title: Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1.
abstract: Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bocioaga
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Iman A
authors.initials: IA
authors.lastname: El Gheriany
authors.affiliation: School of Civil and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Lion
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Ghiorse
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Shuler
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Hay
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003227:Conjugation, Genetic; D005821:Genetic Techniques; D042743:Leptothrix / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008345:Manganese / Q000378:metabolism*; D010084:Oxidation-Reduction; D011995:Recombination, Genetic; D018741:Replication Origin
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008345:Manganese