PAS domain containing chemoreceptor couples dynamic changes in metabolism with chemotaxis

Abstract

Chemoreceptors provide sensory specificity and sensitivity that enable motile bacteria to seek optimal positions for growth and metabolism in gradients of various physicochemical cues. Despite the abundance of chemoreceptors, little is known regarding the sensory specificity and the exact contribution of individual chemoreceptors to the lifestyle of bacteria. Azospirillum brasilense are motile bacteria that can fix atmospheric nitrogen under microaerophilic conditions. Here, we characterized a chemoreceptor in this organism, named AerC, which functions as a redox sensor that enables the cells to seek microaerophilic conditions that support optimum nitrogen fixation. AerC is a representative of a widespread class of soluble chemoreceptors that monitor changes in the redox status of the electron transport system via the FAD cofactor associated with its PAS domains. In A. brasilense, AerC clusters at the cell poles. Its cellular localization and contribution to the behavioral response correlate with its expression pattern and with changes in the overall cellular FAD content under nitrogen-fixing conditions. AerC-mediated energy taxis in A. brasilense prevails under conditions of nitrogen fixation, illustrating a strategy by which cells optimize chemosensing to signaling cues that directly affect current metabolic activities and thus revealing a mechanism by which chemotaxis is coordinated with dynamic changes in cell physiology.

Fig. 1.

AerC is a chemoreceptor homolog found in the nif/fix gene region of the genome of A. brasilense. (A) Organization of the genomic region around aerC (Upper) and protein domains found in AerC (Lower). The arrows indicate the direction of transcription. (B) Expression of AerC in A. brasilense strain Sp7 (wild type) and its ΔaerC mutant derivative (strain AB301) grown in presence of ammonium and under nitrogen-fixation conditions. An equivalent amount of protein extracted from whole cells was analyzed. AerC expression was detected by using an affinity-purified E. coli anti Aer_2–166 antibody that cross-reacts with a protein of about 40 kDa in Sp7 but not AB301 (indicated by an arrow on the Right). The unidentified low molecular weight cross-reacting band present in all lanes was used as an internal control. The experiment was repeated three times and representative results are shown.

Fig. 2.

AerC–Yfp localization in A. brasilense. The wild-type and mutant derivative strains were grown in presence of ammonium (Left) and under nitrogen-fixation conditions (Right). The AB301 strain is a ΔaerC::Kan derivative of Sp7 and the AB103 strain is a ΔcheOp1::Cm derivative of Sp7 (13) (Table S2). In each panel, DIC images are shown on the Left and fluorescent images on the Right. Representative images are shown.

Fig. 3.

Comparative sequence analysis of the PAS domains from microbial chemoreceptors. (A) A neighbor-joining tree generated from multiple sequence alignment of 1,649 PAS domains. Class I members are shown in brown (designated as PAS_FAD1) and exemplified by the PAS domain from the Aer protein of E. coli. Members of the bipartite subfamily of Aer sensors (34) are shown in light brown. Class II members are shown in blue (designated as PAS_FAD2) and exemplified by the two PAS domains from AerC protein of A. brasilense. Members of the general PAS group are shown in black. (B) Sequence alignment of PAS domains from NifL, Aer, and AerC proteins. Positions that are conserved (>95%) within PAS_FAD1 and PAS_FAD2 classes are highlighted in gray. Positions that are suggested to bind FAD in the NifL protein (16) are indicated by asterisks. A tryptophan residue that is involved in FAD binding in NifL and is conserved (>95%) in both classes of PAS domains is highlighted in yellow.

Fig. 4.

Role of tryptophan residues W77 and W199 of the PAS1 and PAS2 domains of AerC in chemoreceptor function. (A) Chemotaxis in the soft agar plate assay of the wild-type A. brasilense, the AB301 strain carrying an empty pRK415 vector (controls), or complemented with wild-type AerC and AerC alleles expressed from their own promoter on pRK415, after 24 h incubation at 28 °C. The soft agar plates contained malate as the carbon and energy source and ammonium chloride as the source of combined nitrogen. Longer incubation times did not change the results. (B) Aerotaxis in the capillary assay. An equivalent number of cells were inoculated in each capillary tube and the photograph was taken after a 5-min incubation period. The arrow indicates the direction of the air gradient from the air–liquid interface. (C) Effect of AerC on the propensity of cells to aggregate by cell-to-cell interaction and to form clumps. Pictures were taken directly from actively growing cultures in MMAB with 10 mM malate. Magnification, ×400. Representative pictures within a field of view are shown. The arrows point to clumps. (D) Subcellular localization of AerC^W77F–Yfp, AerC^W199F–Yfp, and AerC^W77FW199F–Yfp in the AB301 strain under nitrogen-fixation conditions by fluorescence.

Fig. 5.

Time course of aerotactic band movement in a spatial gradient of oxygen under conditions of nitrogen fixation. (A) An equivalent number of cells were inoculated at the bottom of tubes containing soft agar and a source of carbon and energy and incubated under conditions of nitrogen fixation. Photographs were taken at regular intervals (24, 48, and 72 h postinoculation). There was no band formed during the first 10 h of incubation, after which bands were seen moving up the gradients as shown. A representative photograph is shown. The arrows indicate the center of the aerotactic band in each photograph, which also corresponds to the position where samples were collected (directly from the bands) for analyses shown in panels B, C, and D. The star symbols represent statistically significant differences (P < 0.05). (B) Activity of a PnifH–gusA transcriptional fusion. (C) Activity of a PaerC–gusA transcriptional fusion. (D) Total cytosolic FAD content.