AEM Accepted Manuscript Posted Online 18 March 2016 Appl. Environ. Microbiol. doi:10.1128/AEM.00230-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved. 1 A chemotaxis receptor modulates nodulation during the 2 Azorhizobium caulinodans-Sesbania rostrata symbiosis 3 4 Nan Jiang1, 2, Wei Liu1, Yan Li1, Hailong Wu1, 2, Zhenhai Zhang3, Gladys Alexandre4, 5 Claudine Elmerich5, Zhihong Xie1* 6 1Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of 7 Coastal Zone Research, Chinese Academy of Sciences, Yantai, China 8 2School of Resource and Environment, University of Chinese Academy of Sciences, 9 Beijing, China 10 3School of Mechatronics Engineering, Beijing Institute of Technology, Beijing, China 11 4Biochemistry, Cellular and Molecular Biology Department, University of Tennessee, 12 Knoxville, USA 13 5Institut Pasteur, Paris, France 14 *Corresponding author 15 E-mail address: [email protected] 16 17 Abstract 18 Azorhizobium caulinodans ORS571 is a free-living nitrogen-fixing bacterium, which 19 can induce nitrogen-fixing nodules both on the root and the stem of its legume host 20 Sesbania rostrata. This bacterium which is an obligate aerobe, motioned by a polar 21 flagellum, possesses a single chemotaxis signal transduction pathway. The objective 22 of this work was to examine the role that chemotaxis and aerotaxis play in the 23 lifestyle of the bacterium in free-living and symbiotic conditions. In bacterial 24 chemotaxis, chemoreceptors sense environmental changes and transmit this 25 information to the chemotactic machinery to guide motile bacteria to preferred niches. 26 Here, we characterized a chemoreceptor of A. caulinodans containing an N-terminal 27 PAS domain, named IcpB. IcpB is a soluble heme binding protein that localized at the 28 cell poles. An icpB mutant strain was impaired in sensing oxygen gradients and in 1 29 chemotaxis response to organic acids. Compared to the wild type strain, the icpB 30 mutant strain was also affected in the production of extracellular polysaccharides and 31 impaired in flocculation. When inoculated alone, the icpB mutant induced nodules on 32 S. rostrata, but the nodules formed were smaller and had reduced N2-fixing activity. 33 The icpB mutant failed to nodulate its host when inoculated competitively with the 34 wild type strain. Together, the results identify chemotaxis and sensing of oxygen by 35 IcpB as key regulators of the A. caulinodans-S. rostrata symbiosis. 36 37 Importance 38 Bacterial chemotaxis has been implicated in the establishment of various 39 plant-microbe associations, including that of rhizobial symbionts with their legume 40 host. The exact signal(s) detected by the motile bacteria that guide them to their plant 41 hosts remain poorly characterized. Azorhizobium caulinodans ORS571 is a diazotroph, 42 motile and chemotactic rhizobial symbiont of Sesbania rostrata, where it forms 43 nitrogen-fixing nodules on both the roots and the stems of the legume host. Here we 44 identify a chemotaxis receptor sensing oxygen in A. caulinodans that is critical for 45 nodulation and nitrogen fixation on the stems and roots of S. rostrata. These results 46 identify oxygen sensing and chemotaxis as key regulators of the A. caulinodans-S. 47 rostrata symbiosis. 48 49 Introduction 50 Chemotaxis is a stimulated process enabling motile bacterial species to detect 51 chemical gradients and to move in a benefical direction. The bacterial chemotactic 52 system of Escherichia coli is so far the best understood. This bacterium possesses four 53 attractant-specific transmembrane chemoreceptors, named methyl-accepting 54 chemotaxis proteins (MCPs) (1), as well as a fifth chemoreceptor, Aer, which contains 55 an N-terminal Per-Arnt-Sim (PAS) domain that binds a flavin adenine dinucleotide 56 (FAD) cofactor to sense redox changes (2, 3). The chemoreceptors convey sensory 57 information to the flagellar motors across a complex signal transduction pathway 2 58 encompassing six soluble chemotaxis proteins (named CheA, CheB, CheR, CheY, 59 CheW and CheZ) (4, 5). All chemotaxis receptors have highly similar cytoplasmic 60 domains that are essential for the formation of ternary signaling complexes with the 61 histidine kinase CheA and the adaptor protein CheW. These signaling complexes are 62 large molecular ultrastructures that can be seen at the cell poles by fluorescent 63 labelling of chemotaxis proteins and of chemoreceptors (6). 64 Chemoreceptors are functional signaling proteins located at the input end of 65 the signaling pathway. They detect specific effectors with high specificity and 66 transduce chemotactic signals to the downstream proteins (7, 8). While 67 membrane-bound chemoreceptors represent the largest class of chemotaxis receptors 68 found in bacterial genomes (9, 10), soluble cytoplasmic chemoreceptors are also 69 broadly distributed (11). Soluble chemotaxis receptors either appear to localize with 70 other receptors at the cell poles (12), or they can localize as separate cytoplasmic 71 clusters (13). 72 A. caulinodans ORS571 is a symbiont of the aquatic tropical legume, Sesbania 73 rostrata. A. caulinodans is capable of inducing nodule formation on the roots as well 74 as at stem-located root primordia of the host plant (14, 15). In addition to nitrogen 75 fixation in roots and stem nodules, A. caulinodans ORS571 is capable of fixing 76 nitrogen in the free living state, providing it can locate conditions where oxygen 77 concentrations are very low (14). Chemotaxis plays a key role in the establishment of 78 symbiotic relationships of diverse bacteria with plants (16, 17), but its role in the A. 79 caulinodans-S. rostrata symbiosis has not been investigated. In this work, we 80 characterized a PAS-containing chemoreceptor in A. caulinodans that we named IcpB 81 (internal chemotaxis protein B) and showed that IcpB senses oxygen via a 82 heme-bound cofactor and that it modulates aerotaxis and chemotaxis. We also 83 provided evidence that supports a critical role for IcpB in the establishment of a 84 functional symbiosis between A. caulinodans and its host plant. 85 86 Materials and Methods 3 87 Media, bacterial strains, and growth conditions 88 The bacterial strains and plasmids are listed in Table 1. A. caulinodans 89 ORS571 and its derivatives were grown at 37°C in TY medium (10 g/l tryptone, 5 g/l . 90 yeast extract, and 4 g/l CaCl2 2H2O) (18) or in L3 minimal medium (10 mM KH2PO4, . 91 10 mg/ml DL-sodium lactate, 100 μg/ml MgSO4 7H2O, 50 μg/ml NaCl, 40 μg/ml . 92 CaCl2 2H2O, 5.4 μg/ml FeCl3 6H2O, 5 μg/ml Na2MoO4 2H2O, 2 μg/ml biotin, 4 μg/ml 93 nicotinic acid, and 4 μg/ml pantothenic acid) (19), which was either supplemented 94 with 10 mM NH4Cl (L3 + N medium) or lacked any nitrogen source (L3 – N 95 medium). When indicated in the text, sodium lactate was substituted with other 96 carbon sources as the sole carbon source in L3 medium. The growth medium of A. 97 caulinodans was supplemented with ampicillin (final concentration of 100 μg/ml) and 98 nalidixic acid (final concentration of 25 μg/ml). 99 100 Behavioral assays 101 The soft agar plate and temporal gradient assays for chemotaxis in A. 102 caulinodans were performed essentially as previously described (24), with some 103 modifications. For the soft agar assay, cells were grown to mid-log phase in TY 104 medium, washed and resuspended in chemotaxis buffer (10 mM K2HPO4, 10 mM 105 KH2PO4, 0.1 mM EDTA, pH=7.0) to an OD600nm of ~ 0.6. Aliquots of 5 μl of this 106 bacterial suspension were inoculated at the center of L3 minimal soft agar plates 107 solidified with 0.3% agar and containing different carbon sources added at a final 108 concentration of 10 mM. The inoculated soft agar plates were incubated for 3-5 days 109 at 37°C before being photographed. 110 The temporal assay for aerotaxis was essentially carried out according to the 111 method described by Alexandre et al. (17). A 10-μl drop of bacterial suspension 112 adjusted to an OD600nm = 0.2 was placed on a microscope slide, inside a 113 microchamber that was ventilated with humidified N2 or air gas (flow rate 800 ml 114 min-1). The cell suspension was equilibrated with air for 2 min. After that, the 115 ventilating gas was switched to N2 for 1-3 min and then changed to air again by the 4 116 way of controlling a gas valve. The motion of bacteria was digitally recorded using 117 Cellsens Dimension 1.7 (Olympus Corp.). The time it took for swimming bacteria to 118 return to a pre-stimulus swimming pattern after stimulation was determined by 119 measuring the average reversal frequency (RF) of free-swimming cells, using 120 CellTrak 1.1 (Motion Analysis Corp., SantaRosa, CA). The removal of air caused a 121 transient increase in the RF and the addition of air caused a transient decrease in the 122 RF. Experiments were performed three times, with a minimum of six replicates per 123 sample. 124 125 Flocculation assay 126 Flocculation was estimated using the method described by Burdman et al. 127 (20) with the following modifications. Overnight cultures in liquid TY medium were 128 normalized to an OD600nm of 1.0, and 200 μl were inoculated into 10 ml L3 medium 129 added to a 40-ml conical sterile tube. These conical tubes were incubated vertically in 130 a rotary shaker (180 rpm) at 37°C. After incubation for 24 h and 48 h, the tubes were 131 removed from the shaker and left standing for 30 min. After this period, flocculated 132 cells had settled to the bottom of the tube while the non-flocculated cells remained in 133 suspension. The turbidity of the supernatant (ODs) and the total turbidity (ODt) of the 134 culture obtained after mechanical dispersion of the flocs by treatment in a tissue 135 homogenizer were measured by spectrophotometry as OD600nm.