Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28°C and 35°C.
Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses.
Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species.
In most agricultural soils, nitrogen (N) is the main limiting nutrient and, accordingly, it is often supplied to crops as chemical fertilizers. Significant losses of N-fertilizers occur either by leaching—resulting in eutrophication of rivers, lakes, aquifers— or by denitrification, contributing to global warming
. However, estimates indicate that up to 60% of the N needs of legume crops may be obtained from the biological nitrogen fixation (BNF) process
[2, 3], with significant economic benefits to farmers while mitigating environmental impacts.
Common bean (Phaseolus vulgaris L.) is the most important food legume in South and Central America and in East Africa. It can establish symbiotic relationships with a variety of described and still-to-be-described rhizobial species
. An important limitation to the BNF process involving common bean is the high genetic instability of the symbiotic plasmid of the rhizobial strains, as reported for Rhizobium phaseoli and Rhizobium etli. This instability has been attributed to genomic rearrangements, plasmid deletions and mutations, which are intensified under stressful conditions
[5, 6]. Abiotic stresses such as high soil temperatures, in addition to water deficit, salinity and soil acidity comprise the main factors causing genetic instability
Among common-bean rhizobia, Rhizobium tropici is recognized for its tolerance of environmental stresses, including high temperatures
[7–9]. Within this species, strain PRF 81 (= SEMIA 4080) is known for the high capacity in fixing N2, competitiveness against other rhizobia, and tolerance of environmental stresses; it has been used in commercial inoculants in Brazil since 1998
[10, 11]. More information about the strain, including genetic characterization, is given elsewhere
[10, 12, 13]. The strain is deposited at the “Diazotrophic and Plant Growth Promoting Bacteria Culture Collection” at Embrapa Soja (
Mechanisms of response to stresses are usually highly conserved among bacterial species, and designed for rapid adaptation to environmental and metabolic changes. These conserved responses comprise the expression of molecular chaperones, such as DnaK (and its assistants DnaJ and GrpE), GroEL (and its assistant GroES), and also of small heat-shock proteins
. All are polypeptide-binding proteins implicated in protein folding, protein targeting to membranes, renaturation, and in the control of protein-protein interactions. In addition to conserved responses, some bacterial species also possess specific metabolic adaptations to stressful conditions.
Recently, a draft genome of R. tropici strain PRF 81 revealed several probable genes that may be related to its outstanding symbiotic and saprophytic abilities and also its adaptability to environmental stresses
; elucidation of the whole genome of the strain is now in progress (
http://www.bnf.lncc.br). However, elucidating biological implications of a given genome requires understanding of gene expression; therefore, proteomic studies, complementary to the structural genome, are critical.
Despite the ecological, evolutionary and economic importance of R. tropici, proteomic information about the species is scarce. In addition, the intriguing tolerance to high temperature of R. tropici strains is far from being understood. In this context, our objective with this study was to report a proteomic study of R. tropici strain PRF 81, focusing on the determination of adaptive responses to heat stress.
Bacterial growth conditions
R. tropici strain PRF 81 was pre-cultured in 10-mL aliquots of tryptone-yeast extract medium (TY), at 80 rpm and 28°C, in the dark. The pre-cultures were then transferred to Erlenmeyer flasks containing 200 mL of TY medium and bacteria were grown under two treatment conditions: control (28°C) and with heat stress (35°C). Cells were incubated until the exponential phase of growth was reached (optical density of 0.6 at 600 nm), what took approximately 18 h, with low agitation (80 rpm) to minimize the production of extra-cellular polysaccharides, which can interfere in 2-D gel electrophoresis.
Total protein extraction
Cultures were centrifuged at 5,000 x g, at 4°C and cells were carefully washed with a solution containing 3 mM KCl; 1.5 mM KH2PO4; 68 mM NaCl; and 9 mM NaH2PO4. Washed cells were resuspended in 600 μL of a buffer containing 10 mM Tris–HCl pH 8.0; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM DTT; and 0.5 mM PMSF. Aliquots of 150 μL were stored in ultrafreezer (–80°C) until the analyses.
For whole-cell protein extraction, aliquots were resuspended in lysis buffer containing 9.5 M urea; 2% CHAPS; 0.8% v/v Pharmalyte 4–7; and 1% DTT, and submitted to forty cycles of freezing in liquid N2 and thawing at 37°C, as described by Lery et al.. The lysates were separated from particulate material by centrifugation at 14.000 x g for 90 min, at 4°C.
An additional step of concentration with phenol was done, increasing significantly the quality and reproducibility of the 2-D gels (data not shown). Aliquots (500 μL) of the lysates were homogenized with a solution containing 0.8 mL of Tris-buffered phenol pH 8.0, and 0.8 mL of SDS buffer (0.1 M Tris–HCl pH 8.0; 2% SDS; 5% β-mercaptoethanol; 30% sucrose; 1 mM phenylmethylsulfonyl fluoride, PMSF). The samples were homogenized for 5 min and centrifuged at 16,000 x g for 15 min at 4°C, and the top phenol layer (500 μL) was transferred to a new tube. Proteins were precipitated for 1 h at –20°C with three volumes of pre-cooled 0.1 M ammonium acetate in absolute methanol and then centrifuged (16,000 x g for 15 min at 4°C). The pellet was washed once with pre-cooled methanol and once with pre-cooled 80% v/v acetone, followed by drying.
The pellet was resuspended with the lysis buffer and concentration was determined by Bradford’s method
2-D electrophoresis and visualization
For IEF, lysates were dissolved with DeStreak buffer (GE Healthcare) to a final concentration of 300 μg of protein and 2% v/v IPGphor in 250 μL of solution. IPG-strips (pH 4–7, 13 cm, GE Healthcare) were rehydrated with the protein solution and covered with cover fluid (GE Healthcare). Loaded strips were submitted to focalization in an Ettan IPGphor IEF system (GE Healthcare) for 1 h at 200 V, 1 h at 500 V, a gradient step to 1,000 V for 1 h, a gradient step to 8,000 V for 2 h 30 min, and fixed at 8,000 V for 1 h 30 min. The final Vh was fixed at 24,800. After focusing, strips were equilibrated first for 20 min in 5 mL of TE buffer (50 mM Tris–HCl pH 8.8; 6 M urea; 30% v/v glycerol; 2% w/v SDS; and 0.2% v/v of a 1% solution of bromophenol blue) supplemented with 50 mg DTT and then in TE buffer with 175 mg iodoacetamine, also for 20 min.
2-D electrophoresis was performed on a 12% polyacrylamide gel (18 × 16 cm) in a Ruby SE 600 vertical electrophoresis system (GE Healthcare). The run was carried out for 30 min at 15 mA/gel and 240 min at 30 mA/gel, using the Low Molecular Weight Calibration Kit for SDS Electrophoresis (Amersham Biosciences) to provide standards. For each strain, the extraction procedure and gel electrophoresis were run in triplicates. Gels were fixed overnight with an ethanol-acetic acid solution before being stained with Coomassie Blue PhastGelTM R-350 (GE Healthcare) and scanned (ImageScanner LabScan v5.0).
Gel image analysis and spot selection
Spots were strictly identified in the high-resolution digitalized gel images and analyzed by Image Master 2D Platinum v 5.0 software (GE Healthcare). After background subtraction, ratios of mean normalized spot volumes were calculated and values of related spots were compared between both conditions. All selected spots exhibiting a higher volume in the heat stress condition were statistically evaluated (p ≤ 0.05) upon Student’s t-test, using XLSTAT (Addinsoft, France, add-in to Microsoft Excel).
Sample preparation and MALDI-TOF mass spectrometry
Protein spots showing significant changes in mean normalized volume were excised and processed as described by Chaves et al.. Digestion was achieved with trypsin (Gold Mass Spectrometry Grade, Promega, Madison, WI), at 37°C, overnight.
Tryptic peptides (1 μL) were mixed with saturated solution of α-cyano- 4-hydroxy-cinnamic acid (HCCA) in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA). The mixture was spotted onto a MALDI (matrix assisted laser desorption ionization) sample plate and allowed to crystallize at room temperature. The same procedure was used for the standard peptide calibration mix (Bruker Daltonics). For mass spectra acquisition, a MALDI-TOF-MS (MALDI-time-of-flight in tandem) Autoflex Spectrometer (Bruker Daltonics) was operated in the reflector for MALDI-TOF peptide mass fingerprint (PMF) and in the “LIFT” mode for MALDI-TOF/TOF in the fully manual mode, using FlexControl 3.0 software.
In silico protein identification
PMFs and MS/MS ion spectra generated were searched against the public database NCBInr (National Center for Biotechnology Information non-redundant), using Mascot software v. 2.3 (Matrix Science). For protein searches, performed in the Proteobacteria taxonomic group, monoisotopic masses were used, considering a peptide tolerance of 150 ppm and allowance of one missed cleavage. When MS/MS was carried out, a tolerance of 0.3 Da was acceptable. Carbamidomethylation of cysteine and oxidation of methionine were considered fixed and variable modifications, respectively.
Identifications were validated only when the Mowse (molecular weight search) score was significant, above the recommended cutoff of 52 for PMFs. Searches on the Decoy database were done in the automated mode in the Mascot software, using a random database (NCBInr/Proteobacteria) strategy. Both decoy score and false discovery rates were considered for the validation of the searches of MS and MS/MS data and to measure the quality of the matches (p ≤ 0.05); using this approach false discovery rates were always less than 1%. The spectrometry datasets are available at PRIDE (
http://ebi.ac.uk/pride/) with the experiment accession number 14817.
A set of bioinformatics tools was used for improved characterization of identified proteins. The proteins were fitted into COG (Clusters of Orthologous Groups) categories according to their functional inference, using the COGnitor program (
. Software packages PSORT-B
 and PSLpred
 were used for prediction of subcellular localization.
Results and discussion
2-D electrophoresis and differential spots selection
Several compounds, such as salts, nucleic acids and polysaccharides, may interfere with electrophoretic separation, resulting in streaky 2-D patterns, and thus should be removed. R. tropici PRF 81 produces high amounts of exopolysaccharides (EPS) in vitro and interference with electrophoretic resolution was overcome with a final wash step of the whole protein extract with phenol. In addition, to improve separation resolution, we employed IPG strips with a pH range of 4.0 to 7.0 in the first-dimension electrophoresis, to achieve better protein resolution than with broader-range (pH 3.0 to 10.0) strips (data not shown), in which the proteins remained concentrated in the central part of the gel (pH 5.0 to 7.0).
Using the computer-assisted gel-image analysis software, the majority of the molecular masses associated with the spots ranged between 14 and 97 kDa (Figure
1). The volume of each spot was normalized as a percent of the total volume of all detected spots in the gel. This procedure was followed for all gels and the values generated for each spot were compared between the control (28°C) and the experimental (35°C) treatment, and only well-defined spots present in the three replicates and showing statistically significant differences (p ≤ 0.05) were selected. A significant difference in several relative spot volumes indicated that the elevated temperature led to changes in the proteome of R. tropici PRF 81.
General proteome response to heat stress
Maximum soil temperatures in tropical soils can often exceed 40°C. Optimal temperature of growth of R. tropici species is around 28°C, and although there are reports of tolerance of PRF 81 to 40°C
[9, 10], our preliminary tests have shown that 35°C was the highest temperature that did not affect substantially growth; under higher temperatures, the slower growth rate had critical effects on the proteomic profile (data not shown). Joszefczuk et al. also reported, in a heat stress response experiment with Escherichia coli, that one of the most striking features was the strong influence of high temperatures on the bacterium growth. In addition, contrasting with the majority of the studies about heat stress only with a short period of growth at high temperatures, our study considered a heat stress for the whole period of PRF 81 growth.
In comparison to other common-bean rhizobial species, R. tropici is known for its genetic stability and adaptation to stressful conditions
[8, 9], and, although PRF 81 is an outstanding strain in terms of these properties
[10, 11, 13], little is known of the molecular determinants of its heat tolerance. In order to obtain an overview of the heat responses, we analyzed the cytoplasmic and periplasmic contents and identified the whole-cell protein expression changes when the cells were grown at 35°C. Fifty-nine significantly induced proteins were identified by mass spectrometry, and twenty-six of them were detected exclusively under heat stress conditions. All identified proteins were distributed across fifteen COG functional categories; six fit into the category of general prediction (R), one was classified in the category of unknown function (S) and only one was assigned as “not in COG” (Table
Identified proteins ofRhizobium tropiciPRF 81 whole cell extracts up-regulated after growth at high temperature (35°C)
Organism (best match)
Fold change ratio2
C - Energy production and conversion
Succinyl-coa synthetase beta subunit protein
3.27 ± 0.12
1.65 ± 0.06
F0F1 ATP synthase subunit beta
2.68 ± 0.03
E- Amino acid transport and metabolism
Glutamine synthetase I
2.92 ± 0.08
2.19 ± 0.09
Putative periplasmic substrate binding protein
Opine permease ATP-binding protein
Oligoendopeptidase F protein
Aspartate-B-semialdehyde dehydrogenase protein
1.38 ± 0.043
Diaminobutyrate--pyruvate aminotransferase protein
Branched-chain amino acid aminotransferase
F- Nucleotide transport and metabolism
1.38 ± 0.13
G - Carbohydrate transport and metabolism
2.88 ± 0.37
Alpha amylase catalytic region
H - Coenzyme transport and metabolism
1.70 ± 0.09
Biotin protein ligase
3.98 ± 0.24
I - Lipid transport and metabolism
3-Oxoacyl-(acyl-carrier-protein (ACP)) synthase III domain protein
Beta-ketoadipyl coa thiolase
2.95 ± 0.07
P - Inorganic ion transport and metabolism
2.27 ± 0.07
Cellular processes and signaling
D - Cell cycle control, cell division, chromosome partitioning
Cell division protein
2.42 ± 0.26
Condesin subunit F
T - Signal transduction mechanisms
Protein tyrosine phosphatase
Serine protein kinase protein
2.41 ± 0.19
Putative two component response regulator Nitrogen assimilation regulatory protein
Two component response regulator
1.35 ± 0.10
O - Posttranslational modification, protein turnover, chaperones
Bacterioferritin comigratory protein
3.40 ± 0.26
1.36 ± 0.19
M - Cell wall/membrane/envelope biogenesis
Putative metalloendopeptidase protein
1.31 ± 0.22
Outer membrane lipoprotein
N - Cell motility
1.21 ± 0.16
Information storage and processing
J - Translation, ribosomal structure and biogenesis
Translation elongation factor Ts
1.86 ± 0.02
Elongation factor G
1.98 ± 0.13
2.23 ± 0.09
Translation initiation factor IF-2
1.29 ± 0.09
Elongation factor EF-Tu protein
3.40 ± 0.31
K – Transcription
LysR family transcriptional regulator
AraC family transcriptional regulator
Transcriptional regulator protein
4.71 ± 0.09
Transcriptional regulator GntR family
Transcriptional regulator ROK family
Transcriptional regulator, MarR family
L - Replication, recombination and repair
ATP-dependent RNA helicase protein
2.29 ± 0.14
R - General function prediction only
FeS assembly ATPase SufC
Chloride peroxidase protein
1.59 ± 0.02
2.58 ± 0.14
NADPH-dependent FMN reductase
2.03 ± 0.19
2,5-diketo-D-gluconic acid reductase A
1.34 ± 0.21
Ferredoxin reductase protein
1.48 ± 0.12
S - Function Unknown
Hypothetical protein Avi_3814
1.42 ± 0.34
NO related COG
Hypothetical protein Rleg2_5527
1.57 ± 0.13
1Theoretical/Experimental values. Da: Daltons.
2↑1.00 in the fold change ratio means that the protein was only identified in the experimental condition (35°C).
Matched peptides masses and MS/MS combined results are available in PRIDE (
http://ebi.ac.uk/pride/) under the experiment accession number 14817.
Among the differentially expressed proteins, twenty-five were related to metabolic functions, the majority of them associated with amino acid transport and metabolism (group E) (Table
1), corroborating the proteomic reference map of Bradyrhizobium japonicum strain CPAC 15, a microsymbiont of soybean
, and indicating high metabolic activity even under stressful conditions.
Also within this category, it is worth mentioning that NocP, an opine permease ATP-binding protein, was differentially expressed under high temperature. Opine is a compound released by crown-gall tumors produced by Agrobacterium (=Rhizobium)
, and genes related to its metabolism were detected in the draft genome of PRF 81 and now confirmed at the translational level in our study. Putative genes related to rhizopine metabolism (an opine-like compound) were reported in R. tropici for the first time by our research group
. The ability to catabolize rhizopine appears to enhance the rate at which a strain is able to form nodules when it is in competition with a strain that is unable to catabolize a rhizopine. The mechanism responsible for this enhanced symbiotic ability is still unclear
. Moreover, we were unable to establish an exact association between stressful conditions and opine-like compounds metabolism, but our results presented an indicative of this correlation.
In relation to cellular processes and signaling, thirteen proteins were identified in categories D, T, O, M and N (Table
1). Two of these proteins are known to be correlated with heat tolerance, DnaK and GroEL molecular chaperones
[12, 25]. Two proteins also found in this group were thioredoxin TrxA and bacterioferritin comigratory proteins (Bcp), which have been characterized as oxidative-stress responsive.
Still considering the COG classification, thirteen induced proteins comprised a set related to information storage and processing (Table
1), including transcription regulators and translation factors. The translation factors can act as chaperones in response to heat stress, and more details of this function are discussed below.
Interesting was also the differential expression of VirD4, a TraG-like protein that plays an important role in conjugative transfer showing high similarity to Agrobacterium, and also reported in the draft genome of strain PRF 81
. The transcription of the vir regulon in Agrobacterium tumefaciens is induced by specific plant-phenolic compounds, but also by several other abiotic stimuli, such as low pH and temperatures below 30°C
. VirD4 acts in the translocation of effectors proteins and has been associated with different plant-bacterium interactions, both pathogenic and symbiotic. Also, VirD4 acts in couple DNA processing and transference by conjugation mechanism. Therefore, this protein has a broader role than the action in type IV secretion system. An association between heat stress and type IV secretion system components was described by Zahri et al., since the expression of type IV secretion system in a modified E. coli induced heat shock genes.
Differential expression of the two-component response regulators (NtrX and ChvI)
Two-component systems are composed by a sensor kinase protein that transmits the environmental stimulus to a response regulator protein via phosphorylation. The phosphorylated regulator modulates the expression of the target genes required for the appropriate changes, mediating rapid metabolic responses for adaptation to new conditions
. Interestingly, these two up-regulated proteins in our study (NtrX and ChvI) are the response-regulator components.
NtrX has also been found to be expressed in Gluconacetobacter diazotrophicus, Sinorhizobium (=Ensifer) meliloti, and Mesorhizobium loti. This protein is recognized to be involved in N metabolism and nitrogen fixation, probably acting as a transcriptional activator of genes related to nitrate metabolism
The second two-component system, ChvI, characterized in several bacteria such as S. meliloti and A. tumefaciens, acts in translation regulation of enzymes related to the biosynthesis of the succinoglycan exopolysaccharide (EPSI). In addition to this role, this two-component system signaling is critical for the viability of free-living S. meliloti strains
, by acting in biofilm formation, motility, nutrient utilization and cell protection
[37–39]. It has been reported that the succinoglycan may form a diffusion barrier, protecting against oxidative stress
, suggesting that, in R. tropici PRF 81, in addition to participating in symbiosis signaling, the succinoglycan EPSI plays an important role in heat-stress protection.
Induced molecular chaperones DnaK and GroEL
Temperature is especially harmful to cells because it can damage the structure of macromolecules. Many of the molecular chaperons—such as DnaK and GroEL—are highly conserved in evolution
, preventing and repairing harmful effects. As reported in other proteomic studies
[42–44], DnaK and GroEL were significantly induced in PRF 81 at high temperature.
DnaK is classified according to its molecular weight in the Hsp70 chaperone group, the most versatile chaperone system. In addition to a main role in de novo folding, DnaK has various other functions, including protein transport
, and in the increased stability of RNA polymerase σ32 factor (RpoH), an important component of the heat-shock response in several organisms
At optimal temperature, σ32 factor is rapidly degraded, but if temperature is raised, σ32 stability increases due to its interaction with DnaK chaperone
. Therefore, in response to a sudden increase in temperature, the levels of σ32 in the cell rise, leading to the regulation of transcription of genes encoding other heat-shock proteins, which also contribute to heat tolerance
As described for E. coli, Bacillus cereus and Acinetobacter baumannii, in R. tropici PRF 81 the molecular chaperone GroEL was up-regulated under high temperature. The differential expression of GroEL is critical to thermotolerance, since the chaperone can routinely rescue more than 80% of a denatured protein population
. Essentially, GroEL modulates its affinity for folding intermediates through the binding and hydrolysis of ATP, and the highly coordinated binding and releasing of substrate proteins may lead to recovery of the functional state of the proteins
Induction of chaperone-like proteins: Translation factors
Besides the main function of ensuring gene expression accuracy by transporting the correct codons in the translation process, elongation and initiation factors can also act as chaperones in response to heat stress
[57, 58]. In our study, three elongation factors (EF-Tu, Ef-G and Ef-Ts) and one initiation factor (IF-2) were up-regulated when R. tropici PRF 81 was grown at 35°C (Table
1), indicating the probable involvement of these factors in protein folding and protection, contributing to the thermotolerance of PRF 81.
EF-Tu is highly homologous to cellular GTP-proteins, occupying a key position in translation
. EF-Tu interacts with GTP, aminoacyl-tRNA, ribosomes, and a second factor, EF-Ts, which mediates GDP/GTP exchange on EF-Tu. In addition, Hendrick, and Hartl
 observed that EF-Tu protein may also act as a molecular chaperone, protecting proteins against thermal damage. Studying heat responses, Jacobson and Rosenbuch
 reported that large quantities of EF-Tu molecules in cells might constitute a reservoir of chaperone-like molecules that prevent the aggregation of non-native proteins until permissive renaturation conditions are restored. The shift of the activities of transport of aminoacyl-tRNA to the aminoacyl ribosome site and as chaperone of EF-Tu is dependent on the binding of this factor with GTP or GDP.
Considering the efficiency of chaperone activity,
 showed that the elongation factor EF-Tu when bonded with GDP had greater capacity of stimulating renaturation of enzymes than when interacting with GTP. In contrast, Kudlicki and collaborators
 found that EF-Tu bonded with GDP is less active than when it is bonded with GTP in catalyzing protein renaturation. Still, in that study, the authors reported that the EF-Ts elongation factor plays a similar role as GTP, suggesting that in the presence of these cofactors—EF-Ts or GTP—EF-Tu can perform several rounds of protein renaturation. These divergent studies indicate that the EF-Tu chaperonin activity is dependent on the specific protein in which the protection will be promoted. Interestingly, in our study, both elongation factors—EF-Tu and EF-Ts—were up-regulated under heat stress.
Both the elongation factor EF-G and the initiation factor IF2 were also found to act as chaperone proteins
. These factors are involved in the translocation of ribosomes on mRNA and in the binding of initiator tRNA to the 30 S ribosomal subunit, respectively
. EF-G bound to GDP, instead of to GTP, seems to be more active in the formation of stable complexes with unfolded proteins, assisting in protein folding and renaturation
. Finally, the chaperone properties of EF-Tu, EF-G, and IF2 suggest that translation factors are ancestral protein-folding factors that appeared before chaperones and protein-disulfide isomerases
Cross-talk between heat and oxidative stress
Reactive oxygen species (ROS) are by-products of normal metabolic processes, but at high levels may be lethal for cells. However, in both symbiotic and pathogenic relations, transient production of ROS, detected in the early events of plant-microorganism interactions, may be considered as specific signals during the interaction process
. Previous studies have reported the accumulation of ROS in early stages of Rhizobium/legumes symbiosis establishment
[65–67]. Therefore, the ability of the bacteria to tolerate and overcome the changes in the environment induced by the plant host seems to be important for the establishment of a successful symbiotic interaction
To detoxify ROS, symbiotic bacteria display a multiple antioxidant defense that is required for both the development and the functioning of the symbiosis
. Fernando et al. showed participation of thioredoxin (TrxA) in the reactivation of proteins damaged by oxidative stress, or by other conditions that cause the generation of ROS. These compounds cause covalent modifications in proteins, for example the oxidation of free sulfydryl groups (-SH), forming disulfide bonds (S-S). In this case, thioredoxin transfers reducing power to damaged proteins, restoring their reduced state
. Finally, thioredoxin was synthesized under high-temperature conditions, confirming its induction as a general response to stress
; it is also induced in the early stages of symbiotic interaction in B. japonicum and in the plant interaction with G. diazotrophicus.
Both bacterioferritin (Bfr), a protein related to inorganic ion transport, and bacterioferritin comigratory protein (Bcp), a peroxiredoxin protein, were up-regulated in our study. These proteins have been related to oxidative stress responses, similarly to thioredoxin. The former (Bfr) acts indirectly in defense mechanisms against oxidative damage effects inside the cell, since it transports inorganic ions, for example Fe2+, resulting in the decomposition of the peroxides over-produced during the oxidative stress
. The latter (Bcp) has a protective role in the defensive response to oxidative stress, possibly via up-regulation of total and reduced glutathione levels
In Salmonella typhimurium, the oxidative stress caused by hydrogen peroxide treatment led to the induction of heat shock proteins such as DnaK, while the heat stress induced proteins related with cell protection against the oxidative stress
. Interestingly, when Lenco et al. studied oxidative stress responses from a proteomic perspective, they observed the induction of several heat-responsive proteins, such as GroEL and GroES, as a reflection of regulation of heat-shock protein biosynthesis during bacterial oxidative stress.
We found up-regulation of several proteins responsive to oxidative stress, such as isocitrate dehydrogenase, which plays a key role in NADPH recycling under oxidative stress
[78–80]], also the flavoprotein WrbA, a quinone oxidoreductase with redox activity
[80, 81], among others. These results, added to others reporting the expression of heat responsive proteins during the oxidative stress, suggest a cross-talk between heat stress and oxidative stress responses.
Although most of the proteins involved in responses to heat are highly conserved, the regulatory mechanisms vary among bacterial species. In our study, we have shown differential expression of some conserved heat-responsive proteins, such as DnaK and GroEL. However, we have also reported the up-regulation of proteins involved in a variety of metabolic pathways, including translation factors and oxidative stress-responsive proteins, indicating that the responses of R. tropici strain PRF 81 to heat stress go beyond the induction of heat-shock proteins.
Strain PRF 81 is known for its high efficiency in fixing nitrogen, and in our study two component response regulators (NtrX and ChvI) were induced during the heat stress. These proteins act in the regulation of the nitrogen-fixation-gene expression and in the regulation of the succinoglycan exopolysaccharide (EPSI) production, respectively, showing that, even under stress conditions, PRF 81 retains nitrogen-fixing and symbiosis-establishment potential, which are essential characteristics for agricultural inoculants.
Finally, this proteomic experiment provides valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81 (
http://www.bnf.lncc.br), and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species. It is a useful reminder that R. tropici is an important species of agronomic interest for its capacity to fix nitrogen under tropical stressful conditions, and also demonstrates high resemblance in many genes, and —now also confirmed in many proteins—to those in pathogenic strains of the genus Agrobacterium.
Matrix assisted laser desorption ionization
Time of flight
Clusters of Orthologous Groups
Biological nitrogen fixation
Tryptone-yeast extract medium
Peptide mass fingerprint
Immobilized pH gradient
Reactive oxygen species
Bacterioferritin comigratory protein.
Acknowledgments and funding
The work was partially supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil)/MCT/MAPA (577933/2008) and CPNq-Repensa (562009/2010-1). MALDI-TOF was acquired with resources from Fundação Araucária, in a common project coordinated by Dr. Fábio Pedrosa, at the Federal University of Paraná. D.F. The authors thank Dr. Allan R. J. Eaglesham for suggestions on the manuscript.
Departamento de Biotecnologia, Universidade Estadual de Londrina
Centro Universitário Filadélfia (UNIFIL)
Instituto Agronômico do Paraná (IAPAR)
Vance CP: Symbiotic nitrogen fixation and phosphorus acquisition: plant nutrition in a world of declining renewable resources.Plant Physiol 2001, 127:390–397.PubMedView Article
Graham PH, Vance CP: Legumes: Importance and constraints to greater utilization.Plant Physiol 2003, 131:872–877.PubMedView Article
Saravanan VS, Madhaiyan M, Osborne J, Thangaraju M, Sa TM: Ecological occurrence of Gluconacetobacter diazotrophicus and nitrogen-fixing Acetobacteraceae members: their possible role in plant growth promotion.Microb Ecol 2008, 55:130–140.PubMedView Article
Ribeiro RA, Barcellos FG, Thompson FL, Hungria M: Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity.Res Microbiol 2009, 160:297–306.PubMedView Article
Djordjevic MA, Zurkowski W, Shine J, Rolfe BG: Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti.J Bacteriol 1983, 156:1035–1045.PubMed
Berry JO, Atherly AG: Induced plasmid-genome rearrangements in Rhizobium japonicum.J Bacteriol 1984, 157:218–224.PubMed
Hungria M, Franco AA, Sprent JI: New sources of high temperature tolerant rhizobia for Phaseolus vulgaris.Plant Soil 1993, 149:103–109.View Article
Hungria M, Vargas MAT: Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil.Field Crops Res 2000, 65:151–164.View Article
Martínez-Romero E, Segovia E, Mercante FM, Franco AA, Graham PH, Pardo MA: Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees.Int J System Bacteriol 1991, 41:417–426.View Article
Hungria M, Andrade DS, Chueire LMO, Probanza A, Guttierrez-Mañero FJ, Megías M: Isolation and characterization of new efficient and competitive bean (Phaseolus vulgaris L.) rhizobia from Brazil.Soil Biol Biochem 2000, 32:1515–1528.View Article
Hungria M, Campo RJ, Mendes IC: Benefits of inoculation of common bean (Phaseolus vulgaris) crop with efficient and competitive Rhizobium tropici strains.Biol Fertil Soil 2003, 39:88–93.View Article
Pinto FGS, Chueire LMO, Vasconcelos ATR, Nicolás MF, Almeida LGP, Souza RC, Menna P, Barcellos FG, Megías M, Hungria M: Novel genes related to nodulation, secretion systems, and surface structures revealed by a genome draft of Rhizobium tropici strain PRF 81.Funct Integr Genomics 2009, 9:263–270.PubMedView Article
Pinto FGS, Hungria M, Mercante FM: Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.).Soil Biol Biochem 2007, 39:1851–1864.View Article
Wagner MA, Zahrl D, Rieser G, Koraimann G: Growth phase and cell division dependent activation and inactivation of the σ32 regulon in Escherichia coli.J Bacteriol 2009, 191:1695–1702.PubMedView Article
Lery LM, Coelho A, Von Kruger WM, Gonçalves MS, Santos MF, Valente RH: Protein expression profile of Gluconacetobacter diazotrophicus PAL5, a sugarcane endophytic plant growth-promoting bacterium.Proteomics 2008, 8:1631–1644.PubMedView Article
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.Anal Biochem 1976, 72:248–254.PubMedView Article
Chaves DFS, Souza EM, Monteiro RA, Pedrosa FO: A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78.J Proteomics 2009, 73:50–56.PubMedView Article
Tatusov RL, Galperin M, Natale DA, Koonin EV: The COG database: a tool for genome scale analysis of protein functions and evolution.Nucleic Acids Res 2000, 28:33–36.PubMedView Article
Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FS: PSORTb v.2.0: Expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis.Bioinformatics 2005, 21:617–623.PubMedView Article
Bhasin M, Garg A, Raghava GPS: PSLpred: prediction of subcellular localization of bacterial proteins.Bioinformatics 2005, 21:2522–2524.PubMedView Article
Szymon J, Sebastian K, Gareth C, Jedrzej S, Alvaro C-I, Dirk S, Joachim S, Lothar W: Metabolomic and transcriptomic stress response of Escherichia coli.Mol Syst Biol 2010, 6:364.
Batista JSS, Torres AR, Hungria M: Towards a two-dimensional proteomic reference map of Bradyrhizobium japonicum CPAC 15: spotlighting “hypothetical proteins”.Proteomics 2010, 10:3176–3189.PubMedView Article
Montoya AL, Chilton MD, Gordon MP, Sciaky D, Nester EW: Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumor cells: role of plasmid genes.J Bacteriol 1977, 129:101–107.PubMed
Gordon DM, Ryder MH, Heinrich K, Murphy PJ: An Experimental Test of the Rhizopine Concept in Rhizobium meliloti.Appl Environ Microbiol 1996, 62:3991–3996.PubMed
Rodriguez F, Arsene-Ploetze F, Rist W, Rudiger S, Schneider-Mergener J, Mayer MP, Bukau B: Molecular basis for regulation of the heat shock transcription factor σ32 by the DnaK and DnaJ chaperones.Mol Cell 2008, 32:347–358.PubMedView Article
Brencic A, Winans SC: (2005) Detection of and response to signals involved in host–microbe interactions by plant-associated bacteria.Microbiol Mol Biol Rev 2005, 69:155–194.PubMedView Article
Zahrl D, Wagner M, Bischof K, Koraimann G: Expression and Assembly of a Functional Type IV Secretion System Elicit Extracytoplasmic and Cytoplasmic Stress Responses inEscherichia coli.J Bacteriol 2006, 188:6611–6621.PubMedView Article
Meletzus D, Zellermann EM, Kennedy C: Identification and characterization of the ntrcBC and ntrYX genes in Acetobacter diazotrophicus. In Biological Nitrogen Fixation for the 21st Century. Edited by: Elmerich C, Kondorosi A, Newton WE. Kluwer Academic Publishers, Dordrecht; 1998:125–126.
Capela D, Barloy-Hubler F, Gouzy J, Bothe G, Ampe F, Batut J, Boistard P, Becker A, Boutry M, Cadieu E, Dréano S, Gloux S, Godrie T, Goffeau A, Kahn D, Kiss E, Lelaure V, Masuy D, Pohl T, Portetelle D, Pühler A, Purnelle B, Ramsperger U, Renard C, Thébault P, Vandenbol M, Weidner S, Galibert F: Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021.Proc Natl Acad Sci 2001, 98:9877–9882.PubMedView Article
Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, Watanabe A, Idesawa K, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A, Mochizuki Y, Nakayama S, Nakazaki N, Shimpo S, Sugimoto M, Takeuchi C, Yamada M, Tabata S: Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.DNA Res 2000, 7:331–338.PubMedView Article
Ishida ML, Assumpção MC, Machado HB, Benelli EM, Souza EM, Pedrosa FO: Identification and characterization of the two component NtrY/NtrX regulatory system in Azospirillum brasilense.Braz J Med Biol Res 2002, 35:651–661.PubMedView Article
Pawlowski K, Klosse U, de Bruijn FJ: Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism.Mol Gen Genomics 1991, 231:124–138.View Article
Chen EJ, Sabio EA, Long SR: The periplasmic regulator ExoR inhibits ExoS/ChvI two-component signaling in Sinorhizobium meliloti.Mol Microbiol 2008, 69:1290–1303.PubMedView Article
Yuan ZC, Liu P, Saenkham P, Kerr K, Nester EW: Transcriptome profiling and functional analysis of Agrobacterium tumefaciens reveals a general conserved response to acidic conditions (pH 55) and a complex acidmediated signaling involved in Agrobacterium–plant interactions.J Bacteriol 2008, 190:494–507.PubMedView Article
Cheng HP, Walker GC: Succinoglycan production by Rhizobium meliloti is regulated through the ExoS-ChvI two-component regulatory system.J Bacteriol 1998, 180:20–26.PubMed
Fujishige NA, Kapadia NN, de Hoff PL, Hirsch AM: Investigations of Rhizobium biofilm formation.FEMS Microbiol Ecol 2006, 56:195–206.PubMedView Article
Wells DH, Chen EJ, Fisher RF, Long SR: ExoR is genetically coupled to the ExoS-ChvI two-component system and located in the periplasm of Sinorhizobium meliloti.Mol Microbiol 2007, 64:647–664.PubMedView Article
Yao SY, Luo L, Har KJ, Becker A, Rüberg S, Yu GQ, Zhu JB, Cheng HP: Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production.J Bacteriol 2004, 186:6042–6049.PubMedView Article
Davies BW, Walker GC: Identification of novel Sinorhizobium meliloti mutants compromised for oxidative stress protection and symbiosis.J Bacteriol 2007, 189:2110–2113.PubMedView Article
Gupta RS: Evolution of the chaperonin families (Hsp60, Hsp10, and Tcp-1) of proteins and the origin of eukaryotic cells.Mol Microbiol 1995, 15:1–11.PubMedView Article
Movahedi S, Waites W: A two-dimensional protein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation.J Bacteriol 2000, 182:4758–4763.PubMedView Article
Münchbach M, Nocker A, Narberhaus F: Multiple small heat shock proteins in rhizobia.J Bacteriol 1999, 181:83–90.PubMed
Janakiraman A, Fixen KR, Gray AN, Niki H, Goldberg MB: A genome-scale proteomic screen identifies a role for DnaK in chaperoning of polar autotransporters in Shigella.J Bacterioly 2009, 191:6300–6311.View Article
Hartl FU, Hayer-Hartl M: Converging concepts of protein folding in vitro and in vivo.Nature Struct Mol Biol 2009, 16:574–581.View Article
Bukau B: Regulation of the Escherichia coli heat shock response.Mol Microbiol 1993, 9:671–680.PubMedView Article
Georgopoulos C, Liberek K, Zylicz M, Ang D: The Biology of Heat Shock Proteins and Molecular Chaperones: Monograph 26. Cold Spring Harbor Laboratory, Cold Spring Harbor; 1994:209–249.
Yura T: Regulation and conservation of the heat-shock transcription factor sigma32.Genes Cells 1996, 1:277–284.PubMedView Article
Alexandre A, Oliveira S: Most heat-tolerant rhizobia show high induction of major chaperone genes upon stress.FEMS Microbiol Ecol 2011, 75:28–36.PubMedView Article
Gamer J, Multhaup G, Tomoyasu T, McCarty JS, Rudiger S, Schonfeld HJ, Schirra C, Bujard H, Bukau BA: A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates the activity of the Escherichia coli heat shock transcription factor sigma32.EMBO J 1996, 15:607–617.PubMed
Gross CA: Function and regulation of the heat shock proteins. In Escherichia coli and Samonella. Edited by: Neidhard FC. ASM Press, Washington DC; 1996:1382–1399.
Fayet O, Ziegelhoffer T, Georgopoulos C: The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures.J Bacteriol 1989, 171:1379–1385.PubMed
Periago PM, Van Schaik W, Abee T, Wouters JA: Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579.Appl Environ Microbiol 2002, 68:3486–3495.PubMedView Article
Cardoso K, Gandra RF, Wisniewski ES, Osaku CA, Kadowaki MK, Felipach-Neto V, Haus LFA, Simão RCG: DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii.J Med Microbiol 2010, 59:1061–1068.PubMedView Article
Goloubinoff P, Christeller JT, Gatenby AA, Lorimer GH: Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP.Nature 1989, 342:884–889.PubMedView Article
Sigler PB, Xu Z, Rye HS, Burston SG, Fenton WA, Horwich AL: Structure and function in GroEL mediated protein folding.Annu Rev Biochem 1998, 67:581–608.PubMedView Article
Caldas TD, Yaagoubi A, Richarme G: Chaperone properties of bacterial elongation factor EF-Tu.J Biol Chem 1998, 273:11478–11482.PubMedView Article
Jacobson GR, Rosenbuch JP: Abundance and membrane association of elongation factor Tu in E. coli.Nature 1976, 261:23–26.PubMedView Article
Kudlicki W, Coffman A, Kramer G, Hardesty B: Renaturation of rhodanese by translational elongation factor (EF) Tu - protein refolding by EF-Tu flexing.J Biol Chem 1997, 272:32206–32210.PubMedView Article
Grunberg-Manago M: Escherichia coli and Salmonella typhimurium Cellular and Molecular biology. 2nd edition. Edited by: Neidhardt FC. ASM Press, Washington, DC; 1996:1432–1457.
Nanda AK, Andrio E, Marino D, Pauly N, Dunand C: Reactive Oxygen Species during Plant-microorganism Early Interactions.J Integr Plant Biol 2010, 52:195–204.PubMedView Article
Kopcinska J: Localization of Reactive Oxygen species During Symbiosis of Early clover and Rhizobium leguminosarum bv Trifolii.Acta Biologica Cracoviensia Series Botanica 2009, 51:93–98.
Meilhoc E, Boscari A, Bruand C, Puppo A, Brouquisse R: Nitric Oxide in Legume-Rhizobium Symbiosis.Plant Sci 2011, 181:573–581.PubMedView Article
Peleg-Grossman S, Melamed-Book N, Levine A: ROS production during symbiotic infection suppresses pathogenesis-related gene expression.Plant Signal Behav 2012, 7:409–416.PubMedView Article
Normand P, Lapierre P, Tisa LS, Gogarten JP: Genome characteristics of facultatively symbioticFrankiasp. strains reflect host range and host plant biogeography.Genome Res 2007, 17:7–15.PubMedView Article
Pauly N, Pucciariello C, Mandon K, Innocenti G: Reactive oxygen and nitrogen species and glutathione: key players in the legume–Rhizobium symbiosis.J Exp Bot 2006, 57:1769–1776.PubMedView Article
Fernando MR, Nanri H, Yoshitake S, Nagato-Kuno K, Minakami S: Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.Eur J Biochem 1992, 209:917–922.PubMedView Article
Cabiscol E, Tamarit J, Ros J: Oxidative stress in bacteria and protein damage by reactive oxygen species.Internatl Microbiol 2000, 3:3–8.
Scharf C, Riethdorf S, Ernst H, Engelmann S, Volker U, Hecker M: Thioredoxin is an essential protein induced by multiple stresses in Bacillus subtilis.J Bacteriol 1998, 180:1869–1877.PubMed
Batista JSS, Hungria M: Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains.J Proteomics 2012, 75:1211–1219.View Article
Kang G, Park E, Kim K, Lim C: Overexpression of bacterioferritin comigratory protein (Bcp) enhance viability and reduced glutathione level in the fission yeast under stress.J Microbiol 2009, 47:60–67.PubMedView Article
Morgan RW, Christman MF, Jacobson FS, Storz G, Ames BN: Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins.Proc Nati Acad Sci USA 1986, 83:8059–8063.View Article
Lenco J, Pavkova I, Hubalek M, Stulik J: Insights into the oxidative stress response in Francisella tularensis LVS and its mutant DiglC1 + 2 by proteomics analysis.FEMS Microbiol Lett 2006, 246:47–54.View Article
Marino D, González EM, Frendo P, Puppo A, Arrese-Igor C: NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP+-dependent isocitrate dehydrogenase.Planta 2007, 225:413–421.PubMedView Article
Brown SM, Upadhya R, Shoemaker JD, Lodge JK: Isocitrate dehydrogenase is important for nitrosative stress resistance in Cryptococcus neoformans, but oxidative stress resistance is not dependent on glucose-6-phosphate dehydrogenase.Eukaryot Cell 2010, 9:971–980.PubMedView Article
Patridge EV, Ferry JG: WrbA from Escherichia coli and Archaeoglobus fulgidusIs an NAD(P)H: quinone oxidoreductase.J Bacteriol 2006, 188:3498–3506.PubMedView Article
Andrade SLA, Patridge EV, Ferry JG, Einsle O: Crystal structure of the NADH:quinone oxidoreductase WrbA from Escherichia coli.J Bacteriol 2007, 189:9101–9107.PubMedView Article
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