Part:BBa_K4462020

PccoN1

Extended 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, which controls a variety of metabolic functions related to the aerobic-to-anaerobic transition and is active under hypoxic conditions. The ANR regulator is homologous to the Escherichia coli FNR (fumarate and nitrate reductase regulator) protein, which senses oxygen concentration through the oxidation state of iron in a [4Fe–4S]²⁺or a [2Fe–2S]²⁺ cluster bound to four conserved Cysteine residues. In E. coli, under low oxygen conditions, this cluster undergoes reduction and converts FNR to the dimeric active form. The active FNR binds through a helix-turn helix motif at the C-terminal end to conserved -40 regions (termed FNR-boxes) in FNR-dependant promoters and regulates transcription. This mechanism is conserved across FNR family regulators, 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. In the interest of being conservative, we opted to amplify a ~400bp region starting from the transcription start site from the KT2440 genome for characterisation. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 38
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 51
  
• 1000
  COMPATIBLE WITH RFC[1000]