, and walking. Physical examination showed that both upper limb vertebral tracts were damaged and both lower extremity motor nerves were damaged. Electrophysiology suggested motor axonal neurogenic damage. Brain magnetic resonance imaging demonstrated leukodystrophy. Sural nerve biopsy suggested mild axonal damage. Skeletal muscle biopsy suggested neurogenic skeletal muscle damage. Genetic testing suggested that there was a heterozygous mutation at the shear site of the AARS gene. An AARS mutation may cause dHMN associated with pyramidal tract signs.

Anti-alanyl tRNA positive antisynthase syndrome with Kaposi sarcoma.

We report a rare case of antisynthase syndrome (ASS) complicated with Kaposi sarcoma, analyze its clinical characteristics, and review the literature on the topic. An 80-year-old male patient developed fever, cough, and shortness of breath. Lung high-resolution computed tomography showed nonspecific interstitial pneumonia in both lungs, and myositis antibody examination showed strongly positive anti-alanyl tRNA synthase (PL-12) antibodies. Based on these findings, the patient was diagnosed with ASS. After full-dose glucocorticoid treatment, the symptoms of fever and cough were relieved, but skin thickening and pigmentation in both feet were observed. We confirmed Kaposi sarcoma through skin pathology and immunohistochemical examination of the bottom of the patient's feet, and the patient was transferred to a cancer hospital for radiotherapy. ASS presents with some skin changes that might lead to misdiagnosis. ASS complicated with Kaposi sarcoma is rare, and to our knowledge, this is the first case reported in China.

AARS2 Compound Heterozygous Variants in a Case of Adult-Onset Leukoencephalopathy With Axonal Spheroids and Pigmented Glia.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), usually referred to as hereditary diffuse leukoencephalopathy with spheroids or pigmentary orthochromatic leukodystrophy, is genetically caused by CSF1R mutations. AARS2 was recently confirmed to be another causative gene in a series of CSF1R-negative ALSP cases. We report a case of adult-onset leukoencephalopathy with ALSP with AARS2 variants. A 34-year-old woman presented with 2 years of motor and cognitive deterioration with severely impaired cortical functions and rigid spasticity. Brain magnetic resonance imaging showed a confluent, patchy, and predominantly frontoparietal, periventricular pattern of white matter lesions, with relatively preserved subcortical U-fibers. Brain biopsy revealed axonal spheroids, severe demyelination and pigmented macrophages. Genetic analyses revealed compound heterozygous c.1691T>C and c.179C>A variants in the AARS2 gene. CSF1R mutation testing was negative. Our findings proved adult-onset leukoencephalopathy with spheroids and pigmented glia to be a genetically heterogeneous disease entity. The selective brain involvement without ovarian failure might be a new subtype in AARS2 mutations related to ALSP.

Overexpression of human mitochondrial alanyl-tRNA synthetase suppresses biochemical defects of the mt-tRNAAla mutation in cybrids.

Mutations of mitochondrial transfer RNAs (mt-tRNAs) play a major role in a wide range of mitochondrial diseases because of the vital role of these molecules in mitochondrial translation. It has previously been reported that the overexpression of mitochondrial aminoacyl tRNA synthetases is effective at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs in cells. Here we report a detailed analysis of the suppressive activities of mitochondrial alanyl-tRNA synthetase (AARS2) on mt-tRNAAla 5655 A>G mutant. Mitochondrial defects in respiration, activity of oxidative phosphorylation complexes, ATP production, mitochondrial superoxide, and membrane potential were consistently rescued in m.5655A>G cybrids upon AARS2 expression. However, AARS2 overexpression did not result in a detectable increase in mutated mt-tRNAAla but caused an increase incharged mt-tRNAAla in mutant cybrids, leading to enhanced mitochondrial translation. This indicated that AARS2 improved the aminoacylation activity in the case of m.5655A>G, rather than having a stabilizing effect on the tRNA structure. The data presented in this paper deepen our understanding of the pathogenesis of mt-tRNA diseases.

Novel cancer gene variants and gene fusions of triple-negative breast cancers (TNBCs) reveal their molecular diversity conserved in the patient-derived xenograft (PDX) model.

Despite the improved 5-year survival rate of breast cancer, triple-negative breast cancer (TNBC) remains a challenge due to lack of effective targeted therapy and higher recurrence and metastasis than other subtypes. To identify novel druggable targets and to understand its unique biology, we tried to implement 24 patient-derived xenografts (PDXs) of TNBC. The overall success rate of PDX implantation was 45%, much higher than estrogen receptor (ER)-positive cases. Immunohistochemical analysis revealed conserved ER/PR/Her2 negativity (with two exceptions) between the original and PDX tumors. Genomic analysis of 10 primary tumor-PDX pairs with Ion AmpliSeq CCP revealed high degree of variant conservation (85.0%-96.9%) between primary and PDXs. Further analysis showed 44 rare variants with a predicted high impact in 36 genes including Trp53, Pten, Notch1, and Col1a1. Among them, we confirmed frequent Notch1 variant. Furthermore, RNA-seq analysis of 24 PDXs revealed 594 gene fusions, of which 163 were in-frame, including AZGP1-GJC3 and NF1-AARSD1. Finally, western blot analysis of oncogenic signaling proteins supporting molecular diversity of TNBC PDXs. Overall, our report provides a molecular basis for the usefulness of the TNBC PDX model in preclinical study.

Editing activity for eliminating mischarged tRNAs is essential in mammalian mitochondria.

Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.

Novel AARS2 gene mutation producing leukodystrophy: a case report.

AARS2 gene (NM_020745.3) mutations result in two different phenotypic diseases: infantile mitochondrial cardiomyopathy and late-onset leukoencephalopathy. The patient's first symptoms appeared at the age of 18 years with behavioral changes and psychiatric problems. Some years later, extrapyramidal symptoms, cognitive impairment, nystagmus, dysarthria and pyramidal symptoms also developed. The brain magnetic resonance imaging (MRI) indicated extensive white matter abnormalities. The diagnosis of AARS2 gene mutations causing leukodystrophy was confirmed by genetic testing. Segregation analysis confirmed the compound heterozygous state of the patient. Histological examination of the biopsy did not prove specific pathological alterations. The clinical phenotype of our patient was compared with seven previously described patients suffering from leukoencephalopathy caused by AARS2 mutations. We have documented a new, nonsense AARS2 gene mutation (c.578T>G, p.Leu193*) and a known missense mutation (c.595C>T, p.Arg199Cys) associated with leukoencephalopathy in a male patient. Clinical features, imaging characteristics and genetic testing are presented, and histological data from an AARS2-related leukodystrophy patient are described for the first time.

Thymidine kinase 2 and alanyl-tRNA synthetase 2 deficiencies cause lethal mitochondrial cardiomyopathy: case reports and review of the literature.

Cardiomyopathy is a common manifestation in neonates and infants with mitochondrial disorders. In this study, we report two cases manifesting with fatal mitochondrial hypertrophic cardiomyopathy, which include the third known patient with thymidine kinase 2 deficiency and the ninth patient with alanyl-tRNA synthetase 2 deficiency. The girl with thymidine kinase 2 deficiency had hypertrophic cardiomyopathy together with regression of gross motor development at the age of 13 months. Neurological symptoms and cardiac involvement progressed into severe myopathy, psychomotor arrest, and cardiorespiratory failure at the age of 22 months. The imaging methods and autoptic studies proved that she suffered from unique findings of leucoencephalopathy, severe, mainly cerebellar neuronal degeneration, and hepatic steatosis. The girl with alanyl-tRNA synthetase 2 deficiency presented with cardiac failure and underlying hypertrophic cardiomyopathy within 12 hours of life and subsequently died at 9 weeks of age. Muscle biopsy analyses demonstrated respiratory chain complex I and IV deficiencies, and histological evaluation revealed massive mitochondrial accumulation and cytochrome c oxidase-negative fibres in both cases. Exome sequencing in the first case revealed compound heterozygozity for one novel c.209T>C and one previously published c.416C>T mutation in the TK2 gene, whereas in the second case homozygozity for the previously described mutation c.1774C>T in the AARS2 gene was determined. The thymidine kinase 2 mutations resulted in severe mitochondrial DNA depletion (to 12% of controls) in the muscle. We present, for the first time, severe leucoencephalopathy and hepatic steatosis in a patient with thymidine kinase 2 deficiency and the finding of a ragged red fibre-like image in the muscle biopsy in a patient with alanyl-tRNA synthetase 2 deficiency.

Analysis of Mutations in AARS2 in a Series of CSF1R-Negative Patients With Adult-Onset Leukoencephalopathy With Axonal Spheroids and Pigmented Glia.

IMPORTANCE: Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a frequent cause of adult-onset leukodystrophy known to be caused by autosomal dominant mutations in the CSF1R (colony-stimulating factor 1) gene. The discovery that CSF1R mutations cause ALSP led to more accurate prognosis and genetic counseling for these patients in addition to increased interest in microglia as a target in neurodegeneration. However, it has been known since the discovery of the CSF1R gene that there are patients with typical clinical and radiologic evidence of ALSP who do not carry pathogenic CSF1R mutations. These patients include those in whom the pathognomonic features of axonal spheroids and pigmented microglia have been found. Achieving a genetic diagnosis in these patients is important to our understanding of this disorder. OBJECTIVE: To genetically characterize a group of patients with typical features of ALSP who do not carry CSF1R mutations. DESIGN, SETTINGS, AND PARTICIPANTS: In this case series study, 5 patients from 4 families were identified with clinical, radiologic, or pathologic features of ALSP in whom CSF1R mutations had been excluded previously by sequencing. Data were collected between May 2014 and September 2015 and analyzed between September 2015 and February 2016. MAIN OUTCOMES AND MEASURES: Focused exome sequencing was used to identify candidate variants. Family studies, long-range polymerase chain reaction with cloning, and complementary DNA sequencing were used to confirm pathogenicity. RESULTS: Of these 5 patients, 4 were men (80%); mean age at onset of ALSP was 29 years (range, 15-44 years). Biallelic mutations in the alanyl-transfer (t)RNA synthetase 2 (AARS2) gene were found in all 5 patients. Frameshifting and splice site mutations were common, found in 4 of 5 patients, and sequencing of complementary DNA from affected patients confirmed that the variants were loss of function. All patients presented in adulthood with prominent cognitive, neuropsychiatric, and upper motor neuron signs. Magnetic resonance imaging in all patients demonstrated a symmetric leukoencephalopathy with punctate regions of restricted diffusion, typical of ALSP. In 1 patient, brain biopsy demonstrated axonal spheroids and pigmented microglia, which are the pathognomonic signs of ALSP. CONCLUSIONS AND RELEVANCE: This work indicates that mutations in the tRNA synthetase AARS2 gene cause a recessive form of ALSP. The CSF1R and AARS2 proteins have different cellular functions but overlap in a final common pathway of neurodegeneration. This work points to novel targets for research and will lead to improved diagnostic rates in patients with adult-onset leukoencephalopathy.

Revised phylogeny of Bacteroidetes and proposal of sixteen new taxa and two new combinations including Rhodothermaeota phyl. nov.

Members of the phylum Bacteroidetes, which was originally defined as a monophyletic branch encompassing the genera Cytophaga, Flavobacterium and Bacteroides (CFB), are widely studied due to their importance in environmental and gut microbiology. As a consequence, the number of species names with standing in nomenclature has doubled in the past five years. In this study, a revision of an earlier phylogeny of Bacteroidetes has been performed using the 16S rRNA gene as a backbone in combination with the 23S rRNA gene, as well as multilocus sequence analysis (MLSA) of 29 orthologous protein sequences, and indels in the sequences of the beta subunit of the F-type ATPase and the alanyl-tRNA synthetase. In addition, taxonomic data for Bacteroidetes has been updated by considering the orphan species list, signature nucleotides in the 16S rRNA sequence, the list of outlier species, and discrepancies with the current taxonomy at the genus rank level. As a result, seven new taxa are proposed within Bacteroidetes (Chitinophagia classis nov., Chitinophagales ord. nov., Crocinitomicaceae fam. nov., Odoribacteraceae fam. nov., Hymenobacteraceae fam. nov., Thermonemataceae fam. nov. and Persicobacteraceae fam. nov.), as well as one new phylum Rhodothermaeota phyl. nov. that contains two classes, two orders, four families and a new genus with two new combinations.

A novel AARS mutation in a family with dominant myeloneuropathy.

OBJECTIVE: To determine the genetic cause of neurodegeneration in a family with myeloneuropathy. METHODS: We studied 5 siblings in a family with a mild, dominantly inherited neuropathy by clinical examination and electrophysiology. One patient had a sural nerve biopsy. After ruling out common genetic causes of axonal Charcot-Marie-Tooth disease, we sequenced 3 tRNA synthetase genes associated with neuropathy. RESULTS: All affected family members had a mild axonal neuropathy, and 3 of 4 had lower extremity hyperreflexia, evidence of a superimposed myelopathy. A nerve biopsy showed evidence of chronic axonal loss. All affected family members had a heterozygous missense mutation c.304G>C (p.Gly102Arg) in the alanyl-tRNA synthetase (AARS) gene; this allele was not identified in unaffected individuals or control samples. The equivalent change in the yeast ortholog failed to complement a strain of yeast lacking AARS function, suggesting that the mutation is damaging. CONCLUSION: A novel mutation in AARS causes a mild myeloneuropathy, a novel phenotype for patients with mutations in one of the tRNA synthetase genes.

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.

The split genes of Nanoarchaeum equitans are an ancestral character.

The introns early hypothesis predicts that introns were fundamental in assembling the first genes. In Nanoarchaeum equitans some genes are split into two. If these split genes were the ancestral forms, as suggested by the introns early hypothesis, then the end-beginning of the two parts of the split protein in a multiple alignment with the orthologous proteins from the Eukarya and Arachaea domains should make a clear prediction on where the intron in the homologous eukaryotic gene should be positioned. The analysis has shown that the introns are in this position, which is therefore predictable on the basis of the split proteins of N. equitans. This corroborates the hypothesis that the split genes of N. equitans are the plesiomorphic forms of these genes. If true, this would show that the origin of genes was polyphyletic as the monophyletic origin hypothesis would deny the existence, in a real organism, of these ancestral (split) genes, which imply that they were assembled late on and after the domains of life were established. Furthermore, it would seem that hyperthermophily is also an ancestral trait because it is linked to a split gene in N. equitans.

Positional recognition of a tRNA determinant dependent on a peptide insertion.

The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized. Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position. This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala). Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem. Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate. Larger or smaller deletions were ineffective. The crystal structure of the orthologous A. aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition.

Genomic clustering of tRNA-specific adenosine deaminase ADAT1 and two tRNA synthetases.

Human tRNA-specific adenosine deaminase (hADAT1) specifically converts A37 in the anticodon loop of human tRNA(Ala) to inosine via a hydrolytic deamination mechanism. The enzyme is related to a family of RNA editing enzymes (ADARs) specific for pre-mRNA, and it has been cloned based on its sequence homology to the catalytic domain of ADARs. In the present study we have analyzed the 5'-flanking sequence of the murine ADAT1 gene, revealing that the first transcribed exon is located 1.1 kb downstream from the polyadenylation site of lysyl tRNA synthetase (KARS). The close proximity is conserved in the human genome with an intergenic distance of 5.5 kb. We determined the complete cDNA sequence as well as exon/intron organization of murine KARS. Significant sequence similarities between KARS and ADAT1 are apparent within their substrate interaction domains. Radiation hybrid panel analysis mapped human ADAT1 and human KARS to region q22.2--22.3 of Chromosome (Chr) 16 with alanyl tRNA synthetase (AARS) positioned centromeric to the KARS and ADAT1 genes. 16q22--24 has recently been recognized as a susceptibility candidate locus for several autoimmune inflammatory diseases. The clustering of three tRNA specific genes, of which two are specific for tRNA(Ala), may indicate their evolutionary relatedness or common factors involved in regulating their expression.

The phylogeny of proteobacteria: relationships to other eubacterial phyla and eukaryotes.

The evolutionary relationships of proteobacteria, which comprise the largest and phenotypically most diverse division among prokaryotes, are examined based on the analyses of available molecular sequence data. Sequence alignments of different proteins have led to the identification of numerous conserved inserts and deletions (referred to as signature sequences), which either are unique characteristics of various proteobacterial species or are shared by only members from certain subdivisions of proteobacteria. These signature sequences provide molecular means to define the proteobacterial phyla and their various subdivisions and to understand their evolutionary relationships to the other groups of eubacteria as well as the eukaryotes. Based on signature sequences that are present in different proteins it is now possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low-G+C Gram-positive-->high-G+C Gram-positive-->Deinococcus-Thermus (green nonsulfur bacteria)-->cyanobacteria-->Spirochetes-->Chlamydia-Cytophaga-Aquifex -green sulfur bacteria-->Proteobacteria-1 (epsilon and delta)-->Proteobacteria-2 (alpha)-->Proteobacteria-3 (beta)-->Proteobacteria-4 (gamma). An unexpected but important aspect of the relationship deduced here is that the main eubacterial phyla are related to each other linearly rather than in a tree-like manner, suggesting that the major evolutionary changes within Bacteria have taken place in a directional manner. The identified signatures permit placement of prokaryotes into different groups/divisions and could be used for determinative purposes. These signatures generally support the origin of mitochondria from an alpha-proteobacterium and provide evidence that the nuclear cytosolic homologs of many genes are also derived from proteobacteria.

tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin.

In previous studies we demonstrated that mutations in the genes cysB, cysE, and cls (nov) affect resistance of Escherichia coli to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307-311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766-1771, 1995). In this work we expand this list with mutations in rpoN (the gene for RNA polymerase subunit sigma54) and the tRNA synthetase genes alaS, argS, ileS, and leuS. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the cls gene diminishes resistance to novobiocin but not to mecillinam.

RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria.

A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity and extreme thermostability of purified A. pyrophilus RNA polymerase were verified. Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide. Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (beta and beta') showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A. pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits beta and beta', A. pyrophilus was placed within the Gram-negative bacteria below the epsilon subdivision of the Proteobacteria. No support was found for the 16S rRNA-based hypothesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies. This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms.