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1. Genome editing in ubiquitous freshwater Actinobacteria

   https://doi.org/10.1128/aem.00865-24  

   Bairagi, Nachiketa; Keffer, Jessica L; Heydt, Jordan C; Maresca, Julia A (November 2024, Applied and Environmental Microbiology)

   Bose, Arpita (Ed.)

   ABSTRACT Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show thatRhodoluna lacicolaandAurantimicrobium photophilum, which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities. IMPORTANCETo advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments. 

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2. CaeNDR, the Caenorhabditis Natural Diversity Resource

   https://doi.org/10.1093/nar/gkad887  

   Crombie, Timothy A.; McKeown, Ryan; Moya, Nicolas D.; Evans, Kathryn S.; Widmayer, Samuel J.; LaGrassa, Vincent; Roman, Natalie; Tursunova, Orzu; Zhang, Gaotian; Gibson, Sophia B.; et al (October 2023, Nucleic Acids Research)

   Abstract Studies of model organisms have provided important insights into how natural genetic differences shape trait variation. These discoveries are driven by the growing availability of genomes and the expansive experimental toolkits afforded to researchers using these species. For example, Caenorhabditis elegans is increasingly being used to identify and measure the effects of natural genetic variants on traits using quantitative genetics. Since 2016, the C. elegans Natural Diversity Resource (CeNDR) has facilitated many of these studies by providing an archive of wild strains, genome-wide sequence and variant data for each strain, and a genome-wide association (GWA) mapping portal for the C. elegans community. Here, we present an updated platform, the Caenorhabditis Natural Diversity Resource (CaeNDR), that enables quantitative genetics and genomics studies across the three Caenorhabditis species: C. elegans, C. briggsae and C. tropicalis. The CaeNDR platform hosts several databases that are continually updated by the addition of new strains, whole-genome sequence data and annotated variants. Additionally, CaeNDR provides new interactive tools to explore natural variation and enable GWA mappings. All CaeNDR data and tools are accessible through a freely available web portal located at caendr.org. 

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3. Promoter Architecture Differences among Alphaproteobacteria and Other Bacterial Taxa

   https://doi.org/10.1128/mSystems.00526-21  

   Myers, Kevin S.; Noguera, Daniel R.; Donohue, Timothy J. (August 2021, mSystems)

   Faust, Karoline (Ed.)

   ABSTRACT Much of our knowledge of bacterial transcription initiation has been derived from studying the promoters of Escherichia coli and Bacillus subtilis . Given the expansive diversity across the bacterial phylogeny, it is unclear how much of this knowledge can be applied to other organisms. Here, we report on bioinformatic analyses of promoter sequences of the primary σ factor (σ 70 ) by leveraging publicly available transcription start site (TSS) sequencing data sets for nine bacterial species spanning five phyla. This analysis identifies previously unreported differences in the −35 and −10 elements of σ 70 -dependent promoters in several groups of bacteria. We found that Actinobacteria and Betaproteobacteria σ 70 -dependent promoters lack the TTG triad in their −35 element, which is predicted to be conserved across the bacterial phyla. In addition, the majority of the Alphaproteobacteria σ 70 -dependent promoters analyzed lacked the thymine at position −7 that is highly conserved in other phyla. Bioinformatic examination of the Alphaproteobacteria σ 70 -dependent promoters identifies a significant overrepresentation of essential genes and ones encoding proteins with common cellular functions downstream of promoters containing an A, C, or G at position −7. We propose that transcription of many σ 70 -dependent promoters in Alphaproteobacteria depends on the transcription factor CarD, which is an essential protein in several members of this phylum. Our analysis expands the knowledge of promoter architecture across the bacterial phylogeny and provides new information that can be used to engineer bacteria for use in medical, environmental, agricultural, and biotechnological processes. IMPORTANCE Transcription of DNA to RNA by RNA polymerase is essential for cells to grow, develop, and respond to stress. Understanding the process and control of transcription is important for health, disease, the environment, and biotechnology. Decades of research on a few bacteria have identified promoter DNA sequences that are recognized by the σ subunit of RNA polymerase. We used bioinformatic analyses to reveal previously unreported differences in promoter DNA sequences across the bacterial phylogeny. We found that many Actinobacteria and Betaproteobacteria promoters lack a sequence in their −35 DNA recognition element that was previously assumed to be conserved and that Alphaproteobacteria lack a thymine residue at position −7, also previously assumed to be conserved. Our work reports important new information about bacterial transcription, illustrates the benefits of studying bacteria across the phylogenetic tree, and proposes new lines of future investigation. 

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4. Bacterial Secondary Metabolite Biosynthetic Potential in Soil Varies with Phylum, Depth, and Vegetation Type

   https://doi.org/10.1128/mBio.00416-20  

   Sharrar, Allison M.; Crits-Christoph, Alexander; Méheust, Raphaël; Diamond, Spencer; Starr, Evan P.; Banfield, Jillian F. (June 2020, mBio)

   Davies, Julian E. (Ed.)

   ABSTRACT Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils and whether the genetic potential for production of these metabolites differs with soil depth and vegetation type within a geographic region. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with various vegetation and bedrock characteristics and reconstructed 1,334 metagenome-assembled genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria , Chloroflexi , and candidate phylum “ Candidatus Dormibacteraeota.” Surprisingly, one genome of a candidate phyla radiation (CPR) bacterium coded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy. IMPORTANCE Microbes produce specialized compounds to compete or communicate with one another and their environment. Some of these compounds, such as antibiotics, are also useful in medicine and biotechnology. Historically, most antibiotics have come from soil bacteria which can be isolated and grown in the lab. Though the vast majority of soil bacteria cannot be isolated, we can extract their genetic information and search it for genes which produce these specialized compounds. These understudied soil bacteria offer a wealth of potential for the discovery of new and important microbial products. Here, we identified the ability to produce these specialized compounds in diverse and novel bacteria in a range of soil environments. This information will be useful to other researchers who wish to isolate certain products. Beyond their use to humans, understanding the distribution and function of microbial products is key to understanding microbial communities and their effects on biogeochemical cycles. 

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5. Tip extension and simultaneous multiple fission in a filamentous bacterium

   https://doi.org/10.1073/pnas.2408654121  

   Chimileski, Scott; Borisy, Gary G; Dewhirst, Floyd E; Mark_Welch, Jessica L (September 2024, Proceedings of the National Academy of Sciences)

   Organisms display an immense variety of shapes, sizes, and reproductive strategies. At microscopic scales, bacterial cell morphology and growth dynamics are adaptive traits that influence the spatial organization of microbial communities. In one such community—the human dental plaque biofilm—a network of filamentousCorynebacterium matruchotiicells forms the core of bacterial consortia known as hedgehogs, but the processes that generate these structures are unclear. Here, using live-cell time-lapse microscopy and fluorescent D-amino acids to track peptidoglycan biosynthesis, we report an extraordinary example of simultaneous multiple division within the domainBacteria. We show thatC. matruchotiicells elongate at one pole through tip extension, similar to the growth strategy of soil-dwellingStreptomycesbacteria. Filaments elongate rapidly, at rates more than five times greater than other closely related bacterial species. Following elongation, many septa form simultaneously, and each cell divides into 3 to 14 daughter cells, depending on the length of the mother filament. The daughter cells then nucleate outgrowth of new thinner vegetative filaments, generating the classic “whip handle” morphology of this taxon. Our results expand the known diversity of bacterial cell cycles and help explain how this filamentous bacterium can compete for space, access nutrients, and form important interspecies interactions within dental plaque. 

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