{"doi":"10.1101/2021.07.13.452269","title":"A genetic toolkit for investigating <i>Clavibacter</i> : markerless deletion, permissive site identification and an integrative plasmid","abstract":"Abstract The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advancements of these techniques in Gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens which cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen C. michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and confirmed using whole genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a non-replicating integrative plasmid that enables recombination into a permissive genomic location. Expression of eGFP by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis . These molecular tools are essential advancements for investigating Gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":225670,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9542,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":828393,"name":"Andrea Tang","orcid":"0009-0002-1795-1964","position":1,"is_corresponding":false},{"id":233503,"name":"Gitta Coaker","orcid":"0000-0003-0899-2449","position":2,"is_corresponding":false},{"id":246873,"name":"Danielle M. Stevens","orcid":"0000-0001-5630-137X","position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:54:30.292454Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}