Long hierarchical cascades regulate developmental processes
Biofilm formation and bacterial mobility can be seen as the outcome of specialised cell differentiation pathways. Biofilm formation involves subsequent cellular changes at the morphological and physiological levels resulting in bacterial populations with multiple phenotypes.4,30,31 Furthermore, bacteria living in biofilm communities are present in different developmental stages (at least four defined stages) as has been observed in Cryptoccocus, Pseudomonas, Staphylococcus, Xanthomonas, etc.32–37
The part of E. coli transcriptional cross-regulatory network involved in the control of biofilm formation and motility exhibits a relatively complex topology with several long cascades from CRP, IHF and FNR to downstream specialised TFs (see Table 1 and Fig. 2a). Several of these cascades converge on the master regulators for motility and biofilm formation (FhlCD and CsgD, respectively).
Furthermore, a relatively high proportion of the downstream specialised TFs autoactivate themselves (see below), a feature that is rare at the level of the whole transcriptional network. In addition, we could identify 105 multi-element circuits in the transcriptional regulatory network involving 2 to 14 different elements (including transcription and sigma factors, see Fig. 3 and supplementary material for the complete list of circuits). Interestingly, the information fluxes inside these circuits follow a frequent route in the network with the order CsgD > Cspa > HNS > GadX > RpoS > IHF. From IHF the regulatory flow diverge in two main directions: IHF > RpoH > RpoD and IHF > FIS > CRP. In line with this observation, CRP and RpoD are the major distributors, whereas CsgD and GadX are the main collectors of information. These multi-element circuits inside the cross-regulatory network are novel observations. The functional relevance of these regulatory structures remains to be assessed experimentally. Tentatively, these circuits may implement a feedback between the presence of different stresses and the basic machinery for replication and growth.
CsgD, the master regulator for biofilm formation, is directly involved in 28 of these long circuits, suggesting a particular tight coupling of CsgD activity with the intracellular status. In contrast, FlhCD, the master compound regulator for motility and chemotaxis, is known to be regulated by nine other TFs but has not yet been reported to regulate any other TF.
Note that the motility module has its own sigma factor, FliA, regulated by FlhCD. FliA is required for the transcription of the genes required for the last part of flagella development and for chemotaxis machinery.38,39 In contrast, the genes for biofilm development are transcribed by the housekeeping sigma 70 and RpoS, the sigma factor expressed in response to general stress.40
The execution of such long regulatory cascades requires time. Indeed, complete flagella assembly may take a generation time or longer.41–43 The occurrence of positively autoregulated TFs at several intermediate steps enables informed decisions about the cellular/environmental condition. In some conditions, cellular duplication might be faster than the conclusion of a long regulatory cascade. This implies that bacterial populations likely consist of mixtures of bacteria with transcriptional programs at different levels in long regulatory cascades.