## 3 Regulation of DAPG Biosynthesis
### Regulation by Translational Repressors of the TetR Family  
The _phlF_ and _phlH_ genes encode two pathway-specific regulators from the TetR family. The TetR family regulators consist of an N-terminal DNA-binding domain and a C-terminal domain [44]. These regulators bind to palindromic repeated sequences localized upstream of the target gene, repressing its expression. In most cases, the C-terminal domain interacts with one or several ligands, which subsequently reduce the ability of the regulator to bind DNA [44]. PhlF has been shown to repress _phl_ACBD_ expression by binding, as a dimer, to an inverted repeated sequence (_phlO_) localized downstream of the _phlA_ transcriptional start site [45; 46]. DAPG and MAPG positively regulate _phl_ACBD_ expression by modulating PhlF activity [15; 45]. These two molecules can dissociate the PhlF-_phlO_ complex in a concentration-dependent manner and prevent further binding of PhlF to _phO_[45]. This suggests that DAPG and MAPG could act as ligands for PhlF. As for PhlH, it regulates the intracellular concentration levels of DAPG by modulating the expression of _phlG_[47]. PhlH binds (likely as a dimer) to the upstream sequence of _phlG_ and strongly represses its expression, preventing PhlG-mediated degradation of DAPG [47]. DAPG (and to a lesser extent MAPG) interacts physically with PhlH and can dissociate the PhlH-DNA complex, releasing the PhlH-mediated repression of _phlG_.  
PhlF and PhlH regulate DAPG biosynthesis and degradation at different stages of growth. PhlF acts as a repressor of DAPG biosynthesis during the early growth stages. This is evidenced by the fact that DAPG production is observed earlier in a _phlF_ mutant [15; 45; 46]. The growth of the _phlF_ mutant is, however, reduced in the early growth stages compared to the wild type [46]. This suggests that, by repressing DAPG production in the early stages, PhlF enables DAPG-producing _Pseudomonas_ spp. to outcompete and outgrow other microorganisms. On the other hand, PhlH-mediated repression of _phlG_ in the early stages of growth is essential for DAPG production, as a _phlH_ mutant produces very low levels of DAPG [15; 47]. This indicates that PhlH-mediated repression of _phlG_ would normally be abolished later in the growth stages. [PERSON] and colleagues [47] proposed that PhlG promotes bacterial growth in a nutrient-limited environment by reducing the resources allocated to DAPG production in the late growth stages. Indeed, a _AphIG_ mutant exhibited a lower growth rate and cell density in a nutrient-limited medium compared to the wild type, but this difference was not observed in the richer KB medium. Thus, DAPG sequentially promotes its own biosynthesis and degradation by modulating PhIF and PhIH activity. Furthermore, PhIF-mediated repression of _phIACBD_ is abolished sooner during growth when compared to the PhIH-mediated repression of _phIG_. In this regard, we believe that PhIF and PhIH might have differential binding affinities, either for DNA or for MAPG/DAPG to explain this discrepancy. Other factors could also modulate PhIF and PhIH activity.  
Figure 3: Regulation of DAPG biosynthesis in _Pseudomonas_ spp. DAPG production is regulated at the transcriptional level by the TetR repressors PhIH and PhIF, and at the posttranscriptional level via the Gac/Rsm pathway. PhIF binds as a dimer to the operator _phO_, located between _phIF_ and _phIA_ and represses the expression of _phIABCD_. When DAPG and MAPG concentrations increase, MAPG and DAPG interact with PhIF, dissociating the _phO_-PhIF complex and relieving _phIACBD_ transcription. Likewise, PhIH binds to an operator, located between _phIH_ and _phIG_ and represses _phIG_ expression. DAPG and MAPG can dissociate PhIF-DNA binding and relieve _phIG_ expression. Upon reception of an environmental cue, GacS activate GacA by phosphotransfer. GacA activates the transcription of several small non-coding RNAs (RsmX, RsmY and RsmZ), which in turn sequester the RNA-binding proteins RsmA/RsmE, relieving _phIABCD_ expression.