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ABSTRACT Archaeal molecular biology has been a topic of intense research in recent decades as their role in global ecosystems, nutrient cycles, and eukaryotic evolution comes to light. The hypersaline-adapted archaeal speciesHalobacterium salinarumandHaloferax volcaniiserve as important model organisms for understanding archaeal genomics, genetics, and biochemistry, in part because efficient tools enable genetic manipulation. As a result, the number of strains in circulation among the haloarchaeal research community has increased in recent decades. However, the degree of genetic divergence and effects on genetic integrity resulting from the creation and inter-lab transfer of novel lab stock strains remain unclear. To address this, we performed whole-genome re-sequencing on a cross-section of wild-type, parental, and knockout strains in both model species. Integrating these data with existing repositories of re-sequencing data, we identify mutations that have arisen in a collection of 60 strains, sampled from two species across eight different labs. Independent of sequencing, we construct strain lineages, identifying branch points and significant genetic events in strain history. Combining this with our sequencing data, we identify small clusters of mutations that definitively separate lab strains. Additionally, an analysis of gene knockout strains suggests that roughly one in three strains currently in use harbors second-site mutations of potential phenotypic impact. Overall, we find that divergence among lab strains is thus far minimal, though as the archaeal research community continues to grow, careful strain provenance and genomic re-sequencing are required to keep inter-lab divergence to a minimum, prevent the compounding of mutations into fully independent lineages, and maintain the current high degree of reproducible research between lab groups. IMPORTANCEArchaea are a domain of microbial life whose member species play a critical role in the global carbon cycle, climate regulation, the human microbiome, and persistence in extreme habitats. In particular, hypersaline-adapted archaea are important, genetically tractable model organisms for studying archaeal genetics, genomics, and biochemistry. As the archaeal research community grows, keeping track of the genetic integrity of strains of interest is necessary. In particular, routine genetic manipulations and the common practice of sharing strains between labs allow mutations to arise in lab stocks. If these mutations affect cellular processes, they may jeopardize the reproducibility of work between research groups and confound the results of future studies. In this work, we examine DNA sequences from 60 strains across two species of archaea. We identify shared and unique mutations occurring between and within strains. Independently, we trace the lineage of each strain, identifying which genetic manipulations lead to observed off-target mutations. While overall divergence across labs is minimal so far, our work highlights the need for labs to continue proper strain husbandry. 

Abstract Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood.Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin’s cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains. 

Abstract While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeonHaloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation,N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.