**1. Introduction**

Bacteria sense and process environmental signals in order to adapt to changes in their surroundings. These signals are relayed through regulatory networks that adjust the cells' behavior, often through changes in gene expression. The alphaproteobacterium *Dinoroseobacter shibae* is a member of the marine roseobacter group and an aerobic anoxygenic photoheterotrophic bacterium, capable of both aerobic and anaerobic respiration [1]. It can be free-living or an algal symbiont [1] and is a metabolically versatile bacterium able to adapt to changes in its highly dynamic environment. For example, at the end of an algal bloom when the oxygen concentration drops, an alternative terminal electron acceptor such as nitrate can be used for respiration [1,2].

The response to the change from aerobic to anaerobic conditions is controlled by four Crp/Fnr transcriptional regulators in *D. shibae* [3]. Crp/Fnr regulators are widely distributed among bacteria and form a superfamily consisting of 14 phylogenetic subgroups [4]. The versatility of this family is reflected by both the wide range of signals that are sensed, such as temperature [5], oxygen [6], and nitric oxide (NO) [7], and the range of metabolic processes regulated upon activation, which include respiration-related processes and especially the transition between aerobic and anaerobic lifestyles [3,8].

Two well-studied members of this family are the Dnr and Fnr proteins. Dnr proteins bind a heme cofactor that allows for sensing of NO [4,9], while Fnr proteins react to low oxygen tension [4,6]. In *D. shibae*, FnrL and DnrD regulate DnrE and DnrF in a cascade-type network that controls the transition from aerobic to anaerobic growth, heme and carotenoid synthesis, multiple other metabolic processes, and flagellar synthesis [3]. The importance of these regulators in *D. shibae* is well illustrated by the observation that loss of FnrL affects the transcript levels of over 400 genes [3].

Another important regulatory system in *D. shibae* is the CtrA phosphorelay [10]. Like the Crp/Fnr regulators, this phosphorelay integrates an environmental signal, in this case, the autoinducer concentration as an indicator of cell density, and adjusts gene expression in response [11]. This phosphorelay is conserved within the majority of alphaproteobacterial lineages and consists of the histidine kinase CckA, the phosphotransferase ChpT and the transcriptional regulator CtrA [10]. In *D. shibae*, the CtrA phosphorelay is activated by the quorum sensing (QS) signal of the main acyl-homoserine lactone (AHL) synthase (LuxI1) with subsequent regulation of genes for flagellar motility, recombination and competence proteins, a tight adherence (tad) pilus involved in attachment to carbohydrates on the host cells [12], cell cycle control, gene transfer agen<sup>t</sup> (GTA) production, bis-(3--5-)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling, the NO-sensing heme-nitric oxide/oxygen binding domain (HNOX) protein, and the AHL synthases LuxI2 and LuxI3 [11,13,14]. Deletion of *cckA* has been found to abolish the mutualistic interaction between *D. shibae* and its algal host, demonstrating that the CtrA phosphorelay is essential for establishment of this symbiosis, at least partly due to the requirement for flagella [15]. The Crp/Fnr and CtrA phosphorelay networks are connected by their shared regulation of flagellar gene expression and due to their involvement in symbiosis with the host dinoflagellate.

There are three ways bacteria can be exposed to NO. Some bacteria generate NO during denitrification, and this is considered the activator for DnrD in *D. shibae* [3,16]. NO can be produced intracellularly through the oxidization of L-arginine to NO and L- citrulline [17] or via a nitric oxide synthase (NOS) [17,18]. NO released by some eukaryotic organisms can be a form of communication with their symbiotic bacteria and is then typically sensed by HNOX proteins [19]. The HNOX genes are often located adjacent to genes encoding c-di-GMP signaling proteins or histidine kinases. In the context of symbioses, only a few NO-detecting systems have been found that do not involve c-di-GMP signaling but instead directly integrate into QS systems [20–22]. In *D. shibae*, an HNOX protein detects NO and thereupon inhibits the c-di-GMP synthesizing enzyme Dgc1 [23].

The potential for overlap between Crp/Fnr-based regulation and the CtrA phosphorelay also exists in the purple non-sulfur alphaproteobacterium *Rhodobacter capsulatus*. Its CtrA phosphorelay was originally discovered due to its regulation of GTA production [24], but it also affects many other genes such as those associated with flagellar motility, gas vesicles, and c-di-GMP signaling [24,25]. Like *D. shibae*, *R. capsulatus* can switch between aerobic and anaerobic lifestyles, which involves Crp/Fnr regulation, the RegA/B two-component system, and CrtJ [26–28]. Loss of FnrL affects the transcript levels of 20% of *R. capsulatus* genes [29], including 42 that are directly regulated and encode c-di-GMP signaling, gas vesicle, and flagellar proteins, among others [29].

These initial surveys of the activities of redox regulators and the CtrA phosphorelays in *D. shibae* and *R. capsulatus* indicated a potential connection of the regulons. Therefore, we were interested in exploring in more detail the extent to which these regulatory systems interact. We re-analyzed ten available transcriptomic datasets for the two species. Deletion mutants, including those of redox regulators and the CtrA phosphorelay/QS networks, were analyzed to examine the regulon overlap of these systems and to evaluate their potential integration. We also included further analyses of available transcriptomic datasets of wild type strains undergoing physiological changes related to the environmental signals integrated by these regulatory systems.

#### **2. Materials and Methods**

#### *2.1. Datasets Analyzed in this Study*

Ten published and accessible microarray and RNA-seq transcriptomic datasets for chosen gene knockout strains and experiments monitoring responses to changes in environmental conditions were obtained from the NCBI GenBank database (Table 1).



#### *2.2. Processing and Analysis of Datasets*

This study includes four different types of transcriptomic data (Table 1) that could not be processed and analyzed as one dataset. We therefore used the changes in transcript levels (log2 fold change) compared to the controls used in the respective studies (e.g., wild type or time point before changes in the environmental conditions) for each dataset. RNA-seq data from *D. shibae* (reads per gene) and *R. capsulatus* (log2 fold change) were obtained from the respective publications (Table 1).

Agilent microarray datasets were processed using the LIMMA package in R [35]. Background correction was performed with the "normexp" method and an offset of 10. Two-color microarrays were normalized with the "loess" method before quantile normalization. Signals/intensities from spots were averaged.

<sup>A</sup>ffymetrix microarray datasets were processed using the R packages LIMMA, makecdfenv, and affy [35–37]. The CDF environment for GSE18149 was generated using the corresponding CDF file downloaded from GEO (accession GPL9198). Data were normalized with the rma function. A linear fit model was generated for comparison.

In order to analyze the CckA and ChpT regulons, thresholds were set that allowed definition of regulated and non-regulated genes. These thresholds were applied to the log2 fold change in transcript level values in the *cckA* and *chpT* deletion mutants. A gene was not considered regulated when its log2 fold change was between 1 and −1 while a log2 fold change value above 1 or below −1 indicated an affected gene. The analyzed genes were grouped based on published information about their functional categories as described (Supplementary Table S1).
