Table 1 Bacterial strains, plasmids and primers used in this study
Strain or Plasmid | Description1 | Source or Reference |
|---|---|---|
E. coli | Â | Â |
DH10B | F- mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80d lacZ ΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG | [30] |
M. smegmatis | Â | Â |
mc2155 | Electrocompetent wild-type strain of M. smegmatis | [25] |
NP6 | mc2155 ΔpitA | This study |
NP13 | mc2155 ΔpitA carrying pCPitA; Hygr | This study |
Plasmids | Â | Â |
pJEM15 | E. coli-mycobacteria shuttle vector for the creation of transcriptional promoter fusions to lacZ; Kmr | [27] |
pX33 | pPR23 [29] carrying a constitutive xylE marker; Gmr | [13] |
pUHA267 | E. coli vector with mycobacteriophage L5 integrase and attP for integration into L5 attB of mycobacteria; Hygr | AgResearch, Wallaceville, NZ |
pAH1 | pJEM15 harbouring a 750 bp pitA-lacZ fusion; Kmr | This study |
pPitAKO | pX33 harbouring the pitA deletion construct; Gmr, Sacs, ts | This study |
pCPitA | pUHA267 harbouring pitA with its native promoter; Hygr | This study |
pSG10 | pJEM15 harbouring a 500 bp phnD-lacZ fusion; Kmr | [13] |
pSG42 | pJEM15 harbouring a 560 bp pstS-lacZ fusion; Kmr | [13] |
Primers | Â | Â |
PitA1 | AAATTTACTAGTGTCGTCGATGGATTCTTC | This study |
PitA2 | TCAGATCAGGTGAAGTCGAAAGCAAGTG | This study |
PitA3 | CGACTTCACCTGATCTGAAGGAACGTTGA | This study |
PitA4 | AAATTTACTAGTAACGAGGGTGGTAGACAGAC | This study |
PitA5 | AAATTTGCATGCGTGAAGTCGAAAGCAAGTG | This study |
PitA6 | AAATTTGTCGTCGATGGATTCTCC | This study |
cPitAf | AAATTTAAGCTTGTCGTCGATGGATTCTCC | This study |
cPitAr | AAATTTAAGCTTACGTTCCTTCAGATCAGAC | This study |