Oliver, and R. of adaptation during one or more phases of its complex life cycle. Stevenson and Babb (21) previously showed that open reading frame BB0377 was able to complement a deficiency in DH5, thereby supporting a designation for open reading frame BB0377. Furthermore, the addition of cell-free supernatants of DH5 harboring the gene to cultures (at 23 or 34C) induced the differential regulation of 25 to 30 borrelial proteins, ostensibly from the presence of AI-2 in the culture medium (21). However, AI-2-like activity was not detectable in high-density culture supernatants of strain B31, TAME hydrochloride N40, or 297, nor was the expression of the gene in evaluated (21). Nevertheless, it was concluded that the LuxS/AI-2 quorum-sensing system is operative in (21). The objectives of the present study were to examine directly the expression of in expression, and determine whether LuxS was required for mammalian infectivity by the Lyme disease spirochete. Generation of mutants. Infectious strain TAME hydrochloride 297 (12) and all other bacterial strains TAME hydrochloride and plasmid constructs used in this study are listed TAME hydrochloride in Table ?Table1.1. The overall strategy for targeted gene disruption in strain 297 was described previously (8). All PCR primers were designed on the basis of strain B31 sequence information (5) (http://www.tigr.org), and their approximate positions are depicted in Fig. ?Fig.1B.1B. PCR was routinely performed with the Expand High Fidelity PCR System (Roche Diagnostics). First, a 5,014-bp region of the gene with flanking sequences from strain 297 was PCR amplified by using primers priAH156 (5-CTGCTAGTGCCTATTCTGCTATC) and priAH159 (5-GGTTCCTGCAAATGTTGTTCAGAG) and ligated into pGEMT-easy (Promega, Madison, Wis.) to yield pALH330 (Fig. ?(Fig.1A).1A). The gene was then disrupted by inserting an erythromycin resistance marker, (8, 18), into the unique was inserted in an orientation opposite to that of (priAH102 and priAH104 [8]) in combination with priAH166 and priAH168 (see below). Open in a separate window FIG. 1. Construction of the suicide plasmid (pALH340) for disruption of (hatched box) and flanking sequences (only the relevant portion of the plasmid is shown). Two genes, and (gray boxes), along with was insertionally disrupted with (vertical stripes), yielding pALH340. Short arrows indicate approximate primer locations. (C) Agarose gel electrophoresis patterns of amplicons for pALH330 (lanes 1 to 3) and pALH340 (lanes 4 to 6 6). The primers used to generate products were as follows: lanes 1 and 4, priAH166 and priAH168; lanes 2 and 5, priAH104 and priAH166; lanes 3 and 6, priAH102 and priAH168. PCR using combinations of relative to in pALH340 (lanes 5 and 6). TABLE 1. Strains and recombinant CR2 plasmids DH5F? 80d?(Sigma Chemical Co., St. Louis, Mo.). Following 36 h of recovery in the absence of erythromycin, the entire transformation mixture was added to 50 ml of prewarmed BSK-H medium (Sigma) containing erythromycin (0.06 g/ml). Erythromycin-resistant spirochetes could easily be distinguished (via dark-field microscopy) from nonviable (nonmotile) cells after 10 to 14 days of incubation at 37C. No viable spirochetes were detected in a control culture of BSK-H plus erythromycin inoculated with mock-electroporated wild-type bacteria, indicating that spirochetes were efficiently killed and that spontaneous erythromycin resistance did not occur among the population. It was postulated that further selection and enrichment of transformed spirochetes under conditions that more closely mimic the mammalian host environment (as opposed to in vitro growth on solid medium) might assist with the recovery of mutants that retained the ability to infect and replicate within mammalian tissues. To approach this, 10 ml of the erythromycin-resistant mutant pool or a mock-electroporated control pool (both diluted to 1 1,000 spirochetes per ml in fresh BSK-H plus erythromycin) were placed into sterile dialysis membrane chambers (DMCs), which were then implanted into rat peritoneal cavities (1). After 15 days, motile spirochetes were not detectable in DMCs inoculated with mock-electroporated wild-type bacteria, again indicating that initial erythromycin selection efficiently eliminated all wild-type may not be required for the growth of during mammalian host-adapted conditions. Furthermore, PCR analysis of genomic contents from the pool of erythromycin-resistant spirochetes within DMCs did not reveal any amplicons representative.