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Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of Bradyrhizobium diazoefficiens

Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of... RESEARCH ARTICLE crossmark Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of Bradyrhizobium diazoefficiens a b c d b c b Gargi Kulkarni, Nicolas Busset, Antonio Molinaro, Daniel Gargani, Clemence Chaintreuil, Alba Silipo, Eric Giraud, a,e,f Dianne K. Newman Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA ; IRD, Laboratoire des Symbioses Tropicales et b c Méditerranéennes (LSTM), UMR IRD/SupAgro/INRA/UM2/CIRAD, Montpellier, France ; Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Naples, Italy ; d e CIRAD, UMR BGPI, Montpellier, France ; Howard Hughes Medical Institute, Pasadena, California, USA ; Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California, USA G.K. and N.B. contributed equally to this article. ABSTRACT A better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbio- ses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires hopanoid production for optimal fitness. While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis. In contrast, synthesis of extended (C ) hopanoids is required for growth microaerobically and under various stress conditions (high temperature, low pH, high osmolarity, bile salts, oxidative stress, and antimicrobial peptides) in the free-living state and also during symbiosis. These defects might be due to a less rigid mem- brane resulting from the absence of free or lipidA-bound C hopanoids or the accumulation of the C hopanoid diploptene. 35 30 Our results also show that C hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides in Aeschynomene nodules that induce drastic modification in bacterial morphology and physiology. The study of hopanoid mutants in plant symbionts thus provides an opportunity to gain insight into host-microbe interactions during later stages of symbiotic progression, as well as the microenvironmental conditions for which hopanoids provide a fitness advantage. IMPORTANCE Because bradyrhizobia provide fixed nitrogen to plants, this work has potential agronomical implications. An un- derstanding of how hopanoids facilitate bacterial survival in soils and plant hosts may aid the engineering of more robust agro- nomic strains, especially relevant in regions that are becoming warmer and saline due to climate change. Moreover, this work has geobiological relevance: hopanes, molecular fossils of hopanoids, are enriched in ancient sedimentary rocks at discrete inter- vals in Earth history. This is the first study to uncover roles for 2Me- and C hopanoids in the context of an ecological niche that captures many of the stressful environmental conditions thought to be important during (2Me)-hopane deposition. Though much remains to be done to determine whether the conditions present within the plant host are shared with niches of relevance to the rock record, our findings represent an important step toward identifying conserved mechanisms whereby hopanoids con- tribute to fitness. Received 27 July 2015 Accepted 17 September 2015 Published 20 October 2015 Citation Kulkarni G, Busset N, Molinaro A, Gargani D, Chaintreuil C, Silipo A, Giraud E, Newman DK. 2015. Specific hopanoid classes differentially affect free-living and symbiotic states of Bradyrhizobium diazoefficiens. mBio 6(5):e01251-15. doi:10.1128/mBio.01251-15. Editor Frederick M. Ausubel, Mass General Hospital Copyright © 2015 Kulkarni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Eric Giraud, eric.giraud@ird.fr, or Dianne K. Newman, dkn@caltech.edu. variety of plants, including leguminous (1), actinorhizal (2), (LPS) is essential for all stages of rhizobial symbiosis, including Aand land-dwelling (3) plants, rely on bacterial symbiotic part- root hair infection, symbiotic tissue (nodule) establishment, and ners for assimilation of nitrogen, an essential macronutrient. survival within the plant cell (6). Whether and to what extent These symbioses require bacterial invasion of plant tissues and other microbial membrane lipids regulate the establishment and adaptation of the bacterial symbiont to the plant host environ- maintenance of plant-microbe symbioses are unclear. Here, we ment, processes in which several microbial membrane lipids play consider whether hopanoids (7), steroid-like pentacyclic triterpe- key roles. For instance, phosphatidylcholine is critical for efficient noid lipids, support plant-microbe interactions. Hopanoids are nitrogen fixation in several legume-rhizobial partnerships such as the progenitors of hopanes, molecular fossils that exhibit intrigu- soybean-Bradyrhizobium diazoefficiens (formerly named Brady- ing yet poorly understood abundance patterns in the rock record rhizobium japonicum [4]) and alfalfa-Sinorhizobium meliloti (5). (8). In part, our interest in hopanoids derives from a desire to Similarly, an intact outer membrane (OM) lipopolysaccharide interpret ancient biomarkers and the conviction that this requires September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 1 Kulkarni et al. a nuanced understanding of the biological functions of diverse and fixed atmospheric nitrogen produced by bacterial nitrogenase hopanoids in modern niches. (21, 23). Supporting such high levels of bacterial nitrogenase ac- The capacity for hopanoid biosynthesis is statistically enriched tivity requires both extensive host control of bacteroid physiology in the (meta-)genomes of bacteria associated with plants (9), and and establishment of a specific host microenvironment, defined hopanoids have been found in high abundance in membranes of by low oxygen, low pH, hyperosmosis, and oxidative stress (24). well-studied plant symbionts of the Bradyrhizobium (40% of total There is some evidence suggesting that the extent of these envi- lipid extract [TLE]) and Frankia (87%) genera (10, 11). Ho- ronmental stresses varies between plant hosts. For example, panoids promote membrane rigidity (12) and confer protection A. afraspera was recently shown to produce nodule-specific, against numerous stresses, including acidic or alkaline pH, high cysteine-rich antimicrobial peptides (NCR peptides) that induce temperature, high osmolarity, oxidative stress, detergents, and an- differentiation of the bacteroid into an enlarged, elongated, and tibiotics (13–16). They have varied structures, formed via meth- polyploid state, whereas in the soybean host, NCR peptides are ylation (2Me or 3Me), unsaturation, and/or attachment to a absent and bacteroid morphology and ploidy are similar to those ribose-derived side chain (C ) (Fig. 1A; also see Fig. S1 in the of the free-living state (25). Thus, it is likely that the microbial supplemental material) (7). Whether there are functional distinc- adaptations required for survival within root nodules are host tions under specific environmental conditions for diverse ho- specific, and we hypothesized that specific hopanoid mutants may panoid types is unclear, yet some evidence suggests that they have exhibit variable phenotypes in diverse plant hosts. nonoverlapping roles. For example, in Rhodopseudomonas palus- Here, we focus on the phenotypic consequences of the inability tris (17) and Burkholderia cenocepacia (14), C hopanoids are of B. diazoefficiens to produce two specific hopanoid classes, 2Me- critical for OM stability and for resistance to low pH, detergent and C hopanoids. We compare its hopanoid-dependent stress (sodium dodecyl sulfate [SDS]), and polymyxin B, respectively. phenotypes in the free-living state to those of other hopanoid Though the absence of 2Me-hopanoids did not manifest a stress producers. We further explore the fitness effects of 2Me- or C phenotype in previous tests of R. palustris, their biosynthesis is hopanoid production within a natural ecological context: the transcriptionally induced under stress (13). This suggests that symbiotic microenvironment of soybean and A. afraspera cells. 2Me-hopanoids might contribute to stress resistance under con- These studies begin to define the role of 2Me- and C hopanoids ditions yet to be identified in this and other organisms; consistent during the progression of plant-microbe symbioses and provide with this notion, 3Me-hopanoids contribute to late-stationary- insight into microbial membrane factors that facilitate adapta- phase survival in Methylococcus capsulatus (18). In vitro, 2Me- tions to particular microenvironments. hopanoids rigidify membranes of varied compositions (12). How- RESULTS ever, until now, no study has explored whether different hopanoids impact fitness in a natural ecological context. Shc appears to be essential for the survival of B. diazoefficiens. Recently, it was shown that elimination of hopanoid biosyn- To eliminate hopanoid production in B. diazoefficiens and to test thesis in photosynthetic Bradyrhizobium strain BTAi1 impairs its whether a requirement for hopanoids in efficient symbiosis is con- symbiosis with the legume Aeschynomene evenia (15). Because the served between B. diazoefficiens and Bradyrhizobium strain BTAi1, absence of all hopanoids likely had a broad and drastic impact on we first attempted deletion of the gene encoding the enzyme cat- cellular physiology and hence symbiosis, in this study we tested alyzing the first step in hopanoid biosynthesis, squalene hopene specific effects of 2Me- and C hopanoids (7) using B. diazoeffi- cyclase (Shc) (Fig. 1B). We were unable to isolate a shc mutant ciens USDA110, the best-studied Bradyrhizobium strain. In addi- using either the pK18mobsacB-based markerless gene deletion tion to C and C hopanoids (10), B. diazoefficiens makes tetra- method (~400 colonies screened) or the gene replacement strat- 30 35 hymanol, a triterpenoid with a gammacerane skeleton (19) (Fig. 1A; egy with pSUP202pol4 (~1,200 colonies screened) (26). This sug- also see Fig. S1 in the supplemental material). Intriguingly, while gests that Shc might be essential either because hopanoids are most hopanoids are thought to occur free within membranes, the C required for growth and survival of B. diazoefficiens or because hopanoid, (2-Me) 34-carboxyl-bacteriohopane-32,33-diol, was squalene, the substrate of Shc (16), accumulates to toxic levels found to be covalently attached to LPS lipidA, a well-established within the shc mutant. To rule out the latter possibility, we tried player in a broad range of host-microbe interactions, to form a to delete the entire operon encoding squalene-synthesizing en- compound called hopanoid-lipid A (HoLA) (15, 20) (see zymes (hpnCDE), shc (hpnF), and hpnG (which catalyzes the sec- Fig. S1B). ond step in the synthesis of C hopanoids) (17), but again, we B. diazoefficiens exhibits two different lifestyles, free living in were unable to obtain the hpnCDEFG mutant (~150 colonies soil or symbiotic within legume root nodule cells (1, 21). In addi- screened) (Fig. 1B). These results suggest that hopanoid synthesis tion to its native soybean host, B. diazoefficiens can engage in is essential for the survival of B. diazoefficiens under the conditions nitrogen-fixing symbioses with the stems and roots of the tropical used to select the mutants. legume Aeschynomene afraspera (22). In both of the these hosts, B. diazoefficiens hpnP and hpnH mutants are unable to development of the symbiosis progresses through a series of de- make 2Me- and C hopanoids, respectively. To eliminate syn- fined stages: (i) colonization and invasion of host root tissue; (ii) thesis of 2Me- or C hopanoids specifically, we deleted genes internalization of bacteria by plant cells to form an organelle-like predicted to encode the C-2 methylase, hpnP (27), or the first structure called the symbiosome, comprising endosymbiotic bac- enzyme catalyzing the extension of C hopanoids, hpnH (17) terial cells termed “bacteroids” that are surrounded by a plant- (Fig. 1B). As illustrated in Fig. 1C, no methylated hopanoids were derived “peribacteroid” membrane (see Fig. S2 in the supplemen- detected in hpnP mutant TLE using gas chromatography-mass tal material); and (iii) initiation of nitrogen fixation by bacteroids, spectrometry (GC-MS) and liquid chromatography-mass spec- during which there is a high rate of nutrient exchange across the trometry (LC-MS) (see Tables S1 and S2 in the supplemental ma- symbiosome membranes between plant-supplied carbon sources terial) (28, 29). The hpnH mutant does not make any detectable 2 mbio.asm.org September/October 2015 Volume 6 Issue 5 e01251-15 Roles of Hopanoids in Survival of Legume Symbionts C hopanoids, including aminotriol (a and c), BHP-508 (VIII; degradation product of aminotriol), bacteriohopanetetrol (b and e), and adenosylhopane (d). In addition, the hpnH mutant ac- cumulates a 6-fold excess of the HpnH substrate (17), diploptene (IV; wild type [WT], 18  2 g/mg TLE; hpnP mutant, 29 6 g/mg TLE; hpnH mutant, 111  3 g/mg TLE). We also analyzed the presence of HoLA in the mutants using matrix- assisted laser desorption ionization–mass spectrometry (MALDI- MS) (Fig. 2). WT and hpnP mutant lipidA are composed of a mixture of penta- to hepta-acylated species, whereas hpnH mu- tant lipidA is mainly hexa-acylated (see Fig. S1B). In WT and hpnP mutant hepta-acylated species, a C hopanediolic acid is ester linked to hexa-acylated lipidA, and traces of a second ho- panoid substitution are also detected; conversely, thehpnH mu- tant is missing any lipidA-bound hopanoids. Not only do our results confirm the proposed roles of HpnP and HpnH, but they also show that synthesis of C hopanoids is required for HoLA production. C hopanoids contribute to outer membrane rigidity. We employed a fluorescence polarization method by incubating the dye diphenyl hexatriene (DPH) with whole cells to determine whether 2Me- and C hopanoids affect the rigidity of B. diazoef- ficiens membranes at 25°C and 40°C (Fig. 3). Because previous studies of whole cells of different R. palustris hopanoid mutants indicated that the majority of DPH gets incorporated into the OM, we interpret whole-cell polarization values to reflect the ri- gidity of the OM (12, 29). Membranes of all strains were less rigid at the higher temperature. The hpnP mutant membrane was as rigid as the WT membrane at both temperatures, whereas the hpnH mutant membrane was less rigid. Thus, C hopanoids are important for maintaining membrane rigidity in B. diazoefficiens in vivo, in contrast to R. palustris, where thehpnH mutant mem- brane showed rigidity similar to that of the WT, despite the capac- ity of C hopanoids to enhance rigidity in vitro (12). This indi- cates that the fraction of C hopanoids or HoLA in the OM may be greater in B. diazoefficiens than R. palustris. Despite the lack of C hopanoids, thehpnH mutant membrane is morphologically indistinguishable from the WT membrane, as seen in whole-cell cryo-transmission electron microscopy (cryo-TEM) micrographs (see Fig. S3 in the supplemental material). C hopanoids are important for aerobic growth. Does a less rigid membrane affect the fitness of hpnH at different tempera- tures? To address this question, we compared aerobic growth rates of the hpnH mutant at 30°C and 37°C with those of the WT and the hpnP mutant (Fig. 4A and B). The hpnP mutant grows like the WT at both temperatures, whereas the hpnH mutant grows slower at 30°C and is unable to grow at 37°C. These results suggest that C hopanoids are important for growth at ambient temper- Figure Legend Continued FIG 1 (A) Structures of hopanoid and tetrahymanol. B. diazoefficiens makes ylation. (C) GC-MS and LC-MS (inset) total ion chromatograms of total lipid C hopanoids, such as diploptene (C-22C-30) and diplopterol (OH at 30 extracts from aerobically grown B. diazoefficiens strains. For GC-MS, main C-22); C hopanoids, such as bacteriohopanetetrol (BHT; R OH) and ami- 35 2 hopanoid peaks are numbered and the methylated counterparts elute 0.2 to nobacteriohopanetriol (aminotriol; R NH ); and tetrahymanol. All these 2 2 0.5 min earlier. I, pregnane acetate (standard); II, (2Me) hop-17(21)-ene; III, compounds can be methylated at C-2 (2Me, R CH ). (B) Hopanoid biosyn- 1 3 (2Me) hop-x-ene; IV, (2Me) hop-22(29)-ene (diploptene); V, (2Me) hop-21- thetic gene cluster of B. diazoefficiens. In this study, we focused on the genes ene; VI, (2Me) hopan-22-ol (diplopterol); VII, (2Me and 20Me) tetrahy- colored in gray: the shc (squalene hopene cyclase) product catalyzes squalene manol; and VIII, BHP-508. LC-MS: a, aminotriol; b, BHT; c, 2Me-aminotriol; cyclization to hopene, the first reaction in the hopanoid biosynthetic pathway; d, adenosylhopane; e, 2Me-BHT. Lipid analysis for each strain was performed the hpnH product catalyzes addition of adenosine to hopene, the first reaction in triplicate. For chemical structures of hopanoids, refer to Fig. S1A in the in the synthesis of C hopanoids; and the hpnP product catalyzes C-2 meth- 35 supplemental material. (Continued) September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 3 Kulkarni et al. FIG 3 Whole-cell membrane fluidity measurements by fluorescence polar- ization show that rigidity decreases for all strains as temperature increases and that the hpnH mutant membrane is less rigid than that of the WT or the hpnP mutant (**, P  0.01 by Student’s two-tailed t test). Error bars repre- sent the standard deviations from three biological replicates (~22 technical replicates). challenged hpnP and hpnH mutants with a variety of stressors that are relevant during the initiation and progression of symbio- sis, such as hypoxia, acidic pH, high osmolarity, reactive oxygen species, and peptide antibiotics (24, 25). Under hypoxic conditions with 0.5% oxygen, the hpnP mu- tant is unable to attain growth yields as high as those of the WT and the hpnH mutant fails to grow (Fig. 4C). This indicates that in the free-living state 2Me-hopanoids contribute to microaerobic growth and C hopanoids are essential. Using GC-MS, we deter- mined the abundance of these hopanoid types in the WT (see Table S3 in the supplemental material). The amount of 2Me- hopanoids dramatically increased from 33%  2% TLE under oxic conditions to 77% 2% TLE under hypoxic conditions. This is consistent with hopanoid methylation being important to sustain WT levels of microaerobic growth. The only C ho- panoid detectable by GC-MS, BHP-508, increased in abun- dance from 3%  1% TLE for cells grown aerobically to 21% 1% TLE when grown microaerobically, in agreement with a microaerobic growth defect for the hpnH mutant. Under acidic conditions (pH 6), the hpnH mutant is unable to grow (Fig. 4D). The hpnH mutant is also more prone to FIG 2 MALDI-MS analysis of lipidA from B. diazoefficiens strains. LipidA stationary-phase stress, osmotic stressors (NaCl and inositol), and from the WT and the hpnP mutant is composed of a mixture of penta- membrane destabilizers (bile salts and EDTA [17]) than the WT, acylated and hexa-acylated species, whereas hpnH mutant lipidA is mainly hexa-acylated. A C hopanediolic acid is ester linked to hexa-acylated and as evidenced by a reduction in hpnH mutant growth on stressor hepta-acylated lipid A in the WT and the hpnP mutant. The hpnH mutant gradient plates (Fig. 4E). Additionally, disc diffusion assays does not contain any lipidA-bound hopanoids. showed that the hpnH mutant is more sensitive to oxidative (hydrogen peroxide [H O ]), detergent (SDS), and acidic (hydro- 2 2 chloric acid [HCl]) stresses than the WT (Fig. 4F). Because B. di- ature (30°C) and essential for growth at higher temperature azoefficiens is exposed to NCR peptides in A. afraspera, we also (37°C). As shown in our whole-cell membrane fluidity measure- tested the sensitivity of the hpnH mutant to two antimicrobial ments, the higher the temperature, the less rigid the membrane. peptides, polymyxin B (30) and NCR335 from the legume Medi- This might be the reason why C hopanoids are absolutely re- cago truncatula (31). The hpnH mutant displayed a 10-fold- quired to maintain membrane rigidity at 37°C but are dispensable lower MIC (48 g/ml) for polymyxin B than did the WT (512 g/ at 30°C. It is important to note that the phenotypic defect of the ml). In addition, the hpnH mutant was found to be 100-fold hpnH mutant could be due to either the absence of C ho- more susceptible than the WT to NCR335 (Fig. 4G). The hpnP panoids or the lack of downstream products, such as HoLA, and mutant withstood all of the abovementioned stressors as well as even accumulation of the HpnH substrate diploptene, or a com- did the WT, with the exception of acidic stress, where it grew bination of these factors. slower than the WT. 2Me- and C hopanoids are important for microaerobic C hopanoids are required to establish an efficient symbio- 35 35 growth, and C hopanoids are involved in stress tolerance. Ho- sis with A. afraspera but not with soybean. Because hopanoids panoids have been shown to contribute to stress tolerance in di- are required for microaerobic growth and stress tolerance in the verse organisms (13–16). We speculated that such protection free-living state, we hypothesized that they would aid survival would also be seen in B. diazoefficiens. To test this hypothesis, we within the plant microenvironment. To test this, we analyzed the 4 mbio.asm.org September/October 2015 Volume 6 Issue 5 e01251-15 Roles of Hopanoids in Survival of Legume Symbionts symbiotic phenotypes of hpnP and hpnH mutants on two host plants, soybean and A. afraspera. On soybean, at 21 days post inoculation (dpi), both mutants induced fewer nodules and dis- played slightly reduced nitrogenase activity as estimated by the acetylene reduction assay (ARA) relative to the WT (see Fig. S4A to C in the supplemental material). However, these differences were not statistically significant, and cytological analyses done by confocal microscopy and TEM revealed no differences between WT and mutant nodules (see Fig. S4M to X). Thus, under the conditions of these assays, neither 2Me- nor C hopanoids appear to be important for symbiosis between B. diazoefficiens and soy- bean. In contrast, a clear effect of the hpnH mutation could be ob- served when B. diazoefficiens infected A. afraspera. Plants inocu- lated with the hpnH mutant displayed typical nitrogen starva- tion symptoms, including foliage chlorosis, reduced plant growth, and half the acetylene reduction activity of WT- and hpnP mutant-infected plants (Fig. 5A and B). Reduced nitrogen fixation was not due to a decrease in the numbers of nodules, which were comparable in WT- and hpnH mutant-infected plants at 9, 14, and 21 dpi, suggesting that the hpnH mutation does not affect nodule organogenesis (Fig. 5C; see also Fig. S5 in the supplemental material). However, cytological analyses revealed that hpnH mutant nodules displayed several disorders in comparison to WT and hpnP mutant nodules (Fig. 5D to Z=). At the cellular level, hpnH mutant nodules were smaller (Fig. 5D to F) and had pink or, in ~30% of cases, even white central tissue in contrast to WT and hpnP mutant nodules, which were dark pink due to the accumulation of the O carrier leghemoglobin (Fig. 5G to I). The central symbiotic tissue of hpnH mutant nodules was often disorganized and partially in- fected (Fig. 5L, M, and R), as opposed to the fully occupied tissue of WT and hpnP mutant nodules (Fig. 5J, K, P, and T). In some hpnH mutant nodules, the presence of necrotic regions— char- acterized by the accumulation of autofluorescent brown com- pounds— could be seen (Fig. 5M and N). These are likely poly- phenol compounds whose production is associated with plant defense responses (32). Within hpnH mutant nodules, iodine staining also revealed accumulation of starch granules in the non- infected cells surrounding the symbiotic tissue, whereas such granules were rarely observed in WT and hpnP mutant nodules (Fig. 5O). Starch accumulation is indicative of an imbalance be- tween the photosynthates furnished by the plant and the ability of the bacterium to metabolize them, a typical feature of nonfixing or underperforming strains (33, 34). To determine whether hpnH mutant symbiotic defects stem from a problem in the bacterial differentiation process or are due to a damaged membrane, we examined nodule sections by confo- cal microscopy using live-dead staining (35) and TEM to analyze FIG 4 Growth of B. diazoefficiens strains under various stress conditions. (A the ultrastructure of bacteroids. Confocal microscopy revealed to D) Growth of the WT (circles), thehpnP mutant (squares), and thehpnH that all strains, including the hpnH mutant, differentiated prop- mutant (triangles) was monitored as optical density at 600 nm (OD )inPSY at 30°C (A), PSY at 37°C (B), microaerobic PSY with 0.5% O at 30°C (C), and erly into elongated bacteroids, which were, for the majority, via- PSY at pH 6 and 30°C (D). Each curve represents the average of at least three ble, as indicated by the green Syto9 staining (Fig. 5P to U). How- biological replicates, except the microaerobic growth curves, for which a rep- ever, TEM analysis showed that the cell envelope of some hpnH resentative data set out of four trials is shown. (E and F) Growth of B. diazoef- mutant bacteroids was not well delineated and, in a few cases, even ficiens strains under stress as measured in stressor gradient plates with 50 mM broken (Fig. 5Y and Z=). Similar damage was seen in the peribac- NaCl, 500 mM inositol, 0.4% bile salts, or 1 mM EDTA (E) or by disc diffusion assays with 10% SDS, 5.5 M H O , and 2 M HCl (F). Error bars represent teroid membrane that surrounds bacteroids. Deposits of cellular 2 2 standard errors (n  9). *, P  0.05, and **, P  0.01, by Tukey’s honestly material, possibly resulting from the release of plant or bacterial significant difference test. (G) NCR335 sensitivity of B. diazoefficiens strains. cytoplasm, were also observed in the peribacteroid space, suggest- ing a beginning of senescence or perhaps necrosis of symbiotic September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 5 Kulkarni et al. FIG 5 B. diazoefficiens hpnH mutant is impaired in symbiosis with A. afraspera at 21 dpi. (A) Comparison of growth of plants, noninoculated (NI) or inoculated with WT or hpnH or hpnP mutant. (B) Quantification of acetylene reduction activity (ARA) in plants inoculated with WT or hpnH or hpnP mutant. Error bars represent standard errors (n  10). **, P  0.01 by Tukey’s honestly significant difference test. (C) Number of nodules per plant elicited by WT andhpnH andhpnP mutants. (D to M) Aspect of the nodules elicited by WT (D, G, and J),hpnP mutant (E, H, and K), andhpnH mutant (F, I, L, and M). (D to F) Whole roots; bars, 1 mm. (G to I) Cross section of live nodules; bars, 500 m. (J to L) Nodule thin sections viewed by bright-field microscopy; bars, 500 m. (M) The black arrow shows plant defense reactions (necrotic plant cells); bar, 500 m. (N) Aspect of the nodules elicited byhpnH mutant as observed by confocal microscopy using the live-dead kit; bar, 200 m. White arrows show plant defense reactions. (O) Aspect of the nodules elicited by hpnH mutant stained with Lugol; bar, 500 m. White arrows point to starch granules in black. (P to U) Confocal microscopy observations of nodules elicited by WT (P and Q), hpnH (R and S), and hpnP (T and U) strains and stained using Syto9 (green; healthy bacteroids), calcofluor (blue; plant cell wall), and propidium iodide (red; infected plant nuclei and bacteroids with compromised membranes); bars, 200 m (P R, and T) and 20 m (Q, S, and U). (V to Z=) TEM of nodules elicited by WT (V, W, and X) andhpnH mutant (Y, Z, and Z=). (V and Y) Black arrows show symbiososmes. (Z) Cell envelope of somehpnH mutant bacteroids is not well delineated (boldface black arrow), and some deposits of cellular material can be observed in the peribacteroid space (lightface black arrow). (Z=) The boldface black arrow shows bacteroid wall breakdown. The lightface black arrow shows cellular material of unknown origin. Bars, 2 m (V and Y), 0.5 m (W and Z), and 0.2 m (X and Z=). 6 mbio.asm.org September/October 2015 Volume 6 Issue 5 e01251-15 Roles of Hopanoids in Survival of Legume Symbionts bacterial cells (Fig. 5Z and Z=). Such defects were not observed in the abundance of 2Me-hopanes in ancient sediments peaks during the WT (Fig. 5V to X) orhpnP nodules. Taken together, our data oceanic anoxic events (8), and today, C hopanoids appear to be indicate that under these conditions, C hopanoids, but not 2Me- enriched in hypoxic regions of the oceans (40). hopanoids, play an important role in facilitating the fitness of Why do we observe context-dependent hopanoid phenotypes B. diazoefficiens in symbiosis with A. afraspera. in planta? The phenotypic difference of the requirement for C hopanoids in A. afraspera but not in soybean likely stems from DISCUSSION differences between the plants’ intracellular environments. In Bacteria that provide plants with fixed nitrogen represent an at- both plants, the bacterium is exposed to a variety of stresses, in- tractive agronomical alternative to nitrogen fertilizers (36). Re- cluding oxidative, osmotic, and acidic stresses, within the mi- cently, we found a statistically significant correlation between the croaerobic niche of the infected plant cell (24). Although such an presence of hopanoid biosynthetic genes and organisms, metabo- environment is less than ideal for the hpnH mutant, it is able to lisms, and environments known to support plant-microbe inter- colonize as evidenced by its successful symbiosis with the soybean actions (9). This raised the hypothesis that hopanoids could sup- plant. However, unlike soybean, in A. afraspera, B. diazoefficiens port bacterial fitness in the context of symbiosis for certain undergoes terminal differentiation due to the action of NCR pep- organisms. Building on our recent observation that hopanoids tides (25). Because the hpnH mutant is highly sensitive to NCR promote symbiosis between the legume A. evenia and the Brady- peptides, exposure within the host might reduce the viable mutant rhizobium BTAi1 strain (15), we explored the generality of this population by causing cell death or increasing susceptibility to this finding by studying the phylogenetically different and better- and other plant defense mechanisms. Consistent with this hy- known Bradyrhizobium species B. diazoefficiens, focusing on the pothesis, the hpnH mutant only partially infects the A. afraspera roles of two dominant hopanoid classes. In the free-living state of nodule tissue. B. diazoefficiens, 2Me-hopanoids contribute to growth under mi- In A. afraspera, synthesis of C hopanoids is critical for several croaerobic and acidic conditions and C hopanoids are required aspects of the symbiosis, including evasion of plant defense reac- for microaerobic growth and tolerance to diverse stresses found in tions, efficient utilization of plant photosynthates, and nitrogen the symbiotic microenvironment. Consistent with these pheno- fixation. Two reasons why the plant host mounts an immune re- types, C hopanoids are critical for symbiosis between B. diazoef- sponse against the hpnH mutant may be that the altered mutant ficiens and A. afraspera, and yet they are dispensable for symbiosis surface layer, as seen in TEM images, is unable to suppress this with soybean. This intriguing finding suggests that the microen- response (41) and/or the host induces nodule senescence prema- vironment encountered by plant symbionts varies between hosts. turely on detecting an underproductive symbiont (42). Consistent Bradyrhizobium strain BTAi1 and B. diazoefficiens differ phys- with this, nitrogenase activity is reduced in the hpnH mutant iologically in important ways. Shc proteinss from these two strains relative to the WT, a likely consequence of poor cell viability. fall in distinct phylogenetic clades (15). Unlike Bradyrhizobium Similarly, the buildup of plant carbon as starch in hpnH mutant strain BTAi1, B. diazoefficiens is unable to photosynthesize (37, nodules might indicate slow metabolism and/or perturbation of 38). Moreover, B. diazoefficiens infects plants via a Nod factor- membrane transport processes that facilitate bacteroid carbon ac- dependent pathway, whereas Bradyrhizobium strain BTAi1 uses quisition. alternate symbiotic strategies (39). Our inability to delete shc in The global agricultural economy is largely based on nitrogen B. diazoefficiens suggests that hopanoids are essential in this spe- fertilizers, with the United States alone consuming 13,000 tons per cies, in contrast to Bradyrhizobium strain BTAi1, where shc mu- annum (43). However, the usage of nitrogen fertilizers comes with tants are viable. This fundamental difference between the species a price, as their production requires burning of fossil fuels and likely reflects differences in the niches that they inhabit as a con- their runoff from soils leads to surface and groundwater contam- sequence of their metabolic differences and what is required for ination (44). Rhizobia, which naturally fix nitrogen in association survival therein. with common crops, are an environmentally and economically What insights can we gain about 2Me- and C hopanoid func- feasible alternative to nitrogen fertilizers. Our results reveal that tions in natural contexts based on ex planta experiments? This is hopanoids affect the ability of B. diazoefficiens to cope with envi- the first study to identify conditions under which synthesis of ronmental stresses as well as its nitrogen fixation efficiency in a 2Me-hopanoids is important for any cell type. These include hy- plant host-dependent manner. This observation is relevant to in- poxic and acidic conditions that B. diazoefficiens possibly per- terpreting ancient patterns of hopane deposition, which correlate ceives as stress, thus upregulating 2Me-hopanoid production as with paleoenvironmental conditions where nitrogen fixation may has been seen in R. palustris (13) to enhance membrane rigidity have provided a selective advantage (8). In addition, understand- and stability (12). However, it is puzzling that 2Me-hopanoids are ing the roles of hopanoids in bacterial stress resistance and how dispensable under similar conditions within the plant cell, and a they facilitate nitrogen fixation may enable the engineering of ag- priority for future work will be to explain this paradox. Prior work ronomically useful strains with enhanced tolerance to rising tem- in B. cenocepacia has shown that C hopanoids promote stress perature and salinity. tolerance and antibiotic resistance (14). Similarly, we found that synthesis of C hopanoids is important for growth under oxic and hypoxic conditions and for tolerance to diverse stressors. Consis- MATERIALS AND METHODS tent with our whole-cell membrane rigidity measurements, these Bacterial strains and growth conditions. Bacterial strains used in this phenotypes could be due to higher membrane fluidity resulting study are listed in Table S4 in the supplemental material. Escherichia coli from the absence of C hopanoids, the inability to make HoLA, 35 strains were grown in lysogeny broth (LB) (45) at 37°C. B. diazoefficiens and/or the accumulation of the C hopanoid diploptene. We strains were grown at 30°C in either rich medium (peptone-salts-yeast hope to tease apart these possibilities going forward. Intriguingly, extract medium with 0.1% arabinose [PSY] [46] or yeast extract-mannitol September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 7 Kulkarni et al. medium [YM] [47]) or minimal medium (buffered nodulation medium REFERENCES [BNM] [48]). 1. Masson-Boivin C, Giraud E, Perret X, Batut J. 2009. Establishing DNA methods, plasmid construction, and transformation. All plas- nitrogen-fixing symbiosis with legumes: how many rhizobium recipes? mid constructions and primers used in this study are described in Ta- Trends Microbiol 17:458 – 466. http://dx.doi.org/10.1016/ j.tim.2009.07.004. ble S4 in the supplemental material. 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Nat Commun 5:5106. http:// cal Institute (HHMI) to D.K.N.; the Agence Nationale de la Recherche, dx.doi.org/10.1038/ncomms6106. grant “BugsInACell” no. ANR-13-BSV7-0013, to E.G.; and the Italian 16. Welander PV, Hunter RC, Zhang L, Sessions AL, Summons RE, New- Ministry of Education, Universities and Research (PRIN) and Mizutani man DK. 2009. Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1. J Bacteriol 191: Foundation for Glycoscience 2014 to A.M. and A.S. D.K.N. is an HHMI 6145– 6156. http://dx.doi.org/10.1128/JB.00460-09. Investigator. The EM facility where the cryo-TEM micrographs were col- 17. Welander PV, Doughty DM, Wu CH, Mehay S, Summons RE, Newman lected is supported by the Agouron and Beckman foundations. DK. 2012. Identification and characterization of Rhodopseudomonas We thank Raphael Ledermann and Hans-Martin Fischer for providing palustris TIE-1 hopanoid biosynthesis mutants. 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Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of Bradyrhizobium diazoefficiens

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RESEARCH ARTICLE crossmark Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of Bradyrhizobium diazoefficiens a b c d b c b Gargi Kulkarni, Nicolas Busset, Antonio Molinaro, Daniel Gargani, Clemence Chaintreuil, Alba Silipo, Eric Giraud, a,e,f Dianne K. Newman Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA ; IRD, Laboratoire des Symbioses Tropicales et b c Méditerranéennes (LSTM), UMR IRD/SupAgro/INRA/UM2/CIRAD, Montpellier, France ; Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Naples, Italy ; d e CIRAD, UMR BGPI, Montpellier, France ; Howard Hughes Medical Institute, Pasadena, California, USA ; Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California, USA G.K. and N.B. contributed equally to this article. ABSTRACT A better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbio- ses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires hopanoid production for optimal fitness. While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis. In contrast, synthesis of extended (C ) hopanoids is required for growth microaerobically and under various stress conditions (high temperature, low pH, high osmolarity, bile salts, oxidative stress, and antimicrobial peptides) in the free-living state and also during symbiosis. These defects might be due to a less rigid mem- brane resulting from the absence of free or lipidA-bound C hopanoids or the accumulation of the C hopanoid diploptene. 35 30 Our results also show that C hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides in Aeschynomene nodules that induce drastic modification in bacterial morphology and physiology. The study of hopanoid mutants in plant symbionts thus provides an opportunity to gain insight into host-microbe interactions during later stages of symbiotic progression, as well as the microenvironmental conditions for which hopanoids provide a fitness advantage. IMPORTANCE Because bradyrhizobia provide fixed nitrogen to plants, this work has potential agronomical implications. An un- derstanding of how hopanoids facilitate bacterial survival in soils and plant hosts may aid the engineering of more robust agro- nomic strains, especially relevant in regions that are becoming warmer and saline due to climate change. Moreover, this work has geobiological relevance: hopanes, molecular fossils of hopanoids, are enriched in ancient sedimentary rocks at discrete inter- vals in Earth history. This is the first study to uncover roles for 2Me- and C hopanoids in the context of an ecological niche that captures many of the stressful environmental conditions thought to be important during (2Me)-hopane deposition. Though much remains to be done to determine whether the conditions present within the plant host are shared with niches of relevance to the rock record, our findings represent an important step toward identifying conserved mechanisms whereby hopanoids con- tribute to fitness. Received 27 July 2015 Accepted 17 September 2015 Published 20 October 2015 Citation Kulkarni G, Busset N, Molinaro A, Gargani D, Chaintreuil C, Silipo A, Giraud E, Newman DK. 2015. Specific hopanoid classes differentially affect free-living and symbiotic states of Bradyrhizobium diazoefficiens. mBio 6(5):e01251-15. doi:10.1128/mBio.01251-15. Editor Frederick M. Ausubel, Mass General Hospital Copyright © 2015 Kulkarni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Eric Giraud, eric.giraud@ird.fr, or Dianne K. Newman, dkn@caltech.edu. variety of plants, including leguminous (1), actinorhizal (2), (LPS) is essential for all stages of rhizobial symbiosis, including Aand land-dwelling (3) plants, rely on bacterial symbiotic part- root hair infection, symbiotic tissue (nodule) establishment, and ners for assimilation of nitrogen, an essential macronutrient. survival within the plant cell (6). Whether and to what extent These symbioses require bacterial invasion of plant tissues and other microbial membrane lipids regulate the establishment and adaptation of the bacterial symbiont to the plant host environ- maintenance of plant-microbe symbioses are unclear. Here, we ment, processes in which several microbial membrane lipids play consider whether hopanoids (7), steroid-like pentacyclic triterpe- key roles. For instance, phosphatidylcholine is critical for efficient noid lipids, support plant-microbe interactions. Hopanoids are nitrogen fixation in several legume-rhizobial partnerships such as the progenitors of hopanes, molecular fossils that exhibit intrigu- soybean-Bradyrhizobium diazoefficiens (formerly named Brady- ing yet poorly understood abundance patterns in the rock record rhizobium japonicum [4]) and alfalfa-Sinorhizobium meliloti (5). (8). In part, our interest in hopanoids derives from a desire to Similarly, an intact outer membrane (OM) lipopolysaccharide interpret ancient biomarkers and the conviction that this requires September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 1 Kulkarni et al. a nuanced understanding of the biological functions of diverse and fixed atmospheric nitrogen produced by bacterial nitrogenase hopanoids in modern niches. (21, 23). Supporting such high levels of bacterial nitrogenase ac- The capacity for hopanoid biosynthesis is statistically enriched tivity requires both extensive host control of bacteroid physiology in the (meta-)genomes of bacteria associated with plants (9), and and establishment of a specific host microenvironment, defined hopanoids have been found in high abundance in membranes of by low oxygen, low pH, hyperosmosis, and oxidative stress (24). well-studied plant symbionts of the Bradyrhizobium (40% of total There is some evidence suggesting that the extent of these envi- lipid extract [TLE]) and Frankia (87%) genera (10, 11). Ho- ronmental stresses varies between plant hosts. For example, panoids promote membrane rigidity (12) and confer protection A. afraspera was recently shown to produce nodule-specific, against numerous stresses, including acidic or alkaline pH, high cysteine-rich antimicrobial peptides (NCR peptides) that induce temperature, high osmolarity, oxidative stress, detergents, and an- differentiation of the bacteroid into an enlarged, elongated, and tibiotics (13–16). They have varied structures, formed via meth- polyploid state, whereas in the soybean host, NCR peptides are ylation (2Me or 3Me), unsaturation, and/or attachment to a absent and bacteroid morphology and ploidy are similar to those ribose-derived side chain (C ) (Fig. 1A; also see Fig. S1 in the of the free-living state (25). Thus, it is likely that the microbial supplemental material) (7). Whether there are functional distinc- adaptations required for survival within root nodules are host tions under specific environmental conditions for diverse ho- specific, and we hypothesized that specific hopanoid mutants may panoid types is unclear, yet some evidence suggests that they have exhibit variable phenotypes in diverse plant hosts. nonoverlapping roles. For example, in Rhodopseudomonas palus- Here, we focus on the phenotypic consequences of the inability tris (17) and Burkholderia cenocepacia (14), C hopanoids are of B. diazoefficiens to produce two specific hopanoid classes, 2Me- critical for OM stability and for resistance to low pH, detergent and C hopanoids. We compare its hopanoid-dependent stress (sodium dodecyl sulfate [SDS]), and polymyxin B, respectively. phenotypes in the free-living state to those of other hopanoid Though the absence of 2Me-hopanoids did not manifest a stress producers. We further explore the fitness effects of 2Me- or C phenotype in previous tests of R. palustris, their biosynthesis is hopanoid production within a natural ecological context: the transcriptionally induced under stress (13). This suggests that symbiotic microenvironment of soybean and A. afraspera cells. 2Me-hopanoids might contribute to stress resistance under con- These studies begin to define the role of 2Me- and C hopanoids ditions yet to be identified in this and other organisms; consistent during the progression of plant-microbe symbioses and provide with this notion, 3Me-hopanoids contribute to late-stationary- insight into microbial membrane factors that facilitate adapta- phase survival in Methylococcus capsulatus (18). In vitro, 2Me- tions to particular microenvironments. hopanoids rigidify membranes of varied compositions (12). How- RESULTS ever, until now, no study has explored whether different hopanoids impact fitness in a natural ecological context. Shc appears to be essential for the survival of B. diazoefficiens. Recently, it was shown that elimination of hopanoid biosyn- To eliminate hopanoid production in B. diazoefficiens and to test thesis in photosynthetic Bradyrhizobium strain BTAi1 impairs its whether a requirement for hopanoids in efficient symbiosis is con- symbiosis with the legume Aeschynomene evenia (15). Because the served between B. diazoefficiens and Bradyrhizobium strain BTAi1, absence of all hopanoids likely had a broad and drastic impact on we first attempted deletion of the gene encoding the enzyme cat- cellular physiology and hence symbiosis, in this study we tested alyzing the first step in hopanoid biosynthesis, squalene hopene specific effects of 2Me- and C hopanoids (7) using B. diazoeffi- cyclase (Shc) (Fig. 1B). We were unable to isolate a shc mutant ciens USDA110, the best-studied Bradyrhizobium strain. In addi- using either the pK18mobsacB-based markerless gene deletion tion to C and C hopanoids (10), B. diazoefficiens makes tetra- method (~400 colonies screened) or the gene replacement strat- 30 35 hymanol, a triterpenoid with a gammacerane skeleton (19) (Fig. 1A; egy with pSUP202pol4 (~1,200 colonies screened) (26). This sug- also see Fig. S1 in the supplemental material). Intriguingly, while gests that Shc might be essential either because hopanoids are most hopanoids are thought to occur free within membranes, the C required for growth and survival of B. diazoefficiens or because hopanoid, (2-Me) 34-carboxyl-bacteriohopane-32,33-diol, was squalene, the substrate of Shc (16), accumulates to toxic levels found to be covalently attached to LPS lipidA, a well-established within the shc mutant. To rule out the latter possibility, we tried player in a broad range of host-microbe interactions, to form a to delete the entire operon encoding squalene-synthesizing en- compound called hopanoid-lipid A (HoLA) (15, 20) (see zymes (hpnCDE), shc (hpnF), and hpnG (which catalyzes the sec- Fig. S1B). ond step in the synthesis of C hopanoids) (17), but again, we B. diazoefficiens exhibits two different lifestyles, free living in were unable to obtain the hpnCDEFG mutant (~150 colonies soil or symbiotic within legume root nodule cells (1, 21). In addi- screened) (Fig. 1B). These results suggest that hopanoid synthesis tion to its native soybean host, B. diazoefficiens can engage in is essential for the survival of B. diazoefficiens under the conditions nitrogen-fixing symbioses with the stems and roots of the tropical used to select the mutants. legume Aeschynomene afraspera (22). In both of the these hosts, B. diazoefficiens hpnP and hpnH mutants are unable to development of the symbiosis progresses through a series of de- make 2Me- and C hopanoids, respectively. To eliminate syn- fined stages: (i) colonization and invasion of host root tissue; (ii) thesis of 2Me- or C hopanoids specifically, we deleted genes internalization of bacteria by plant cells to form an organelle-like predicted to encode the C-2 methylase, hpnP (27), or the first structure called the symbiosome, comprising endosymbiotic bac- enzyme catalyzing the extension of C hopanoids, hpnH (17) terial cells termed “bacteroids” that are surrounded by a plant- (Fig. 1B). As illustrated in Fig. 1C, no methylated hopanoids were derived “peribacteroid” membrane (see Fig. S2 in the supplemen- detected in hpnP mutant TLE using gas chromatography-mass tal material); and (iii) initiation of nitrogen fixation by bacteroids, spectrometry (GC-MS) and liquid chromatography-mass spec- during which there is a high rate of nutrient exchange across the trometry (LC-MS) (see Tables S1 and S2 in the supplemental ma- symbiosome membranes between plant-supplied carbon sources terial) (28, 29). The hpnH mutant does not make any detectable 2 mbio.asm.org September/October 2015 Volume 6 Issue 5 e01251-15 Roles of Hopanoids in Survival of Legume Symbionts C hopanoids, including aminotriol (a and c), BHP-508 (VIII; degradation product of aminotriol), bacteriohopanetetrol (b and e), and adenosylhopane (d). In addition, the hpnH mutant ac- cumulates a 6-fold excess of the HpnH substrate (17), diploptene (IV; wild type [WT], 18  2 g/mg TLE; hpnP mutant, 29 6 g/mg TLE; hpnH mutant, 111  3 g/mg TLE). We also analyzed the presence of HoLA in the mutants using matrix- assisted laser desorption ionization–mass spectrometry (MALDI- MS) (Fig. 2). WT and hpnP mutant lipidA are composed of a mixture of penta- to hepta-acylated species, whereas hpnH mu- tant lipidA is mainly hexa-acylated (see Fig. S1B). In WT and hpnP mutant hepta-acylated species, a C hopanediolic acid is ester linked to hexa-acylated lipidA, and traces of a second ho- panoid substitution are also detected; conversely, thehpnH mu- tant is missing any lipidA-bound hopanoids. Not only do our results confirm the proposed roles of HpnP and HpnH, but they also show that synthesis of C hopanoids is required for HoLA production. C hopanoids contribute to outer membrane rigidity. We employed a fluorescence polarization method by incubating the dye diphenyl hexatriene (DPH) with whole cells to determine whether 2Me- and C hopanoids affect the rigidity of B. diazoef- ficiens membranes at 25°C and 40°C (Fig. 3). Because previous studies of whole cells of different R. palustris hopanoid mutants indicated that the majority of DPH gets incorporated into the OM, we interpret whole-cell polarization values to reflect the ri- gidity of the OM (12, 29). Membranes of all strains were less rigid at the higher temperature. The hpnP mutant membrane was as rigid as the WT membrane at both temperatures, whereas the hpnH mutant membrane was less rigid. Thus, C hopanoids are important for maintaining membrane rigidity in B. diazoefficiens in vivo, in contrast to R. palustris, where thehpnH mutant mem- brane showed rigidity similar to that of the WT, despite the capac- ity of C hopanoids to enhance rigidity in vitro (12). This indi- cates that the fraction of C hopanoids or HoLA in the OM may be greater in B. diazoefficiens than R. palustris. Despite the lack of C hopanoids, thehpnH mutant membrane is morphologically indistinguishable from the WT membrane, as seen in whole-cell cryo-transmission electron microscopy (cryo-TEM) micrographs (see Fig. S3 in the supplemental material). C hopanoids are important for aerobic growth. Does a less rigid membrane affect the fitness of hpnH at different tempera- tures? To address this question, we compared aerobic growth rates of the hpnH mutant at 30°C and 37°C with those of the WT and the hpnP mutant (Fig. 4A and B). The hpnP mutant grows like the WT at both temperatures, whereas the hpnH mutant grows slower at 30°C and is unable to grow at 37°C. These results suggest that C hopanoids are important for growth at ambient temper- Figure Legend Continued FIG 1 (A) Structures of hopanoid and tetrahymanol. B. diazoefficiens makes ylation. (C) GC-MS and LC-MS (inset) total ion chromatograms of total lipid C hopanoids, such as diploptene (C-22C-30) and diplopterol (OH at 30 extracts from aerobically grown B. diazoefficiens strains. For GC-MS, main C-22); C hopanoids, such as bacteriohopanetetrol (BHT; R OH) and ami- 35 2 hopanoid peaks are numbered and the methylated counterparts elute 0.2 to nobacteriohopanetriol (aminotriol; R NH ); and tetrahymanol. All these 2 2 0.5 min earlier. I, pregnane acetate (standard); II, (2Me) hop-17(21)-ene; III, compounds can be methylated at C-2 (2Me, R CH ). (B) Hopanoid biosyn- 1 3 (2Me) hop-x-ene; IV, (2Me) hop-22(29)-ene (diploptene); V, (2Me) hop-21- thetic gene cluster of B. diazoefficiens. In this study, we focused on the genes ene; VI, (2Me) hopan-22-ol (diplopterol); VII, (2Me and 20Me) tetrahy- colored in gray: the shc (squalene hopene cyclase) product catalyzes squalene manol; and VIII, BHP-508. LC-MS: a, aminotriol; b, BHT; c, 2Me-aminotriol; cyclization to hopene, the first reaction in the hopanoid biosynthetic pathway; d, adenosylhopane; e, 2Me-BHT. Lipid analysis for each strain was performed the hpnH product catalyzes addition of adenosine to hopene, the first reaction in triplicate. For chemical structures of hopanoids, refer to Fig. S1A in the in the synthesis of C hopanoids; and the hpnP product catalyzes C-2 meth- 35 supplemental material. (Continued) September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 3 Kulkarni et al. FIG 3 Whole-cell membrane fluidity measurements by fluorescence polar- ization show that rigidity decreases for all strains as temperature increases and that the hpnH mutant membrane is less rigid than that of the WT or the hpnP mutant (**, P  0.01 by Student’s two-tailed t test). Error bars repre- sent the standard deviations from three biological replicates (~22 technical replicates). challenged hpnP and hpnH mutants with a variety of stressors that are relevant during the initiation and progression of symbio- sis, such as hypoxia, acidic pH, high osmolarity, reactive oxygen species, and peptide antibiotics (24, 25). Under hypoxic conditions with 0.5% oxygen, the hpnP mu- tant is unable to attain growth yields as high as those of the WT and the hpnH mutant fails to grow (Fig. 4C). This indicates that in the free-living state 2Me-hopanoids contribute to microaerobic growth and C hopanoids are essential. Using GC-MS, we deter- mined the abundance of these hopanoid types in the WT (see Table S3 in the supplemental material). The amount of 2Me- hopanoids dramatically increased from 33%  2% TLE under oxic conditions to 77% 2% TLE under hypoxic conditions. This is consistent with hopanoid methylation being important to sustain WT levels of microaerobic growth. The only C ho- panoid detectable by GC-MS, BHP-508, increased in abun- dance from 3%  1% TLE for cells grown aerobically to 21% 1% TLE when grown microaerobically, in agreement with a microaerobic growth defect for the hpnH mutant. Under acidic conditions (pH 6), the hpnH mutant is unable to grow (Fig. 4D). The hpnH mutant is also more prone to FIG 2 MALDI-MS analysis of lipidA from B. diazoefficiens strains. LipidA stationary-phase stress, osmotic stressors (NaCl and inositol), and from the WT and the hpnP mutant is composed of a mixture of penta- membrane destabilizers (bile salts and EDTA [17]) than the WT, acylated and hexa-acylated species, whereas hpnH mutant lipidA is mainly hexa-acylated. A C hopanediolic acid is ester linked to hexa-acylated and as evidenced by a reduction in hpnH mutant growth on stressor hepta-acylated lipid A in the WT and the hpnP mutant. The hpnH mutant gradient plates (Fig. 4E). Additionally, disc diffusion assays does not contain any lipidA-bound hopanoids. showed that the hpnH mutant is more sensitive to oxidative (hydrogen peroxide [H O ]), detergent (SDS), and acidic (hydro- 2 2 chloric acid [HCl]) stresses than the WT (Fig. 4F). Because B. di- ature (30°C) and essential for growth at higher temperature azoefficiens is exposed to NCR peptides in A. afraspera, we also (37°C). As shown in our whole-cell membrane fluidity measure- tested the sensitivity of the hpnH mutant to two antimicrobial ments, the higher the temperature, the less rigid the membrane. peptides, polymyxin B (30) and NCR335 from the legume Medi- This might be the reason why C hopanoids are absolutely re- cago truncatula (31). The hpnH mutant displayed a 10-fold- quired to maintain membrane rigidity at 37°C but are dispensable lower MIC (48 g/ml) for polymyxin B than did the WT (512 g/ at 30°C. It is important to note that the phenotypic defect of the ml). In addition, the hpnH mutant was found to be 100-fold hpnH mutant could be due to either the absence of C ho- more susceptible than the WT to NCR335 (Fig. 4G). The hpnP panoids or the lack of downstream products, such as HoLA, and mutant withstood all of the abovementioned stressors as well as even accumulation of the HpnH substrate diploptene, or a com- did the WT, with the exception of acidic stress, where it grew bination of these factors. slower than the WT. 2Me- and C hopanoids are important for microaerobic C hopanoids are required to establish an efficient symbio- 35 35 growth, and C hopanoids are involved in stress tolerance. Ho- sis with A. afraspera but not with soybean. Because hopanoids panoids have been shown to contribute to stress tolerance in di- are required for microaerobic growth and stress tolerance in the verse organisms (13–16). We speculated that such protection free-living state, we hypothesized that they would aid survival would also be seen in B. diazoefficiens. To test this hypothesis, we within the plant microenvironment. To test this, we analyzed the 4 mbio.asm.org September/October 2015 Volume 6 Issue 5 e01251-15 Roles of Hopanoids in Survival of Legume Symbionts symbiotic phenotypes of hpnP and hpnH mutants on two host plants, soybean and A. afraspera. On soybean, at 21 days post inoculation (dpi), both mutants induced fewer nodules and dis- played slightly reduced nitrogenase activity as estimated by the acetylene reduction assay (ARA) relative to the WT (see Fig. S4A to C in the supplemental material). However, these differences were not statistically significant, and cytological analyses done by confocal microscopy and TEM revealed no differences between WT and mutant nodules (see Fig. S4M to X). Thus, under the conditions of these assays, neither 2Me- nor C hopanoids appear to be important for symbiosis between B. diazoefficiens and soy- bean. In contrast, a clear effect of the hpnH mutation could be ob- served when B. diazoefficiens infected A. afraspera. Plants inocu- lated with the hpnH mutant displayed typical nitrogen starva- tion symptoms, including foliage chlorosis, reduced plant growth, and half the acetylene reduction activity of WT- and hpnP mutant-infected plants (Fig. 5A and B). Reduced nitrogen fixation was not due to a decrease in the numbers of nodules, which were comparable in WT- and hpnH mutant-infected plants at 9, 14, and 21 dpi, suggesting that the hpnH mutation does not affect nodule organogenesis (Fig. 5C; see also Fig. S5 in the supplemental material). However, cytological analyses revealed that hpnH mutant nodules displayed several disorders in comparison to WT and hpnP mutant nodules (Fig. 5D to Z=). At the cellular level, hpnH mutant nodules were smaller (Fig. 5D to F) and had pink or, in ~30% of cases, even white central tissue in contrast to WT and hpnP mutant nodules, which were dark pink due to the accumulation of the O carrier leghemoglobin (Fig. 5G to I). The central symbiotic tissue of hpnH mutant nodules was often disorganized and partially in- fected (Fig. 5L, M, and R), as opposed to the fully occupied tissue of WT and hpnP mutant nodules (Fig. 5J, K, P, and T). In some hpnH mutant nodules, the presence of necrotic regions— char- acterized by the accumulation of autofluorescent brown com- pounds— could be seen (Fig. 5M and N). These are likely poly- phenol compounds whose production is associated with plant defense responses (32). Within hpnH mutant nodules, iodine staining also revealed accumulation of starch granules in the non- infected cells surrounding the symbiotic tissue, whereas such granules were rarely observed in WT and hpnP mutant nodules (Fig. 5O). Starch accumulation is indicative of an imbalance be- tween the photosynthates furnished by the plant and the ability of the bacterium to metabolize them, a typical feature of nonfixing or underperforming strains (33, 34). To determine whether hpnH mutant symbiotic defects stem from a problem in the bacterial differentiation process or are due to a damaged membrane, we examined nodule sections by confo- cal microscopy using live-dead staining (35) and TEM to analyze FIG 4 Growth of B. diazoefficiens strains under various stress conditions. (A the ultrastructure of bacteroids. Confocal microscopy revealed to D) Growth of the WT (circles), thehpnP mutant (squares), and thehpnH that all strains, including the hpnH mutant, differentiated prop- mutant (triangles) was monitored as optical density at 600 nm (OD )inPSY at 30°C (A), PSY at 37°C (B), microaerobic PSY with 0.5% O at 30°C (C), and erly into elongated bacteroids, which were, for the majority, via- PSY at pH 6 and 30°C (D). Each curve represents the average of at least three ble, as indicated by the green Syto9 staining (Fig. 5P to U). How- biological replicates, except the microaerobic growth curves, for which a rep- ever, TEM analysis showed that the cell envelope of some hpnH resentative data set out of four trials is shown. (E and F) Growth of B. diazoef- mutant bacteroids was not well delineated and, in a few cases, even ficiens strains under stress as measured in stressor gradient plates with 50 mM broken (Fig. 5Y and Z=). Similar damage was seen in the peribac- NaCl, 500 mM inositol, 0.4% bile salts, or 1 mM EDTA (E) or by disc diffusion assays with 10% SDS, 5.5 M H O , and 2 M HCl (F). Error bars represent teroid membrane that surrounds bacteroids. Deposits of cellular 2 2 standard errors (n  9). *, P  0.05, and **, P  0.01, by Tukey’s honestly material, possibly resulting from the release of plant or bacterial significant difference test. (G) NCR335 sensitivity of B. diazoefficiens strains. cytoplasm, were also observed in the peribacteroid space, suggest- ing a beginning of senescence or perhaps necrosis of symbiotic September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 5 Kulkarni et al. FIG 5 B. diazoefficiens hpnH mutant is impaired in symbiosis with A. afraspera at 21 dpi. (A) Comparison of growth of plants, noninoculated (NI) or inoculated with WT or hpnH or hpnP mutant. (B) Quantification of acetylene reduction activity (ARA) in plants inoculated with WT or hpnH or hpnP mutant. Error bars represent standard errors (n  10). **, P  0.01 by Tukey’s honestly significant difference test. (C) Number of nodules per plant elicited by WT andhpnH andhpnP mutants. (D to M) Aspect of the nodules elicited by WT (D, G, and J),hpnP mutant (E, H, and K), andhpnH mutant (F, I, L, and M). (D to F) Whole roots; bars, 1 mm. (G to I) Cross section of live nodules; bars, 500 m. (J to L) Nodule thin sections viewed by bright-field microscopy; bars, 500 m. (M) The black arrow shows plant defense reactions (necrotic plant cells); bar, 500 m. (N) Aspect of the nodules elicited byhpnH mutant as observed by confocal microscopy using the live-dead kit; bar, 200 m. White arrows show plant defense reactions. (O) Aspect of the nodules elicited by hpnH mutant stained with Lugol; bar, 500 m. White arrows point to starch granules in black. (P to U) Confocal microscopy observations of nodules elicited by WT (P and Q), hpnH (R and S), and hpnP (T and U) strains and stained using Syto9 (green; healthy bacteroids), calcofluor (blue; plant cell wall), and propidium iodide (red; infected plant nuclei and bacteroids with compromised membranes); bars, 200 m (P R, and T) and 20 m (Q, S, and U). (V to Z=) TEM of nodules elicited by WT (V, W, and X) andhpnH mutant (Y, Z, and Z=). (V and Y) Black arrows show symbiososmes. (Z) Cell envelope of somehpnH mutant bacteroids is not well delineated (boldface black arrow), and some deposits of cellular material can be observed in the peribacteroid space (lightface black arrow). (Z=) The boldface black arrow shows bacteroid wall breakdown. The lightface black arrow shows cellular material of unknown origin. Bars, 2 m (V and Y), 0.5 m (W and Z), and 0.2 m (X and Z=). 6 mbio.asm.org September/October 2015 Volume 6 Issue 5 e01251-15 Roles of Hopanoids in Survival of Legume Symbionts bacterial cells (Fig. 5Z and Z=). Such defects were not observed in the abundance of 2Me-hopanes in ancient sediments peaks during the WT (Fig. 5V to X) orhpnP nodules. Taken together, our data oceanic anoxic events (8), and today, C hopanoids appear to be indicate that under these conditions, C hopanoids, but not 2Me- enriched in hypoxic regions of the oceans (40). hopanoids, play an important role in facilitating the fitness of Why do we observe context-dependent hopanoid phenotypes B. diazoefficiens in symbiosis with A. afraspera. in planta? The phenotypic difference of the requirement for C hopanoids in A. afraspera but not in soybean likely stems from DISCUSSION differences between the plants’ intracellular environments. In Bacteria that provide plants with fixed nitrogen represent an at- both plants, the bacterium is exposed to a variety of stresses, in- tractive agronomical alternative to nitrogen fertilizers (36). Re- cluding oxidative, osmotic, and acidic stresses, within the mi- cently, we found a statistically significant correlation between the croaerobic niche of the infected plant cell (24). Although such an presence of hopanoid biosynthetic genes and organisms, metabo- environment is less than ideal for the hpnH mutant, it is able to lisms, and environments known to support plant-microbe inter- colonize as evidenced by its successful symbiosis with the soybean actions (9). This raised the hypothesis that hopanoids could sup- plant. However, unlike soybean, in A. afraspera, B. diazoefficiens port bacterial fitness in the context of symbiosis for certain undergoes terminal differentiation due to the action of NCR pep- organisms. Building on our recent observation that hopanoids tides (25). Because the hpnH mutant is highly sensitive to NCR promote symbiosis between the legume A. evenia and the Brady- peptides, exposure within the host might reduce the viable mutant rhizobium BTAi1 strain (15), we explored the generality of this population by causing cell death or increasing susceptibility to this finding by studying the phylogenetically different and better- and other plant defense mechanisms. Consistent with this hy- known Bradyrhizobium species B. diazoefficiens, focusing on the pothesis, the hpnH mutant only partially infects the A. afraspera roles of two dominant hopanoid classes. In the free-living state of nodule tissue. B. diazoefficiens, 2Me-hopanoids contribute to growth under mi- In A. afraspera, synthesis of C hopanoids is critical for several croaerobic and acidic conditions and C hopanoids are required aspects of the symbiosis, including evasion of plant defense reac- for microaerobic growth and tolerance to diverse stresses found in tions, efficient utilization of plant photosynthates, and nitrogen the symbiotic microenvironment. Consistent with these pheno- fixation. Two reasons why the plant host mounts an immune re- types, C hopanoids are critical for symbiosis between B. diazoef- sponse against the hpnH mutant may be that the altered mutant ficiens and A. afraspera, and yet they are dispensable for symbiosis surface layer, as seen in TEM images, is unable to suppress this with soybean. This intriguing finding suggests that the microen- response (41) and/or the host induces nodule senescence prema- vironment encountered by plant symbionts varies between hosts. turely on detecting an underproductive symbiont (42). Consistent Bradyrhizobium strain BTAi1 and B. diazoefficiens differ phys- with this, nitrogenase activity is reduced in the hpnH mutant iologically in important ways. Shc proteinss from these two strains relative to the WT, a likely consequence of poor cell viability. fall in distinct phylogenetic clades (15). Unlike Bradyrhizobium Similarly, the buildup of plant carbon as starch in hpnH mutant strain BTAi1, B. diazoefficiens is unable to photosynthesize (37, nodules might indicate slow metabolism and/or perturbation of 38). Moreover, B. diazoefficiens infects plants via a Nod factor- membrane transport processes that facilitate bacteroid carbon ac- dependent pathway, whereas Bradyrhizobium strain BTAi1 uses quisition. alternate symbiotic strategies (39). Our inability to delete shc in The global agricultural economy is largely based on nitrogen B. diazoefficiens suggests that hopanoids are essential in this spe- fertilizers, with the United States alone consuming 13,000 tons per cies, in contrast to Bradyrhizobium strain BTAi1, where shc mu- annum (43). However, the usage of nitrogen fertilizers comes with tants are viable. This fundamental difference between the species a price, as their production requires burning of fossil fuels and likely reflects differences in the niches that they inhabit as a con- their runoff from soils leads to surface and groundwater contam- sequence of their metabolic differences and what is required for ination (44). Rhizobia, which naturally fix nitrogen in association survival therein. with common crops, are an environmentally and economically What insights can we gain about 2Me- and C hopanoid func- feasible alternative to nitrogen fertilizers. Our results reveal that tions in natural contexts based on ex planta experiments? This is hopanoids affect the ability of B. diazoefficiens to cope with envi- the first study to identify conditions under which synthesis of ronmental stresses as well as its nitrogen fixation efficiency in a 2Me-hopanoids is important for any cell type. These include hy- plant host-dependent manner. This observation is relevant to in- poxic and acidic conditions that B. diazoefficiens possibly per- terpreting ancient patterns of hopane deposition, which correlate ceives as stress, thus upregulating 2Me-hopanoid production as with paleoenvironmental conditions where nitrogen fixation may has been seen in R. palustris (13) to enhance membrane rigidity have provided a selective advantage (8). In addition, understand- and stability (12). However, it is puzzling that 2Me-hopanoids are ing the roles of hopanoids in bacterial stress resistance and how dispensable under similar conditions within the plant cell, and a they facilitate nitrogen fixation may enable the engineering of ag- priority for future work will be to explain this paradox. Prior work ronomically useful strains with enhanced tolerance to rising tem- in B. cenocepacia has shown that C hopanoids promote stress perature and salinity. tolerance and antibiotic resistance (14). Similarly, we found that synthesis of C hopanoids is important for growth under oxic and hypoxic conditions and for tolerance to diverse stressors. Consis- MATERIALS AND METHODS tent with our whole-cell membrane rigidity measurements, these Bacterial strains and growth conditions. Bacterial strains used in this phenotypes could be due to higher membrane fluidity resulting study are listed in Table S4 in the supplemental material. Escherichia coli from the absence of C hopanoids, the inability to make HoLA, 35 strains were grown in lysogeny broth (LB) (45) at 37°C. B. diazoefficiens and/or the accumulation of the C hopanoid diploptene. We strains were grown at 30°C in either rich medium (peptone-salts-yeast hope to tease apart these possibilities going forward. Intriguingly, extract medium with 0.1% arabinose [PSY] [46] or yeast extract-mannitol September/October 2015 Volume 6 Issue 5 e01251-15 mbio.asm.org 7 Kulkarni et al. medium [YM] [47]) or minimal medium (buffered nodulation medium REFERENCES [BNM] [48]). 1. Masson-Boivin C, Giraud E, Perret X, Batut J. 2009. Establishing DNA methods, plasmid construction, and transformation. All plas- nitrogen-fixing symbiosis with legumes: how many rhizobium recipes? mid constructions and primers used in this study are described in Ta- Trends Microbiol 17:458 – 466. http://dx.doi.org/10.1016/ j.tim.2009.07.004. ble S4 in the supplemental material. 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Nat Commun 5:5106. http:// cal Institute (HHMI) to D.K.N.; the Agence Nationale de la Recherche, dx.doi.org/10.1038/ncomms6106. grant “BugsInACell” no. ANR-13-BSV7-0013, to E.G.; and the Italian 16. Welander PV, Hunter RC, Zhang L, Sessions AL, Summons RE, New- Ministry of Education, Universities and Research (PRIN) and Mizutani man DK. 2009. Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1. J Bacteriol 191: Foundation for Glycoscience 2014 to A.M. and A.S. D.K.N. is an HHMI 6145– 6156. http://dx.doi.org/10.1128/JB.00460-09. Investigator. The EM facility where the cryo-TEM micrographs were col- 17. Welander PV, Doughty DM, Wu CH, Mehay S, Summons RE, Newman lected is supported by the Agouron and Beckman foundations. DK. 2012. Identification and characterization of Rhodopseudomonas We thank Raphael Ledermann and Hans-Martin Fischer for providing palustris TIE-1 hopanoid biosynthesis mutants. 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