Pantoea Bacteriophage vB_PagS_MED16-A Siphovirus Containing a 2'-Deoxy-7-amido-7-deazaguanosine-Modified DNA.

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.

Reductive Evolution and Diversification of C5-Uracil Methylation in the Nucleic Acids of Mollicutes.

The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup use the ancestral tetrahydrofolate-dependent TrmFO enzyme to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S rRNA. RlmFO has replaced the S-adenosyl-L-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U-modifying enzymes that is highly dynamic and has undergone horizontal transfer.

The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALysUUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Gene Graphics: a genomic neighborhood data visualization web application.

Summary: The examination of gene neighborhood is an integral part of comparative genomics but no tools to produce publication quality graphics of gene clusters are available. Gene Graphics is a straightforward web application for creating such visuals. Supported inputs include National Center for Biotechnology Information gene and protein identifiers with automatic fetching of neighboring information, GenBank files and data extracted from the SEED database. Gene representations can be customized for many parameters including gene and genome names, colors and sizes. Gene attributes can be copied and pasted for rapid and user-friendly customization of homologous genes between species. In addition to Portable Network Graphics and Scalable Vector Graphics, produced representations can be exported as Tagged Image File Format or Encapsulated PostScript, formats that are standard for publication. Hands-on tutorials with real life examples inspired from publications are available for training. Availability and implementation: Gene Graphics is freely available at https://katlabs.cc/genegraphics/ and source code is hosted at https://github.com/katlabs/genegraphics. Contact: katherinejh@ufl.edu or remizallot@ufl.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Discovery of the ß-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. N(6)-methyl-threonylcarbamoyl adenosine (m(6)t(6)A) 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 N(6)-methyl group of m(6)t(6)A in tRNA(Thr) specific for ACY codons. TrmO has a unique single-sheeted ß-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 t(6)A to form m(6)t(6)A in tRNA(Thr). Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m(6)t(6)A was converted to cyclic t(6)A (ct(6)A), suggesting that t(6)A is a common precursor for both m(6)t(6)A and ct(6)A. Furthermore, N(6)-methylation of t(6)A 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 m(6)t(6)A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.

YeiR: a metal-binding GTPase from Escherichia coli involved in metal homeostasis.

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 is linked to zinc depletion. In vitro, the tagged protein binds several Zn(2+) 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 Zn(2+) binding. These results support the predicted connection between the COG0523 P-loop GTPases and roles in metal homeostasis.

Experimental evolution of a facultative thermophile from a mesophilic ancestor.

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.