Molecular Characterization of eutF Mutants ofSalmonella typhimurium LT2 Identifies eutFLesions as Partial-Loss-of-Function tonB Alleles

ABSTRACT The eutF locus of Salmonella typhimuriumLT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization of eutF mutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining the eutFlocus, Δ903 and eutF1115, are partial-loss-of-function tonB alleles. Both mutations were complemented by plasmids containing a wild-type allele of theEscherichia coli tonB gene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carrying eutF alleles. Molecular analysis of the Δ903 allele identified a deletion that resulted in the fusion of the 3′ end of tonB with the 3′ end oftrpA. In-frame translation of the tonB-trpAfusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Δ903 that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut−phenotype caused by allele Δ903 showed that the new mutation was a deletion of two nucleotides at the tonB-trpAfusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine and eut operon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, the eutF locus does not exist.

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Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

Zeitschrift für Naturforschung C ◽

10.1515/znc-1990-0541 ◽

1990 ◽

Vol 45 (5) ◽

pp. 538-543 ◽

Cited By ~ 6

Author(s):

D. Friedberg ◽

J. Seijffers

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Characterization ◽

Amino Acid Level ◽

Acetolactate Synthase ◽

Mutant Gene ◽

Wild Type ◽

E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

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Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

10.1101/2021.03.29.437598 ◽

2021 ◽

Author(s):

Yang-Yang Li ◽

Rong-Jun Cai ◽

Jia-Ying Yang ◽

Tamara L. Hendrickson ◽

Ye Xiang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Second Step ◽

Loss Of Function ◽

Wild Type ◽

Translational Fidelity ◽

Partial Loss ◽

Biophysical Characterization ◽

Minimal Impact ◽

Mutant Strains

AbstractMost bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

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Faculty Opinions recommendation of Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010692.163057 ◽

2002 ◽

Author(s):

Stephen Busby

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Molecular Characterization ◽

Antisense Rna ◽

Direct Repeat ◽

Cell Killing

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Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)84453-8 ◽

1986 ◽

Vol 261 (19) ◽

pp. 8811-8816 ◽

Cited By ~ 2

Author(s):

D C Blomstrom ◽

D Fahey ◽

R Kutny ◽

B D Korant ◽

E Knight

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Cloning ◽

Molecular Characterization

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Molecular Characterization of the CytochromebGene andIn VitroAtovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03956-14 ◽

2015 ◽

Vol 59 (3) ◽

pp. 1818-1821 ◽

Cited By ~ 6

Author(s):

Luicer A. Ingasia ◽

Hoseah M. Akala ◽

Mabel O. Imbuga ◽

Benjamin H. Opot ◽

Fredrick L. Eyase ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Genetic Polymorphism ◽

Molecular Characterization ◽

Mutant Allele ◽

Inhibitory Concentration ◽

Wild Type Allele ◽

Wild Type ◽

Western Kenya ◽

Content Type

ABSTRACTThe prevalence of a genetic polymorphism(s) at codon 268 in the cytochromebgene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227Plasmodium falciparumparasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

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Trans-splicing as a Novel Mechanism to Explain Interallelic Complementation in Drosophila

Genetics ◽

10.1093/genetics/160.4.1481 ◽

2002 ◽

Vol 160 (4) ◽

pp. 1481-1487 ◽

Cited By ~ 2

Author(s):

Fabien Mongelard ◽

Mariano Labrador ◽

Ellen M Baxter ◽

Tatiana I Gerasimova ◽

Victor G Corces

Keyword(s):

Molecular Characterization ◽

Wild Type ◽

Functional Protein ◽

Gypsy Insulator ◽

Type Function ◽

Homologous Chromosomes ◽

Trans Splicing ◽

Interallelic Complementation

AbstractTwo mutant alleles of the same gene, each located in one of the two homologous chromosomes, may in some instances restore the wild-type function of the gene. This is the case with certain combinations of mutant alleles in the mod(mdg4) gene. This gene encodes several different proteins, including Mod(mdg4)2.2, a component of the gypsy insulator. This protein is encoded by two separate transcription units that can be combined in a trans-splicing reaction to form the mature Mod(mdg4)2.2-encoding RNA. Molecular characterization of complementing alleles shows that they affect the two different transcription units. Flies homozygous for each allele are missing the Mod(mdg4)2.2 protein, whereas wild-type trans-heterozygotes are able to synthesize almost normal levels of the Mod(mdg4)2.2 product. This protein is functional as judged by its ability to form a functional insulator complex. The results suggest that the interallelic complementation in the mod(mdg4) gene is a consequence of trans-splicing between two different mutant transcripts. A conclusion from this observation is that the trans-splicing reaction that takes place between transcripts produced on two different mutant chromosomes ensures wild-type levels of functional protein.

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Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2372-2382.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2372-2382

Author(s):

K M Arndt ◽

S L Ricupero ◽

D M Eisenmann ◽

F Winston

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Repeat ◽

Single Amino Acid ◽

Wild Type ◽

Dna Binding Specificity ◽

Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil

Ciência Rural ◽

10.1590/0103-8478cr20141810 ◽

2015 ◽

Vol 45 (12) ◽

pp. 2197-2200 ◽

Cited By ~ 2

Author(s):

Thor Vinícius Martins Fajardo ◽

Monique Bezerra Nascimento ◽

Marcelo Eiras ◽

Osmar Nickel ◽

Gilvan Pio-Ribeiro

Keyword(s):

Amino Acid ◽

Molecular Characterization ◽

Molecular Analysis ◽

Nucleotide Sequences ◽

Amino Acid Sequences ◽

Causal Agent ◽

Ringspot Virus ◽

Prunus Necrotic Ringspot Virus ◽

First Time

ABSTRACT: There is no molecular characterization of Brazilian isolates of Prunus necrotic ringspot virus (PNRSV), except for those infecting peach. In this research, the causal agent of rose mosaic was determined and the movement (MP) and coat (CP) protein genes of a PNRSV isolate from rose were molecularly characterized for the first time in Brazil. The nucleotide and deduced amino acid sequences of MP and CP complete genes were aligned and compared with other isolates. Molecular analysis of the MP and CP nucleotide sequences of a Brazilian PNRSV isolate from rose and others from this same host showed highest identities of 96.7% and 98.6%, respectively, and Rose-Br isolate was classified in PV32 group.

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