Background The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times. Results Two of the clones recovered from this enrichment process, sur2 and sur3, exhibited significantly increased survival relative to wild-type bacteria. In coinfection experiments, the ratio of sur2 colonies to wild-type colonies was 1:1 at 0 hours but increased to 20:1 at 24 hours post phagocytosis. The ratio of sur3 colonies to wild-type colonies was 1:1 at 0 hours and 5:1 at 24 hours. The M. tuberculosis ORFs responsible for increased survival were shown to be Rv0365c for the sur2 clone and Rv2235 for the sur3 clone. These ORFs encode proteins with as-of-yet unknown functions. Conclusions We identified two M. tuberculosis ORFs which may be involved in the ability of tubercle bacilli to survive in macrophages.

Background In global gene expression profiling experiments, variation in the expression of genes of interest can often be hidden by general noise. To determine how biologically significant variation can be distinguished under such conditions we have analyzed the differences in gene expression when Bacillus subtilis is grown either on methionine or on methylthioribose as sulfur source. Results An unexpected link between arginine metabolism and sulfur metabolism was discovered, enabling us to identify a high-affinity arginine transport system encoded by the yqiXYZ genes. In addition, we tentatively identified a methionine/methionine sulfoxide transport system which is encoded by the operon ytmIJKLMhisP and is presumably used in the degradation of methionine sulfoxide to methane sulfonate for sulfur recycling. Experimental parameters resulting in systematic biases in gene expression were also uncovered. In particular, we found that the late competence operons comE, comF and comG were associated with subtle variations in growth conditions. Conclusions Using variance analysis it is possible to distinguish between systematic biases and relevant gene-expression variation in transcriptome experiments. Co-variation of metabolic gene expression pathways was thus uncovered linking nitrogen and sulfur metabolism in B. subtilis.

Background In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus. Results Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene). Conclusions We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.

Background The first two steps in the capping of cellular mRNAs are catalyzed by the enzymes RNA triphosphatase and RNA guanylyltransferase. Although structural and mechanistic differences between fungal and mammalian RNA triphosphatases recommend this enzyme as a potential antifungal target, it has not been determined if RNA triphosphatase is essential for the growth of fungal species that cause human disease. Results We show by classical genetic methods that the triphosphatase (Pct1) and guanylyltransferase (Pce1) components of the capping apparatus in the fission yeast Schizosaccharomyces pombe are essential for growth. We were unable to disrupt both alleles of the Candida albicans RNA triphosphatase gene CaCET1, implying that the RNA triphosphatase enzyme is also essential for growth of C. albicans, a human fungal pathogen. Conclusions Our results provide the first genetic evidence that cap synthesis is essential for growth of an organism other than Saccharomyces cerevisiae and they validate RNA triphosphatase as a target for antifungal drug discovery.

Culture presence/absence of M. avium and M. intracellulare by sample qPCR M. avium and M. intracellular detection and quatification by sample. This dataset is associated with the following publication: Pfaller, S., D. King, J. Mistry, and M. Donohue. Occurrence Revisited - Mycobacterium avium and Mycobacterium intracellulare in Potable Water in the USA. Applied Microbiology and Biotechnology. Springer, New York, NY, USA, 106: 2715–2727, (2022).

The dataset contains the first draft genome sequence of the type strain of Mycobacterium chimaera, Fl-0169. This dataset is associated with the following publication: Pfaller, S., V. Tokarev, C. Kessler, C. McLimans, V. Gomez-Alvarez, J. Wright, D. King, and R. Lamendella. Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169. Genome Announcements. American Society for Microbiology, Washington, DC, USA, 5(8): e01620-16, (2017).

Background Methylthioadenosine, the main by-product of spermidine synthesis, is degraded in Bacillus subtilis as adenine and methylthioribose. The latter is an excellent sulfur source and the precursor of quorum-sensing signalling molecules. Nothing was known about methylthioribose recycling in this organism. Results Using trifluoromethylthioribose as a toxic analog to select for resistant mutants, we demonstrate that methylthioribose is first phosphorylated by MtnK, methylthioribose kinase, the product of gene mtnK (formerly ykrT), expressed as an operon with mtnS (formerly ykrS) in an abundant transcript with a S-box leader sequence. Although participating in methylthioribose recycling, the function of mtnS remained elusive. We also show that MtnK synthesis is boosted under starvation condition, in the following decreasing order: carbon-, sulfur- and nitrogen-starvation. We finally show that this enzyme is part of the family Pfam 01633 (choline kinases) which belongs to a large cluster of orthologs comprizing antibiotic aminoglycoside kinases and protein serine/threonine kinases. Conclusions The first step of methylthioribose recycling is phosphoryltaion by MTR kinase, coded by the mtnK (formerly ykrT) gene. Analysis of the neighbourhood of mtnK demonstrates that genes located in its immediate vicinity (now named mtnUVWXYZ, formerly ykrUVWXYZ) are also required for methylthioribose recycling.

Modeling data and analysis scripts generated during the current study are available in the github repository: https://github.com/USEPA/CompTox-MIEML. RefChemDB is available for download as supplemental material from its original publication (PMID: 30570668). LINCS gene expression data are publicly available and accessible through the gene expression omnibus (GSE92742 and GSE70138) at https://www.ncbi.nlm.nih.gov/geo/ . This dataset is associated with the following publication: Bundy, J., R. Judson, A. Williams, C. Grulke, I. Shah, and L. Everett. Predicting Molecular Initiating Events Using Chemical Target Annotations and Gene Expression. BioData Mining. BioMed Central Ltd, London, UK, issue}: 7, (2022).