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1. Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

   https://doi.org/10.1186/s12864-020-07134-4  

   Wangsanuwat, Chatarin; Heom, Kellie A.; Liu, Estella; O’Malley, Michelle A.; Dey, Siddharth S. (October 2020, BMC Genomics)

   Abstract BackgroundRNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. ResultsEMBR-seq results in 90% of the sequenced RNA molecules from anE. coliculture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. ConclusionsEMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples. 

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2. The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar

   https://doi.org/10.1038/s41564-021-01003-w  

   DeHart, Tanner G.; Kushelman, Mara R.; Hildreth, Sherry B.; Helm, Richard F.; Jutras, Brandon L. (December 2021, Nature Microbiology)

   Abstract Peptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N- acetylglucosamine (Glc N Ac) β-(1–4)-linked to N -acetylmuramic acid (Mur N Ac). Borrelia burgdorferi , the tick-borne Lyme disease pathogen, produces glycan chains in which Mur N Ac is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain Glc N Ac–Glc N Ac. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility. 

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3. De novo cholesterol biosynthesis in bacteria

   https://doi.org/10.1038/s41467-023-38638-8  

   Lee, Alysha K.; Wei, Jeremy H.; Welander, Paula V. (May 2023, Nature Communications)

   Abstract Eukaryotes produce highly modified sterols, including cholesterol, essential to eukaryotic physiology. Although few bacterial species are known to produce sterols, de novo production of cholesterol or other complex sterols in bacteria has not been reported. Here, we show that the marine myxobacteriumEnhygromyxa salinaproduces cholesterol and provide evidence for further downstream modifications. Through bioinformatic analysis we identify a putative cholesterol biosynthesis pathway inE. salinalargely homologous to the eukaryotic pathway. However, experimental evidence indicates that complete demethylation at C-4 occurs through unique bacterial proteins, distinguishing bacterial and eukaryotic cholesterol biosynthesis. Additionally, proteins from the cyanobacteriumCalothrixsp. NIES-4105 are also capable of fully demethylating sterols at the C-4 position, suggesting complex sterol biosynthesis may be found in other bacterial phyla. Our results reveal an unappreciated complexity in bacterial sterol production that rivals eukaryotes and highlight the complicated evolutionary relationship between sterol biosynthesis in the bacterial and eukaryotic domains. 

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4. Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes

   Lacroix, Benoit; Citovsky, Vitaly. (January 2018, Current topics in microbiology and immunology)

   Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research. 

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5. Heat Stress of Algal Partner Hinders Colonization Success and Alters the Algal Cell Surface Glycome in a Cnidarian-Algal Symbiosis

   https://doi.org/10.1128/spectrum.01567-22  

   Maruyama, Shumpei; Mandelare-Ruiz, Paige E.; McCauley, Mark; Peng, Wenjing; Cho, Byeong Gwan; Wang, Junyao; Mechref, Yehia; Loesgen, Sandra; Weis, Virginia M. (May 2022, Microbiology Spectrum)

   Kormas, Konstantinos Aristomenis (Ed.)

   ABSTRACT Corals owe their ecological success to their symbiotic relationship with dinoflagellate algae (family Symbiodiniaceae). While the negative effects of heat stress on this symbiosis are well studied, how heat stress affects the onset of symbiosis and symbiont specificity is less explored. In this work, we used the model sea anemone, Exaiptasia diaphana (commonly referred to as Aiptasia), and its native symbiont, Breviolum minutum , to study the effects of heat stress on the colonization of Aiptasia by algae and the algal cell-surface glycome. Heat stress caused a decrease in the colonization of Aiptasia by algae that were not due to confounding variables such as algal motility or oxidative stress. With mass spectrometric analysis and lectin staining, a thermally induced enrichment of glycans previously found to be associated with free-living strains of algae (high-mannoside glycans) and a concomitant reduction in glycans putatively associated with symbiotic strains of algae (galactosylated glycans) were identified. Differential enrichment of specific sialic acid glycans was also identified, although their role in this symbiosis remains unclear. We also discuss the methods used to analyze the cell-surface glycome of algae, evaluate current limitations, and provide suggestions for future work in algal-coral glycobiology. Overall, this study provided insight into how stress may affect the symbiosis between cnidarians and their algal symbionts by altering the glycome of the symbiodinian partner. IMPORTANCE Coral reefs are under threat from global climate change. Their decline is mainly caused by the fragility of their symbiotic relationship with dinoflagellate algae which they rely upon for their ecological success. To better understand coral biology, researchers used the sea anemone, Aiptasia, a model system for the study of coral-algal symbiosis, and characterized how heat stress can alter the algae's ability to communicate to the coral host. This study found that heat stress caused a decline in algal colonization success and impacted the cell surface molecules of the algae such that it became more like that of nonsymbiotic species of algae. This work adds to our understanding of the molecular signals involved in coral-algal symbiosis and how it breaks down during heat stress. 

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