Patrick C. Thiaville

USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus

ABSTRACT 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 Kochs 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.

Genotype Is Correlated with but Does Not Predict Virulence of Vibrio vulnificus Biotype 1 in Subcutaneously Inoculated, Iron Dextran-Treated Mice

ABSTRACT 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.

Global translational impacts of the loss of the tRNA modification t6A in yeast

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.

Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A), a universal modification of tRNA

The tRNA modification field has a rich literature covering biochemical analysis going back more than 40 years, but many of the corresponding genes were only identified in the last decade. In recent years, comparative genomic-driven analysis has allowed for the identification of the genes and subsequent characterization of the enzymes responsible for N6-threonylcarbamoyladenosine (t6A). This universal modification, located in the anticodon stem-loop at position 37 adjacent to the anticodon of tRNAs, is found in nearly all tRNAs that decode ANN codons. The t6A biosynthesis enzymes and synthesis pathways have now been identified, revealing both a core set of enzymes and kingdom-specific variations. This review focuses on the elucidation of the pathway, diversity of the synthesis genes, and proposes a new nomenclature for t6A synthesis enzymes.

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Significance The discovery of a novel modification system that inserts 7-deazaguanine derivatives in DNA, modifications thought until now to occur only in RNA, is an excellent illustration of the power of biological evolution to alter the ultimate function not only of the distinct proteins but also of entire metabolic pathways. The extensive lateral transfer of the gene cluster responsible for this modification highlights its significance as a previously unrecognized foreign DNA defense system that bacteria and phages use to protect their genomes. The characterization of these DNA modification pathways also opens the door to novel tools to manipulate nucleic acids. The discovery of ∼20-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 ∼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 preQ0 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.

Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice

Vibrio vulnificus is Gram‐negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran‐treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony‐forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran‐treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid‐containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12–15 h infection period were not significantly different in iron dextran‐treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non‐iron dextran‐treated mice with clinical strains demonstrated that the greatest effect of iron dextran‐treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran‐treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15–28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram‐negative bacteria.

Discovery of the β-barrel–type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs

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.

Cross kingdom functional conservation of the core universally conserved threonylcarbamoyladenosine tRNA synthesis enzymes.

ABSTRACT Threonylcarbamoyladenosine (t6A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t6A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t6A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332–6346, 2013, http://dx.doi.org/10.1093/nar/gkt322). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t6A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t6A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli. The data revealed a potential for TC-AMP channeling in the t6A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t6A and bring additional advancement in our understanding of the reaction mechanism.

Essentiality of threonylcarbamoyladenosine (t(6)A), a universal tRNA modification, in bacteria.

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.

A Genetic Investigation of the KEOPS Complex in Halophilic Archaea

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.