TABLE 3.

PCR primer pairs used in this study.

	PCR, sequencing and RT-PCR
Primer name	Primer sequence
fHbp cloning (restriction sites underlined)
PacI-fHbp-for	5′-GCGCAATTAATTAATTGCTTCTTTGACCTGCC-3′
PmeI-fHbp-rev	5′-ACCTGTTTAAACAATGGTTATTGCTTGGCG-3′
pGCC4 sequencing primers
pGCC4-fwd	5′-AGACATCCGCCAAACCATCC-3′
pGCC4-rev	5′-TGCTTCCGGCTGTTGTGTGG-3′
RT-PCR primers
fHbp-for	5′-GTTTCGCAACCATCTTCCCG-3′
fHbp-rev	5′-GACTTTATCCGTCAAATCGA-3′
recA-for	5′-GAAGAGGTATTGGCAACGA-3′
recA-rev	5′- CGGATTTGTTGATGATGTCG-3′
Bacterial two-hybrid (restriction sites underlined)
BamHIfwd_MC58FHbp	5′-GCGAGGATCCATGACTAGGAGTAAACCTGTGAATC-3′
EcoRIrev_MC58FHbp	5′-GATCGAATTCTTATTGCTTGGCGGCAGGCCGATATG-3′
BamHIfwd_L91543FHbp	5′-GCGAGGATCCATGCCGTCTGAACCGTTGTTCGGACGGC-3′
EcoRIrev_L91543FHbp	5′-GATCGAATTCTTACTGCTTGGCGGCAAGACCGATATGG-3′
PstIfwd_SecA	5′-CGATCTGCAGATGCTGACAAACATTGCCAAGAAAATC-3′
SmaIreverse_SecA	5′-CGACCCGGGTTAAGCCAGTTTGCCGTGGCATTG-3′
Site-directed mutagenesis (incorporated mutations italicized)
SP1_Fwd	5′-CTTCTGCTGCCTTTCTCTGACCG-3′
SP1_Rev	5′-GCAGTTCGGTTCACAGGT-3′
SP2_Fwd	5′-TTCTCTGACCACTGCCCTGATTC-3′
SP2_Rev	5′-AAGCAGCAGAAGGCAGTT-3′
Deletion of fHbp by SOEing
HA1_FHbp_Fwd	5′-GATAGAATTCCGAGTATGCAGCTTTG-3′
Rev_HA1_FHbp	5′-GATGATGGTTGCCATTGTGAAAATGCCGTCC-3′
Kan_fwd	5′-TTCACAATGGCAACCATCATCGATG-3′
Kan_rev	5′-AAACCTTTCAGACGGCATGTAATGCTCTGCC-3′
HA2_FHbp_fwd	5′-ATGCCGTCTGAAAGGTTTACTCCTAGTCATACG-3′
HA2_FHbp_rev	5′-CTTAGGATCCCCACGGCGCATACAAATTC-3′
Deletion of slam by SOEing (kan sequence in bold, HA sequence underlined, overlapping sequence italicized)
HA1_0313_Fwd	5′-GATAGAATTCAGGCGCAGTTTACCTACTTG-3′
HA1_0313_rev	5′-GATGATGGTTGTATCAATCGGCGGATTGTATC-3′
0313_Kan_fwd	5′-GATACAATCCGCCGATTGATACAACCATCATCGATG-3′
0313_Kan_rev	5′-AACAGCAATTCAGACGGCATGTAATGCTCTGCC-3′
HA2_0313_fwd	5′-ATGCCGTCTGAATTGCTGTTCCTTTTCGGAGG-3′
HA2_0313_rev	5′-CTTAGGATCCGAACGGCTTATGGCTTTGGGAC-3′
Disruption of Lnt
Lnt_fwd	5′-GGCAGGAGATATGCGCTAAG-3′
Lnt_rev	5′-GTACTGGTCGCCCACAACCT-3′