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Lemon, K. P.

Publications and source records attributed to Lemon, K. P..

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SyngenicDNA: stealth-based evasion of restriction-modification barriers during bacterial genetic engineering

Restriction-modification (RM) systems hinder the use of genetic approaches in the vast majority of bacteria. Here, we describe a systematic approach to adapt genetic tools for use in bacteria that are genetically intractable or poorly tractable owing to active RM defenses. In this process, we determine the genome and methylome of a bacterial strain and use this information to define the bacteriums RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent SyngenicDNA tool and propagate the tool as novel minicircle plasmids, termed SyMPL tools, before transformation. Using SyngenicDNA and SyMPL tools, we achieved a profound, >100,000- fold, improvement in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus demonstrating the efficacy of these approaches for evading RM systems. The SyngenicDNA and SyMPL approaches are effective, flexible, and should be broadly applicable in microbial genetics. We expect these will facilitate a new era of microbial genetics free of the restraints of restriction-modification barriers.

microbiology

New insights into human nostril microbiome from the expanded Human Oral Microbiome Database (eHOMD): a resource for species-level identification of microbiome data from the aerodigestive tract

The expanded Human Oral Microbiome Database (eHOMD) is a comprehensive microbiome database for sites along the human aerodigestive tract that revealed new insights into the nostril microbiome. The eHOMD provides well-curated 16S rRNA gene reference sequences linked to available genomes and enables assignment of species-level taxonomy to most NextGeneration sequences derived from diverse aerodigestive tract sites, including the nasal passages, sinuses, throat, esophagus and mouth. Using Minimum Entropy Decomposition coupled with the RDP Classifier and our eHOMD V1-V3 training set, we reanalyzed 16S rRNA V1-V3 sequences from the nostrils of 210 Human Microbiome Project participants at the species level revealing four key insights. First, we discovered that Lawsonella clevelandensis, a recently named bacterium, and Neisseriaceae [G-1] HMT-174, a previously unrecognized bacterium, are common in adult nostrils. Second, just 19 species accounted for 90% of the total sequences from all participants. Third, one of these 19 belonged to a currently uncultivated genus. Fourth, for 94% of the participants, two to ten species constituted 90% of their sequences, indicating nostril microbiome may be represented by limited consortia. These insights highlight the strengths of the nostril microbiome as a model system for studying interspecies interactions and microbiome function. Also, in this cohort, three common nasal species (Dolosigranulum pigrum and two Corynebacterium species) showed positive differential abundance when the pathobiont Staphylococcus aureus was absent, generating hypotheses regarding colonization resistance. By facilitating species-level taxonomic assignment to microbes from the human aerodigestive tract, the eHOMD is a vital resource enhancing clinical relevance of microbiome studies.\n\nIMPORTANCEThe eHOMD (ehomd.org) is a valuable resource for researchers, from basic to clinical, who study the microbiomes, and the individual microbes, in health and disease of body sites in the human aerodigestive tract, which includes the nasal passages, sinuses, throat, esophagus and mouth, and the lower respiratory tract. The eHOMD is an actively curated, web-based, open-access resource. eHOMD provides the following: (1) species-level taxonomy based on grouping 16S rRNA gene sequences at 98.5% identity, (2) a systematic naming scheme for unnamed and/or uncultivated microbial taxa, (3) reference genomes to facilitate metagenomic, metatranscriptomic and proteomic studies and (4) convenient cross-links to other databases (e.g., PubMed and Entrez). By facilitating the assignment of species names to sequences, the eHOMD is a vital resource for enhancing the clinical relevance of 16S rRNA gene-based microbiome studies, as well as metagenomic studies.

microbiology