The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ 0 ) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2'-deoxy-preQ 0 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 ∼150 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 preQ 0 derivative, 2'-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, 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.DNA modification | restriction-modification | 7-deazaguanine | comparative genomics | queuosine H ypermodifications of DNA requiring more than one synthetic enzyme are not as prevalent and chemically diverse as RNA hypermodifications, but around a dozen have been identified in DNA to date (1). The functions of most DNA hypermodifications are still not known, but some have roles in protection against restriction enzymes, whereas others affect thermal stability temperature, DNA packaging, or transcription regulation (2). For example, the hypermodified DNA base β-D-glucosyl-hydroxymethyluracil, or base J, is an epigenetic factor that regulates Pol II transcription initiation in kinetoplastids of trypanosomes (3). The recently discovered phosphorothioate (PT) modification of the DNA backbone in bacteria was found to perform different functions in different organisms (4-6). In Salmonella Cerro 87, PT occurs on each strand of a GAAC/GTTC motif as part of a restriction-modification (R-M) system, whereas in Vibrio cyclitrophicus FF75, which lacks PT restriction enzymes, PT occurs on one strand of C ps CA motifs, and the function remains unclear (6). In 2013, Iyer et al. described the computational prediction of 12 novel DNA hypermodificat...
The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.
Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.Vibrio vulnificus is a halophilic bacterium present naturally in estuarine waters and often contaminates oysters and other shellfish (for a review, see reference 15). V. vulnificus is an opportunistic pathogen of humans, causing primary septicemia and wound infection in susceptible individuals, and is the leading cause of reported seafood-related deaths in the United States. In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage. Mortality rates for susceptible individuals who develop fulminating primary septicemia are greater than 50% (17). Skin infections can lead to severe cellulitis, necrotizing fasciitis, and myositis requiring surgical debridement of infected tissues or amputation of the limb (4, 29, 42). Therapeutic intervention is often difficult since death can occur in less than 24 h after contact with the bacteria. In a mouse model of infection, V. vulnificus replicates extremely rapidly in host tissues (40, 41) and kills host cells including neutrophils (41).Over 20 years of genetic analysis, only a few virulence factors have been identified and confirmed by using the molecular version of Koch's postulates (15). Among the confirmed virulence factors are capsular polysaccharide (49), acquisition of iron (34, 52), type IV p...
Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t6A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNAIle-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t6A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t6A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t6A in tRNAs. Thus, although t6A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.
Vibrio vulnificus is the leading cause of reported deaths from infections related to consumption of seafood in the United States. Affected predisposed individuals frequently die rapidly from sepsis. Otherwise healthy people can experience severe wound infection, which can lead to sepsis and death. A question is why, with so many people consuming contaminated raw oysters, the incidence of severe V. vulnificus disease is low. Molecular typing systems have shown associations of V. vulnificus genotypes and the environmental or clinical source of the strains, suggesting that different genotypes possess different virulence potentials. We examined 69 V. vulnificus biotype 1 strains that were genotyped by several methods and evaluated them for virulence in a subcutaneously inoculated iron dextran-treated mouse model. By examining the relationships between skin infection, systemic liver infection, and presumptive death (a decrease in body temperature), we determined that liver infection is predicated on severe skin infection and that death requires significant liver infection. Although most strains caused severe skin infection, not every strain caused systemic infection and death. Strains with polymorphisms at multiple loci (rrn, vcg, housekeeping genes, and repetitive DNA) designated profile 2 were more likely to cause lethal systemic infection with more severe indicators of virulence than were profile 1 strains with different polymorphisms at these loci. However, some profile 1 strains were lethal and some profile 2 strains did not cause systemic infection. Therefore, current genotyping schemes cannot strictly predict the virulence of V. vulnificus strains and further investigation is needed to identify virulence genes as markers of virulence.Vibrio vulnificus is a halophilic bacterium that is naturally present in estuarine waters and contaminates oysters and other shellfish. It is an opportunistic pathogen of humans that causes primary septicemia and wound infection in susceptible hosts and is the leading cause of reported seafood-related deaths due to infection in the United States (for a review, see reference 17). V. vulnificus strains are divided into three biotypes, with human disease caused predominantly by biotype 1 and disease of eels caused predominantly by biotype 2. Biotype 3 was recently isolated from wound infections in Israel and represents an emerging form of this species (3). Several factors have been definitively shown to contribute to virulence of V. vulnificus, including capsular polysaccharide (60), the ability to acquire iron (38, 45, 61), type IV pili (46), flagella (37, 47), RTX toxin (30, 35, 39), and others (17, 27). To date, no single factor has been identified that can distinguish between naturally occurring virulent and less virulent isolates of V. vulnificus.In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage (reviewed in reference 17). Primary septicemia is characterized by high fever, chills, hypotension, and septic shock. ...
Methylation is a versatile reaction involved in the synthesis and modification of biologically active molecules, including RNAs. N6-methyl-threonylcarbamoyl adenosine (m6t6A) is a post-transcriptional modification found at position 37 of tRNAs from bacteria, insect, plants, and mammals. Here, we report that in Escherichia coli, yaeB (renamed as trmO) encodes a tRNA methyltransferase responsible for the N6-methyl group of m6t6A in tRNAThr specific for ACY codons. TrmO has a unique single-sheeted β-barrel structure and does not belong to any known classes of methyltransferases. Recombinant TrmO employs S-adenosyl-L-methionine (AdoMet) as a methyl donor to methylate t6A to form m6t6A in tRNAThr. Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m6t6A was converted to cyclic t6A (ct6A), suggesting that t6A is a common precursor for both m6t6A and ct6A. Furthermore, N6-methylation of t6A enhanced the attenuation activity of the thr operon, suggesting that TrmO ensures efficient decoding of ACY. We also identified a human homolog, TRMO, indicating that m6t6A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.
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