MenT nucleotidyltransferase toxins extend tRNA acceptor stems and can be inhibited by asymmetrical antitoxin binding

Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity

“Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics

How does sub-cellular localization affect the fate of bacterial mRNA?

Recently a number of seminal studies have revealed that both sequence and spatio-temporal factors govern RNA decay in bacteria, which is crucial for regulation of gene expression. Ribonucleases have been described that not only exhibit sequence preferences, but also are sub-cellularly localised. Furthermore, the RNA itself is distributed in an organised manner and does not diffuse freely or randomly within the bacterial cells. Thus, even within the sub-micrometer distances of the bacterial intra-cellular space, the positions of the enzymes and their substrates are kept in check. Adding to this complexity is the secondary structure and sequence specificity that many, perhaps all, ribonucleases exhibit, including those that are responsible for "general" RNA degradation. In this review, the implications of these novel findings are discussed and specific examples from Staphylococcus aureus are analysed

An Essential Factor for High Mg2+ Tolerance of Staphylococcus aureus

Internal bacterial concentration of Mg2+, the most abundant divalent cation in living cells, is estimated to be in the single millimolar range. However, many bacteria will thrive in media with only micromolars of Mg2+, by using a range of intensely studied and highly efficient import mechanisms, as well as in media with very high magnesium concentration, presumably mediated by currently unknown export mechanisms. Staphylococcus aureus has a particularly high Mg2+ tolerance for a pathogen, growing unimpaired in up to 770 mM Mg2+, and we here identify SA0657, a key factor in this tolerance. The predicted domain structure of SA0657 is shared with a large number of proteins in bacteria, archaea and even eukarya, for example CorB from Salmonella and the human CNNM protein family. One of the shared domains, a CBS pair potentially involved in Mg2+ sensing, contains the conserved Glycine326 which we establish to be a key residue for SA0657 function. In light of our findings, we propose the name MpfA, Magnesium Protection Factor A, for SA0657

New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus

We have developed a range of vectors for allelic replacements in Staphylococcus aureus to facilitate genetic work in this opportunistic pathogen. The central feature of the vector range is a selection/counterselection system that takes advantage of the 5-fluoroorotic acid (FOA) resistance and pyrimidine prototrophy caused by the loss and gain, respectively, of the pyrF and pyrE genes. This system allows for stringent counterselection of the vectors during the second homologous recombination of a classic allelic replacement. The basic vector pRLY2, which contains the pyrFE genes from Bacillus subtilis, was combined with chloramphenicol, erythromycin, and tetracycline resistance genes and four different versions of nonreplicative or conditionally replicative origins of replication. The choice between these 12 different pRLY vectors allows for high versatility and ensures that the vectors can be used in virtually any genetic background. Finally, as proof of concept, we present six deletions or modifications of components in the S. aureus degradosome as well as the operon containing the cshB DEAD box helicase

EMOTE-conv: A Computational Pipeline to Convert Exact Mapping of Transcriptome Ends (EMOTE) Data to the Lists of Quantified Genomic Positions Correlated to Related Genomic Information

The determination of 5'-ends of RNA molecules is important for understanding various steps of gene expression and regulation in all organisms, such as transcription initiation, RNA maturation, and degradation. While previous methods like Phosphorylation Assay By Ligation of Oligonucleotides, Rapid Amplification of cDNA Ends, Capped Analysis of Gene expression, tag RNA-seq and differential RNA-seq have their own specifications and limitations, Exact Mapping Of Transcriptome Ends (EMOTE) assay has been designed to determine the 5'-ends of RNAs on a transcriptome-wide scale. EMOTE-conv exploits the raw sequence reads generated from the EMOTE assay which is, to the best of our knowledge, the only method that can map the exact RNA 5'-ends of of all types on a transcriptome wide scale. It converts EMOTE data into the quantified list of genomic positions that corresponds to the 5'-end of RNA, signifying 5'-base RNA and the other related genomic information. EMOTE-conv is platform-independent, user-friendly and easy-to-use. It can be used with the data generated from other sequencing platforms with a converter as well as the data generated from any organism, species or strains. The EMOTE-conv software is available at: http://sourceforge.net/projects/emotecon

Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2

The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS) elements and a single nonautonomous mobile element, SM2. For each of these, the element was identified and shown to be present at its original genomic position, consistent with a progressive increase in the copy numbers of the mobile elements. In addition, several base pair substitutions, as well as small deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries were defined using a specially developed “in vitro library” strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while the other involved the inversion of a 25-kb region. Both were bordered by IS elements and were inferred to have arisen through recombination events. The results underline the caution required in working experimentally with an organism such as S. solfataricus with a continually changing genome

Transcriptome-Wide Analyses of 5′-Ends in RNase J Mutants of a Gram-Positive Pathogen Reveal a Role in RNA Maturation, Regulation and Degradation

The long RNA (ribonucleic acid) chains are key intermediates in the transfer of information from the genes on chromosomes to the production of protein. An RNA copy (mRNA) is transcribed from the gene, and this copy is then translated into protein by a complex molecular machine called the ribosome. The amount of mRNA copies of a given gene is therefore important for how much of the corresponding protein can be generated. This “pool” of mRNA depends on how many copies are made per second, but also on how many copies disappear due to degradation. In this study, we examine mutants of the bacterial pathogen Staphylococcus aureus, which have lost one or both of their RNase J genes (ribonuclease J). These mutants grow relatively fine under standard laboratory conditions, but will stop growing if they are stressed by alternative food sources or temperatures equivalent to a high fever (42°C). We show that the RNase J enzymes are major players in the degradation of RNA by removing one link at a time in the RNA-chains, thus influencing the pool of mRNAs in the cell. Furthermore, certain RNAs are processed to stable and active forms by RNase J, instead of being degraded