Chromosome Transfer in Rhodobacter spharoides : Hfr Formation and Genetic Evidence for Two Unique Circular Chromosomes

Abstract

A 600-bp oriT-containing DNA fragment from the Jtkodobacter sphaeroides 2.4.1 S tactor (oriT,) (A. Suwanto and S. Kaplan, J. Bacterial. 174: 1124-1134, 1992) 1r.r s shown to proniotc polarized cl~romosomalt ransfer when providled in cis. A Kmr-oriTs-scvR-sacB (KTS) DNA cassette was constrricted by inserting oriT,-SUCK-sacR into a pUTmini-Tn5 Kml derivative. Witli this delivery system, KTS appeared to be randomly inserted into the genome of R. splraeroides, generating mutant strains !vliicli also gained ttie ability to act as I-tfr donors. An AseI site in the Kmr cartridge (from Tn903) and Drat alrd StlaBI sites in SUCK-sacB(t he levansucrasc gene fro111 Bacillirs siibtilis) \rrere employed to localize the KTS insertion definitively by pulsed-field gel electrophoresis. The orientation of oriT, at the sit: of irisertior~w as delerniined by Southern Iiybridization analysis. Interrupted mating esperiinc~itps erforti~edw ith soine of the Hfr strains exhibited a gradient of marker transfer and further provided genetic evidence for I lie circularity and presence of two chronioso~i~allin kage groups in this bacterium. Tlie genetic and envir ~nnieiitacl onditions for optimized ~iiatingb et\rreenK . splraeroidess trains \\,ere also dclfned. The results presented l~erea nd our phyrical niap of the K. sphaeroides 2.1.1 gelionie are discussed in light of the presence of two cl romosomes.