<scp>TbsP</scp> and <scp>TrmB</scp> jointly regulate <i>gapII</i> to influence cell development phenotypes in the archaeon <i>Haloferax volcanii</i>

Hackley, Rylee K.; Hwang, Sungmin; Herb, Jake T.; Bhanap, Preeti; Lam, Katie; Vreugdenhil, Angie; Darnell, Cynthia L.; Pastor, Mar Martinez; Martin, Johnathan H.; Maupin‐Furlow, Julie A.; Schmid, Amy K.

doi:10.1111/mmi.15225

Cited by 3 publications

(1 citation statement)

References 87 publications

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“…This type of morphogenic transformation occurs in a variety of conditions, including for motility, early-log phase growth, and in response to micronutrient depletion 18,22 . CetZ1 is also expected to be essential in other conditions that promote rod development or cell elongation 23,24 . Based on monomer and protofilament crystal structures 18 , and the common properties of TSF proteins 25 , CetZs are predicted to polymerize through binding GTP, forming protofilaments and potentially higher-order assemblies like those of tubulin and FtsZ.…”

Section: Introductionmentioning

confidence: 99%

Dynamic self-association of archaeal tubulin-like protein CetZ1 drivesHaloferax volcaniimorphogenesis

de Silva,

Shinde,

Brown

et al. 2024

Preprint

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Tubulin superfamily (TSF) proteins include the well-known eukaryotic tubulin and bacterial FtsZ families, and lesser-known archaeal CetZ family. In eukaryotes and bacteria, GTP-dependent polymerization and self-association of tubulin and FtsZ protofilaments are integral to the formation of cytoskeletal structures with essential roles in cell division, growth, and morphology. Archaeal CetZs are implicated in the control of cell shape and motility through unknown mechanisms. Here, we reveal a sequence of subcellular localization patterns of CetZ1, the prototypical member of the CetZ family, during stages ofHaloferax volcaniirod cell development, in which it plays an essential role. Like tubulin and FtsZ, we found that CetZ1 formed GTP-dependent polymers in vitro, which appear to associate laterally as irregular polymer bundles. Mutations targeting regions predicted to mediate self-association and dynamic turnover of CetZ1, including the longitudinal (GTPase T7 and T4 loops) and lateral assembly interfaces, perturbed or altered rod shape development and subcellular assembly and dynamics, and caused corresponding effects on polymerization in vitro. Remarkably, a conspicuous amphipathic protrusion in the large microtubule (M-) loop, a characteristic of the CetZ1 subfamily, also strongly influenced function and assembly. Our findings reveal the importance of dynamic CetZ1 self-association in cellular morphogenesis involving multiple regions of the TSF fold, including tubulin- and FtsZ-like structural characteristics and CetZ1-specific features. Furthermore, they support a mechanism involving CetZ1 dynamic guidance of cell envelope-associated structures that reshape the cell during morphogenesis.

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Section: Introductionmentioning

confidence: 99%

Dynamic self-association of archaeal tubulin-like protein CetZ1 drivesHaloferax volcaniimorphogenesis

de Silva,

Shinde,

Brown

et al. 2024

Preprint

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CetZ1-dependent assembly and positioning of the motility machinery inhaloarchaea

Brown,

Islam,

Ruan

et al. 2024

Preprint

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The tubulin-like CetZ proteins are archaeal cytoskeletal proteins that contribute to cell shape and swimming motility in the halophilic archaeon Haloferax volcanii. Currently it is unknown whether CetZs contribute to motility solely through their control of cell shape or in other ways too. Here, we used cryo-electron and fluorescence microscopy to observe H. volcanii cell surface filaments and the localisation of the motility machinery, respectively, in strains with cetZ1 or cetZ2 deletion, overexpression, and polymerisation-defective mutant backgrounds. Our results show that CetZ1 has an important role at the poles of mature motile rod cells for the assembly of key motility proteins, including ArlD1, a constituent of the archaellum base, and the chemotaxis sensory array adapter CheW1 and signal transducer CheY. Importantly, overproduction of CetZ1 and CetZ1-mTurquoise2 inhibited motility and reduced the frequency of localisation of the motility machinery markers but did not affect rod-shape in swimming cells. Our data suggest that CetZ1 acts as a polar cytoskeletal structure that orchestrates the assembly and positioning of both major components of the motility machinery. The multifunctionality and dynamic redeployment of CetZ1 during motile cell development is reminiscent of eukaryotic cytoskeletal proteins and the roles of tubulin at the base of the eukaryotic flagellum.

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Genomic re-sequencing reveals mutational divergence across genetically engineered strains of model archaea

Soborowski,

Hackley,

Hwang

et al. 2024

Preprint

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Because archaea are the evolutionary ancestors of eukaryotes, archaeal molecular biology has been a topic of intense recent research. The hypersaline adapted archaeal speciesHalobacterium salinarumandHaloferax volcaniiserve as important model organisms because facile tools enable genetic manipulation. As a result, the number of strains in circulation among the haloarchaeal research community has increased over the last few decades. However, the degree of genetic divergence and effects on genetic integrity during inter-lab transfers remain unclear. To address this question, 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 2 species across 8 different labs. Independent of sequencing, we construct strain lineages, identifying branch points and significant genetic effects 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 1 in 3 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 in the haloarchaeal research community.Data SummaryNovel sequencing data for this project was submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and can be found under bioproject accession PRJNA1120443. SRA accessions for previously published sequencing data are available in supplementary table 1. R code for performing analysis and generating figures is available athttps://github.com/andrew-soborowski/halophile_genome_resequencing.Impact StatementArchaea are important due to their shared evolutionary history with eukaryotes. As the archaeal research community grows, keeping track of the genetic integrity of archaeal 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.

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TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

Cited by 3 publications

References 87 publications

Dynamic self-association of archaeal tubulin-like protein CetZ1 drivesHaloferax volcaniimorphogenesis

Dynamic self-association of archaeal tubulin-like protein CetZ1 drivesHaloferax volcaniimorphogenesis

CetZ1-dependent assembly and positioning of the motility machinery inhaloarchaea

Genomic re-sequencing reveals mutational divergence across genetically engineered strains of model archaea

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