Proteomic analysis of Rhizobium favelukesii LPU83 in response to

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Proteomic analysis of Rhizobium favelukesii LPU83 in response to acid stress Juliet F. Nilsson, Lucas G. Castellani, Walter O. Draghi, Julieta Pérez Giménez, Gonzalo A. Torres Tejerizo, and Mariano Pistorio J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00275 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Journal of Proteome Research

Proteomic analysis of Rhizobium favelukesii LPU83 in response to acid stress. Juliet F. Nilsson1, Lucas G. Castellani1, Walter O. Draghi1, Julieta Pérez Giménez1, Gonzalo A. Torres Tejerizo1 and Mariano Pistorio1*. 1IBBM

(Instituto de Biotecnología y Biología Molecular), CCT-La Plata, CONICET,

Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calles 49 y 115 (1900). La Plata, Argentina *Corresponding author: Mariano Pistorio E-mail: [email protected]

ABSTRACT. Acid soils constitute a severe problem for leguminous crops mainly through a disturbance in rhizobium-legume interactions. Rhizobium favelukesii—an acid-tolerant rhizobium able to nodulate alfalfa—is highly competitive for nodule occupation under acid conditions, but inefficient for biologic nitrogen fixation. In this work, we obtained a general description of the acid-stress response of R. favelukesii LPU83 by means of proteomics by comparing the total proteome profiles in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. Thus, a total of 336 proteins were identified with a significant differential expression, 136 of which species were

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significantly overexpressed and 200 underexpressed in acidity. An in-silico functional characterization with those respective proteins revealed a complex and pleiotropic response by these rhizobia involving components of oxidative phosphorylation, glutamate metabolism, and peptidoglycan biosynthesis, among other pathways. Furthermore, a lower permeability was evidenced in the acid-stressed cells along with several overexpressed proteins related to γaminobutyric-acid metabolism, such as the gene product of livK, which gene was mutated. This mutant exhibited an acid-sensitive phenotype in agreement with the proteomics results. We conclude that both γ-aminobutyric-acid metabolism and a modified cellular envelope could be relevant to acid tolerance in R. favelukesii.

KEY WORDS: Acid-stress, Rhizobium, proteomics, GABA, alfalfa, acid soils, livK, membranepermeability, FBN, acid-tolerance.

1. INTRODUCTION Leguminous plants can develop symbiotic interactions with compatible rhizobia that culminate in the formation of root nodules1,2. The rhizobia belong to diverse phylogenetic taxa included in the α- and ß-proteobacteria classes, but have in common the capability of inducing nodules on the roots of the appropriate legume host3. These structures contain differentiated rhizobia, known as bacteroids, which reduce atmospheric nitrogen to ammonia, an assimilable form for utilization by either the bacteria or the plant2. These symbiotic interactions play an essential role in agricultural-production systems through an enrichment of the soils with nitrogen that maximizes crop yields, thus decreasing the need for using chemical fertilizers that are harmful to the environment.


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Medicago sativa (alfalfa) is the most widely cultivated forage legume for cattle encompassing about 32 million hectares worldwide4. The growth and persistence of alfalfa is impaired in moderately acid soils. This impairment is highly relevant because 179 million hectares of arable land contain acid soils5. The low performance of alfalfa and other leguminous plants in acid soils is attributable to several conditions that affect the host plant, their rhizobia, and the symbiotic interactions6-8. Ensifer meliloti and Ensifer medicae, both microsymbiont of alfalfa, are efficient for biologic nitrogen fixation, but are sensitive to low pHs, especially E. meliloti7,9,10. Therefore, from an empirical point of view, the acid tolerance of rhizobia, when occurring, has been considered as a phenotypic characteristic that greatly facilitates the establishment of symbiosis under acidic conditions11. Many studies on the genes involved in the acid-stress response have been undertaken with E. medicae WSM419. By using Tn5-mutant libraries a functionally diverse set of pH-related genes have been identified—namely, actA, actR/S, actP, exoH, phrR and lpiA12-18. In addition, Tiwari et al.19 generated a pool of random promotor fusions to the gusA gene; which constructions revealed that cytochrome synthesis, potassium-ion cycling, lipid biosynthesis, and transport processes are key components of the E. medicae WSM419 acid-stress response. Through a proteomics analysis, Reeve et al.20 obtained the profiles of proteins that were either persistently or only transiently acid-expressed in E. medicae WSM419; suggesting that folding, proteolysis, and transport processes were critical functions in E. medicae for growth in acidity. Another study, revealed that a short-term exposure of the E. meliloti 1021 to low pH was sufficient to induce significant transcriptional changes in diverse rhizobial genes related to various cellular functions21, and Draghi et al.22 reported a combined analysis of the cultural, proteomic, transcriptomic, and metabolic responses of E. meliloti 2011 growing under controlled acid stress in a chemostat. Their work indicated


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alterations mainly in the proteins and genes associated with the bacterial surface, in the amount of exopolysaccharide secreted, in the central carbon metabolism, and in respiration. The genetic evidence acquired to date indicates that acid tolerance in rhizobia is generally a multigenic phenotype. Nevertheless, examples can be cited where the overexpression of certain stressresponse genes, such as groEL and cbb3 oxidase, have been useful in improving rhizobial symbiotic performance under suboptimal conditions 23 Although alfalfa is a highly specific plant with respect to its symbiotic partner, rhizobial strains other than E. meliloti and E. medicae can also nodulate this plant species. Rhizobium favelukesii is an acid-tolerant rhizobium that nodulates alfalfa and other leguminous plants24,25, though inefficient for nitrogen fixation25-27. In addition, this rhizobium outcompetes E. meliloti in the nodulation of alfalfa in acid soils thus constituting an agricultural inconvenience in soils where the former rhizobium is intended for nodulation26. Hence, from a practical point of view, R. favelukesii provides a system for studying the natural mechanisms that bacterium could employ for pH resistance. Moreover, an understanding of the genetic response to acid stress in R. favelukesii constitutes a potentially powerful basis for effecting a rational improvement in E. meliloti performance at low pH for increasing biologic nitrogen fixation in acid soils. The study reported here was, thus, aimed at elucidating the determinants involved in the acidstress response of R. favelukesii through an omics approach. We accordingly analyzed the proteome response to acid stress of the characteristic strain R. favelukesii LPU83 through nanoflow ultrahigh-performance liquid chromatography coupled to a quadrupole Orbitrap™ mass spectrometer. In addition, we evaluated the relevance to acid-tolerance of two overexpressed proteins identified in this study in the phenotypical behavior of R. favelukessi growing at low pH.


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2. MATERIALS AND METHODS Bacterial culture and growth conditions Rhizobium favelukesii LPU83 and mutants (Table S1) were cultivated in glutamatesucrose (GS) minimal medium28 at 28 ºC and 160 rpm. The medium pH was buffered with a combination of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] and MES [2-(Nmorpholino) ethanesulfonic acid] both at 10 Mm29. Growth at different pHs was evaluated through measurements of the optical density at 600 nm (OD600nm) as a function of time. The inocula were grown in tryptone–yeast-extract (TY) medium30 and the growth kinetics (28 ºC, 200 rpm) analyzed by monitoring OD600 in a microplate reader (BMG Labtech, Germany). For the solid media, 15 g of agar was added per liter of TY medium. The final antibiotic concentrations per ml of either medium were: 50 µg kanamycin (Km) for Escherichia coli or 400 µg streptomycin (Sm) and 120 µg neomycin (Nm) for R. favelukesii.

Protein extraction Rhizobium favelukesii LPU83 was cultivated at pH 7 and pH 4.6 up to an O.D600nm of 0.5. An OD600nm of 0.5 in the flask (spectrophotometer cuvettes, optical path length:: 1 cm) is equal to an OD600nm of 0.25 in the microplate reader (Fig. 1, microplates of 24 wells, optical path length: 0.516 cm). A volume of 400 ml of the cultures was centrifuged at 11,000 x g for 30 min at 4 ºC and washed twice with low-salt washing buffer (3 mM KCl, 1.5 mM KH2PO4, 68 mM NaCl, 9 mM NaH2PO4; 31. The pellet was resuspended in approximately 10 ml of 10 mM Tris-


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Buffer pH 8.5 with the protease inhibitor phenylmethylsulfonyl fluoride. The samples were transferred to 2-ml tubes with glass beads (a mixture of diameters 0.1 to 0.3 mm) and disrupted with a Precellys™ tissue homogenizer (2 cycles of 5,000 rpm for 20 seconds, Bertin Instruments). The samples were then centrifuged at 10,000 x g for 20 min at 4 ºC and the supernatants transferred to new tubes for a subsequent 1 h incubation with DNase (RQ1 DNase, PROMEGA) and RNase A at 37 ºC. The samples were next ultracentrifuged at 100,000 x g for 1 h at 4 ºC. For each cell culture two kinds of protein samples were obtained, a fraction enriched in membrane protein and another in cytosolic protein. The pellets containing the membrane proteins were resuspended in rehydration buffer (7 M urea, 2 M thiourea, 10 % [v/v] isopropanol, 2 % [v/v] Triton X100), while the cytosolic proteins were precipitated with 4 volumes of cold acetone overnight at –20 ºC and stored until processed. Finally, those cytosolic proteins were centrifuged at 10,000 x g at 4 ºC for 20 min, followed by a washing with 90 % (v/v) acetone, and resuspended in rehydration buffer (7 M urea, 2 M thiourea). The total protein content of the samples was determined by the Bradford assay with Coomassie Brilliant blue32. Mass spectrometry and data analysis For the proteomic fractions enriched in membrane proteins, two biological replicates, each consisting of 3 technical replicates, were analyzed for both acidic and neutral conditions. For the fractions enriched in cytosolic proteins, 3 biological replicates with a single technical replicate each were used. First, protein samples were reduced with dithiotreitol (10 mM in 50 mM ammonium bicarbonate) at 56 ºC for 60 min and then alkylated with iodoacetamide (20 mM in 50 mM ammonium bicarbonate) at room temperature in the dark. The proteins were precipitated with 0.2 volumes of trichloroacetic acid at –20 ºC for 2 h, then centrifuged at 14,000 x g for 10 min and the pellets washed three times with acetone at –20 ºC before storage in the


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cold. The pellets were resuspended in 50 mM ammonium bicarbonate, digested with trypsin (Promega V5111) and the peptides desalted with Zip-Tip™ C18 columns (Millipore). Finally, the protein samples were analyzed by nanoflow ultrahigh-performance liquid chromatography (Thermo Scientific, model EASY-nLC 1000) with an EASY-Spray™ column (P/N ES801, C18, 2 µm, 100 A, 50 µm x 15 cm, serial number: 10433446, temperature 50°C. Thermo Scientific) coupled to a quadrupole Orbitrap™ mass spectrometer (Thermo Scientific, model Q-Exactive). A volume of 2 µl was injected and the column flow rate set to 300 nl/min. The gradient used was from 95 % solution A (0.1 % [v/v] formic acid in water) and 5 % solution B (0.1 % [v/v] formic acid in acetonitrile) to 80 % B in 185 min, 20–32 % in 44 min, 32–95 % in 1 min, and 95 % B for 10 min. A voltage of 3.5 kV was used for electrospray ionization. Resolution of full MS was 70,000 and tandem MS-MS was 17,500. Threshold for precursor ion selection: 10,000. The number of precursors selected for tandem MS in each scan cycle was 15. The mass window for precursor ion selection was 1.6 m/z. Normalized collision energy was 27. Charge state screening settings: all charges (excepted +1 and unassigned). Dynamic exclusion parameters: 20.0 seconds. This analysis was performed by the Center of Chemical and Biological Studies by Mass Spectrometry (Centro de Estudios Químico y Biológicos por Espectrometría de Masa [CEQUIBIEM], National University of Buenos Aires). The Andromeda search engine in the MaxQuant software [version 1.5.3.30]33, was used to process the mass spectra and to identify the peptides. Searches were performed throughout the Rhizobium

favelukesii

LPU83

protein-sequence

database

(https://www.uniprot.org/proteomes/UP000019443, UniProt). The peptide identification was made with a false-discovery rate (FDR) of 1 %34, and peptides were likewise assigned to proteins with a protein FDR of 1 %. For the first search a precursor ion-mass tolerance of 20 ppm was


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used that allowed for m/z retention-time recalibration of precursor ions that were then subjected to a main search at a precursor ion-mass tolerance of 4.5 ppm and a product ion-mass tolerance of 0.02 Da. The search parameters included up to two missed cleavages at trypsin/P on the sequence, and the oxidation of methionine as a dynamic modification. A minimum of two unique peptides was used to identify a protein.

Label-free quantitation was performed via the

corresponding module found in MaxQuant [version 1.5.3.30]35. All the biological replicates under each condition were included in the experimental design of label-free protein-level quantitation (LFQ) and the normalization performed by means of unique and razor-peptide features corresponding to identifications filtered with respective FDRs of 0.01 and 0.05 for peptides and proteins, respectively. The statistical analysis and visualization were performed with the Perseus software (version 1.5.6.0, Max Planck Institute, Germany)36. The statistical significance of the relative quantitative changes between the conditions of neutral and acid pH in replicate data was determined by the Student t test at a p value of

Proteomic analysis of Rhizobium favelukesii LPU83 in response to

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