**3. Results**

#### *3.1. Overlap of the Crp*/*Fnr and CtrA Regulons in* Dinorosebacter shibae

The possible interaction between the Crp/Fnr regulator and CtrA phosphorelay networks was first assessed using transcriptomic datasets for regulator deletion mutants. The changes of transcript levels of known Crp/Fnr- and CtrA-controlled traits revealed a strong overlap of both regulons, with the regulator-encoding genes themselves affected by losses of the other regulators (Figure 1). Under both aerobic and anaerobic conditions, the loss of *dnrD* or *dnrF* resulted in increased transcript levels of the CtrA phosphorelay, QS, flagellar motility, tad pilus, competence and recombination, gene transfer agen<sup>t</sup> (GTA), *divL* and c-di-GMP signaling genes (Figure 1A). In all datasets, the GTA genes showed comparatively small changes in transcript levels (Figure 1), probably as a result of a small subpopulation actually expressing these genes [13]. Only the loss of *dnrF* led to a change in gene expression between aerobic and anaerobic conditions, since a greater increase in the transcript levels could be observed under anaerobic conditions for most of its regulon (Figure 1C). The loss of *fnrL* or *dnrE* resulted in increased transcript levels of *ctrA*, *cckA*, *chpT*, *luxI1*, *luxR1*, and *luxR2* but had little to no effect on the downstream CtrA regulon (Figure 1B).

**Figure 1.** Transcriptomic data for genes in selected functional groups in different knockout strains. The four Crp/Fnr regulator knockouts were grown under aerobic (ae) or anaerobic (an) conditions. The log2 fold changes compared to the respective wild type (WT) (**A**,**B**) or against themselves grown at different conditions, are shown (**C**). The CtrA phosphorelay and quorum sensing system knockouts were grown aerobically to the stationary phase and compared to the WT (**D**). The functional group assignments on the right are based on published information as described in Supplementary Table S1. Note: the Δ*luxI1* strain retains a portion of the gene that can therefore result in mapped reads.

Almost all examined genes showed an opposite pattern in the CtrA phosphorelay and *luxI1* mutants (Figure 1D) compared to *dnrD* and *dnrF* (Figure 1A). Most of the genes showed decreased transcript levels in strains lacking any of the CtrA phosphorelay genes, with the exceptions of the Crp/Fnr regulators where the largest increase was found for *dnrF* (Figure 1D). Loss of *luxI1* resulted in increased transcript levels for *fnrL*, *dnrD*, and *dnrF*, but no changes were observed for *dnrE* (Figure 1D).

#### *3.2. The Role of ChpT in Signal Integration*

In *D. shibae*, deletion of neither *ctrA* nor *cckA* had an influence on expression of *chpT*, whereas the loss of either *ctrA* or *chpT* resulted in decreased expression of *cckA* (Figure 1D) [11]. However, all three CtrA phosphorelay component genes showed reduced transcript levels in the absence of the AHL synthase *luxI1* (Figure 1D) [13], whereas loss of the Crp/Fnr regulators resulted in increased transcript levels of these genes (Figure 1A,B). Therefore, in contrast to *ctrA* and *ccka*, *chpT* is not regulated by the CtrA phosphorelay itself, but by other factors that can thereby control the phosphorylation state of CtrA. These findings also sugges<sup>t</sup> that *chpT* transcription is regulated oppositely by QS and the Crp/Fnr regulators.

This is supported by binding site predictions for FnrL [3] that sugges<sup>t</sup> it binds at the promoter of *chpT* and *clpX*, which encodes a protease known to cleave CtrA [3,38,39]. Deletion of *fnrL* strongly increased the expression of *chpT* but only resulted in minimal changes for *clpX* (Figure 1B). Binding site prediction for the Dnr regulators did not find any binding sites near *clpX* or the CtrA phosphorelay genes [3].

It was previously found that more genes were a ffected by the loss of *chpT* than *cckA* [11], suggesting ChpT regulates some genes independent of CckA and that a di fferent kinase might regulate its activity and thereby a ffect downstream gene expression. Among the genes a ffected by the loss of *chpT* but not *cckA*, *dnrF* was the most upregulated gene during exponential growth while *lexA* and *recA* were among those most downregulated genes in both exponential and stationary phases (Figure 2). Although there was a small increase in transcript levels of *dnrF* in the *cckA* deletion strain during exponential growth, it did not pass the threshold we defined (see Materials and Methods). These findings sugges<sup>t</sup> a link between *dnrF* and *chpT*.

Additional discrepancies between CckA and ChpT are apparent from their opposing e ffects on the *nap* gene cluster during exponential growth (Figure 2A), although this is not maintained in stationary phase (Figure 2B). In exponential phase, loss of *cckA* led to decreased transcript levels of the *nap* gene cluster, while the loss of *ctrA* and *chpT* led to increased levels (Figure 2A). This cluster is the only denitrification cluster activated by FnrL but repressed by the three Dnr regulators [3]. Interestingly, transcript levels of all four denitrification gene clusters were increased in the AHL synthase knockout Δ*luxI2* but were una ffected in Δ*luxI1* (Figure 2C).

#### *3.3. Time-Resolved Evaluation of Environmental Changes and the Regulation of c-di-GMP Signaling Genes*

Interactions between the networks in *D. shibae* were further analyzed using time-resolved transcriptomic datasets. These were collected following the switch from aerobic to anaerobic conditions in wild type cells (Figures 3A and 4A) [31], following the external addition of AHL autoinducer to the AHL synthase mutant Δ*luxI1* (Figures 3B and 4B) [13], and through the culture growth phases for Δ*luxI1* in the absence of AHL (Figure 4C) [14].

**Figure 2.** Comparison of CtrA phosphorelay, Crp/Fnr regulator, and denitrification gene expression control by CtrA phosphorelay and LuxI1/2 synthases during exponential and stationary growth phases. Samples for the *ctrA*, *cckA*, and *chpT* knockout mutants were analyzed at mid-exponential (OD 0.4) (**A**) and stationary (six hours after onset of stationary phase) (**B**) phases of growth. The Δ*luxI1* data were obtained during stationary phase, six hours after the onset of stationary phase, and the Δ*luxI2* data were obtained during the mid-exponential growth phase (**C**).

Upon the shift to anaerobic conditions, all three *dnr* genes showed an immediate increase in transcript levels for 30 min and then stayed constant, whereas those of *fnrL* decreased (Figure 4A). These changes corresponded with increased transcript levels of the denitrification gene clusters, with the *nap* cluster showing a slightly different pattern than the *nir* and *nos* clusters (Figure 3A). Slight increases were observed for the c-di-GMP signaling, flagellar, tad pilus, and QS genes (Figure 3A). Four of the five c-di-GMP signaling genes showed increased transcript levels following the transfer to an anaerobic environment, whereas *dgc2* showed a slight decrease (Figure 4A).

The addition of AHL to the Δ*luxI1* strain led to increased transcript levels for all CtrA- and QS-controlled genes (Figure 3B). This included the CtrA phosphorelay and c-di-GMP signaling genes, with *dgc2* showing the largest increase (Figure 4B). No effect was visible for the Crp/Fnr regulator-encoding genes (Figure 4B) and only a minor increase of the *nap* gene cluster was observed among the denitrification genes (Figure 3B).

Due to the increased transcript levels observed for CtrA regulon genes in the *dnrD* and *dnrF* deletion strains, it was expected that the same genes would also be decreased under anaerobic conditions. Instead, it turned out that the change from aerobic to anaerobic conditions (Figure 3) resulted in increased transcript levels for these genes. However, this increase was small, and effects were not observed for some genes that appeared to be controlled by the individual regulators based on the knockout transcriptomic data (Figure 1). This included the regulation of the CtrA phosphorelay genes by the Crp/Fnr regulators. Vice versa, loss of the CtrA phosphorelay genes indicated their repression of Crp/Fnr regulator gene expression (Figure 1D), but the contrary was observed in the respective physiological datasets where the Crp/Fnr regulators seem to be upregulated (Figure 3B). Notably however, in both physiological datasets, *dgc2* stands out as distinctly affected compared to other c-di-GMP signaling genes (Figure 4A,B). Also, in the non-induced Δ*luxI1* culture, no influence of the QS null mutant on the Crp/Fnr regulators was observed, but the CtrA phosphorelay and c-di-GMP signaling genes were down-regulated (Figure 4C).

**Figure 3.** Time-resolved transcriptomic analysis for genes in selected groups in response to environmental changes. (**A**) Gene expression changes after the shift to anaerobic growth compared to aerobic conditions. (**B**) Gene expression after external addition of autoinducer 3-oxo C14 HSL to the QS synthase null mutant (Δ*luxI1*).

Interestingly, in contrast to *fnrL*, *dgc2*, and *chpT*, the other Crp/Fnr regulators, c-di-GMP signaling, and CtrA phosphorelay genes all decreased at the onset of the stationary phase (Figure 4C). Moreover, analysis of the Crp/Fnr knockout data showed that the loss of *dnrF* or *dnrD* resulted in increased transcript levels of four of the c-di-GMP signaling genes under anaerobic growth conditions, with only *dgc2* being unaffected (Figure S1A). Loss of *luxI1* and the CtrA phosphorelay genes resulted in decreased transcripts for all five genes (Figure S1B,C), although the effects on *dgc2* were smaller than for the other genes in the stationary phase (Figure S1C).

**Figure 4.** Time- and density-resolved transcript levels in three different conditions for three groups of regulators. The expression profiles of the CtrA phosphorelay genes (top), c-di-GMP signaling genes (middle), and four Crp/Fnr regulator-encoding genes (bottom) are plotted. The changes in transcript levels were monitored after the switch from aerobic to anaerobic growth over a time period of 120 min (**A**), after the external addition of autoinducer (3-oxo C14 HSL) to the synthase null mutant (Δ*luxI1*) over a period of 180 min (**B**), and during logarithmic (samples 1–5) and stationary (sample 6) phases of growth as determined by optical density (**C**).

#### *3.4. E*ff*ects on the CtrA Regulon during Coculture of Dinoroseobacter shibae and Its Algal Host*

In the two-phase interaction of *D. shibae* with its dinoflagellate host *Prorocentrum minimum* [14,40], a mutualistic growth phase (0 to 21 days of cocultivation) is followed by a pathogenic growth phase (21 to 30 days of cocultivation) that leads to death of the algae [15]. Analysis of the transcriptomic data of *D. shibae* during cocultivation showed an overall increase in the transcription for the CtrA regulon genes during the transition between the two phases (day 24 compared to day 18), followed by a decrease during the late-pathogenic phase, after 30 days (Supplementary Figure S2).

Of the CtrA phosphorelay genes, only *chpT* remained upregulated during the pathogenic interaction. Evaluation of the denitrification gene clusters showed strong variation among these genes (Supplementary Figure S2), likely arising from overall low expression levels, and this made it difficult to draw any conclusions.

#### *3.5. RegA Activates the CtrA Regulon in Rhodobacter capsulatus*

Next, we asked if the observed overlap between redox regulators and the CtrA phosphorelay system is conserved in another member of the family *Rhodobacteraceae*. For *R. capsulatus*, transcriptomic data were available for knockout mutants of *ctrA*, *cckA*, and the known redox regulator-encoding genes *fnrL*, *regA*, and *crtJ*. We identified three additional Crp/Fnr regulator-encoding genes in this bacterium based on blast searches (RCAP\_rcp00107, RCAP\_rcc01561, RCAP\_rcc03255), but these genes showed no evidence of differential regulation in any of the analyzed datasets and we did not consider them further. A blast search also identified a homologue (RCAP\_rcc02630) of the HNOX-encoding gene of *D. shibae* (Dshi\_2815). This gene encodes a protein with a predicted heme nitric oxide/oxygen binding (HNOB) domain and is located adjacent to a c-di-GMP signaling gene (RCAP\_rcc02629) that was recently demonstrated to affect GTA production and motility in *R. capsulatus* [41]. When bound to NO, the HNOX homologue in *D. shibae* inhibits the activity of the diguanylate cyclase Dgc1, which is encoded by the neighboring gene [23].

FnrL is the only Crp/Fnr regulator that has been studied in *R. capsulatus* [29]. Its loss did not result in any large changes in transcript levels for the examined traits under anaerobic phototrophic conditions (Figure 5), and the same was observed for the loss of *crtJ*, which encodes a transcription factor that controls numerous photosynthesis and cytochrome genes [32] (Figure 5). RegA is the response regulator of the RegB/A two-component system that controls photosynthesis, nitrogen and carbon fixation, denitrification, and respiration genes in response to oxygen availability [26]. In contrast to *fnrL* and *crtJ*, we found that the loss of *regA* resulted in a strong decrease in transcript levels of the CtrA regulon genes (Figure 5), indicating that RegA acts as a direct or indirect activator of these genes. Like the genes involved in regulation of photosynthesis and the change between aerobic/anaerobic lifestyle in *D. shibae*, loss of *regA* affected *chpT* the most among the CtrA phosphorelay genes in *R. capsulatus*. Loss of the CtrA phosphorelay genes had no influence on transcription of *fnrL*, *regA*, *regB*, or the other putative Crp/Fnr regulator-encoding genes (Supplementary Figure S3). A comparison of photosynthetic anaerobic growth and aerobic cultivation in *R. capsulatus* showed the CtrA-regulated traits have reduced transcript levels under anaerobic conditions (Figure 5).

**Figure 5.** Effects of growth conditions and three regulator knockouts on the transcript levels of eight categorized groups of genes in *Rhodobacter capsulatus*. The microarray-based transcriptomic data for aerobic versus anaerobic growth in the wild type and for three mutants, *fnrL*, *regA*, and *crtJ*, compared to the wild type are shown.
