Part:BBa_K4462021

Panr

Truncated version of the ANR-regulated, hypoxia-activated promoter of ccoN1 from Pseudomonas putida KT2440 (Ugidos et al., 2008)

Usage and Biology

Transcriptional regulation with respect to oxygen availability in Pseudomonas species is principally achieved through the ANR (anaerobic regulation of arginine deaminase and nitrate reduction) global regulator. The dimeric active form of ANR carries a [4Fe–4S]²⁺ cluster and binds to the conserved ANR-binding box (5′-TTGATNNNNATCAA-3′) that is typically located in the -40 region of regulated promoters. Upon exposure to oxygen, this iron-sulphur cluster is partly destroyed and the ANR loses its DNA binding and gene regulatory ability. Depending on the position of its binding site, ANR can activate or repress transcription under hypoxic conditions where it exists in its active form.

The Pseudomonas putida KT2440 terminal oxidase Cbb3-1 is known to be activated under low oxygen tension. In particular, transcription of the gene ccoN1 undergoes a ~500-fold increase under hypoxic conditions in an ANR-dependent manner. The promoter of ccoN1 (hereafter, PccoN1) contains a putative ANR-binding box at the -40.5 region and was selected as a potential candidate for characterisation. The -10 and -35 elements are recognised by RNA polymerase bound to sigma-70, with the -10 region closely matching the P. putida consensus. This part is a truncated 58bp version of PccoN1 that contains the -10 and -35 regions and the putative -40.5 ANR-binding box, which in theory should be induced by ANR under hypoxic conditions. We intended to use a Pseudomonas-compatible hypoxic promoter in our project to restrict the expression of Cytochrome P450cam to hypoxic conditions to enhance halocarbon biodegradation. While ANR is structurally similar to the E. coli FNR regulatory protein and interacts with the FNR-binding box, the induction of a promoter with FNR-binding sequences by ANR is relatively inefficient compared to the induction of a promoter bearing ANR-binding sequences. This part is thus a replacement for promoters like (BBa_K1123000) in Pseudomonas species.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]