Table 2.

Primer pairs used in this study ^a

Primer pair	PCR Product/Description	Primer sequences
P1: HVO_0665 preKO BamHI RV1	Primers anneal ~500 bp upstream and downstream of hvo_0665; used with H26 genomic DNA as template to generate pJAM2819 (pre-deletion) by ligation into HindIII to BamHI sites of pTA131	5’- ATGAAGCTTAACGCGAGTCTCCTGTGGGCGCTCGG-3'
P2: HVO_0665 HindIII preKO FW1		5’-ATTGGATCCGACGCGCGCACCTCGCCGTTC-3’
P4: HVO_0665 3’-inverse	Used to generate plasmid pJAM2820 (Δhvo_0665) by inverse PCR using pJAM2819 as a template	5’-TCCCGCGCCGGCCGACGACTGA-3’
P3: HVO_0665 5’-inverse		5’-TCCGTCGCGTCGGTGAAGCCGTCGAACGACAT-3’
P7: HVO_0665 700 bp confirm FW	Anneal ~700 bp upstream and downstream of hvo_0665; used to confirm Δhvo_0665	5’-GCTCGGCGGGGCGAACACG-3
P8: HVO_0665 700 bp confirm RV		5’-GTGACCCACGAGACGACCCACGCG-3’
P5: HVO_0665 NdeI	Used to screen for Δhvo_0665 and generate pJAM2821 (in trans complement of Δhvo_0665)	5’-GGGCGGCATATGTCGTTCGACGGCTTCAC-3’
P6: HVO_0665 KpnI		5’-TTGGTACCGTCGTCGGCCGGCGC-3’
P7: C165A THI4 FW	Used to generate pJAM2822 for synthesis of the site-directed variant HvThi4 C165A	5’- CGCGAACTCACGGCGGTCGACCCCATC-3’
P8: C165A THI4 RV		5’- GATGGGGTCGACCGCCGTGAGTTCGCG-3’

^aItalic in the sequences of primers P1/P2 and P7/P8 indicates the introduced BamHI and HindIII sites for ligation into pTA131 and mutation resulting to Cys to Ala conversion, respectively.