Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase

Table 1

Primers, strains, and plasmids used.

(a)

Primer	Sequence
Upppx	GTCCATATGGACTTGCAAAGC
Dwnppx	TGCTTACCGCACGTTGAGGC
UpppxN	CATATGCACCAGCACGAGGAC
DwnppxC	CTAAGCGCCGACGAGGTCG
Up 304	GTCCATATGGACTTGCAAAGC
DWN 304	GACCAGTTAGTCGTAGAGAAC
E126A	CTCCGGCCGCGAGGCAGCCCGCCTGATC
D149A	CCGGCTGGTCAGCGCCATCGGCGGCGGC
G151A	GGTCAGCGACATCGCCGGCGGCAGCACCG
S154A	CATCGGCGGCGGCGCCACCGAGTTCATC
E156A	CGGCGGCAGCACCGAGTTCATCATCGGCC

(b)

Strain or plasmid	Genotype and/or description	Reference or source
P. aeruginosa PAO1	Wild type	WUCG (2005)
E. coli XL10-Gold	TetΔ(mcrA)183  Δ(mcrCB-hsdSMR-mrr) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lacHte [F′proAB lacI^qZΔM15 Tn10 (Tet) Amy Cam]	Stratagene
E. coli BL21-CodonPlus	E. coli B F- dcmompThsdS(rB- mB-) gal	Promega
pCR 2.1-TOPO	Amp^rKm^r; vector for TA cloning of PCR products	Invitrogen
pET-15b	Amp^r, T7 promoter, multiple cloning sites, His-tag coding sequence	Qiagen
pCR-ppx	1.5 Kb EcoRI/NdeI fragment containing the ppx gene cloned into pCR 2.1-TOPO	This study
pCR-Nppx	0.94 Kb EcoRI/NdeI fragment containing the ppx gene cloned into pCR 2.1-TOPO	This study
pCR-N303ppx	0.94 Kb EcoRI/NdeI fragment containing the N-terminal domain of ppx gene cloned into pCR 2.1-TOPO	This study
pCR-Cppx	0.56 Kb EcoRI/NdeI fragment containing the ppx gene cloned into pCR 2.1-TOPO	This study
pET-ppx	1.5 Kb EcoRI/NdeI fragment containing the ppx gene cloned into pET-15b	This study
pET-Nppx	0.94 Kb EcoRI/NdeI fragment containing the N-terminal domain of ppx gene cloned into pET-15b	This study
pET-N303ppx	0.94 Kb EcoRI/NdeI fragment containing the N-terminal domain of ppx gene cloned into pET-15b	This study
pET-Cppx	0.56 Kb EcoRI/NdeI fragment containing the ppx gene cloned into pET-15b	This study

A pair of complementary primers was used to create each mutant. The sequence of the sense strand is shown. Changes in the sequences are shown in boldface type and underlined.