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Appl Environ Microbiol. Jul 2010; 76(13): 4510–4520.
Published online May 7, 2010. doi:  10.1128/AEM.02007-09
PMCID: PMC2897426

Characterization of the NifA-RpoN Regulon in Rhizobium etli in Free Life and in Symbiosis with Phaseolus vulgaris[down-pointing small open triangle]

Abstract

The NifA-RpoN complex is a master regulator of the nitrogen fixation genes in alphaproteobacteria. Based on the complete Rhizobium etli genome sequence, we constructed an R. etli CFN42 oligonucleotide (70-mer) microarray and utilized this tool, reverse transcription (RT)-PCR analysis (transcriptomics), proteomics, and bioinformatics to decipher the NifA-RpoN regulon under microaerobic conditions (free life) and in symbiosis with bean plants. The R. etli NifA-RpoN regulon was determined to contain 78 genes, including the genes involved in nitrogen fixation, and the analyses revealed 42 new NifA-RpoN-dependent genes. More importantly, this study demonstrated that the NifA-RpoN regulon is composed of genes and proteins that have very diverse functions, that play fundamental and previously less appreciated roles in regulating the normal physiology of the cell, and that have important functions in providing adequate conditions for efficient nitrogen fixation in symbiosis. The R. etli NifA-RpoN regulon defined here has some components in common with other NifA-RpoN regulons described previously, but the vast majority of the components have been found only in the R. etli regulon, suggesting that they have a specific role in this bacterium and particular requirements during nitrogen fixation compared with other symbiotic bacterial models.

Rhizobium etli is a nitrogen-fixing soil bacterium that is able to form a root nodule symbiosis with leguminous plants, specifically Phaseolus vulgaris (common bean), one of the most important crops in Mexico and Latin America (51). The R. etli-P. vulgaris symbiosis is a complex and biologically important relationship in which the bacterium fixes atmospheric nitrogen and the plant provides carbon compounds and protection against some environmental stresses. The detailed mechanisms that allow this interaction are not completely understood (7). In R. etli CFN42 and other nitrogen-fixing symbiotic bacteria, NifA-RpoN is a master regulator of nitrogen fixation genes, and NifA plays a central role in ensuring expression of the nitrogen fixation apparatus during symbiosis. The NifA protein belongs to the enhancer-binding protein family of transcriptional regulators that activate gene expression in concert with RNA polymerase containing the specialized sigma factor σ54 (RpoN), which allows the polymerase core to recognize −24/−12 promoters (14).

The R. etli CFN42 nifA gene is located on the symbiotic plasmid (pCFN42d) (23). Similar to R. etli CFN42, R. etli CNPAF512 has two rpoN genes encoding the alternative sigma factor σ54, which are differentially regulated during symbiosis and free-living growth (40). During free-living growth RpoN1, encoded on the chromosome, is required for growth on several nitrogen and carbon sources (41). When rpoN2, which is located on the symbiotic plasmid, is inactivated, there is a sharp decrease in nitrogen fixation (40), indicating that this gene has an essential role in bacteroids (16, 17). The NifA enhancer-binding protein controls transcriptional activation of rpoN2 under free-living microaerobic conditions and during symbiosis (15). The rpoN2 gene of R. etli CNPAF512 is orthologous to the R. etli CFN42 rpoN2 (rpoNd) gene, a gene located on pCFN42d.

In symbiotic diazotrophs, such as Sinorhizobium meliloti, transcription of nifA and fix genes is controlled predominantly by the oxygen-responsive two-component FixL-FixJ system. In S. meliloti NifA, along with FixK, controls 19 symbiotic targets (6). In Bradyrhizobium japonicum RegS-RegR controls the NifA-RpoN regulon, which regulates 65 targets for nitrogen fixation and other processes in microaerobiosis and anaerobiosis (free-living conditions) (25, 26). In Rhizobium leguminosarum, nifA is autoregulated (37), while in Azorhizobium caulinodans it is under the direct control of FixK (32).

In R. etli, nifA gene expression has features markedly distinct from the features in other rhizobia, and no genetic elements involved in its regulation have been identified. However, NifA-dependent gene activation occurs only at low oxygen concentrations (43, 55). To date, only a few targets, including nifHDK, iscN nifUS, fixABC, prxS-rpoN2, melA, and bacS, have been reported to be members of the R. etli NifA-RpoN2 regulon during symbiosis with P. vulgaris (15, 17, 27, 31, 43, 55).

Using the R. etli CFN42 genome sequence (24), we designed and constructed a microarray to analyze transcription of the NifA regulon of R. etli during symbiosis. The microarray chip contains 70-base (70-mer) oligonucleotides representing all 6,034 open reading frames (ORFs) that have been detected in the genome sequence of this organism.

The main goal of this work was to identify and more strictly define the R. etli NifA-RpoN regulon. This was done by comparing wild-type strain CFN42 with a knockout mutant lacking the NifA regulatory protein grown under microaerobic free-living conditions and during symbiosis. This study is the first study of R. etli that used global approaches, such as transcriptomics (DNA microarray and real-time reverse transcription [RT]-PCR), in combination with proteomics and bioinformatics. The regulon described here includes some genes that are shared with other NifA-RpoN regulons. However, more of the components found in this work have been found only in the R. etli CFN42 regulon and have specific functions in this species.

MATERIALS AND METHODS

Design and construction of the R. etli microarray.

The R. etli complete array oligonucleotide set contains 6,034 arrayable 70-mers representing the genome of strain CFN42 and was designed using the following NCBI reference sequences: NC 007761 (chromosome), NC 007761 (pCFN42f), NC 007765 (pCFN42e), NC 007764 (pCFN42c), NC 007763 (pCFN42b), NC 007762 (pCFN42a), and NC 004041 (pCFN42d) (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/Rhizobium_etli_CFN_42/).

The 70-mer oligonucleotides were designed by using several ad hoc programs written in PERL and other software, as follows. The first program read the DNA sequences corresponding to each of the annotated genes of the R. etli CFN42 genome. If the size of the gene permitted, the program then cut 50 bases from both the 5′ and 3′ ends. After this, the program scanned, base by base, each possible 70-mer of the sequence to ensure that there was no single-base repeat longer than 7 bp and the melting temperature (Tm) was 80 ± 5°C (the formula used for calculating the Tm was Tm = 81.5 + 16.6 × log[Na+] + 41 × [G+C]/length − 500/length, where [Na+] was 0.1 M and the length was 70). The resulting candidate oligonucleotides were compared with the complete genome sequence using NCBI BLASTALL (BLASTALL-PBLASTN) (1) with a maximum e value of 1.0 (−e 1.0) and no masking of low-information segments (−F F). The BLAST results were used to ensure that no candidate oligonucleotide could cross-hybridize in the microarray. We used the program PALINDROME from the EMBOSS software suite (44, 49) to find hairpins and filter out any candidate oligonucleotides with potential hairpins more than 9 bases long. Our programs also ensured that no oligonucleotide had more than 20 bases in common with any other coding sequence (Table (Table11).

TABLE 1.
Oligonucleotide set selection criteria and characteristics of the R. etli CFN42 microarray

When two or more oligonucleotide sequences for the same ORF satisfied all of the selection rules, the oligonucleotide was selected with the following parameters: a Tm close to 80°C and minimum cross-hybridization identity. A control that included 4 positive controls and 12 negative controls was also designed using the Drosophila melanogaster genome. All oligonucleotides were commercially synthesized without modification by MWG-Biotech (Ebersberg, Germany), and 50 μM solutions were prepared using Micro Spotting solution (ArrayIt Brand Products) and spotted in duplicate onto SuperAmine-coated slides (25 by 75 mm; TeleChem International, Inc.) using 24 columns and 22 rows per grid (the total number of grids was 24) using a high-speed robot at the microarray facility at Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Finally, the slides were fixed at 80°C for 4 h. For prehybridization, the slides were rehydrated with water vapor at 60°C and fixed with two cycles of UV light (1,200 J). After 2 min of boiling at 92°C, the slides were washed with 95% ethanol for 1 min and prehybridized in 5× SSC, 0.1% SDS, 1% bovine serum albumin (BSA) for 1 h at 42°C (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were washed and dried prior to hybridization (35, 50).

Bacterial strains and growth conditions.

The bacterial strains used were R. etli wild-type strain CFN42 (24) and mutant strain CFNX247 (ΔnifA::ΩSp/Sm) (22). For growth under microaerobic conditions, cultures were incubated in 150-ml bottles that were closed with an airtight stoppers and flushed with several volumes of an oxygen-argon (1:99, vol/vol) mixture. The cultures were grown with shaking (200 rpm) for 10 h at 30°C as previously described by Girard et al. (21, 22).

Plant experiments.

Three-day-old P. vulgaris cv. Negro Jamapa seedlings were inoculated with R. etli CFN42 or the nifA mutant strain as previously described by Peralta et al. (45). Eleven days postinoculation (dpi), nodules were picked from the roots and immediately frozen in liquid nitrogen and stored at −70°C until they were used. Bacteria were isolated from nodules, and their identities were verified by using antibiotic resistance.

RNA isolation, synthesis of labeled cDNA, and microarray hybridization.

Microarray experiments were carried out using three independently isolated RNA preparations from independent cultures and sets of plants. Approximately 3 g of nodules was immersed in liquid nitrogen and macerated. Total RNA was isolated by acid hot phenol extraction as described previously by de Vries et al. (13). For microaerobic free-living conditions, 50-ml portions of bacterial cell cultures were collected, and total RNA was isolated using an RNeasy minikit (Qiagen, Hilden, Germany). The RNA concentration was determined by measuring the absorbance at 260 nm. The integrity of RNA was determined by running samples on a 1.3% agarose gel. Ten micrograms of RNA was differentially labeled with Cy3-dCTP and Cy5-dCTP using a CyScribe first-strand cDNA labeling kit (Amersham Biosciences). Pairs of Cy3- and Cy5-labeled cDNA samples were mixed and hybridized with the array as described by Hegde et al. (28). After the arrays were washed, they were scanned using a pixel size of 10 μm with a Scan Array Lite microarray scanner (Perkin-Elmer, Boston, MA).

DNA microarray analysis.

Spot detection, determination of mean signals and mean local background intensities, image segmentation, and signal quantification were performed for the microarray images using the Array-Pro Analyzer 4.0 software (Media Cybernetics, L.P.). Microarray data were analyzed with the genArise software, which was developed in the Computing Unit of the Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (http://www.ifc.unam.mx/genarise/). The local background value was subtracted from the intensity of each spot, and Lowess normalization was applied to the slide. The normalized ratio of the expression of the experimental sample to the expression of the control was calculated with genArise software for each experimental replicate. This software identifies differentially expressed genes by calculating an intensity-dependent z score. It uses a sliding window algorithm to calculate the mean and standard deviation within a window surrounding each data point and determines a z score which measures the number of standard deviations that a data point is from the mean: zi = [Ri·mean(R)]/sd(R), where zi is the z score for each element, mean(R) is the mean log ratio, Ri is the log ratio for each element, and sd(R) is the standard deviation of the log ratio. Using this criterion, the elements in all experiments with a z score of >2 standard deviations were considered significantly differentially expressed genes. To eliminate dye effects, labeling with different dyes was performed for single replicates. However, no significant differences between Cy3 and Cy5 in incorporation, fluorescence yield, or stability were observed.

The data sets for all genes that were significantly downregulated in the NifA mutant strain, including all expression level ratios, are shown in the supplemental material. The complete data set for the transcriptome analysis has been deposited in the GEO database (http://www.ncbi.nlm.nih.gov/geo.html) under accession number GPL10081 for the platform, accession number GSE20440 for the microaerobiosis data, and accession number GSE2063 for the symbiosis data.

In silico analysis.

In order to identify putative regulatory binding sites in the upstream regions (UTRs) of the coding sequences (CDSs) of the R. etli genome, we compared a set of regulatory binding sites previously identified for NifA and RpoN in R. etli and other nitrogen-fixing symbiotic bacteria (4, 23, 40, 42) with the UTRs of R. etli. The MEME v 3.5.0 program (2) was used to find motifs (sequence patterns that occurred repeatedly in a group of sequences) in the regulatory binding sites and to construct search matrices, which represented the probability that each possible letter was at each position in the pattern. With this set of matrices, we searched for putative regulatory binding sites in the UTRs (from bp −500 to bp 200 with respect to the ATG transcriptional start codon) of R. etli, using the MAST v 3.5.0 program (2, 3). The search was conducted iteratively by adding the discovered putative regulatory binding sites to the previously described binding sites to construct a new set of matrices and repeating the search until there was convergence (when no more putative regulatory binding sites were found).

Proteomics methodology.

Bacteroids were purified from root nodules by centrifugation through self-generated Percoll gradients (48). Bacteroid proteins were obtained by sonication at 24 kHz for 5 cycles consisting of 1 min on and 1 min off at 4°C in a Vibra Cell (Sonics, United States) in the presence of a 106-protease inhibitor (Complete tablets; Roche Diagnostics GmbH, Mannheim, Germany). To further limit proteolysis, protein isolation was performed using phenol extraction (30). To solubilize and obtain completely denatured and reduced proteins, pellets were dried and resuspended as previously reported (19). Prior to electrophoresis samples were mixed with 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 2 mM tributhyl phosphine (TBP), 2% ampholytes, and 60 mM dithiothreitol (DTT). The methods used for sample preparation, analytical and preparative two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), and image analysis have been described previously (18), and pH gradients were determined by using a 2D SDS-PAGE standard (Sigma, United States). For the first dimension approximately 500 μg of total protein was loaded. The gels were stained with Coomassie blue R-250, and protein spots on all of the gels were detected at a resolution of 127 by 127 μm using a PDI image analysis system and PD-Quest software (Protein Databases, Inc., Huntington Station, NY). We were interested in spots that showed at least a 2-fold change and met the conditions of a statistical Student test (level of significance, 95%). Selected spots from Coomassie blue-stained preparative 2D gels were excised manually and prepared for mass spectrum analysis (19). All experiments were performed three times. Mass spectra were obtained using a Bruker Daltonics Autoflex (Bruker Daltonics, Billerica, MA) operated in the delayed extraction and reflectron mode. Spectra were externally calibrated using a peptide calibration standard (Bruker Daltonics 206095). Peak lists of the tryptic peptide masses were generated and searched against the NCBI nr databases or with Rhizobase (http://bacteria.kazusa.or.jp/rhizo/) using the Mascot search program (Matrix Science, Ltd., London United Kingdom).

Real-time RT-PCR.

We used real-time quantitative PCR to obtain an independent assessment of the expression of selected genes. The cDNA used for microarrays or freshly prepared cDNA was used as a template for real-time PCR. The following primers were used: nifHa-RE1SP00308f (5′-GGG CAG AAG ATC CTG ATC GT-3′) and nifHa-RE1SP00308r (5′-ATC TCC TGG GCC TTG TTC TC-3′) for the nifH gene; nifKb-RE1SP00200f (5′-CCG GAA TAC AGG CAG ATG CT-3′) and nifKb-RE1SP00200r (5′-CTC CTT GAA GTG ACG CGA CA-3′) for the nifK gene; rpoN-RE1SP00218f (5′-CCA GCT CAT CGC GTC TAT TC-3′) and rpoN-RE1SP00218r (5′-CGA AAG GTT GCC TGT GTC AC-3′) for the rpoN2 gene; yp017-RE1SP00100 (5′-GAC GAC CGA CGA CTA TTT CA-3′) for the prxS gene; virB4-RE1SP00146f (5′-GGC GTT TCC TTC GAG ACA TC-3′) and virB4-RE1SP00146r (5′-CAA GTT CGG TAT CGG ATC GG-3′) for the virB4d gene; yhd00040-RE1SP00079f (5′-GAT CGA TCT GGC ACG TAT GT-3′) and yhd00040-RE1SP00079r (5′-TAG ACT CTC GGC AAG ACG TT-3′) for the yhd00040 gene; flgKch-RECH00680f (5′-CAC GCA GAG CAG TGT CGT AT-3′) and flgKch-RECH00680r (5′-GGC GAC GTT TCA TAG TCG TT-3′) for the flgKch gene; ypf00267-REPF00546f (5′-TCT ACT GGC TCA CGA ACA CG-3′) and ypf00267-REPF00546r (5′-TTG TCG AGC CTG TTG AAG TG-3′) for the ypf00267 gene; yha00045-RHEA00136F (5′-TCG AGC GCT GGC AAT CTG GC-3′) and yha00045-RHEA00136R (5′-ACA GCC GGT TGT CGC CGT TG-3′) for the yha00045 gene; and hisC-RE1SP0000233f (5′-CGA TGG CGA GAC AGC TAA AT-3′) and hisC-RE1SP0000233r (5′-ATC ATC GCA ACG CTA TCT CC-3′) for the hisCd gene. Each reaction mixture contained 12.5 μl SYBR green PCR master mixture (Applied Biosystems), 3.5 μl H2O, forward and reverse primers in 5 μl, and the template in 4 μl. PCRs were performed with the ABI Prism 7700 sequence detection system (Applied Biosystems) using the following program: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The dissociation protocol was 95°C for 15 s and 60°C for 20 s, followed by ramp from 60°C to 95°C for 20 min. The transcript of the histidinol phosphate aminotransferase protein (hisCd) was used as an internal (unregulated) reference for relative quantification. This gene was selected as a reference because its expression is constitutive under all of the conditions tested (free living and symbiosis) (data not shown) and, additionally, it is in the pSym replicon, which contains practically all members of the NifA regulon. We also used rpoA as a reference and obtained similar results. The results of real-time RT-PCR were analyzed using the ΔΔCT method (52), and the data are expressed below as relative levels of expression. All reactions were done in triplicate.

RESULTS AND DISCUSSION

R. etli whole-genome array.

The R. etli CFN42 oligonucleotide (70-mer) microarray was designed on the basis of the genome sequence and annotation published by Gonzalez et al. (24). This array comprises a collection of 6,051 probes, 6,034 of which are from the annotated predicted coding sequences (CDSs). A total of 5,776 (95.72%) of the CDSs were represented by a unique probe set and satisfied all of the selection criteria mentioned in Materials and Methods (Table (Table1).1). Only 258 (4.28%) oligonucleotides did not satisfy one or more of these criteria (Table (Table1).1). A total of 148 genes were not represented by exclusive probe sets, mostly because their sequences were too similar to other sequences. Similar oligonucleotide set results were obtained for the S. meliloti oligonucleotide set array (Array Ready Oligo Set version 1.0; Qiagen, Germany), for which 95.3% of the oligonucleotides met all of the criteria and 4.7% of the oligonucleotides did not meet the criteria (Qiagen). The reliable microarray described here is a useful tool for functional genomics studies of one of the most agriculturally important bacteria.

Transcriptome analysis of the NifA regulon in symbiosis.

To dissect the NifA-RpoN regulon under symbiotic conditions, we first analyzed rpoN gene expression in both strains during symbiosis by using real-time RT-PCR. Previously, two differentially regulated rpoN genes were identified in R. etli. The first copy of the rpoN gene, rpoN1, is required for housekeeping functions and allows cells to grow on several nitrogen and carbon sources under free-living conditions. The second copy (rpoN2) is essential for symbiotic nitrogen fixation (15). Using real-time RT-PCR, we analyzed rpoN2 expression in both strains (the wild-type and nifA mutant strains) during symbiosis (Fig. (Fig.11 a) and observed that in R. etli CFN42 rpoN2 expression depends on the NifA protein because it did not occur in the R. etli nifA mutant under free-living and symbiotic conditions (Fig. (Fig.1a1a and and1b).1b). Similar data were reported previously by Michiels et al. (40) and Dombrecht et al. (15) for R. etli CNPAF512, and, in agreement with these reports, we obtained for R. etli wild-type strain CFN42 a messenger PCR product that included the prxS and rpoN2 genes (Fig. (Fig.1c1c).

FIG. 1.
(a and b) Relative levels of transcripts in R. etli wild-type and nifA mutant strains in symbiosis (a) and under microaerobic culture conditions (b). The hisCd transcript was used as an internal (unregulated) reference for relative quantification. (c) ...

To examine the NifA-RpoN regulon under symbiotic conditions, the transcriptomes of bacteroids isolated from 11-dpi nodules of P. vulgaris infected with either the wild type or the nifA mutant strain were compared. We selected this time point for our study because (i) Puppo and colleagues previously found that determinate nodules like bean nodules are initiated by meristem cells in the outer cortex, but cell division stops 10 days after infection, when nitrogen fixation beings (47), (ii) Valderrama et al. (55) found that the nitrogenase genes in R. etli are induced only 10 days after inoculation of the bacterium onto bean roots, and (iii) there are not drastic changes in the physiology of the nifA mutant at 11 dpi like those observed in Fix nodules obtained at later times, for which early senescence and in most cases cell death have been documented (9, 22). We demonstrated that there was nodule integrity and bacteroid viability by using electron microscopy and confocal microscopy with fluorescent dyes (propidium iodide) in nodules inoculated with the nifA mutant strain at 11 dpi (data not shown).

Total RNA was extracted from bacteroids, and cDNA was synthesized, differentially labeled with the fluorophore Cy3 (RNA from bacteroids of the R. etli wild-type strain) or Cy5 (RNA from bacteroids of the R. etli nifA mutant strain), and then hybridized to a Rhizobium_etli_CFN42_6051_v1.0 DNA microarray, as described in Materials and Methods. Three biological replicates with one dye swap were performed. We found that 75 genes showed reduced expression in the nifA mutant in symbiosis if we established a z score log2 ratio cutoff of ≥2.0 using the geneArise software (see Table S1 in the supplemental material). When the physical organization of the induced genes was analyzed in detail, we found that some of these genes are members of operons (iscN-nifSUW, fixABCX, and cpxP1P2P3-yhd00049-cpxP4 operons) and some are monocistronic units (yha00045, ypd00055, and yhd00073).

To support the conclusion that NifA is involved in transcriptional regulation of the potential target genes, we measured the differential gene expression by using real-time RT-PCR assays. As expected, the mRNA levels of practically all of the genes tested (nifH, nifK, virB4d, and yha00045) were clearly lower in the nifA mutant strain than in the wild-type strain (Fig. (Fig.1a).1a). On the other hand, only three genes were upregulated in the nifA mutant compared to the wild-type strain in symbiosis (see Table S2 in the supplemental material).

In R. etli, as in other rhizobia, nifHDK and other genes involved in nitrogen fixation, like fixABCX, are controlled by the oxygen-responsive NifA activator protein (22, 55). Our transcriptome analysis results for symbiosis showed that all of these genes were downregulated in bacteroids of the nifA mutant strain. However, the most significant finding was the suggestion that 36 genes not reported previously in other bacterial NifA regulons are controlled by NifA (Table (Table22).

TABLE 2.
Stringent and extended sets of genes in the R. etli NifA-RpoN regulona

Comparison of microaerobically grown wild-type and nifA cells.

To obtain a more complete picture of the genes whose expression is regulated by NifA-RpoN, we carried out a new set of microarray experiments in which we compared the global transcriptional profiles of the wild-type strain and the NifA mutant strain grown under free-living microaerobic conditions as described in Materials and Methods. By using a z score ratio cutoff of ≥2 for genes whose expression changed significantly in each of three microarray replicates, we identified 38 genes whose levels of transcript expression were lower in the nifA mutant than in the wild type (see Table S3 in the supplemental material). We also validated some of the results for genes such as flgKch, ypf00267, yhd00040, and yha00045 using real-time RT-PCR; in contrast, rpoN2 and nifH were found to be differentially expressed only when RT-PCR was used (Fig. (Fig.1a1a and and1b1b).

When both approaches (microarrays and real-time RT-PCR) were used, the flgKch and ypf00267 genes were found to be downregulated in the nifA mutant strain in microaerobiosis but not in symbiosis. It has been suggested that flgKch is part of the flgEKL-flaF-flbT-flgDch operon (24), but using microarrays we did not detect expression of the other members of this operon. Regarding incomplete operon expression in microaerobisis, Bobik et al. (6) reported that in some cases it is not possible to detected expression of all members of an operon using microarrays due to low levels of expression. However, a Northern blot analysis showed that there was only one band corresponding to the size of flgKch mRNA, 1,487 bp (data not show). Similar results were obtained for the cpxA gene, which is a member of the probable cpxA-ypd0045 operon, and for iscN of the iscN-nifUSW operon. Similar to the results of the experiments under symbiotic conditions, in which we detected 3 genes that were induced in the nifA mutant strain, under microaerobic conditions we detected induced expression of 37 genes in the mutant, which were distributed throughout the partitioned genome of R. etli CFN42 (see Table S4 in the supplemental material); this might have been a result of indirect regulatory effects originating from the mutant genotype.

As mentioned above, in our microarray analysis we considered only genes whose expression in three replicates was constant. However, when the data were analyzed separately, we observed that almost all of the genes detected as part of the NifA regulon in symbiosis were induced differentially under microaerobic conditions. These results and those obtained for B. japonicum (25) and S. meliloti (5) indicate that microaerobic conditions for free-living growth are probably not compatible with maximal expression of rhizobial NifA proteins. As we mentioned above and in agreement with previous reports (40), using RT-PCR we observed that in R. etli rpoN2 expression in symbiosis and under microaerobic conditions depends on the NifA protein (Fig. (Fig.1a1a and and1b).1b). The level of amino acid identity of RpoN1 and RpoN2 is 56%, and this could suggest that RpoN1 has less capacity for binding to RpoN2 promoters and vice versa. Moreover, in Rhodobacter capsulatus (46) RpoN1 and RpoN2 have different targets, determined in part by the capacity of each protein to bind to its corresponding site. Specificity is determined mainly by region 1, and in R. etli there is a major difference between RpoN1 and RpoN2 in this region. On the other hand, it has been reported that rpoN1 expression is reduced during microaerobiosis and symbiosis, suggesting that in addition to RpoN1, RpoN2 negatively regulates rpoN1 expression (40). Another feature that affects gene expression compared with expression during microaerobiosis is the very low oxygen concentration in the nodule (20, 34), which has been proposed to produce differential regulation in the FixLJ-FixK2-FixK1 cascade of B. japonicum (39), of which NifA is a part.

We propose that NifA has greater stability and activity in symbiosis, which leads to increased expression of RpoN2, and as a result of the targets that were identified in each experimental replicate, we suggest that the putative targets of RpoN1 are not expressed in the absence of the rpoN1 product. We assumed that we did not use the best conditions for NifA-RpoN2 function in microaerobiosis, and while we detected some targets under our conditions, the expression of NifA-RpoN2 was not as stable as it was in symbiosis, which caused the replicates to vary substantially.

In silico search for RpoN and NifA binding sites.

The array results reported above suggest that the effect of NifA on gene expression could be due to direct or indirect regulation of the corresponding promoters by NifA. In order to obtain insight into which promoters are regulated directly and which promoters are regulated indirectly, a genome-wide DNA motif search was performed.

The bacterial alternative sigma factor RpoN recognizes and binds to a −24/−12-type promoter with the following consensus sequence: 5′TGGCACG-N4-TTGCW-3′, where the −24 GG and the −12 GC dinucleotides are indicated by bold type (4). RpoN binds to these promoter regions and interacts with the NifA protein that recognizes and binds to a specific upstream sequence, TGT-N10-ACA; all of the sequences used to locate NifA and RpoN sites and sequence logos (11) created with these sequences are shown in Fig. S1 in the supplemental material and in Table Table33.

TABLE 3.
Regulatory RpoN-NifA binding sites detected in the genome of R. etli CFN42 using the MEME-MAST program and manually

Using the MEME and MAST programs, the search was restricted to the regions from bp −500 to bp 200 from the start codons of annotated genes. A total of 154 RpoN binding sites and 97 NifA binding sites were predicted (see Table S5 in the supplemental material); 17 of these sites had both motifs, while 18 additional binding sites were detected manually (7 sites with NifA binding motifs, 2 sites with RpoN binding motifs, and 9 sites with both binding motifs), resulting in a total of 35 targets (Table (Table3).3). The genome locations of the RpoN- and NifA-dependent sequence elements identified were cross-examined using the genes identified by the transcriptome analysis that had a z score cutoff of 2.0. As shown in Table Table3,3, we concluded that of the 35 NifA-RpoN sites identified, 33 (94.2%) are apparently functional.

In symbiosis a total of 61 genes associated with both motifs showed significant changes in microarray experiments, and these genes are members of 14 operons and 12 monocistronic units (Table (Table2).2). In contrast, there were 5 genes that were differentially expressed in the NifA mutant strain but for which no NifA- or RpoN-dependent regulatory elements could be found (see Table S6 in the supplemental material). In microaerobiosis we identified 38 genes whose levels of transcript expression were lower in the nifA mutant than in the wild-type strain (see Table S3 in the supplemental material). However, for 9 genes neither a NifA binding site nor an RpoN binding site was detected (see Table S6 in the supplemental material), and just 19 targets (25 genes) were identified as targets that had both motifs, which corresponds to 65.7% of the total number of genes whose expression was reduced in the nifA mutant in microaerobiosis; additionally, in 4 more cases only one of the motifs was present. It is relevant that the expression of 19 of the 25 genes with both putative transcription factor binding sites was reduced in the nifA mutant in symbiosis. On the other hand, the expression of virB4d, which also possessed putative NifA and RpoN motifs, was reduced in the nifA mutant background in symbiosis as determined by microarrays and RT-PCR assays, in contrast to the findings for microaerobiosis (Fig. (Fig.1a1a and and1b).1b). Also, NifA and RpoN regulatory binding sites were predicted to be in the promoter region of yhd00040, but by using microarray analysis a low level of expression in the nifA mutant strain in microaerobiosis but not in symbiosis was found, which is not consistent with the results of a real-time RT-PCR analysis, in which we detected differential expression under both conditions (Fig. (Fig.1a1a and and1b).1b). Since real-time RT-PCR is a more sensitive technique than microarray assays, it is possible that we detected expression of this gene only by the former method.

A number of NifA-dependent genes were detected only under microaerobic conditions (yhd00040, flgKch, thrB, ype00167, yhch00663, and ypf00267), suggesting that some NifA regulation could be specific for the free-living state of the bacterium. Nevertheless, some of the genes whose expression was not detected in symbiosis were expressed at low levels under microaerobic conditions. Consequently, we cannot exclude the possibility that the lack of detection of these genes under symbiotic conditions might actually have been due to a technical limitation, given that the overall sensitivity of the arrays is better with free-living RNAs than with symbiotic RNAs. For this reason, whether NifA contributes to biochemical functions specific for free-living R. etli remains to be determined.

In our opinion, these data define the NifA regulon in R. etli more reliably than other NifA regulon studies have, because our analysis included results obtained both under free-living (microerobic) conditions and in symbiosis. On the other hand, we found that the nodA and ypd0010 genes, containing both NifA and RpoN binding sites, were not differentially expressed in symbiosis or microaerobiosis (data not shown), which could suggest that there is an alternative NifA-dependent regulation process. nodA is also negatively regulated by NolR (10, 33), and a search for NolR consensus sites in the R. etli genome sequence using MEME and MAST identified three motifs (data not shown), one of which is located upstream of nodA. In addition, nolR is transcribed significantly in symbiosis and microaerobiosis (data not shown). Also, the findings for the ypd00010-yhd00018 operon, which contained both promoter sites but did not exhibit differential expression either in symbiosis or under free-living conditions, suggest that there is an alternative NifA-dependent regulation process.

Identification of NifA-regulated proteins in symbiosis by proteome analysis.

Bacteroid extracts of wild-type strain CFN42 and the nifA mutant strain were obtained and analyzed by 2D PAGE. Three independent biological samples from each strain were used for protein profiling. For each sample, 2D gel electrophoresis was carried out using a pH range of 4 to 8, and the Coomassie blue-stained gels were analyzed using PD-Quest software. On average, more than 800 protein spots were observed in each 2D electrophoresis gel prepared for both strains. In this study we selected only spots that showed at least a 2-fold change and met the conditions of a statistical Student test (significance level, 95%). Seventy-eight polypeptide spots that were present in the wild-type strain gel and not in the nifA mutant gel were identified as probable NifA-regulated protein spots (Fig. (Fig.22 a and 2b). In contrast, 18 polypeptide spots were found only in the nifA mutant strain gel (data not shown). A total of 41 proteins that were differentially expressed in the wild-type strain and the nifA mutant were identified on the basis of their tryptic peptide masses using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis (Table (Table44 ). The genes encoding 24 (58%) of these proteins were associated with NifA and RpoN motifs, and there were significant severalfold changes in the expression of these proteins when transcriptomics experiments were performed. These proteins included 7 different electrophoretic variants of NifD, 4 electrophoretic variants of MelA, 4 electrophoretic variants of Yha00045, 3 electrophoretic variants of NifH, 2 electrophoretic variants of NifK, and one electrophoretic variant each of PrxS, RpoN2, CpxP2, and Yhd00105 (Fig. (Fig.22 and Table Table4).4). However, using 2D PAGE it was not possible to identify proteins like FdxN, IscN, NifW, and FdxB, which were members of a group of 22 proteins identified with microarrays as members of the NifA-RpoN regulon, because they had isoelectric points or molecular weights outside the electrophoretic resolution range utilized in this work (see Table S7 in the supplemental material). Our proteomics experiments confirmed that elements such as CpxP2, Yha00045, and Yhd00105, which are proposed for the first time in this work to be regulated by NifA-RpoN, are members of the NifA-RpoN regulon (which was suggested by transcriptomics findings and the presence of RpoN and NifA binding motifs). Also, in several cases we identified isoforms of the same protein, suggesting that there are regulatory posttransductional modifications. Detection of these modifications is an additional advantage of the proteomics approach used in this work, and for some of these proteins posttransductional modifications have not been reported previously. Further analysis is required to identify the types of modifications in these proteins. Conversely, the results for 17 proteins found only in the wild-type strain (see Table S8 in the supplemental material) did not correlate with either the predicted motif results or transcriptomics analysis results. We know that NifA-dependent σ54 motifs can differ from consensus sites, as recently shown using gel retardation analysis and detailed mutagenesis of the functional NifA promoter of the hupSL genes of R. leguminosarum bv. viciae (36); thus, experiments to confirm that these elements are members of NifA-RpoN regulon are necessary.

FIG. 2.
Proteome profiles of 11-dpi bacteroids from the R. etli wild-type (a) and nifA mutant (b) strains. The numbered protein spots were excised from the gel of the wild-type strain and were identified using MALDI-TOF mass spectrometry. Three independent experiments ...
TABLE 4.
NifA-RpoN regulon proteins identified using MALDI-TOF mass spectrometrya

Global characterization of the R. etli NifA-RpoN regulon.

Previous studies documented that there is an imperfect correlation between the motif scores and fold changes, so promoter sequence data alone are not sufficient to predict transcription efficiency (25). Because of this, to delineate the R. etli NifA-RpoN regulon (stringent set), we selected as members of this regulon (stringent set) only genes that had significant fold changes in transcriptomics experiments (symbiosis and/or free life) and/or whose product was detected by proteomics and that contained RpoN- and NifA-dependent binding sites, which resulted in identification of a total of 67 genes; 42 of these genes were symbiosis specific, 19 were found under both symbiosis and microaerobic (free life) conditions, and 6 were microaerobiosis specific (Table (Table2).2). The extended set included genes that exhibited differential microarray expression (z score ratio cutoff, ≥2) in the nifA mutant strain, in contrast to the wild type in symbiosis or microaerobiosis, but contained only NifA or RpoN motifs. In this category there were 11 genes, 2 of which were detected by microarrays specifically in microaerobiosis. Additionally, similar to the results of the proteomics analysis, in which 17 proteins were found only in the wild-type strain, 5 genes were differentially expressed under symbiosis conditions and 9 genes were differentially expressed under microaerobic conditions in this strain; however, no NifA- and/or RpoN-dependent regulatory elements could be found for these genes (see Table S6 in the supplemental material), suggesting that (i) there may be indirect regulation, (ii) NifA and RpoN promoters are too divergent from the promoters used in our analysis, (iii) there were artifacts of the microarray method (transcriptomics), and/or (iv) there was basal induction or no induction of some genes in ineffective nodules (symbiosis). We also did not consider genes in theoretical operons which did not meet the criteria for the ≥2.0-change cutoff in gene expression in the microarray analysis members of this regulon.

In agreement with the findings of Hu et al. (29) and Curatti et al. (12), the NifA-RpoN stringent regulon of R. etli described in this work contains all of the necessary elements required to fix nitrogen, including nifHDK, nifB, nifE, nifN, nifX, nifW, nifU, and nifS. Moreover, we confirmed the presence of the genes previously reported to be NifA-RpoN dependent in R. etli, including bacS, prxS, rpoN2, and iscN (16, 31, 43), and the presence of operons predicted in silico, including the iscN-nifUSW (16), nifHDKE1-yhd00103-yhd00102-fdxB, nifHDK3E2NX, and nifHD2-hemNd2-yhd00090 operons. For the last three operons, RNA transcripts that were the correct length were detected in Northern blots (data not shown). In summary, the NifA-RpoN regulon (extended set) of R. etli was determined to contain 78 genes, including 50 genes in 15 operons and 28 monocistronic genes.

Comparison of NifA-RpoN regulons in nitrogen-fixing bacteria.

The NifA-RpoN regulons in different rhizobia have been characterized previously (6, 25, 26). In S. meliloti, 19 of the genes regulated by FixJ appeared to be NifA-regulated genes, and analysis of the intergenic sequence upstream of these genes revealed the presence of NifA boxes (TGT-N10-ACA) in five targets (6). In B. japonicum, the NifA-RpoN regulon is composed of 65 elements, including 32 regulated genes with significant NifA and RpoN binding sites (stringent set), 2 manually identified RpoN and NifA sites, and 31 regulated genes with significant RpoN binding sites, but it has no NifA binding site (extended set) (26). Remarkably, the total NifA regulon in R. etli described in this work consisted of 67 genes in the stringent set and 78 genes in the extended set.

The majority (90%) of the members of the R. etli NifA-RpoN regulon (stringent set) occur only on the symbiotic plasmid (60 genes are on pCFN42d, 4 genes are localized on the chromosome, and one gene each is on pCFN42a, pCFN42e, and pCFN42f). R. etli has a small symbiotic region compared with other species. In S. meliloti, the 19 NifA-dependent genes described previously are on pSymA (6), a replicon five times larger than pCFN42d, and in B. japonicum the symbiotic island contains 34 genes regulated by NifA (stringent set) and is twice as large as pCFN42d. To date, the R. etli NifA-RpoN regulon (stringent set) is the largest NifA regulon that has been described, and its members are located in a smaller proportion of the genome. The yha00045 gene, which contains functional NifA and RpoN binding sites, which was determined by proteomic and microarrays studies to be expressed only in the wild-type strain, and whose expression under symbiosis and microaerobic conditions was validated by RT-PCR, is the only R. etli regulon gene detected (stringent set) on pCFN42a. This replicon has been reported to be indispensable for the transfer of the symbiotic plasmid (8, 54), and both plasmids have lower G+C contents (58%) than the rest of the R. etli genome (61.5%) (24). These data suggest that both replicons were acquired during the same event, and our data support the hypothesis that there is a coordinated relationship between pCFN42a and pCFN42d.

The R. etli NifA-RpoN regulon (stringent set) contains at least one gene in each of 16 functional classes (53); in comparison, the members of the B. japonicum regulon belong to only 12 classes (extended set), and the members of the S. meliloti regulon (stringent set) belong to only 6 functional classes. Classical genes necessary for nitrogen fixation, like the nifHDKENX and nifB-fdxN-nifZ operons and fixA, are present in all regulons. Additionally, we observed 21 other genes that are present in both the B. japonicum and R. etli NifA-RpoN regulons; in comparison, there are only 13 genes that are present in both the R. etli and S. meliloti regulons. Hence, 43 (54.4%), 49 (76.6%), and 6 (31.6%) of the genes were present only in the R. etli, B. japonicum, and S. meliloti regulons, respectively, which indicates that there are important differences and specificities in these regulons.

Interestingly, in the R. etli stringent set 47% of the genes were categorized as hypothetical genes, conserved hypothetical genes, or genes with unknown functions (see Fig. S2 in the supplemental material). For the 35 remaining genes, functional categories, such as energy production and conversion (14 elements), were significantly overrepresented. Also present were genes encoding products involved in functions like transport (divided into six clusters of orthologous groups of genes [COGS], as well as inorganic ion transport), metabolism (see Fig. S2 in the supplemental material), and secondary metabolite biosynthesis, transport, and metabolism (see Fig. S2 in the supplemental material), and there were three or four elements in each COG (see Fig. S2 in the supplemental material).

For several coding regions with unknown functions, some of the proteins contained domains that indicated that they are involved in various important physiological functions; these coding regions included ypd00053 (multidrug resistance efflux pump domain), ypd00054 (putative bacteriocin/antibiotic exporters), and ypd00055 (similar to Mesorhizobium loti mll6943, which contains a domain conserved in aquaporin proteins, some of which are involved in urea, methylammonium, lactic acid, or ammonia transport) (38). Experimental demonstration of these roles would provide further insights into the NifA-RpoN regulon in R. etli.

The abundance of hypothetical genes that are components of the R. etli NifA-RpoN regulon is not surprising since the NifA-RpoN regulon of B. japonicum is composed of 25 hypothetical genes (26), but only two of these genes (bir1954 and bir2725) had sequence identity (51% and 57%, respectively) with ORFs in R. etli. However, neither of these genes was determined by us to be part of the R. etli NifA-RpoN regulon. This result confirmed that the expression of genes for symbiosis is different in B. japonicum and R. etli.

Conclusion.

One of the purposes of this study was to develop and evaluate a relatively low-cost whole-genome DNA microarray system for R. etli. Here, we describe the most complete characterization to date of the NifA-RpoN regulon, the first regulon in rhizobia described using transcriptomic profiling, proteome analysis, and bioinformatics. We show that there are 67 members of the stringent set in the R. etli NifA-RpoN regulon and 78 members in the extended set. Our studies, done in symbiosis and in free life, revealed 42 new NifA-RpoN-dependent genes. The R. etli NifA-RpoN regulon contains components that are also present in other bacterial regulons described previously, although the majority of the components described here are present only in the R. etli regulon, which suggests that there are specific requirements for nitrogen fixation in different bacterial models.

In the past, NifA has been characterized largely as a regulator of nitrogen fixation, a finding which was confirmed in the present work. More importantly, however, this study demonstrates that the NifA-RpoN regulon is composed of genes with very diverse functions. Our results reveal that in addition to providing adequate conditions for efficient nitrogen fixation, NifA also plays a fundamental and heretofore less appreciated role in regulation of the normal physiology of the cell.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Michael F. Dunn and David Krogmann for useful scientific comments; Sandra Contreras, Raúl Noguez, and Miguel Elizalde for MALDI-TOF protein identification; María de Lourdes Girard and Nicolas Gómez-Hernández for technical advice concerning microaerobic experiments; and Oliver Castillo, J. L. Zitlalpopoca, and Hadau Sánchez for plant experiments and greenhouse support.

This research was supported by DGAPA-UNAM grant IN222707 and by CONACYT grants 60641 and ING206512. E.S.B. was a recipient of a Ph.D. studentship from the Consejo Nacional de Ciencia y Tecnología (México).

Footnotes

[down-pointing small open triangle]Published ahead of print on 7 May 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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