TABLE 1.

Plasmids, strains, and primers used in this study

Plasmid, strain, or primer	Genotype, description, or sequence	Reference, source, or comment
Plasmids
pFNLTP6 gro-gfp	Shuttle plasmid with LVS groE operon promoter upstream of gfp, Kmr Apr	28
p6-gro	Shuttle plasmid derivative with groE promoter, Kmr Apr	This study
p6-gro-hfq	Shuttle plasmid derivative with groE promoter upstream of hfq, Kmr Apr	This study
pPV	oriT sacB, Apr Cmr, Francisella suicide plasmid	19
pPV-hfq	pPV carrying hfq deletion fragment for mutagenesis of LVS	This study
pDMK2	mobRP4oriR6KsacB, Kmr, Francisella suicide vector	K. Kadzhaev, unpublished
pDMK-hfq	pDMK2 carrying hfq deletion fragment for mutagenesis of FCS200	This study
pCR4TOPO	PCR cloning vector, Ampr Kmr	Invitrogen
pCR2.1TOPO	PCR cloning vector, Ampr Kmr	Invitrogen
Strains
F. tularensis LVS	F. tularensis subsp. holarctica live vaccine strain, Pmr	A. Sjöstedt
F. tularensis Δhfq mutant	LVS strain with deletion of codons 5 to 71 of hfq	This study
F. tularensis FSC200	F. tularensis subsp. holarctica, clinical isolate	Human ulcer, Sweden
F. tularensis FSC768	FSC200 with deletion of codons 9 to 113 of hfq	This study
E. coli DH5α	F− φ80lacZΔM15 endA1 recA1 hsdR17 supE44 thi-1 gyrA96 relA1 (lacZYA-argF)U169	Invitrogen
E. coli TOP10	(F−) mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 deoR araD139 (Δara-leu)7697 galU galK rpsL (Smr) endA1 nupG	Invitrogen
E. coli S17-1	(F−) RP4-2-Tc::Mu aph::Tn7 recA, Smr	Lab collection
E. coli S17-1λpir	(F−) RP4-2-Tc::Mu aph::Tn7 recA λpir lysogen, Smr	Lab collection
Primers
Hfq-AF	5′- TGGGTCGACTACAGCCAAGCCAACTTTACAAGa	Mutant construction
Hfq2	5′-TATTCTTGACATTATCTCACTTC	Mutant construction
Hfq-BR	5′-CGGCTAGCAAGATCAGACTCTATAGCCTCTTCb	Mutant construction
Hfq3	5′-GAAGTGAGATAATGTCAAGAATAGTTTATAGCTCTTTTAATCCATAc	Mutant construction
Hfq_FNdeI	5′-GGGAATTCCATATGTTGCTAGAATATACGCGAAGd	Complementing plasmid
Hfq_RBamHI	5′-CGCGGATCCTTACTCGTGAATATTACCTTCe	Complementing plasmid
5up-HfqA	5′-AATTAATGCAGAAATTATTAGTG	Mutant verification
3down-HfqB	5′-CTTTTTCAACCTGCTCAATGTAGC	Mutant verification
Hfq-F22	5′-CAAGACCCGTTCTTAAATGCT	RT-PCR
Hfq-R79	5′-CTAGATATACAGATACGCTAAC	RT-PCR
Hfq-R133	5′-CGATACAGAATTGGTCAAAAGC	RT-PCR/5′ RACE
Hfq-R194	5′-GCTGGAACTATAGTAGAAATAG	RT-PCR, 5′ RACE
miaA-F870	5′-GCTATGGAGAACGAAACTAAAGA	RT-PCR
HflX-R170	5′-GATATCTGGCTCAGGATGG	RT-PCR
Hfq200-A_for	5′-AACAGTCCAGGGTGACTAAAATCGAGCCAAT	Mutant construction
Hfq200-B_rev	5′-ATATTACTCGTGTGACATTATCTCACTTCCTTTTA	Mutant construction
Hfq200-C_for	5′-GAGATAATGTCACACGAGTAATATATCTTTTCTTTf	Mutant construction
Hfq200-D_rev	5′-TAACGTCGACTAACACCAAGCTCATACATCT	Mutant construction
Hfq200-E_For	5′-GCGGAAAGAAGATAGATAAGA	Mutant verification
Hfq200-F_Rev	5′-TATCTTCGACTCCTGTTATTTC	Mutant verification
TUL4-435	5′-GCTGTATCATCATTTAATAAACTGCTG	44
TUL4-863	5′-TTGGGAAGCTTGTATCATGGCACT	44
hel_F	5′-GGGATGTCGCCTTTTGATTTTC	qRT-PCRg
hel_R	5′-CTCTTTTGTCCCTTGTGCTTGC	qRT-PCRg
iglC_F	5′-TGGGTGTTTCAAAAGATGGAATG	qRT-PCR
iglC_R	5′-TGCGCAACATACTGGCAAGC	qRT-PCR
FTL_0994_F	5′-CGCTAATGCAATCGTTGATGG	qRT-PCR
FTL_0994_R	5′-CCGGTGACATGATCAACCTCAT	qRT-PCR
FTL_0120_F	5′-CAAGCACCTCATTTGCTTCGAT	qRT-PCR
FTL_0120_R	5′-TTCTATTGCTTGAGCATCATCTTTTTG	qRT-PCR
pdpB_F	5′-TGAATCCGAAACCAATTCATCT	qRT-PCR
pdpB_R	5′-GGCGCCCACAATGACTTATC	qRT-PCR
FTL_0317_F	5′-GTCGTGCGGACCAAACAAAA	qRT-PCR
FTL_0317_R	5′-CGCAGCATCAGTTGTGAGATTG	qRT-PCR
katG_F	5′-ATACGCCGCAACACAAATGG	qRT-PCR
katG_R	5′-CATCCCCATAGCTCGGAAGG	qRT-PCR
iglA_F	5′-TCAAAAGGAAGATCTGTGGATGC	qRT-PCR
iglA_R	5′-CAAAGTTTGGTGCCTCAAAATCA	qRT-PCR
iglB_F	5′-GGAATGGGTATGGTGGCAAA	qRT-PCR
iglB_R	5′-GGATGCTCAAGCAAAGCTTCAA	qRT-PCR

^aThe SalI site is underlined.

^bThe NheI site is underlined.

^cThe sequence overlapping Hfq2 is underlined.

^dThe NdeI site is underlined.

^eThe BamHI site is underlined.

^fThe sequence that overlaps part of Hfq₂₀₀-B_rev is underlined.

^gSee reference 7.