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1. Expanding the capabilities of MuGENT for large-scale genetic engineering of the fastest-replicating species, Vibrio natriegens

   https://doi.org/10.1128/spectrum.03964-23  

   Glasgo, Liz D; Lukasiak, Katie L; Zinser, Erik R (June 2024, Microbiology Spectrum)

   Cardona, Silvia T (Ed.)

   ABSTRACT The fastest replicating bacteriumVibrio natriegensis a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential ofV. natriegensand collectively expand upon its utility as an emerging model organism for synthetic biology. IMPORTANCEVibrio natriegensis an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of theV. natriegensmodel system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing “clean” derivative cells. Finally, we show the full extent of this technique’s capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to theVibriocommunity and specifically to the advancement ofV. natriegensas a model system. 

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2. Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system

   https://doi.org/10.1002/dvdy.32  

   Stahl, Bethany_A; Peuß, Robert; McDole, Brittnee; Kenzior, Alexander; Jaggard, James_B; Gaudenz, Karin; Krishnan, Jaya; McGaugh, Suzanne_E; Duboue, Erik_R; Keene, Alex_C; et al (April 2019, Developmental Dynamics)

   Abstract BackgroundAstyanax mexicanusis a well‐established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. ResultsHere, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficientTol2system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population ofAstyanax. To define specific cell‐types, a Cntnap2‐mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. ConclusionThe lines provide proof‐of‐principle for the application ofTol2transgenic technology inA. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype‐phenotype gap. 

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3. Hypothesized life cycle of the snow algae Chlainomonas sp. (Chlamydomonadales, Chlorophyta) from the Cascade Mountains, USA

   https://doi.org/10.1111/jpy.13454  

   Matsumoto, Maya; Hanneman, Clare; Camara, A_G; Krueger‐Hadfield, Stacy_A; Hamilton, Trinity_L; Kodner, Robin_B (May 2024, Journal of Phycology)

   Abstract Chlainomonas(Chlamydomonadales, Chlorophyta) is one of the four genera of snow algae known to produce annual pink or red blooms in alpine snow. NoChlainomonasspecies have been successfully cultured in the laboratory, but diverse cell types have been observed from many field‐collected samples, from multiple species. The diversity of morphologies suggests these algae have complex life cycles with changes in ploidy. Over 7 years (2017–2023), we observed seasonal blooms dominated by aChlainomonasspecies from late spring through the summer months on a snow‐on‐lake habitat in an alpine basin in the North Cascade Mountains of Washington, USA. The Bagley LakeChlainomonasis distinct from previously reported species based on morphology and sequence data. We observed a similar collection of cell types observed in otherChlainomonasspecies, with the addition of swarming biflagellate cells that emerged from sporangia. We present a life cycle hypothesis for this species that links cell morphologies observed in the field to seasonally available habitat. The progression of cell types suggests cells are undergoing both meiosis and fertilization in the life cycle. Since the life cycle is the most fundamental biological feature of an organism, with direct consequences for evolutionary processes, it is critical to understand how snow algal life cycles will influence their responses to changes in their habitat driven by climate warming. For microbial taxa that live in extreme environments and are difficult to culture, temporal field studies, such as we report here, may be key to creating testable hypotheses for life cycles. 

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

   https://doi.org/10.1111/mmi.15225  

   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.; et al (January 2024, Molecular Microbiology)

   Abstract Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt‐loving organismHaloferax volcaniiexhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related speciesHalobacterium salinarumby activating the expression of enzyme‐coding genes in the gluconeogenesis metabolic pathway. InHbt. salinarum, TrmB‐dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions inHfx. volcanii. The ∆trmBstrain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we nametrmBsuppressor, or TbsP (a.k.a. “tablespoon”). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild‐type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose‐dependent transcription ofgapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients inHfx. volcanii. 

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5. PECTIN METHYLESTERASE51 Affects Stomatal Dimensions, Rosette Area, and Root Length in Arabidopsis thaliana

   https://doi.org/10.1002/pld3.70066  

   Dunham, Angelica L.; Tamadaddi, Chetana; Marshall, Rayna; Anderson, Charles T. (April 2025, Plant Direct)

   ABSTRACT Pectins are abundant in the cell walls of eudicot plants and have been implicated in determining the development and biomechanics of stomatal guard cells, which expand and contract dynamically to open and close stomatal pores on the plant surface, modulating photosynthesis and water transport. Pectic homogalacturonan is delivered to the cell wall in a methylesterified form but can be demethylesterified in the wall by pectin methylesterases, increasing both its ability to form crosslinks via calcium and its susceptibility to degradation by endogenous pectinases. Although a few pectin methylesterases have been implicated in stomatal development and function, this large family of proteins has not been fully characterized with respect to how they modulate stomatal guard cells. Here, we characterized the function of PECTIN METHYLESTERASE51 (PME51), a pectin methylesterase–encoding gene that is expressed in developing guard cells, in stomatal morphogenesis in seedlings and adult plants ofArabidopsis thaliana. OverexpressingPME51led to smaller adult plants with smaller stomatal complexes and subtle changes in initial responses to opening and closure stimuli, whereas knocking outPME51resulted in smaller stomatal complexes and longer roots in seedlings. We observed changes in pectin labeling in knockout and overexpression plants that imply a specific function for PME51 in modulating the degree of methylesterification for homogalacturonan. Together, these findings expand our understanding of how pectin modification by pectin methylesterases affects the development and function of stomatal guard cells, which must maintain a balance of strength and flexibility to optimize plant growth. 

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