Novel genomic island modifies DNA with 7-deazaguanine derivatives

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[subscript 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[subscript 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[subscript 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.Deutsche ForschungsgemeinschaftSingapore-MIT Alliance in Research and Technology (SMART

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

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

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

Nucleoside analysis analysis in wild type <i>Haloferax volcanii</i> and the Δ<i>pcc1</i> mutant cells.

<p>tRNAs were extracted from each strain and hydrolyzed to nucleosides. The nucleoside content was determined by HPLC with detection by UV/Vis at 254 nm. Analyses were performed in triplicate from independent cultures. <b>A</b>. HPLC chromatographs of nucleosides from wild-type, wild-type with synthesized t⁶A added, or Δ<i>pcc1</i>. t⁶A elutes at 24 minutes. <b>B</b>. Comparison of the t⁶A peak area of wild-type and Δ<i>pcc1</i>. The ratios of Ψ-modified base/m₂²G were used to normalize tRNA concentrations across samples with t⁶A peak area of wild-type set at 100%. Δ<i>pcc1</i> contains approximately 19% less t⁶A than wild-type (<i>P</i> = 0.02).</p

Flow cytometry analysis of <i>Haloferax volcanii</i> H195 and H133 Δ<i>pcc1</i> mutant cells.

<p>H195 (based on H133, with a <i>bga</i>Ha-Bb deletion, and a <i>leuB</i>- <i>Ag1</i> allele, which has flow cytometry profiles highly similar to H133– data not shown) <b>A.</b> Cell size as determined by forward light scatter of H195, and of Δ<i>pcc1</i> (HAN16). <b>B.</b> Nucleic acid content as determined by acridine orange fluorescence of H195 (wild-type) and in Δ<i>pcc1</i> (HAN16).</p

Strains used in this study.

<p>Strains used in this study.</p