Interactions among Redox Regulators and the CtrA Phosphorelay in Dinoroseobacter shibae and Rhodobacter capsulatus

Abstract

Bacteria employ regulatory networks to detect environmental signals and respond appropriately, often by adjusting gene expression. Some regulatory networks influence many genes, and many genes are affected by multiple regulatory networks. Here, we investigate the extent to which regulatory systems controlling aerobic-anaerobic energetics overlap with the CtrA phosphorelay, an important system that controls a variety of behavioral processes, in two metabolically versatile alphaproteobacteria, Dinoroseobacter shibae and Rhodobacter capsulatus. We analyzed ten available transcriptomic datasets from relevant regulator deletion strains and environmental changes. We found that in D. shibae, the CtrA phosphorelay represses three of the four aerobic-anaerobic Crp/Fnr superfamily regulator-encoding genes (fnrL, dnrD, and especially dnrF). At the same time, all four Crp/Fnr regulators repress all three phosphorelay genes. Loss of dnrD or dnrF resulted in activation of the entire examined CtrA regulon, regardless of oxygen tension. In R. capsulatus FnrL, in silico and ChIP-seq data also suggested regulation of the CtrA regulon, but it was only with loss of the redox regulator RegA where an actual transcriptional effect on the CtrA regulon was observed. For the first time, we show that there are complex interactions between redox regulators and the CtrA phosphorelays in these bacteria and we present several models for how these interactions might occur.

Keywords: Alphaproteobacteria; ChpT; Crp/Fnr; Dnr; RegA; Rhodobacteraceae; gene transfer agent; motility; nitric oxide; quorum sensing.

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 ΔluxI₁ strain retains a portion of the gene that can therefore result in mapped reads.

Figure 2

Comparison of CtrA phosphorelay, Crp/Fnr regulator, and denitrification gene expression control by CtrA phosphorelay and LuxI_1/2 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 ΔluxI₁ data were obtained during stationary phase, six hours after the onset of stationary phase, and the ΔluxI₂ data were obtained during the mid-exponential growth phase (C).

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 (ΔluxI₁).

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 (ΔluxI₁) 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).

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.

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.