Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii

Table 1 Strains and plasmids used in this study ^a

Strain, plasmid or primer	Description	Source or reference
*E. coli*
TOP10	F⁻ recA1 endA1 hsdR17(r_K ⁻ m_K ⁺) supE44 thi-1 gyrA relA1	Invitrogen
GM2163	F⁻ ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 rpsL136 dam13::Tn9 xylA5 mtl-1 thi-1 mcrB1 hsdR2	New England Biolabs
XL1-Blue	endA1 gyrA96(nal ^R) thi-1 recA1 relA1 lac glnV44 F'[::Tn10 proAB ⁺ lacI ^q Δ(lacZ)M15] hsdR17(r_K ⁻ m_K ⁺)	Stratagene
*Hfx. volcanii*
DS70	wild-type isolate DS2 cured of plasmid pHV2	[10]
H26	DS70 ΔpyrE2	[9]
NC1011	H26 Δthi4	This study
HM1052	H26 ΔubaA	[11]
Plasmids
pTA131	Ap^r; pBluescript II carries P_fdx-pyrE2 with MCS	[9]
pJAM202c	Ap^r; Nv^r; Hfx. volcanii-E. coli shuttle plasmid, empty vector control	[12]
pJAM809	Ap^r; Nv^r; pJAM202c carries P2_rrnA-hvo_1862-strepII	[13]
pJAM2819	Ap^r; pTA131-based plasmid for pre-deletion of hvo_0665	This study
pJAM2820	Ap^r; pTA131-based plasmid for Δhvo_0665	This study
pJAM2821	Ap^r; Nv^r; pJAM809-derived carries P2_rrnA-hvo_0665-strepII (which replaced hvo_1862-strepII)	This study
pJAM2822	Ap^r; Nv^r; pJAM2821-derived carries P2_rrnA-hvo_0665 C165A-strepII	This study

^aAbbreviations: Ap^r, ampicillin resistance; Nv^r, novobiocin resistance; −strepII, C-terminal StrepII-tag fusion coding sequence (preceded by a KpnI site that facilitated generation of the C-terminal StrepII fusion with GlyThr linker). HvThi4 corresponds to HVO_0665, the gene locus tag number of GI: 292654829.

ISSN: 1471-2180