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N-Methylation of the peptide backbone confers pharmacologically beneficial characteristics to peptides that include greater membrane permeability and resistance to proteolytic degradation. The borosin family of ribosomally synthesized and post-translationally modified peptides offer a post-translational route to install amide backbone α-N-methylations. Previous work has elucidated the substrate scope and engineering potential of two examples of type I borosins, which feature autocatalytic precursors that encode N-methyltransferases that methylate their own C-termini in trans. We recently reported the first discrete N-methyltransferase and precursor peptide from Shewanella oneidensis MR-1, a minimally iterative, type IV borosin that allowed the first detailed kinetic analyses of borosin N-methyltransferases. Herein, we characterize the substrate scope and resilient regiospecificity of this discrete N-methyltransferase by comparison of relative rates and methylation patterns of over 40 precursor peptide variants along with structure analyses of nine enzyme-substrate complexes. Sequences critical to methylation are identified and demonstrated in assaying minimal peptide substrates and non-native peptide sequences for assessment of secondary structure requirements and engineering potential. This work grants understanding towards the mechanism of substrate recognition and iterative activity by discrete borosin N-methyltransferases., (© 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. Histidine kinases play an essential role in the regulation of multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the highly conserved catalytic and adenosine triphosphate-binding (CA) domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted in vitro structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain. We found that these compounds, which inhibit the model histidine kinase, HK853 from Thermotoga maritima, have anti-virulence activities inPseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Erin Carlson reports financial support and equipment, drugs, or supplies were provided by University of Minnesota Twin Cities. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation-associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca 2+ -activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation-associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa , reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Amide peptide backbone methylation is a characteristic post-translational modification found in a family of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) called borosins. Previously, we bioinformatically identified >1500 putative borosin pathways in bacteria; however, none of the pathways were associated with a known secondary metabolite. Through in-depth characterization of a borosin pathway in Shewanella oneidensis MR-1, we have now identified a bacterially derived borosin natural product named Shewanellamide A. Borosin identification was facilitated by the creation and analysis of a series of precursor variants and crystallographic interrogation of variant precursor and methyltransferase complexes. Along with assaying two proteases from S. oneidensis , probable boundaries for proteolytic maturation of the metabolite were projected and confirmed via comparison of S. oneidensis knockout and overexpression strains. All in all, the S. oneidensis natural product was found to be a 16-mer linear peptide featuring two backbone methylations, establishing Shewanellamide A as one of the few borosin metabolites yet identified, and the first from bacteria., Competing Interests: M. F. F. is an inventor on patent WO2017EP58327, US20190112583A1, WO2021168399, and US-20230090771-A1. All other authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed 'split borosins'), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis. A series of enzyme-precursor complexes reveal multiple conformational states for both α-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs., (© 2021. The Author(s).)

There is a need for standardized, efficient, and practical sampling methods to support large population-based studies of the internal and external epithelial microbiomes of the bovine udder. The primary objective of this study was to evaluate different sampling devices for the isolation of microbial DNA originating from the internal and external teat epithelium. Secondary objectives were to survey and compare the microbial diversity of external and teat canal epithelial microbiomes using amplicon and shotgun metagenomic sequencing approaches. To address these objectives, we enrolled a convenience sample of 24 Holstein dairy cows and collected samples from the external epithelium at the base of udder, the external teat barrel epithelium, the external teat apex epithelium, and the teat canal epithelium. Extracted DNA was quantified and subjected to PCR amplification of the V4 hypervariable region of the 16S rRNA gene and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA). A subset of samples was subjected to a shallow shotgun metagenomic assay on the Illumina HiSeq platform. For samples collected from the external teat epithelium, we found that gauze squares consistently yielded more DNA than swabs, and Simpson's reciprocal index of diversity was higher for gauze than for swabs. The teat canal epithelial samples exhibited significantly lower diversity than the external sampling locations, but there were no significant differences in diversity between teat apex, teat barrel, and base of the udder samples. There were, however, differences in the microbial distribution and abundances of specific bacteria across external epithelial surfaces. The proportion of shotgun sequence reads classified as Bos taurus was highly variable between sampling locations, ranging from 0.33% in teat apex samples to 99.91% in teat canal samples. These results indicate that gauze squares should be considered for studying the microbiome of the external epithelium of the bovine udder, particularly if DNA yield must be maximized. Further, the relative proportion of host to non-host DNA present in samples collected from the internal and external teat epithelium should be considered when designing studies that utilize shotgun metagenomic sequencing., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Ground beef can be a reservoir for a variety of bacteria, including spoilage organisms, and pathogenic foodborne bacteria. These bacteria can exhibit antimicrobial resistance (AMR) which is a public health concern if resistance in pathogens leads to treatment failure in humans. Culture-dependent techniques are commonly used to study individual bacterial species, but these techniques are unable to describe the whole community of microbial species (microbiome) and the profile of AMR genes they carry (resistome), which is critical for getting a holistic perspective of AMR. The objective of this study was to characterize the microbiome and resistome of retail ground beef products labeled as coming from conventional or raised without antibiotics (RWA) production systems. Sixteen ground beef products were purchased from 6 retail grocery outlets in Fort Collins, CO, half of which were labeled as produced from cattle raised conventionally and half of products were from RWA production. Total DNA was extracted and isolated from each sample and subjected to 16S rRNA amplicon sequencing for microbiome characterization and target-enriched shotgun sequencing to characterize the resistome. Differences in the microbiome and resistome of RWA and conventional ground beef were analyzed using the R programming software. Our results suggest that the resistome and microbiome of retail ground beef products with RWA packaging labels do not differ from products that do not carry claims regarding antimicrobial drug exposures during cattle production. The resistome predominantly consisted of tetracycline resistance making up more than 90% of reads mapped to resistance gene accessions in our samples. Firmicutes and Proteobacteria predominated in the microbiome of all samples (69.6% and 29.0%, respectively), but Proteobacteria composed a higher proportion in ground beef from conventionally raised cattle. In addition, our results suggest that product management, such as packaging type, could exert a stronger influence on the microbiome than the resistome in consumer-ready products. Metagenomic analyses of ground beef is a promising tool to investigate community-wide shifts in retail ground beef. Importantly, however, results from metagenomic sequencing must be carefully considered in parallel with traditional methods to better characterize the risk of AMR in retail products., (Copyright © 2020 Doster, Thomas, Weinroth, Parker, Crone, Arthur, Schmidt, Wheeler, Belk and Morley.)