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. 2014 Dec 11;81(1):220–230. doi: 10.1128/AEM.02720-14

FIG 5.

FIG 5

Determination of the promoter activities of the pnpA, pnpB, pnpC, and pnpR operons and their derivatives. (A) Expression of promoter-lacZ translational fusions containing approximately 300-bp promoters of above the four operons in PaW340(pBBR1mcs-2) without PnpR. The β-galactosidase activities were determined in the above strains containing pCMgfp-lpAlacZ (lpA in the figure), pCMgfp-lpBlacZ (lpB), pCMgfp-lpClacZ (lpC), and pCMgfp-lpRlacZ (lpR) with or without PNP. (B) Expression of promoter-lacZ translational fusions containing approximately 300-bp promoters of the above four operons in PaW340(pBBR1-tacpnpR) with PnpR expression in cis. The β-galactosidase activities were determined in the above strain containing pCMgfp-lpAlacZ (lpA in the figure), pCMgfp-lpBlacZ (lpB), pCMgfp-lpClacZ (lpC), and pCMgfp-lpRlacZ (lpR) with or without PNP. There was a significant difference in promoter activity with or without PNP among the above four strains (P < 0.001, paired-samples test). (C) Expression of promoter-lacZ translational fusions containing 200-bp promoters of the above four operons in PaW340(pBBR1-tacpnpR) with PnpR expression in cis. The β-galactosidase activities were determined in the above strain containing pCMgfp-spAlacZ (spA in the figure), pCMgfp-spBlacZ (spB), pCMgfp-spClacZ (spC), and pCMgfp-spRlacZ (spR) with or without PNP. There was a significant difference in promoter activity with or without PNP among the above four strains (P < 0.001, paired-samples test). (D) Expression of promoter-lacZ translational fusions containing 200-bp mutant promoters of the above four operons in PaW340(pBBR1-tacpnpR) with PnpR expression in cis. The β-galactosidase activities were determined in the above strain containing pCMgfp-spAmlacZ (spAm in the figure), pCMgfp-spBmlacZ (spBm), pCMgfp-spCmlacZ (spCm), and pCMgfp-spRmlacZ (spRm) with or without PNP. There was a significant difference in the promoter activity between regulatory binding sites (RBSs) of the pnpA, pnpB, and pnpR operons and their mutated sites from strain PaW340(pBBR1-tacpnpR) grown on LB plus MM (1:4) with PNP or without PNP (P < 0.001, paired-samples test).