RESUMO
Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: (1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and (2) queuosine precursor transporter (QPTR), a transporter protein that imports Q precursors. Organisms such as the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, MS analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens - from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 confers fitness advantages when B. henselae is growing outside a mammalian host.
Assuntos
Bartonella henselae , Nucleosídeo Q , Nucleosídeo Q/metabolismo , Nucleosídeo Q/genética , Bartonella henselae/genética , Bartonella henselae/metabolismo , Bartonella henselae/enzimologia , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Evolução Molecular , Especificidade por Substrato , Guanina/análogos & derivadosRESUMO
SUMMARYDeazaguanine modifications play multifaceted roles in the molecular biology of DNA and tRNA, shaping diverse yet essential biological processes, including the nuanced fine-tuning of translation efficiency and the intricate modulation of codon-anticodon interactions. Beyond their roles in translation, deazaguanine modifications contribute to cellular stress resistance, self-nonself discrimination mechanisms, and host evasion defenses, directly modulating the adaptability of living organisms. Deazaguanine moieties extend beyond nucleic acid modifications, manifesting in the structural diversity of biologically active natural products. Their roles in fundamental cellular processes and their presence in biologically active natural products underscore their versatility and pivotal contributions to the intricate web of molecular interactions within living organisms. Here, we discuss the current understanding of the biosynthesis and multifaceted functions of deazaguanines, shedding light on their diverse and dynamic roles in the molecular landscape of life.
Assuntos
Bacteriófagos , Produtos Biológicos , Guanina/análogos & derivados , Anticódon , RNA de Transferência/química , RNA de Transferência/genética , Bactérias/genéticaRESUMO
Capturing the published corpus of information on all members of a given protein family should be an essential step in any study focusing on specific members of that family. Using a previously gathered dataset of more than 280 references mentioning a member of the DUF34 (NIF3/Ngg1-interacting Factor 3) family, we evaluated the efficiency of different databases and search tools, and devised a workflow that experimentalists can use to capture the most information published on members of a protein family in the least amount of time. To complement this workflow, web-based platforms allowing for the exploration of protein family members across sequenced genomes or for the analysis of gene neighbourhood information were reviewed for their versatility and ease of use. Recommendations that can be used for experimentalist users, as well as educators, are provided and integrated within a customized, publicly accessible Wiki.
Assuntos
Genoma , Sequência de BasesRESUMO
Capturing the published corpus of information on all members of a given protein family should be an essential step in any study focusing on specific members of that said family. Using a previously gathered dataset of more than 280 references mentioning a member of the DUF34 (NIF3/Ngg1-interacting Factor 3), we evaluated the efficiency of different databases and search tools, and devised a workflow that experimentalists can use to capture the most published information on members of a protein family in the least amount of time. To complement this workflow, web-based platforms allowing for the exploration of protein family members across sequenced genomes or for the analysis of gene neighborhood information were reviewed for their versatility and ease of use. Recommendations that can be used for experimentalist users, as well as educators, are provided and integrated within a customized, publicly accessible Wiki.
RESUMO
Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: 1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and 2) Queuosine Precursor Transporter (QPTR), a transporter protein that imports Q precursors. Organisms like the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, mass spectrometry analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens-from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 has fitness advantages when B. henselae is growing outside a mammalian host.
RESUMO
Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.
Assuntos
Bacteriófagos , Guanina , Bactérias/genética , Bacteriófagos/genética , DNA/genética , Guanina/análogos & derivadosRESUMO
Over the last 25 years, biology has entered the genomic era and is becoming a science of 'big data'. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages. Such a large gap in knowledge hampers all aspects of biological enterprise and, thereby, is standing in the way of genomic biology reaching its full potential. A brainstorming meeting to address this issue funded by the National Science Foundation was held during 3-4 February 2022. Bringing together data scientists, biocurators, computational biologists and experimentalists within the same venue allowed for a comprehensive assessment of the current state of functional annotations of protein families. Further, major issues that were obstructing the field were identified and discussed, which ultimately allowed for the proposal of solutions on how to move forward.
Assuntos
Genômica , Proteínas , Sequência de Bases , Biologia Computacional , Genoma , Anotação de Sequência MolecularRESUMO
The DNA in bacterial viruses collectively contains a rich, yet relatively underexplored, chemical diversity of nucleobases beyond the canonical adenine, guanine, cytosine, and thymine. Herein, we review what is known about the genetic and biochemical basis for the biosynthesis of complex DNA modifications, also called DNA hypermodifications, in the DNA of tailed bacteriophages infecting Escherichia coli and Salmonella enterica. These modifications, and their diversification, likely arose out of the evolutionary arms race between bacteriophages and their cellular hosts. Despite their apparent diversity in chemical structure, the syntheses of various hypermodified bases share some common themes. Hypermodifications form through virus-directed synthesis of noncanonical deoxyribonucleotide triphosphates, direct modification DNA, or a combination of both. Hypermodification enzymes are often encoded in modular operons reminiscent of biosynthetic gene clusters observed in natural product biosynthesis. The study of phage-hypermodified DNA provides an exciting opportunity to expand what is known about the enzyme-catalyzed chemistry of nucleic acids and will yield new tools for the manipulation and interrogation of DNA.
Assuntos
Bacteriófagos , Salmonella enterica , Bacteriófagos/genética , DNA , Escherichia coli/genética , TiminaRESUMO
Members of the DUF34 (domain of unknown function 34) family, also known as the NIF3 protein superfamily, are ubiquitous across superkingdoms. Proteins of this family have been widely annotated as "GTP cyclohydrolase I type 2" through electronic propagation based on one study. Here, the annotation status of this protein family was examined through a comprehensive literature review and integrative bioinformatic analyses that revealed varied pleiotropic associations and phenotypes. This analysis combined with functional complementation studies strongly challenges the current annotation and suggests that DUF34 family members may serve as metal ion insertases, chaperones, or metallocofactor maturases. This general molecular function could explain how DUF34 subgroups participate in highly diversified pathways such as cell differentiation, metal ion homeostasis, pathogen virulence, redox, and universal stress responses.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Genômica , Metais/metabolismo , Chaperonas Moleculares/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Análise por Conglomerados , Biologia Computacional , Escherichia coli/metabolismo , Ácido Fólico/biossíntese , GTP Fosfo-Hidrolases/química , Homeostase , Íons , Proteínas Ferro-Enxofre/metabolismo , Domínios Proteicos , PublicaçõesRESUMO
A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using Pantoea agglomerans strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a gene encoding a preQ0 DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC-MS/MS analysis indicates 2'-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of Pantoea phages. Thus, the data presented in this study provide new information on Pantoea-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.
Assuntos
DNA Viral , Genoma Viral , Guanosina , Fases de Leitura Aberta , Pantoea/virologia , Siphoviridae , Proteínas Virais , DNA Viral/genética , DNA Viral/metabolismo , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Siphoviridae/genética , Siphoviridae/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Phage predation constitutes a major mortality factor for bacteria in aquatic ecosystems, and thus, directly impacts nutrient cycling and microbial community dynamics. Yet, the population dynamics of specific phages across time scales from days to months remain largely unexplored, which limits our understanding of their influence on microbial succession. To investigate temporal changes in diversity and abundance of phages infecting particular host strains, we isolated 121 phage strains that infected three bacterial hosts during a Baltic Sea mesocosm experiment. Genome analysis revealed a novel Flavobacterium phage genus harboring gene sets putatively coding for synthesis of modified nucleotides and glycosylation of bacterial cell surface components. Another novel phage genus revealed a microdiversity of phage species that was largely maintained during the experiment and across mesocosms amended with different nutrients. In contrast to the newly described Flavobacterium phages, phages isolated from a Rheinheimera strain were highly similar to previously isolated genotypes, pointing to genomic consistency in this population. In the mesocosm experiment, the investigated phages were mainly detected after a phytoplankton bloom peak. This concurred with recurrent detection of the phages in the Baltic Proper during summer months, suggesting an influence on the succession of heterotrophic bacteria associated with phytoplankton blooms.
Assuntos
Bacteriófagos , Chromatiaceae , Bacteriófagos/genética , Ecossistema , Flavobacterium , Processos HeterotróficosRESUMO
In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.
Assuntos
Bacteriófagos/genética , DNA/genética , Motivos de Nucleotídeos/genética , Pirimidinonas/farmacologia , Pirróis/farmacologia , Bacteriófagos/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , DNA/efeitos dos fármacos , Enzimas de Restrição-Modificação do DNA/efeitos dos fármacos , Escherichia coli/virologia , Edição de Genes , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Sequenciamento por Nanoporos , Motivos de Nucleotídeos/efeitos dos fármacos , Siphoviridae/genéticaRESUMO
Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G+), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2'-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2'-deoxy-7-amido-7-deazaguanine (dADG), 2'-deoxy-7-cyano-7-deazaguanine (dPreQ0) and 2'-deoxy-7- aminomethyl-7-deazaguanine (dPreQ1). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2'-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.
Assuntos
Vírus de Archaea/genética , Bacteriófagos/genética , Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Guanina/análogos & derivados , Bacteriófagos/metabolismo , Guanina/metabolismo , Pirimidinonas/metabolismo , Pirróis/metabolismoRESUMO
Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2'-deoxyinosine, while the firehammerviruses replace dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.
Assuntos
Bacteriófagos/genética , Campylobacter jejuni/virologia , Desoxiguanosina/genética , Inosina/genética , Vias Biossintéticas/genética , DNA Viral/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Genoma Viral , Inosina/análogos & derivados , Inosina/metabolismo , Myoviridae/genética , FilogeniaRESUMO
The lytic Escherichia coli siphophage BRET was isolated from a chicken obtained at a local market in Abidjan, Côte d'Ivoire. Its linear genome sequence consists of 59,550 bp (43.4% GC content) and contains 88 predicted genes, including 4 involved in archaeosine biosynthesis. Phage BRET is related (95% nucleotide identity) to Enterobacteria phage JenK1.
RESUMO
The 7-deazapurine derivatives, 2'-deoxy-7-cyano-7-deazaguanosine (dPreQ0 ) and 2'-deoxy-7-amido-7-deazaguanosine (dADG) are recently discovered DNA modifications encoded by the dpd cluster found in a diverse set of bacteria. Here we identify the genes required for the formation of dPreQ0 and dADG in DNA and propose a biosynthetic pathway. The preQ0 base is a precursor that in Salmonella Montevideo, is synthesized as an intermediate in the pathway of the tRNA modification queuosine. Of the 11 genes (dpdA - dpdK) found in the S. Montevideo dpd cluster, dpdA and dpdB are necessary and sufficient to synthesize dPreQ0 , while dpdC is additionally required for dADG synthesis. Among the rest of the dpd genes, dpdE, dpdG, dpdI, dpdK, dpdD and possibly dpdJ appear to be involved in a restriction-like phenotype. Indirect competition for preQ0 base led to a model for dADG synthesis in which DpdA inserts preQ0 into DNA with the help of DpdB, and then DpdC hydrolyzes dPreQ0 to dADG. The role of DpdB is not entirely clear as it is dispensable in other dpd clusters. Our discovery of a minimal gene set for introducing 7-deazapurine derivatives in DNA provides new tools for biotechnology applications and demonstrates the interplay between the DNA and RNA modification machineries.
Assuntos
Vias Biossintéticas/genética , DNA Bacteriano/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biossíntese , Salmonella enterica/genética , Salmonella enterica/metabolismo , Família MultigênicaRESUMO
Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redß protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.
RESUMO
Seven-deazapurine modifications were thought to be highly specific of tRNAs, but have now been discovered in DNA of phages and of phylogenetically diverse bacteria, illustrating the plasticity of these modification pathways. The intermediate 7-cyano-7-deazaguanine (preQ0) is a shared precursor in the pathways leading to the insetion of 7-deazapurine derivatives in both tRNA and DNA. It is also used as an intermediate in the synthesis of secondary metabolites such as toyocamacin. The presence of 7-deazapurine in DNA is proposed to be a protection mechanism against endonucleases. This makes preQ0 a metabolite of underappreaciated but central importance.
Assuntos
DNA/metabolismo , RNA/metabolismo , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , DNA/química , DNA/genética , Eucariotos/genética , Eucariotos/metabolismo , Transferência Genética Horizontal , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Redes e Vias Metabólicas , Nucleosídeo Q/química , Nucleosídeo Q/metabolismo , RNA/química , RNA/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
DUF1537 is a novel family of kinases identified by comparative genomic approaches. The family is widespread and found in all sequenced plant genomes and 16% of sequenced bacterial genomes. DUF1537 is not a monofunctional family and contains subgroups that can be separated by phylogenetic and genome neighborhood context analyses. A subset of the DUF1537 proteins is strongly associated by physical clustering and gene fusion with the PdxA2 family, demonstrated here to be a functional paralog of the 4-phosphohydroxy-l-threonine dehydrogenase enzyme (PdxA), a central enzyme in the synthesis of pyridoxal-5'-phosphate (PLP) in proteobacteria. Some members of this DUF1537 subgroup phosphorylate l-4-hydroxythreonine (4HT) into 4-phosphohydroxy-l-threonine (4PHT), the substrate of PdxA, in vitro and in vivo. This provides an alternative route to PLP from the toxic antimetabolite 4HT that can be directly generated from the toxic intermediate glycolaldehyde. Although the kinetic and physical clustering data indicate that these functions in PLP synthesis are not the main roles of the DUF1537-PdxA2 enzymes, genetic and physiological data suggest these side activities function has been maintained in diverse sets of organisms.
Assuntos
Fosfotransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Fosfotransferases/genéticaRESUMO
The discovery of â¼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2'-deoxy-preQ0 and 2'-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in â¼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction-modification role for the cluster in Enterobacteriaceae. Another preQ0 derivative, 2'-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9 g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.