The essential coenzyme NAD plays important roles in metabolic reactions and cell regulation in all organisms. As such, NAD synthesis has been investigated as a source for novel antibacterial targets. Cross-species genomics-based reconstructions of NAD metabolism in group A streptococci (GAS), combined with focused experimental testing in Streptococcus pyogenes, led to a better understanding of NAD metabolism in the pathogen. The predicted niacin auxotrophy was experimentally verified, as well as the essential role of the nicotinamidase PncA in the utilization of nicotinamide (Nm). PncA is dispensable in the presence of nicotinate (Na), ruling it out as a viable antibacterial target. The function of the “orphan” NadC enzyme, which is uniquely present in all GAS species despite the absence of other genes of NAD de novo synthesis, was elucidated. Indeed, the quinolinate (Qa) phosphoribosyltransferase activity of NadC from S. pyogenes allows the organism to sustain growth when Qa is present as a sole pyridine precursor. Finally, the redundancy of functional upstream salvage pathways in GAS species narrows the choice of potential drug targets to the two indispensable downstream enzymes of NAD synthesis, nicotinate adenylyltransferase (NadD family) and NAD synthetase (NadE family). Biochemical characterization of NadD confirmed its functional role in S. pyogenes, and its potential as an antibacterial target was supported by inhibition studies with previously identified class I inhibitors of the NadD enzyme family. One of these inhibitors efficiently inhibited S. pyogenes NadD (sp.NadD) in vitro (50% inhibitory concentration [IC50], 15 μM), exhibiting a noncompetitive mechanism with a Ki of 8 μM.
A comparative genomic analysis predicted that many members of the under-characterized COG0523 subfamily of putative P-loop GTPases function in metal metabolism. In this work we focused on the uncharacterized Escherichia coli protein YeiR by studying both the physiology of a yeiR mutant and the in vitro biochemical properties of YeiR expressed as a fusion with the maltose-binding protein (YeiR-MBP). Our results demonstrate that deletion of yeiR increases the sensitivity of E. coli to EDTA or cadmium and this phenotype in linked to zinc depletion. In vitro, the tagged protein binds several Zn2+ ions with nanomolar affinity and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group, but GTP hydrolysis is enhanced by Zn2+ binding. These results support the predicted connection between the COG0523 P-loop GTPases and roles in metal homeostasis.
GTPase; zinc homeostasis; metal-binding; COG0523; cadmium
Archaeosine (G+) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G+ precursor, 7-cyano-7-deazaguanine (preQ0), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G+ by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G+ lack ArcS homologs. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified: 1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC, the enzyme that produces preQ0; 2) QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQ0 to 7-aminomethyl-7-deazaguanine. Here we show that these two protein families are able to catalyze the formation of G+ in a heterologous system. Structure and sequence comparisons of crenarchaeal and euryarchaeal arcTGTs suggest the crenarchaeal enzymes have broader substrate specificity. These results led to a new model for the synthesis and salvage of G+ in Crenarchaeota.
The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function.
Queuosine; archaeosine; tetrahydrofolate; biopterin; tRNA modification; riboflavin; 6-pyruvoyl-tetrahydropterin synthase
A comparative genomic analysis of the recently sequenced human body louse unicellular endosymbiont Candidatus Riesia pediculicola with a reduced genome (582 Kb), revealed that it is the only known organism that might have lost all post-transcriptional base and ribose modifications of the tRNA body, retaining only modifications of the anticodon-stem-loop essential for mRNA decoding. Such a minimal tRNA modification set was not observed in other insect symbionts or in parasitic unicellular bacteria, such as Mycoplasma genitalium (580 Kb), that have also evolved by considerably reducing their genomes. This could be an example of a minimal tRNA modification set required for life, a question that has been at the center of the field for many years, especially for understanding the emergence and evolution of the genetic code.
tRNA; maturation; translation; modified nucleosides; comparative genomics
The availability of over 3000 published genome sequences has enabled the use of comparative genomic approaches to drive the biological function discovery process. Classically, one used to link gene with function by genetic or biochemical approaches, a lengthy process that often took years. Phylogenetic distribution profiles, physical clustering, gene fusion, co-expression profiles, structural information and other genomic or post-genomic derived associations can be now used to make very strong functional hypotheses. Here, we illustrate this shift with the analysis of the DUF71/COG2102 family, a subgroup of the PP-loop ATPase family.
The DUF71 family contains at least two subfamilies, one of which was predicted to be the missing diphthine-ammonia ligase (EC 220.127.116.11), Dph6. This enzyme catalyzes the last ATP-dependent step in the synthesis of diphthamide, a complex modification of Elongation Factor 2 that can be ADP-ribosylated by bacterial toxins. Dph6 orthologs are found in nearly all sequenced Archaea and Eucarya, as expected from the distribution of the diphthamide modification. The DUF71 family appears to have originated in the Archaea/Eucarya ancestor and to have been subsequently horizontally transferred to Bacteria. Bacterial DUF71 members likely acquired a different function because the diphthamide modification is absent in this Domain of Life. In-depth investigations suggest that some archaeal and bacterial DUF71 proteins participate in B12 salvage.
This detailed analysis of the DUF71 family members provides an example of the power of integrated data-miming for solving important “missing genes” or “missing function” cases and illustrates the danger of functional annotation of protein families by homology alone.
This article was reviewed by Arcady Mushegian, Michael Galperin and L. Aravind.
Diphthamide; Vitamin B12; Amidotransferase; Comparative genomics
KEOPS is an important cellular complex conserved in Eukarya, with some subunits conserved in Archaea and Bacteria. This complex was recently found to play an essential role in formation of the tRNA modification threonylcarbamoyladenosine (t6A), and was previously associated with telomere length maintenance and transcription. KEOPS subunits are conserved in Archaea, especially in the Euryarchaea, where they had been studied in vitro. Here we attempted to delete the genes encoding the four conserved subunits of the KEOPS complex in the euryarchaeote Haloferax volcanii and study their phenotypes in vivo. The fused kae1-bud32 gene was shown to be essential as was cgi121, which is dispensable in yeast. In contrast, pcc1 (encoding the putative dimerizing unit of KEOPS) was not essential in H. volcanii. Deletion of pcc1 led to pleiotropic phenotypes, including decreased growth rate, reduced levels of t6A modification, and elevated levels of intra-cellular glycation products.
Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wild-type fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.
Nearly 2200 genomes encoding some 6 million proteins have now been sequenced. Around 40% of these proteins are of unknown function even when function is loosely and minimally defined as “belonging to a superfamily”. In addition to in silico methods, the swelling stream of high-throughput experimental data can give valuable clues for linking these “unknowns” with precise biological roles. The goal is to develop integrative data-mining platforms that allow the scientific community at large to access and utilize this rich source of experimental knowledge. To this end, we review recent advances in generating whole-genome experimental datasets, where this data can be accessed, and how it can be used to drive prediction of gene function.
DksA is a global transcriptional regulator that directly interacts with RNA polymerase (RNAP) and, in conjunction with an alarmone ppGpp, alters transcription initiation at target promoters. DksA proteins studied to date contain a canonical Cys4 Zn-finger motif thought to be essential for their proper folding and thus activity. In addition to the canonical DksA protein, the Pseudomonas aeruginosa genome encodes a closely-related paralog DksA2 that lacks the Zn-finger motif. Here, we report that DksA2 can functionally substitute for the canonical DksA in vivo in Escherichia coli and P. aeruginosa. We also demonstrate that DksA2 affects transcription by the E. coli RNAP in vitro similarly to DksA. The dksA2 gene is positioned downstream of a putative Zur-binding site. Accordingly, we show that dksA2 expression is repressed by the presence of exogenous Zn, deletion of Zur results in constitutive expression of dksA2, and Zur binds specifically to the promoter region of dksA2. We also found that deletion of dksA2 confers a growth defect in the absence of Zn. Our data suggest that DksA2 plays a role in Zn homeostasis and serves as a back-up copy of the canonical Zn-dependent DksA in Zn poor environments.
DksA; Pseudomonas aeruginosa; Zur; zinc
The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.
As the molecular adapters between codons and amino acids, transfer-RNAs are pivotal molecules of the genetic code. The coding properties of a tRNA molecule do not reside only in its primary sequence. Posttranscriptional nucleoside modifications, particularly in the anticodon loop, can modify cognate codon recognition, affect aminoacylation properties, or stabilize the codon-anticodon wobble base pairing to prevent ribosomal frameshifting. Despite a wealth of biophysical and structural knowledge of the tRNA modifications themselves, their pathways of biosynthesis had been until recently only partially characterized. This discrepancy was mainly due to the lack of obvious phenotypes for tRNA modification–deficient strains and to the difficulty of the biochemical assays used to detect tRNA modifications. However, the availability of hundreds of whole-genome sequences has allowed the identification of many of these missing tRNA-modification genes. This chapter reviews the methods that were used to identify these genes with a special emphasis on the comparative genomic approaches. Methods that link gene and function but do not rely on sequence homology will be detailed, with examples taken from the tRNA modification field.
Like other forms of engineering, metabolic engineering requires knowledge of the components (the ‘parts list’) of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell’s parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of ‘unknown’ proteins and ‘orphan’ enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the ‘missing parts list’ problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life’s machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.
comparative genomics; metabolic reconstruction; orphan enzyme; pathway hole; unknown protein
With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment.
Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The ΔtyrA strain was, as expected, prototrophic for tyrosine, whereas the ΔtyrA ΔfolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.
GTP cyclohydrolase I (GCYH-I) is an essential Zn2+-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn2+-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn2+ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn2+-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.
The bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for protein synthesis. It is homologous to the eukaryotic translation initiation factor 5A (eIF5A). A highly conserved eIF5A lysine is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for eIF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a spermidine derivative in Escherichia coli. However, the biosynthesis pathway of this modification in the bacterial EF-P is yet to be elucidated.
Presentation of the Hypothesis
Here we propose a potential mechanism for the post-translational modification of EF-P. By using comparative genomic methods based on physical clustering and phylogenetic pattern analysis, we identified two protein families of unknown function, encoded by yjeA and yjeK genes in E. coli, as candidates for this missing pathway. Based on the analysis of the structural and biochemical properties of both protein families, we propose two potential mechanisms for the modification of EF-P.
Testing the hypothesis
This hypothesis could be tested genetically by constructing a bacterial strain with a tagged efp gene. The tag would allow the purification of EF-P by affinity chromatography and the analysis of the purified protein by mass spectrometry. yjeA or yjeK could then be deleted in the efp tagged strain and the EF-P protein purified from each mutant analyzed by mass spectrometry for the presence or the absence of the modification. This hypothesis can also be tested by purifying the different components (YjeK, YjeA and EF-P) and reconstituting the pathway in vitro.
Implication of the hypothesis
The requirement for a fully modified EF-P for protein synthesis in certain bacteria implies the presence of specific post-translational modification mechanism in these organisms. All of the 725 bacterial genomes analyzed, possess an efp gene but only 200 (28%) possess both yjeA and yjeK genes. In the other organisms, EF-P may be modified by another pathway or the translation machinery must have adapted to the lack of EF-P modification. Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway.
This article was reviewed by Céline Brochier-Armanet, Igor B. Zhulin and Mikhail Gelfand. For the full reviews, please go to the Reviewers' reports section.
Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.
COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, cobW, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family.
Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, Chlamydomonas reinhardtii.
This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s) might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of COG0523 and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.
Folates have a key role in metabolism, and the folate-dependent generation of DNA precursors in the form of deoxythymidine 5′-phosphate is particularly important for the replication of malaria parasites. Although Plasmodium falciparum can synthesize folate derivatives de novo, a long-standing mystery has been the apparent absence of a key enzyme, dihydroneopterin aldolase, in the classical folate biosynthetic pathway of this organism. The discovery that a different enzyme, pyruvoyltetrahydropterin synthase, can produce the necessary substrate for the subsequent step in folate synthesis raises the question of whether this solution is unique to P. falciparum. Bioinformatic analyses suggest otherwise and indicate that an alternative route to folate could be widespread among diverse microorganisms and could be a target for novel drugs.
Queuosine (Q) and archaeosine (G+) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNAGUN in Eukarya and Bacteria, while G+ is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q0) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in Escherichia coli by folE, is also the first enzyme of pre-Q0 biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an E. coli ΔfolE strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a Haloferax volcanii strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G+. These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.
Threonylcarbamoyladenosine (t6A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNAThr lacking only the t6A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t6A in vivo.
In part due to the existence of simple methods for its cultivation and
genetic manipulation, Haloferax volcanii is a major
archaeal model organism. It is the only archaeon for which the whole
set of post-transcriptionally modified tRNAs has been sequenced,
allowing for an in silico prediction of all RNA
modification genes present in the organism. One approach to check
these predictions experimentally is via the construction of targeted
gene deletion mutants. Toward this goal, an integrative “Gateway
vector” that allows gene deletion in H.
volcanii uracil auxotrophs was constructed. The vector was
used to delete three predicted tRNA modification genes: HVO_2001
(encoding an archaeal transglycosyl tranferase or arcTGT), which is
involved in archeosine biosynthesis; HVO_2348 (encoding a newly
discovered GTP cyclohydrolase I), which catalyzes the first step
common to archaeosine and folate biosynthesis; and HVO_2736 (encoding
a member of the COG1444 family), which is involved in
formation. Preliminary phenotypic analysis of the deletion mutants was
conducted, and confirmed all three predictions.
Archaea; GTP-cyclohydrolase I; halophile; tRNA-modification
Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes.
By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions.
The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.