AcfR is a REC + HTH_LuxR response regulator of the NarL/FixJ family
To identify AcfR (WP_012168725), we analysed the domain architecture and phylogenetic relationship of AcfR and some proteins containing an HTH DNA-binding domain. First, the protein sequence was entered in SMART (http://smart.embl.de/), which indicated that it contains an N-terminal signal receiver domain REC (SM00448) and an HTH_LuxR DNA-binding domain (SM00421) (Supplemental Fig. 1a). Second, the amino-acid sequences of AcfR and some well-studied proteins containing the HTH_LuxR DNA-binding domain were aligned by BioEdit software. As shown in Fig. 1, LuxR-type proteins contained highly conserved HTH_LuxR domains and C-terminal autoinducer-binding domains (six conserved amino acid residues). However, AcfR and the other types of HTH_LuxR proteins (FixJ, NarL, UhpA, RcsB, and CitB) contain the REC domain and HTH_LuxR domain.
Furthermore, the evolutionary relationship between AcfR and several proteins that contain REC + HTH_LuxR domains was analysed. For phylogenetic reconstruction, these proteins were aligned with the ClustalW program and the phylogenetic tree was created with MEGA-X software by the neighbor-joining method. As shown in Fig. 2, AcfR was grouped into a cluster with NarL/FixJ family response regulators. The typical QS regulator LuxR of Vibrio fischeri served as an outgroup. These NarL/FixJ family proteins shared the signal receiver domain REC in the N-terminus and the HTH_LuxR domain in the C-terminus. Furthermore, the amino acid sequences of AcfR and FixJ family regulators were aligned (Fig. 3). Amino acid sequence analysis showed that the similarity between AcfR and response regulator (RR) of Azorhizobium sp. (WP_133864704), FixJ of S. meliloti and A. caulinodans ORS571 was 97.20, 41.20, 44.02%, respectively. These results suggested that AcfR belongs to the FixJ family of two-component response regulators.
To elucidate understand the biological function of AcfR, a protein-protein interaction network was constructed using the STRING database (https://string-db.org/) [14]. As shown in Supplementary Fig. 1b, a total of 8 proteins were predicted to interact with AcfR in the genome of A. caulinodans, including FixL, two AAA family ATPase proteins, two PAS domain S-box proteins, a two-component sensor histidine kinase, a GHKL domain-containing protein, and a magnesium-translocating P-type ATPase. Interestingly, six of eight proteins were predicted to be histidine kinases with HisKA and HATPase_c domains (Supplementary Fig. 2). These results suggested that AcfR, as an RR of the two-component regulatory system, may be involved in histidine kinase signal transduction.
acfR mutation does not affect bacterial growth
To further characterize the regulatory function of AcfR, we generated an acfR deletion mutant strain (ΔacfR) of A. caulinodans by gene homologous recombination and constructed its complemented strain (ΔacfR-C). To confirm whether this mutation affects bacterial growth under normal conditions, the dynamic growth curves of the wild-type (WT), mutant, and complemented strains were tested at various stages of growth. The results indicated that the relative growth rates of the mutant and complemented strains were not significantly different from that of the WT strain (Supplementary Fig. 3), indicating that deletion of the acfR gene did not significantly affect the normal growth of bacteria.
AcfR regulated motility and exopolysaccharide production in free-living state
To further investigate the functions of AcfR in bacterial motility behaviour, the swimming activity of the WT, mutant (ΔacfR), and complemented (ΔacfR-C) strains was tested on 0.3% soft agar plates. Bacterial cultures of these strains were inoculated in the middle of soft agar plates. The plates were incubated for 2–3 days at 37 °C. As shown in Fig. 4, the mutant strain exhibited decreased swimming motility ability compared to that of the WT on L3 plates, with sodium lactate (Fig. 4a) or glycine (Fig. 4b) serving as the sole carbon sources. The motility-deficient phenotype in the mutant was able to be rescued by the complemented strain ΔacfR-C. These results indicated that AcfR positively modulates the cells swimming motility behaviour of A. caulinodans ORS571.
Congo red is often used to detect exopolysaccharide (EPS) production in bacteria [15]. Figure 5 shows that the EPS production levels of the WT, mutant, and complemented strains on Congo red plates. There were no significant differences in the colony morphology and total EPS production among the WT, ΔacfR, and ΔacfR-C strains when they were grown on Congo red plates with sodium lactate as the sole carbon source. However, the colony morphology and the total EPS production were significantly different (P ≤ 0.01) between the ΔacfR and WT cells. The ΔacfR mutant produced less “black pigmented” (Fig. 5a) and total EPS (Fig. 5b) than did the WT when grown on L3 medium containing glycine as the sole carbon source. These results indicated that AcfR was involved in the secretion process of EPS and that different carbon sources might influence the regulation of AcfR on the EPS phenotype.
ΔacfR mutant strain is impaired in competitive nodulation of the host plants
To investigate whether the motility and EPS formation phenotypic defects of the ΔacfR mutant also affect symbiosis characteristics with the host plant, competitive nodulation assays were performed by analysing the levels of nodulation on the host plant. To test whether ΔacfR possessed a competitive disadvantage when competing with WT, cultures of WT and ΔacfR were mixed at 1:1, 1:5, and 1:10 ratios and subsequently incubated with S. rostrata roots and stems. The ratio of the number of wild-type cells to the number of mutant cells in the inoculum was determined by cell counts performed before mixing. At the end of each experiment (usually 35 days), the ratio of nodules induced by the wild-type or the mutant strain was determined by PCR to detect the colonies that grew from surface-sterilized crushed nodules on TY agar plates. The results shown in Fig. 6 demonstrate that the WT formed more nodules (by five- to sixfold) than the mutant strain when inoculated on roots and stems at a 1:1 ratio. With increases in the ratio of the inoculated mutant strain (WT:ΔacfR = 1:5 or 1:10), the nodule occupancy also increased. These results indicated that the competitive nodulation ability of the mutant strain was dramatically weakened compared with that of the WT. However, the number of nodules induced by ΔacfR-C was not significantly different compared with that of the WT when inoculated at a 1:1 ratio, indicating that the competitive nodulation ability was restored in the complemented strain. Therefore, we conclude that the AcfR regulator is essential for normal nodulation competitiveness during symbiosis on S. rostrata.