ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation by Bacillus amyloliquefaciens SQR9

Abstract

Efficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficial Bacillus strains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD⁺/NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of the qoxABCD and ctaCDEF operons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governing Bacillus biofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and other Bacillus rhizobacteria used in agriculture.IMPORTANCEBacillus spp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand, Bacillus is the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555-565, 2015, https://doi.org/10.1111/1462-2920.12527; L. S. Cairns, L. Hobley, and N. R. Stanley-Wall, Mol Microbiol 93:587-598, 2014, https://doi.org/10.1111/mmi.12697; H. Vlamakis, C. Aguilar, R. Losick, and R. Kolter, Genes Dev 22:945-953, 2008, https://doi.org/10.1101/gad.1645008; S. S. Branda, A. Vik, L. Friedman, and R. Kolter, Trends Microbiol 13:20-26, 2005, https://doi.org/10.1016/j.tim.2004.11.006; C. Aguilar, H. Vlamakis, R. Losick, and R. Kolter, Curr Opin Microbiol 10:638-643, 2007, https://doi.org/10.1016/j.mib.2007.09.006; S. S. Branda, J. E. González-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter, Proc Natl Acad Sci U S A 98:11621-11626, 2001, https://doi.org/10.1073/pnas.191384198). However, the identification and sensing of environmental signals triggering Bacillus biofilm formation need further research. Here, we report that the oxygen deficiency signal inducing Bacillus biofilm formation is sensed by the ResDE two-component regulatory system. Our results not only revealed the novel regulatory function of the ResDE two-component regulatory system but also identified the sensing system of a biofilm-triggering signal. This knowledge can help to enhance the biofilm formation and root colonization of plant-beneficial Bacillus strains and also provide new insights of bacterial biofilm formation regulation.

Keywords: Bacillus; biofilm; respiration; root colonization; two-component regulatory system.

FIG 1

Colony structures of Bacillus amyloliquefaciens SQR9, ΔresD, ΔresE strains under different oxygen concentrations and transcriptional analysis of extracellular matrix genes and resD-E under a limited oxygen condition. (A) Colony wrinkling increased with decreasing oxygen concentration. A high oxygen concentration suppressed wrinkling, while the responses of colonies to the different oxygen concentrations were blocked with deletion of resD or resE. Transcriptional levels of epsD and tapA (B) and resD-E (C) were upregulated under oxygen-limiting conditions relative to that under the normal oxygen concentration. The recA gene was used as an internal reference gene. Bars represent the standard deviations of data from three biological replicates. (D) P_resD-gfp showed intense fluorescence under oxygen-limiting conditions. (E) NAD⁺/NADH ratios for the wild-type SQR9 strain grown for 48 h on solid biofilm-inducing medium under the indicated oxygen concentrations. The NAD⁺/NADH ratios are the averages from six colonies for each oxygen concentration.

FIG 2

Isothermal titration calorimetry assay of ResE-NAD⁺ and ResE-NADH interactions. The upper panels show the heat changes observed upon the addition of 40 μl of a 1.05 mM NAD⁺ (A) or NADH (B) solution in PBS buffer (pH 7.4) into a 70 μM ResE protein solution in the same buffer. The lower panels show the integrated heat changes of each injection plotted against the molar ratio of NAD⁺ and NADH to the ResE protein.

FIG 3

Transcriptional levels of biofilm-related and cytochrome complex synthesis genes in ΔresD and ΔresE mutants. (A) The transcriptional levels of spo0A, abrB, sinI, epsD, and tapA in res mutants relative to that of the wild-type strain evaluated by qPCR. Transcriptional levels of ctaCDEF and ctaB (B), qcrABC (C), and qoxABCD (D) operons in res mutants relative to those of the wild-type strain evaluated by qPCR. The recA gene was used as an internal reference gene. Bars represent standard deviations of data from three biological replicates. RQ, relative quantification.

FIG 4

Electrophoretic mobility shift assays (EMSAs) and DNase I footprint assays of ResD-ctaCDEF operon and ResD-qoxABCD operon promoters' interactions. (A, B) EMSAs in which end-labeled DNAs (indicated at the bottom of each figure) were mixed with purified ResD at the following concentrations: 0, 0.28, 0.7, 1.4, and 2.1 μM. (C, D) Electropherograms showing the protected regions of the qoxABCD and ctaCDEF operon promoters after digestion with DNase I following an incubation in the absence or the presence of 3.2 μg and 0.8 μg ResD protein, respectively.

FIG 5

Colony structures of Δcta, Δqcr, and Δqox mutants under different oxygen concentrations and the transcriptional and BTH assays. (A) A lack of the qoxABCD operon blocks the response of colonies to different oxygen concentrations, while a deficiency of either the ctaCDEF or qcrABC operon does not. (B) The transcriptional levels of spo0A, abrB, sinI, kinB, epsD, and tapA in a Δqox mutant relative to that in the wild-type strain evaluated by qPCR. The recA gene was used as an internal reference gene. Bars represent standard deviations of data from three biological replicates. (C) BTH analysis to study the interaction between cytochrome aa₃ and KinB. The positive interaction between QoxABCD and KinB is shown by the blue colonies and the quantification of the β-galactosidase activity (1,400 to 1,500 Miller units). The strains harboring plasmids (pKT25-zip plus pUT18C-zip) and empty plasmids (pKNT25 plus pUT18) are the positive and negative controls, respectively. The LytT strain was also used as a negative control. Dashed line indicates the threshold limit that defines a positive (≥700 Miller units) or a negative (<700 Miller units) interaction signal according to the manufacturer's instructions (EuroMedex). The results represent the means from three independent experiments. Asterisks denote significant differences according to Duncan's multiple range tests at a P value of <0.05.

FIG 6

Model for the two-component regulatory system ResDE mediation of biofilm formation by the control of the electron transport chain under oxygen-limiting conditions (see description in the text).