Publications by Colleges and Departments (MSU - Bozeman)
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Item Discovering unknown associations between prokaryotic receptors and their ligands(Proceedings of the National Academy of Sciences, 2023-11) Dlakić, MensurThe motility of microorganisms through the environment is driven by chemical gradients: They move towards nutrients and away from signals that indicate unfavorable conditions. This chemotaxis is mediated by transmembrane chemoreceptors that recognize one or many target molecules. In most cases, the encounter with a ligand is recorded by a periplasmic sensor domain, which in turn transmits a signal through the membrane to a cytoplasmic signaling domain (1). Under conditions of environmental stress, these signaling cascades may induce profound lifestyle changes from planktonic cells to a biofilm or from active to inactive cells (2). While it is relatively straightforward to annotate most prokaryotic chemoreceptors from the ever-increasing number of sequenced genomes and environmental samples, the identity of their binding partners is often not clear from protein sequence. As the specificity of downstream signaling events is determined by the sensor domain, it is critical to learn about novel pairings between ligands and their receptors. Using a range of computational and experimental approaches, Cerna-Vargas et al. show in PNAS that a subset of a wide-spread group of dCache_1 receptors evolved to recognize various types of biological amines.Item Bacteroides thetaiotaomicron, a Model Gastrointestinal Tract Species, Prefers Heme as an Iron Source, Yields Protoporphyrin IX as a Product, and Acts as a Heme Reservoir(Bacteroides thetaiotaomicron, a Model Gastrointestinal Tract Species, Prefers Heme as an Iron Source, Yields Protoporphyrin IX as a Product, and Acts as a Heme Reservoir, 2023-03) Meslé, Margaux M.; Gray, Chase R.; Dlakić, Mensur; DuBois, Jennifer L.Members of the phylum Bacteroidetes are abundant in healthy gastrointestinal (GI) tract flora. Bacteroides thetaiotaomicron is a commensal heme auxotroph and representative of this group. Bacteroidetes are sensitive to host dietary iron restriction but proliferate in heme-rich environments that are also associated with colon cancer. We hypothesized that B. thetaiotaomicron may act as a host reservoir for iron and/or heme. In this study, we defined growth-promoting quantities of iron for B. thetaiotaomicron. B. thetaiotaomicron preferentially consumed and hyperaccumulated iron in the form of heme when presented both heme and nonheme iron sources in excess of its growth needs, leading to an estimated 3.6 to 8.4 mg iron in a model GI tract microbiome consisting solely of B. thetaiotaomicron. Protoporphyrin IX was identified as an organic coproduct of heme metabolism, consistent with anaerobic removal of iron from the heme leaving the intact tetrapyrrole as the observed product. Notably, no predicted or discernible pathway for protoporphyrin IX generation exists in B. thetaiotaomicron. Heme metabolism in congeners of B. thetaiotaomicron has previously been associated with the 6-gene hmu operon, based on genetic studies. A bioinformatics survey demonstrated that the intact operon is widespread in but confined to members of the Bacteroidetes phylum and ubiquitous in healthy human GI tract flora. Anaerobic heme metabolism by commensal Bacteroidetes via hmu is likely a major contributor to human host metabolism of the heme from dietary red meat and a driver for the selective growth of these species in the GI tract consortium.Item The Pseudomonas aeruginosa RpoH (σ32) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance(MDPI AG, 2023-01) Williamson, Kerry S.; Dlakić, Mensur; Akiyama, Tatsuya; Franklin, Michael J.The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.Item Sulfur cycling and host-virus interactions in Aquificales-dominated biofilms from Yellowstone’s hottest ecosystems(Springer Nature, 2021-10) McKay, Luke J.; Nigro, Olivia D.; Dlakić, Mensur; Luttrell, Karen M.; Rusch, Douglas B.; Fields, Matthew W.; Inskeep, William P.Modern linkages among magmatic, geochemical, and geobiological processes provide clues about the importance of thermophiles in the origin of biogeochemical cycles. The aim of this study was to identify the primary chemoautotrophs and host–virus interactions involved in microbial colonization and biogeochemical cycling at sublacustrine, vapor-dominated vents that represent the hottest measured ecosystems in Yellowstone National Park (~140 °C). Filamentous microbial communities exposed to extreme thermal and geochemical gradients were sampled using a remotely operated vehicle and subjected to random metagenome sequencing and microscopic analyses. Sulfurihydrogenibium (phylum Aquificae) was the predominant lineage (up to 84% relative abundance) detected at vents that discharged high levels of dissolved H2, H2S, and CO2. Metabolic analyses indicated carbon fixation by Sulfurihydrogenibium spp. was powered by the oxidation of reduced sulfur and H2, which provides organic carbon for heterotrophic community members. Highly variable Sulfurihydrogenibium genomes suggested the importance of intra-population diversity under extreme environmental and viral pressures. Numerous lytic viruses (primarily unclassified taxa) were associated with diverse archaea and bacteria in the vent community. Five circular dsDNA uncultivated virus genomes (UViGs) of ~40 kbp length were linked to the Sulfurihydrogenibium metagenome-assembled genome (MAG) by CRISPR spacer matches. Four UViGs contained consistent genome architecture and formed a monophyletic cluster with the recently proposed Pyrovirus genus within the Caudovirales. Sulfurihydrogenibium spp. also contained CRISPR arrays linked to plasmid DNA with genes for a novel type IV filament system and a highly expressed β-barrel porin. A diverse suite of transcribed secretion systems was consistent with direct microscopic analyses, which revealed an extensive extracellular matrix likely critical to community structure and function. We hypothesize these attributes are fundamental to the establishment and survival of microbial communities in highly turbulent, extreme-gradient environments.Item The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases(American Association for the Advancement of Science, 2021-10) Altae-Tran, Han; Kannan, Soumya; Demircioglu, F. Esra; Oshiro, Rachel; Nety, Suchita P.; McKay, Luke J.; Dlakić, Mensur; Inskeep, William P.; Makarova, Kira S.; Macrae, Rhiannon K.; Koonin, Eugene V.; Zhang, FengIscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.