Table 1 Strains, plasmids, and primers used in this study.

   F. tularensis

LVS

F. tularensis subsp. holartica live vaccine strain

Karen Elkins

1581d

LVS with FTL_1581 disruption, Hyg^R

This study

1664d

LVS with FTL_1664 (deoB) disruption, Hyg^R

This study

   E. coli

XL10-Gold

Δ(mcrA)183 Δ (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F' proAB lacI^qZDM15 Tn10 (Tet^R) Amy Cam^R]

Stratagene

DH5α

F-φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (r_k-, m_k+) phoA supE44 λ-thi-1 gyrA96 relA1

Invitrogen

JM109

endA1, recA1, gyrA96, thi, hsdR17 (r_k^-, m_k⁺), relA1, supE44, Δ(lac-proAB), [F' traD36, proAB, laqI^qZΔM15]

Promega

Plasmids

   pFNLTP8

Francisella shuttle plasmid, Ap^R, Km^R

[63]

   pRK2013

Helper plasmid for triparental mating, Km^R

[64]

   pMP615

Francisella shuttle plasmid, Hyg^R

[65]

   pMQ131hyg

F. tularensis suicide vector, pBBR1 ori, oriT, contains the hyg cassette driven by the groEL promoter from pMP615, oriT, Km^R, Hyg^R

This study

   pMQ131hyg1581d

pMQ131hyg with the central 560 base pair region of FTL_1581

This study

   pMQ131hyg1664d

pMQ131hyg with the central 900 base pair region of FTL_1664 (deoB)

This study

   pFTL_1581

Broad-host-range vector, pC194 ori, contains cloned FTL_1581 along with 600 base pairs upstream and 100 base pairs downstream of this gene, complementing plasmid, Ap^R, Cam^R

This study

   pF8AX

pFNLTP8 Δnpt, Ap^R

This study

   pF8CAT

pF8AX with cat, Ap^R, Cam^R

This study

   pFTL_1664

pF8CAT with cloned FTL_1664 (deoB) along with 600 base pairs upstream and 100 base pairs downstream of this gene, complementing plasmid

This study

Primers

   1581_560F

5'-ATGGATCCTGAGCTAAATGATGCTTTAGTATCTC-3'

Invitrogen

   1581_560R

5'-ATGGTACCAAGACGACATAGCCACG-3'

Invitrogen

   1664_900F

5'-ATGGATCCGAACCTGGAGCAGTTGAAT-3'

Invitrogen

   1664_900R

5'-ATGGTACCTAAGAAAGTTGCGGAATATAATAGATG-3'

Invitrogen

   1581_clone_up

5'-GATCGGATCCAGGTCAATCAGGAGTTGG-3'

Invitrogen

   1581_clone_down

5'-GATCGGTACCCACCTATTTGAATTAAAAAGAAGTTTATACAC-3'

Invitrogen

   1664_clone_up

5'-GATCGGATCCGATGGCTATGGTATATCTTCGG-3'

Invitrogen

   1664_clone_down

5'-GATCGGTACCACCGAGAGAATTTCTCGC-3'

Invitrogen

   F8AgeI

5'-CATTAGACCGGTGCGAAACGATCCTCATCCTGTC-3'

Invitrogen

   F8XhoI

5'-Phosphorylated – CATTAGCTCGAGGGAAGAGTATGAGTATTCAAC-3'

Invitrogen

   XhoICAT

5'-CATGCTCGAGTTATAAAAGCCAGTCATTAGGCC-3'

Invitrogen

   AgeICAT

5'-CATGACCGGTATGAACTTTAATAAAATTGATTTAGACAATTGG-3'

Invitrogen