**4. Discussion**

#### *4.1. The Crp*/*Fnr and CtrA*/*QS Regulons Overlap in Dinoroseobacter shibae*

Our analysis revealed an inverse regulatory crosstalk between the Crp/Fnr and CtrA systems in *D. shibae*. We found the denitrification gene clusters and Crp/Fnr regulator genes, especially *dnrF*, to be

part of the CtrA phosphorelay and LuxI2 regulons. *DnrE* was a ffected exclusively by loss of LuxI2, whereas loss of LuxI1 only had minor e ffects on *fnrL*, *dnrD*, and *dnrF* and no e ffect on *dnrE*. In addition to their regulation by LuxI1, which signals cell density, the Crp/Fnr regulators integrate oxygen and NO levels and a ffect all three CtrA phosphorelay genes.

Until now, overlapping regulation by the Crp/Fnr and CtrA systems has only been noted in *D. shibae* for flagellar genes and *recA* [3,12,13], and to our knowledge this level of regulatory interaction has not been reported for alphaproteobacteria. However, a comparable connection between QS and Crp/Fnr regulators has been documented for the gammaproteobacterium *Pseudomonas aeruginosa* where the regulons of the FnrL homolog Anr and QS synthase LasR overlap. Here, denitrification genes are induced by Anr and inhibited by LasR. Additionally, in the absence of *lasR*, Anr regulates production of the QS molecule 4-hydroxy-2-alkylquinoline [42]. At the protein level, nitrite reductase (NirS), a flagellar protein (FliC), and the chaperone DnaK form a complex that influences flagellar formation and motility and thus creates a link between denitrification and motility [43]. In cystic fibrosis infections, *P. aeruginosa* is exposed to ambient conditions with low oxygen tension. The intracellular levels of c-di-GMP increase, which leads to biofilm formation. These conditions also lead to an increase in mutations in the QS transcriptional regulator-encoding gene *lasR*. As *lasR* deletion strains grow to higher cell densities and have higher denitrification rates, it has been suspected that these mutations increase the fitness of the population during infection [44–46].

Combined, these observations indicate that there may be a more widely conserved interaction of Crp/Fnr regulators and QS in proteobacteria. The CtrA phosphorelay is unique to alphaproteobacteria, indicating a potential independent evolution of this regulatory crosstalk in this lineage.

#### *4.2. Inverse Control of the CtrA Regulon by RegA and Anaerobic Photosynthetic Growth Conditions in Rhodobacter capsulatus*

In *R. capsulatus*, the regulons of the redox-responsive two-component system RegA/B [47] and the CtrA phosphorelay overlap. Interestingly, *chpT* stands out because it is the only CtrA phosphorelay gene that is regulated by RegA. Similar to Dnr and Fnr in *D. shibae*, RegA controls the expression of photosynthesis and respiration genes [26]. ChIP-seq with RegA identified binding sites adjacent to several genes also targeted by CtrA: RCAP\_rcc02857 (a c-di-GMP signaling gene involved in GTA production) and its divergently transcribed neighbor (RCAP\_rcc02856), RCAP\_rcc02683 (a chemotaxis gene), and *dksA* (a *dnaK* suppressor gene) [34].

As in *D. shibae*, transcriptomic data from a *fnrL* deletion strain showed no e ffects on the CtrA-controlled traits outside of the CtrA phosphorelay genes themselves. However, ChIP-seq and in silico binding site predictions [29] sugges<sup>t</sup> FnrL binding adjacent to *divL*, *dnaK*, *recA*, flagellar gene clusters, the RcGTA capsid protein-encoding gene, and c-di-GMP signaling genes (including those affecting RcGTA production [41]). Similarly, ChIP-seq with CrtJ [48], a regulator controlling expression of multiple genes involved in photosynthesis, also revealed a connection to the CtrA phosphorelay. Even though the observed transcript level changes in the *crtJ* mutant were small, binding was found adjacent to *ctrA*, *clpX*, a *luxR* family gene*, dnaA*, *spoT*, *ftsZ*, and the first gene in the GTA structural gene cluster (RCAP\_rcc01682) under aerobic and anaerobic cultivation. Binding sites adjacent to *dnaK* and two flagellar genes (*flgB* and *flaA*) were identified under aerobic and anaerobic conditions, respectively.

In *D. shibae*, deletions of the Crp/Fnr regulator-encoding genes indicated an inhibition of the CtrA regulon, but the physiological changes detected by these regulators (switch from aerobic to anaerobic conditions) showed a tendency towards activation of the CtrA regulon. The same was observed for the deletion mutants of the CtrA phosphorelay components and their regulation of the Crp/Fnr regulator genes. In *R. capsulatus*, we could observe a similar pattern but in reverse for regulation of the CtrA regulon by RegA. While the *regA* knockout indicated activation of the CtrA regulon, the switch to anaerobic photosynthetic growth conditions showed an inhibition. This is probably indicative of a more complex interaction among these regulatory systems. However, the *regA* deletion transcriptomic

data are supported by in vivo motility tests that showed reduced swimming ability of the Δ*regA* strain [26].

#### *4.3. Integration of Crp*/*Fnr Regulation into the CtrA Phosphorelay and Regulon*

In *D. shibae*, CtrA binding site predictions and expression data for *ctrA* and *cckA* sugges<sup>t</sup> that CtrA directly regulates its own expression and that of *cckA*, but not *chpT* [13]. Therefore, *chpT* transcription must be regulated from outside of the CtrA phosphorelay and upstream of CtrA. Both, regulatory control of *chpT* and signal integration upstream of CtrA is known for LuxI1 [11]. A similar situation might be possible for Crp/Fnr signal integration due to their regulation of *chpT* (Figure 6A). Since *chpT* is the only RegA-regulated CtrA phosphorelay gene in *R. capsulatus* (and it has a RegA binding site), it seems to play a central role here, too. However, there are also RegA binding sites associated with *clpX* and other genes of the CtrA regulon [26]. Interestingly, the Dnr/Fnr binding site in the *nosR2* promoter in *D. shibae* has the sequence 5--TTAAC-N4-GTCAA-3- [3], which shares a half-site binding motif with CtrA 5--TTAAC-N5-GTTAAC-3- [11]. Previously, comparison between transcriptional regulation and the presence of full and half-site motifs revealed the potential importance of half-site motifs for transcriptional control by CtrA in *R. capsulatus* [34]. Thus, CtrA and Fnr regulators might interact with some of the same/overlapping sequences (Figure 6B).

**Figure 6.** Possible mechanisms of integration of the Crp/Fnr and CtrA systems. (**A**) The LuxI1 and Crp/Fnr signals could be integrated into the CtrA phosphorelay via *chpT* regulation, which does not happen via CckA or CtrA. (**B**) Shared binding site motifs for Crp/Fnr regulators and CtrA might allow direct integration of the NO/oxygen signal into the CtrA regulon. (**C**) An additional histidine kinase (CcsA) has been reported to phosphorylate ChpT in another bacterium, and this could integrate the Crp/Fnr signals and disconnect CckA from the integration. (**D**) Phosphorylation of the Dgc2 receiver domain likely regulates the enzyme's diguanylate cyclase activity and thereby alters the intracellular levels of c-di-GMP, which are known to affect the CtrA regulon.

A distinct role for ChpT is supported by the observation that loss of *chpT* or *ctrA* but not *cckA* results in decreased transcript levels of *dnrF*. It is possible that ChpT integrates signals from more than one kinase into its regulation of CtrA. To our knowledge, the only other instance of a histidine kinase affecting phosphorylation of CtrA via ChpT is CcsA from *Sphingomonas melonis* [49]. Potential homologues of CcsA are encoded in *D. shibae* (Dshi\_1893) and *R. capsulatus* (RCAP\_rcc02545), but effects on transcript levels of these genes were not observed in any of the analyzed datasets. This does not exclude their involvement but also does not allow us to draw further conclusions (Figure 6C).

#### *4.4. Crp*/*Fnr Regulation of the CtrA Regulon is Largely Independent of Oxygen Tension*

Among the Crp/Fnr regulators, only loss of the NO-sensing DnrF resulted in higher inhibition activity of the CtrA system under anaerobic conditions. In *P. aeruginosa*, swimming motility is controlled anaerobically and aerobically and it was suggested that NirS promotes motility in multiple ways, at the protein level or via signaling pathways, depending on oxygen availability [50]. Regulation of QS traits by both NO and oxygen was also found in the interaction of *Vibrio fischeri* with the light organ of its squid host. Here, NO released by the host's immune system regulates the symbionts' settlement via biofilm production while the host's control of oxygen availability regulates bacterial bioluminescence in a circadian manner [51–53]. However, since the Crp/Fnr knockout and physiological change transcriptomic data have opposite effects on the CtrA phosphorelay (inhibition indicated by the knockouts and activation by the shift to anaerobic growth), it is difficult to determine the role of oxygen on the CtrA phosphorelay. In *R. capsulatus*, the knockout transcriptomic data were supported by in vivo experiments, so if the knockout transcriptomic data also reflect the actual CtrA regulon in *D. shibae*, the Crp/Fnr regulators have an inhibitory effect on the CtrA phosphorelay and its regulon. It is known that *Dinoroseobacter* establishes a mutualistic symbiosis with its dinoflagellate host via the CtrA phosphorelay and by means of flagella. It is possible this interaction is repressed towards the end of an algal bloom when oxygen concentrations change, resulting in downregulation of flagella (and other CtrA-regulated traits) via Crp/Fnr regulation.

#### *4.5. The Role of c-di-GMP*

Multiple eukaryotic hosts are known to use NO for communication with microbial symbionts. In some of the characterized systems, NO is sensed by HNOX proteins, which then control c-di-GMP signaling proteins or histidine kinases encoded by genes adjacent to the HNOX-encoding gene. For example, in *Vibrio harveyi*, the HNOX-neighboring histidine kinase phosphorylates the QS transcription regulator LuxU [20], and in *D. shibae*, HNOX inhibits the c-di-GMP signaling enzyme Dgc1 [23]. However, *D. shibae* also has a second c-di-GMP synthesizing enzyme, Dgc2. During adaptation to anaerobic cultivation and at the onset of stationary phase, *dgc2* transcriptional patterns were similar to *chpT* and *fnrL*. The transcript levels of these three genes plateaued, whereas those of the other c-di-GMP signaling, CtrA phosphorelay and Crp/FnrL genes decreased. A unique regulation of *dgc2* was also observed in the *dnrF*, *dnrD*, *cckA*, and *chpT* knockout strains. Thus, both networks (Crp/Fnr and CtrA phosphorelay) regulate *dgc2* and affect its expression in a similar manner as a response to the onset of stationary phase.

The role of *dgc2* in the CtrA phosphorelay and FnrL networks and how it might connect both remain to be clarified. For example, it is possible that phosphorylation of the receiver domain of Dgc2 regulates its c-di-GMP synthase activity. As a result, regulation by the Crp/Fnr or CtrA phosphorelay systems could have different effects on the shared traits (Figure 6D).
