Table 2. Primer sequences and restriction enzyme sites integrated. Primer names caf-F caf-R F1-dn-F F1-dn-R F1-up-F F1-up-R M-dn-F M-dn-R A-dn-F F1-F Enzyme sites SacI PstI EcoRV PstI NheI SmaI SmaI XbaIa XbaI SacI Primer sequencesb,c GAGCTCCGTAAGGAGGTTAAGC CTGCAGTGAACCTATTATATTGCTTCGCGC GATATCGTAACCGTATCTAACCAATAATCC CTGCAGTGAACCTATTATATTGCTTCGCGC ATCGTTAAACATTGCTAGCGAGGAATACGCC CCCGGGAGTGGTGCTTGCAGTTAAATCTG CCCGGGTTGGATCGTTTGTATTCC TTGTAAGGATGATAGGCATGGC TCTAGACGGTGTCTATTTGACTGGACTAC GAGCTCATTATTCGATAGAGGTAATATATG Note: aThe restriction enzyme site XbaI locates downstream of this primer. bThe sequences of the restriction enzyme sites integrated in the primers are bolded. cPrimer sequences are based on template of Y. pestis plasmid pFra (accession No. X61996). doi:10.1371/journal.pone.0036283.t002 Lexington, Kentucky, USA). The conditions used for growing KIM6+ derived strains were 27uC without shaking in BHI liquid medium unless otherwise indicated. Restriction endonucleases, T4 DNA ligase, and Taq DNA polymerase were purchased from New England BioLabs (Beverly, MA) unless otherwise noted. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Genetic manipulations were conducted by using the method as described elsewhere [10]. Plasmid DNA was extracted by using a Qiagen Miniprep Kit (Valencia, CA). DNA fragments were purified and extracted from agarose gel slices, using Qiagen Gel Extraction Kit. Competent E. coli and S. Typhimurium cells were made in 10% glycerol and transformed by electroporation. Construction of plasmids Plasmids pHF and pY. The genes of caf1Mcaf1Acaf1 from plasmid pF1 [10] were amplified with the primers of caf1-F and caf1-R (Table 2), and the DNA fragment was Topo cloned (Invitrogen, Carlsbad, CA). After sequencing for identification with the original caf DNA sequence, the correct clone was used for subcloning. The caf1Mcaf1Acaf1 DNA fragment with a length of 4153 bps was placed downstream of promoter PM of plasmid pHC [16] to generate pHF (Figure 1B). Plasmid pY was derived from pHC by using ScaI to remove the cfa/I operon, and the vector fragment was then self-ligated (Figure 1C). Interim plasmid pSMA. Prior to pSA and pSM construction, an interim plasmid pSMA was constructed (Figure 4A). Plasmid pSMA was derived from pHF by deleting its caf1 gene inner DNA sequence. The caf1 gene downstream sequence was amplified with a pair of primers of F1-dn-F+F1-dn- R, and its upstream sequence was amplified with another pair of primers of F1-up-F+F1-up-R (Table 2). The two DNA fragments were subjected to Topo cloning and sequencing to confirm no mutation was introduced, and then they were fused at the EcoRV and SmaI sites since both are blunt ends. A total of 405 bps was deleted from caf1 gene, and this fusion DNA fragment was placed between NheI and PstI sites in pHF. This plasmid was termed caf Runs as a Double-Edged Sword during Infection pSMA. Based on pSMA, the plasmids pSA and pSM were constructed as follows: Plasmids pSA and pSM. The DNA sequence downstream caf1M was amplified with a pair of primers of M-dn-F+M-dn-R (Table 2). After sequencing identification, it was digested with SmaI and XbaI, and this 287 bps DNA fragment was inserted between the EcoRV and XbaI sites in pSMA. Thus, the 660 bps inner DNA sequence of caf1M was deleted in-frame. The new plasmid was termed pSA (Figure 4B). The DNA sequence downstream caf1A was amplified with a pair of primers of A-dn- F+caf1-R (Table 2). After sequencing confirmation and digested with XbaI and PstI, this 993 bps DNA fragment was installed between the XbaI and PstI sites in pSMA. The 2121 bps inner DNA sequence of caf1A was deleted in-frame. This new plasmid was termed pSM (Figure 4C). Plasmid pSF1. The caf1 gene from pF1 was amplified by a pair of primers of F1-F+caf1-R (Table 2). Fidelity of the 773 bps caf1 gene was verified by sequencing, and it was then inserted into pHF between SacI and PstI sites. Thus, the 4153 bps caf operon was replaced by the caf1 gene. The new plasmid was termed pSF1 (Figure 4D). Western blot analysis of Caf1 expression To verify F1 protein expression and yield, P1-pF1, -pHF, and - pY were inoculated from 280uC freezer to LB agar and allowed to grow overnight at 37uC. Bacteria were then inoculated into liquid LB media with the cell OD600 adjusted to ,0.1. They were incubated at 37uC with shaking at 150 rpm. At 4, 8 and 12 hrs post-inoculation, cultures were centrifuged at 13,000 g for 15 min at 4uC to collect whole cells. Secreted F1 capsular proteins contained in supernatants were precipitated by ammonium sulfate added to 30% saturation as previously described [30]. Supernatant protein pellets were mixed with their corresponding whole cell pellets of P1-pF1, -pHF, and -pY. Samples were subjected to Western blot analysis as previously described [10], and each well was loaded with an equivalent number of CFUs of P1-pF1, -pHF, and -pY cells. The F1 protein yields were quantified by densitometric analysis as previously described [31]. Evaluation of survivability of recombinant Salmonella and Yersinia strains in RAW264.7 macrophages RAW264.7 macrophage cell line (American Type Culture Collection) was used for evaluating the capability of infection and replication for strains P1-pF1, -pHF, -pSA, -pSF1, -pSM, and -pY. Infections were conducted as previously described [32]. 1.256106 RAW264.7 cells/well without antibiotics were allowed to adhere to plastic in 24-well microtiter dishes (BD-Labware, Franklin Lakes, NJ) at 37uC with 5% CO2. Wells were washed, and the nonadherent cells were collected and counted to determine cell numbers that remained plastic-adherent. After overnight culture, cells were infected with bacteria to macrophage ratio of 1:1 for 1 hr at 37uC. Wells were washed twice and then incubated with 50 mg/ml of gentamicin for 30 minutes at 37uC. After washing twice, as described, fresh complete medium containing 2.5 mg/ml of gentamicin was added, and cells were incubated for either an additional 8 or 24 hrs. Next, the macrophages were water lysed, and the bacteria were placed on LB agar plates after a series of dilutions. After incubation overnight at 37uC, the bacterial CFUs were determined. To determine whether the recombinant Yersinia strains of KIM6+/pF1, pHF, pSA, pSF1, and pSM were capable of surviving as robustly as pY in vitro, a macrophage infection assay was conducted as previously described [33]. Briefly, log phase cultures of KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e36283