Acinetobacter baylyi strain ADP1 is a gram-negative bacterium normally studied because of its high competence for genetic transformation and its ability to catabolize plant-derived aromatic compounds. A previous study has identified that the gene cluster ACIAD1969-ACIAD1952 contains genes that may be responsible for potassium tellurite resistance, as well as other proteins that are “hypothetical.” Our goal was to use bioinformatics to investigate this gene cluster and to determine whether it played a role in potassium tellurite resistance as well as twitching motility. Our results indicate that the gene cluster is actually composed of four different operons that play a role in tellurite resistance. We also found that the gene cluster was most likely inherited from horizontal gene transfer, as it is not found in any other Acinetobacter strains. Furthermore, all genes except ACIAD1956, ACIAD1962 and ACIAD1964 are responsible for potassium tellurite resistance in ADP1 and all mutants exhibit twitching motility defects. Our results indicate that the genes in the gene cluster ACIAD1969-ACIAD1952 encode proteins and should no longer be considered “hypothetical.”
Reproductive mechanisms play a vital role in a species’ ability to proliferate and evolve. The complex and dynamic mating strategies that yeast species employ to effectively proliferate provide insight into how various reproductive models operate—comparing species with these unique capabilities can illuminate how sex and reproduction have evolved over time. The methylotrophic yeast Ogataea polymorpha, like many yeast species, exhibits asexual and sexual reproductive capabilities and can undergo mating-type switching before mating. Mating-type switching is a genetic process governed by the MAT locus, and recent investigations have characterized the structure and function of the locus in several species. However, unlike other species, the molecular mechanisms and specific environmental conditions required for mating and mating-type switching in methylotrophic yeast are poorly understood and contemporary testing protocols are time consuming. Here, we began creation of a high throughput assay to quantify mating and mating-type switching frequencies with flow cytometry. We designed three flow cytometry-based assays, including (1) utilizing nuclear DNA staining and cell cycle arrest to identify variations in ploidy indicative of mating, (2) bilateral mating with N- and C-terminus ends of Green Fluorescent Protein (GFP) in mating partners to track mating frequencies, and (3) molecular fluorescent tagging at mating-type specific genes of the MAT locus with genes for GFP and Red Fluorescent Protein (RFP) such that mating-type specific gene expression can be indicated via fluorescence to observe mating-type switching frequencies. Nuclear DNA staining protocols in O. polymorpha produced indistinguishable cell cycle histogram plots, indicating a need for an adapted DNA staining protocol for the species. Bilateral mating is effective at quantifying mating frequencies in Saccharomyces cerevisiae but fails to work effectively in O. polymorpha. Transformations for MAT locus molecular fluorescent tagging are in progress and have yet to be tested on the flow cytometer. Complete development of these assays will streamline the process of studying the genetic and environmental conditions in which yeast reproduce. Establishing more efficient methods to investigate the molecular dynamics of mating and mating-type switching will further our understanding of how reproduction has evolved across yeast species.