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1. Microbial communities associated with the black morel Morchella sextelata cultivated in greenhouses

   https://doi.org/10.7717/peerj.7744  

   Benucci, Gian Maria ; Longley, Reid ; Zhang, Peng ; Zhao, Qi ; Bonito, Gregory ; Yu, Fuqiang ( January 2019 , PeerJ)

   Morels ( Morchella spp.) are iconic edible mushrooms with a long history of human consumption. Some microbial taxa are hypothesized to be important in triggering the formation of morel primordia and development of fruiting bodies, thus, there is interest in the microbial ecology of these fungi. To identify and compare fungal and prokaryotic communities in soils where Morchella sextelata is cultivated in outdoor greenhouses, ITS and 16S rDNA high throughput amplicon sequencing and microbiome analyses were performed. Pedobacter , Pseudomonas , Stenotrophomonas , and Flavobacterium were found to comprise the core microbiome of M. sextelata ascocarps. These bacterial taxa were also abundant in the soil beneath growing fruiting bodies. A total of 29 bacterial taxa were found to be statistically associated to Morchella fruiting bodies. Bacterial community network analysis revealed high modularity with some 16S rDNA operational taxonomic unit clusters living in specialized fungal niches (e.g., pileus, stipe). Other fungi dominating the soil mycobiome beneath morels included Morchella , Phialophora , and Mortierella . This research informs understanding of microbial indicators and potential facilitators of Morchella ecology and fruiting body production. 

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2. Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers

   https://doi.org/10.1002/biot.202000336  

   Datta, Payel ; Yan, LuFeng ; Awofiranye, Adeola ; Dordick, Jonathan S. ; Linhardt, Robert J. ( November 2020 , Biotechnology Journal)

   Heparosan is a non‐sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered‐cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep‐3, an eliminase fromFlavobacterium heparinum) and heparanase Bp (Hep‐Bp, a hydrolase fromBurkholderia pseudomallei). The partial digestion ofE. coliK5 heparosan with purified His‐tagged Hep‐3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2‐DP8) are completely digested with purified GST‐tagged Hep‐Bp and analyzed using gel permeation chromatography. Hep‐Bp specifically cleaves the linkage betweend‐glucuronic acid (GlcA) andN‐acetyl‐d‐glucosamine (GlcNAc) but not the linkage between 4‐deoxy‐α‐L‐threo‐hex‐4‐enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc‐GlcA‐GlcNAc). This method successfully demonstrated the substrate selectivity of Hep‐BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.

    

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3. Ribosomes lacking bS21 gain function to regulate protein synthesis in Flavobacterium johnsoniae

   https://doi.org/10.1093/nar/gkad047  

   McNutt, Zakkary A. ; Roy, Bappaditya ; Gemler, Bryan T. ; Shatoff, Elan A. ; Moon, Kyung-Mee ; Foster, Leonard J. ; Bundschuh, Ralf ; Fredrick, Kurt ( February 2023 , Nucleic Acids Research)

   Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3’ tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU’-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < –13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria.

    

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4. A coevolution experiment between Flavobacterium johnsoniae and Burkholderia thailandensis reveals parallel mutations that reduce antibiotic susceptibility

   https://doi.org/10.1099/mic.0.001267  

   Chodkowski, John L. ; Shade, Ashley ( February 2023 , Microbiology)

   One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic or acquired mechanisms. Here, we performed a coevolution experiment to understand the long-term consequences of antibiotic production and antibiotic susceptibility for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain, Burkholderia thailandensis E264, and the antibiotic-inhibited environmental strain, Flavobacterium johnsoniae UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We observed that the F. johnsoniae ancestor could tolerate the B. thailandensis -produced antibiotic through efflux mechanisms, but that the coevolved lines had reduced susceptibility. We then sequenced genomes from the coevolved and monoculture F. johnsoniae lines, and uncovered mutational ramifications for the long-term antibiotic exposure. The coevolved genomes from F. johnsoniae revealed four potential mutational signatures of reduced antibiotic susceptibility that were not observed in the evolved monoculture lines. Two mutations were found in tolC : one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Deleting the 33 bp from tolC in the F. johnsoniae ancestor reduced antibiotic susceptibility, but not to the degree observed in coevolved lines. Furthermore, the accA mutation matched a previously described mutation conferring resistance to B. thailandensis -produced thailandamide. Analysis of B. thailandensis transposon mutants for thailandamide production revealed that thailandamide was bioactive against F. johnsoniae, but also suggested that additional B. thailandensis -produced antibiotics were involved in the inhibition of F. johnsoniae . This study reveals how multi-generational interspecies interactions, mediated through chemical exchange, can result in novel interaction-specific mutations, some of which may contribute to reductions in antibiotic susceptibility. 

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5. Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli

   https://doi.org/10.3389/fmolb.2021.787388  

   McNutt, Zakkary A. ; Gandhi, Mai D. ; Shatoff, Elan A. ; Roy, Bappaditya ; Devaraj, Aishwarya ; Bundschuh, Ralf ; Fredrick, Kurt ( December 2021 , Frontiers in Molecular Biosciences)

   The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli , a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae , a Bacteroidia which rarely does. In E. coli , 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli . In F. johnsoniae , deletion of three ribosomal RNA ( rrn ) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli , consistent with SD usage in the two organisms. 

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