The isolate which did not possess the plasmid was further verified for curing by PCR amplification of 5 genes or ORFs, senB (forward primer 5′- GCA GAT TCG CGT TTT GAG CA-3′ and reverse primer 5′- CGG check details ATC TTT CAA CGG GAT GG-3′), scsD (forward primer 5′- CAT ACG CTG GAC GGG GAA AC-3′ and reverse primer 5′-GAC GCT CTC CCC TTC CGA CT-3′), traU (forward primer 5′- TTC CTT CTC GCC GGT CAT GT-3′ and reverse primer 5′- CCA GCG AGA GCG GGA AAA TA-3′), transposase (forward primer 5′- GCT TCG GGA ACG CTG TAA CG-3′ and reverse primer 5′- AGA AGG CTG CGG TGC TGA AG-3′), pRS218_113 (forward primer 5′- TGG GGG CTG AAA ACC AGA GA-3′ and reverse primer 5′- ACC GAA GGC ACG AAC TGC AT-3′), and ycfA (forward

primer 5′- CGC CTG GTG GTG AAG

GAA AG-3′ and reverse primer 5′- GAC CAC CTC CCG CAG AAC AC-3′) of pRS218. Isolates that did not possess all of the five genes/ORFs were considered to be cured of pRS218. The plasmid complementation was performed using conjugation as described previously [41]. The main obstacle for complementation was the absence of an antibiotic resistance marker in pRS218 NVP-HSP990 solubility dmso which could have been used for subsequent selection. Therefore, pRS218 was first tagged with cat using the one step inactivation method [39]. Briefly, the cat was amplified using pKD3 plasmid and primers consisted of 36 nucleotides extensions at 5′ and 3′ ends of a putative noncoding region of pRS218 located between base pairs 591 and 831 in the plasmid sequence (Forward primer 5′-CGC CTT CGC GTT GCT CAG TTG TCC AAC CCC GGA AAC GTG TAG GCT GGA GCT GCT TC-3′ and reverse primer 5′-CTC CTC AAT ACT CAA ACA GGG ATC GTT TCG CAG Idoxuridine AGG ACA TAT GAA TAT CCT CCT TAG-3′). Purified PCR product was electroporated to E. coli RS218 carrying the Red helper plasmid pKD119 to construct the pRS218::cat. The temperature sensitive pKD119 plasmid was removed

from pRS218::cat by growing at 42°C followed by screening for tetracycline sensitivity. The E. coli RS218 carrying pRS218::cat was then used as the donor to perform mating experiments. Escherichia coli DH5α was used as an intermediate recipient to transfer pRS218::cat from the donor strain to the recipient plasmid-cured strain. Bacterial growth curve Bacteria were grown in LB broth at 37°C with shaking overnight. Cultures were diluted to 1:100 with LB broth, tissue culture medium or M9 medium with 10 μg/ml niacin and incubated at 37°C with shaking. Optical density at 600 nm (OD600) was taken in triplicate for every 20 min for 6 hrs. The OD values from each time point were averaged and graphed to obtain a growth curve. In vitro invasion assay Invasion assays were performed using hCMEC/D3 cells provided by Dr. Weksler B, Cornell University, NY. The hCMEC/D3 cells were grown in endothelial basal medium (Lonza, Walkersville, MD) containing 5% fetal bovine serum (PAA The Cell Culture Company, Piscataway, NJ), 1.4 μM hydrocortisone (Sigma-Aldrich, St. Louis, MO.