Sensitive Genome-Wide Screen for Low Secondary Enzymatic Activities: The YjbQ Family Shows Thiamin Phosphate Synthase Activity

Morett, Enrique; Saab‐Rincón, Gloria; Olvera, Leticia; Olvera, Maricela; Flores, Humberto; Grande, Ricardo

doi:10.1016/j.jmb.2007.12.017

Cited by 23 publications

(16 citation statements)

References 54 publications

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“…The high sensitivity of the selection method of thiamin phosphate auxotrophy complementation of an E. coli ΔthiE strain allowed us to identify an enzyme, YjbQ, with a cryptic TPS activity. 5 Here, we applied this selection strategy to the evolution of TPI to TPS activity, resulting in the identification of a series of mutants that clearly showed a growth complementation phenotype of this strain. It is worth to mention that the presence of yjbQ in the chromosome is not enough to complement TPS activity.…”

Section: Discussionmentioning

confidence: 99%

Evolutionary Walk between (β/α)8 Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase

Saab‐Rincón

Olvera

et al. 2012

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 99%

Evolutionary Walk between (β/α)8 Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase

Saab‐Rincón

Olvera

et al. 2012

Journal of Molecular Biology

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“…In these experiments, conditionally auxotrophic bacterial strains are transformed with plasmid-encoded libraries of ORFs. The libraries are screened for genes that can suppress the phenotype of the original mutation, provided they are present in high copy number (17)(18)(19) or overexpressed (20,21). Previously, we used the multicopy suppression approach in an attempt to assess the prevalence of promiscuity, on a proteome-wide scale.…”

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confidence: 99%

Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli

Soo

Hanson‐Manful²,

Patrick³

2010

Proc. Natl. Acad. Sci. U.S.A.

112

110

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Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (≤16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004-17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations.antibiotic resistance | evolutionary innovation | molecular evolution | protein promiscuity | phenotype microarray

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“…The predicted ThiR regulons in Thermococcus spp. also include the YjbQ family proteins (encoded by a putative operon with thiR) that were previously characterized for their low thiamine phosphate synthase activity (26).…”

Section: Resultsmentioning

confidence: 99%

A Novel Transcriptional Regulator Related to Thiamine Phosphate Synthase Controls Thiamine Metabolism Genes in Archaea

Rodionov

Leyn

et al. 2017

J Bacteriol

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Thiamine (vitamin B 1 ) is a precursor of thiamine pyrophosphate (TPP), an essential coenzyme in the central metabolism of all living organisms. Bacterial thiamine biosynthesis and salvage genes are controlled at the RNA level by TPPresponsive riboswitches. In Archaea, TPP riboswitches are restricted to the Thermoplasmatales order. Mechanisms of transcriptional control of thiamine genes in other archaeal lineages remain unknown. Using the comparative genomics approach, we identified a novel family of transcriptional regulators (named ThiR) controlling thiamine biosynthesis and transport genes in diverse lineages in the Crenarchaeota phylum as well as in the Halobacteria and Thermococci classes of the Euryarchaeota. ThiR regulators are composed of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which is similar to the archaeal thiamine phosphate synthase ThiN. By using comparative genomics, we predicted ThiR-binding DNA motifs and reconstructed ThiR regulons in 67 genomes representing all above-mentioned lineages. The predicted ThiR-binding motifs are characterized by palindromic symmetry with several distinct lineage-specific consensus sequences. In addition to thiamine biosynthesis genes, the reconstructed ThiR regulons include various transporters for thiamine and its precursors. Bioinformatics predictions were experimentally validated by in vitro DNA-binding assays with the recombinant ThiR protein from the hyperthermophilic archaeon Metallosphaera yellowstonensis MK1. Thiamine phosphate and, to some extent, TPP and hydroxyethylthiazole phosphate were required for the binding of ThiR to its DNA targets, suggesting that ThiR is derepressed by limitation of thiamine phosphates. The thiamine phosphate-binding residues previously identified in ThiN are highly conserved in ThiR regulators, suggesting a conserved mechanism for effector recognition.IMPORTANCE Thiamine pyrophosphate is a cofactor for many essential enzymes for glucose and energy metabolism. Thiamine or vitamin B 1 biosynthesis and its transcriptional regulation in Archaea are poorly understood. We applied the comparative genomics approach to identify a novel family of regulators for the transcriptional control of thiamine metabolism genes in Archaea and reconstructed the respective regulons. The predicted ThiR regulons in archaeal genomes control the majority of thiamine biosynthesis genes. The reconstructed regulon content suggests that numerous uptake transporters for thiamine and/or its precursors are encoded in archaeal genomes. The ThiR regulon was experimentally validated by DNA-binding assays with Metallosphaera spp. These discoveries contribute to our understanding of metabolic and regulatory networks involved in vitamin homeostasis in diverse lineages of Archaea.KEYWORDS Archaea, comparative genomics, regulon reconstruction, thiamine metabolism, transcriptional regulation

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Sensitive Genome-Wide Screen for Low Secondary Enzymatic Activities: The YjbQ Family Shows Thiamin Phosphate Synthase Activity

Cited by 23 publications

References 54 publications

Evolutionary Walk between (β/α)8 Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase

Evolutionary Walk between (β/α)8 Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase

Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli

A Novel Transcriptional Regulator Related to Thiamine Phosphate Synthase Controls Thiamine Metabolism Genes in Archaea

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