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Journal of Bacteriology, August 2004, p. 5384-5391, Vol. 186, No. 16
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.16.5384-5391.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Indole-3-Acetic Acid Biosynthesis Is Deficient in Gluconacetobacter diazotrophicus Strains with Mutations in Cytochrome c Biogenesis Genes

Sunhee Lee,^1, M. Flores-Encarnación,² M. Contreras-Zentella,² L. Garcia-Flores,² J. E. Escamilla,² and Christina Kennedy^1*

Division of Plant Pathology and Microbiology, Department of Plant Sciences, The University of Arizona, Tucson, Arizona 85721,¹ Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Mexico D.F., Mexico²

Received 4 May 2004/ Accepted 17 May 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gluconacetobacter diazotrophicus is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of G. diazotrophicus unable to fix nitrogen, suggested that there are two beneficial effects of G. diazotrophicus on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by G. diazotrophicus, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn5 mutants, released only 6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA^– phenotype was not linked to Tn5. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome c biogenesis (ccm, for cytochrome c maturation). The G. diazotrophicus ccm operon was sequenced; the individual ccm gene products were 37 to 52% identical to ccm gene products of Escherichia coli and equivalent cyc genes of Bradyrhizobium japonicum. Although several ccm mutant phenotypes have been described in the literature, there are no reports of ccm gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the ccm genes of G. diazotrophicus are involved in cytochrome c biogenesis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gluconacetobacter diazotrophicus is a nitrogen-fixing endophyte commonly isolated from Saccharum L. (sugarcane) and occasionally from Ipomoea batatas (sweet potato), Pennissetum purpureum, Ananas comosus [L.] Merr. (pineapple), and Coffea arabica (coffee) (13, 20, 34, 38). ¹⁵N-isotope dilution experiments suggest that up to 80% of sugarcane nitrogen (N) can be derived from atmospheric nitrogen gas, presumably through bacterial nitrogen fixation (see reference 38 for a review). Additionally, Sevilla et al. showed that G. diazotrophicus has two potential beneficial effects on sugarcane: one probably dependent on nitrogen fixation and the other possibly through microbial production of a plant growth-promoting substance (40). Since G. diazotrophicus is known to produce indole-3-acetic acid (IAA), with particularly high amounts produced by strain PAl5 (used in the plant inoculation experiments of Sevilla et al. [40]), we speculated that IAA production may explain the plant growth promotion of sugarcane by G. diazotrophicus.

Biosynthesis of IAA is not limited to higher plants. Organisms such as bacteria, fungi, and algae are able to make physiologically active IAA that may have pronounced effects on plant growth and development. Many bacteria isolated from the rhizosphere have the capacity to synthesize IAA in vitro in the presence or absence of physiological precursors, mainly tryptophan (Trp) (12, 33). Microbial isolates from the rhizosphere of different crops appear to have a greater potential to synthesize and release IAA as secondary metabolites because of the relatively rich supply of substrates (6, 12). In addition, numerous pathogens are active producers of IAA and cause abnormal cell enlargement in infected plants (4, 33).

Production of IAA by microbial isolates varies greatly among different species and strains and depends on the availability of substrate(s). Different biosynthetic pathways for IAA production exist, sometimes in parallel in the same organism (33). For many years it was assumed that Trp was the only precursor of IAA. However, work with tryptophan-auxotrophic mutants and isotope labeling have established that IAA biosynthesis can occur via a tryptophan-independent route (30, 35), although in the presence of Trp microbes release greater quantities of IAA and related compounds.

The pathways for conversion of Trp to IAA can involve deamination, decarboxylation, and/or hydrolysis reactions. In higher plants and most microorganisms, the indole-3-pyruvic acid (IpyA) pathway is the main one for IAA synthesis, whereas other pathways operate in certain species (the indole-3-acetamide pathway, the tryptamine pathway, and the indole-3-acetonitrile pathway). The formation of IpyA from Trp is catalyzed by multispecific aminotransferases, followed by spontaneous or enzymatic decarboxylation to indole-3-acetaldehyde (IAAld), which is then oxidized by an IAAld oxidase to IAA. As a side reaction, IpyA may be reduced to indole-3-lactic acid (ILA) by lactate dehydrogenase, which requires NADH. Indole-3-ethanol (TOL) is the product of a side reaction from IAAld (25, 33).

In this study, the G. diazotrophicus genome was randomly mutagenized with Tn5 to obtain a G. diazotrophicus IAA^– mutant with reduced ability to produce IAA compared to the wild type. This approach revealed a surprising discovery, that cytochrome c biogenesis genes are required for a large proportion, 90%, of the IAA produced in G. diazotrophicus. We describe here for the first time the cloning of genes involved in cytochrome c biogenesis from G. diazotrophicus and demonstrate the involvement of these genes in both respiratory electron transport and IAA production.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, vectors, and growth conditions. The bacterial strains and plasmids used are presented in Table 1. G. diazotrophicus wild-type PAl5 (ATCC 49037) and ccm mutants were maintained in either DYGS or LGIP medium (40). Escherichia coli strains were grown at 37°C in Luria-Bertani broth. Antibiotics for G. diazotrophicus were added at the following concentrations (in micrograms per milliliter): tetracycline, 100; kanamycin, 200; streptomycin, 700. For the isolation of membranes, cells were grown aerobically at 30°C in 3 liters of LGIP medium supplemented with 1.0 mM (NH₄)₂SO₄.

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TABLE 1. Bacterial strains and plasmids used in this study

Mutagenesis. Transposon mutants were generated by conjugation of strain PAl5 with E. coli S17-1 carrying the suicide plasmid pSUP1021, which contains a Tn5 transposon that confers kanamycin resistance. Conjugations were performed on DYGS (pH 6) plates and incubated for 36 h at 30°C. After conjugation the cells were resuspended in LGI medium, diluted, and plated onto DYGS medium with kanamycin. Transconjugants were picked and stored as libraries in 96-well microtiter plates. The site-directed insertion mutants were generated using the fragment (streptomycin) as described previously (41).

Tn5 screening. Tn5-containing transconjugants were grown in minimal LGI medium supplemented with tryptophan (100 µg/ml) as an IAA precursor in 96-well microtiter plates, and after 4 days of incubation the amount of IAA produced by each transconjugant was determined using Salkowasky reagent (18). IAA mutant candidates were further characterized by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis.

HPLC analysis. Extraction and HPLC of G. diazotrophicus cultures for IAA were performed according to the methods of Costacurta et al. (11) with slight modifications. Bacterial cultures (20 ml) were made cell free by centrifugation at 3,000 x g and filtration, and supernatants were extracted three times with ethyl acetate after adjusting the pH to 2.8. Five- to 15-µl aliquots of the filtered extracts were injected into an Alltech, type Econosphere C185U column (250 by 4.6 mm) equipped with a differential UV detector absorbing at 280 nm. The isocratic solvent used for reverse-phase chromatography was acetonitrile-glacial acetic acid (1%) in water (10:90). The flow rate was adjusted to 1 ml/min. Peak retention times were compared with those of chemically synthesized IAA standards and quantified by comparison of peak areas.

Analytical methods. Preparation of membrane proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), staining for heme, spectral analysis of cytochromes, and determination of dehydrogenase activities and respiratory activities were performed as described by Flores-Encarnacion et al. (16) with a few modifications. Briefly, cells in 3 liters of liquid culture were obtained after 36 h of growth with shaking at 250 rpm. Cells were pelleted by centrifugation and then washed twice with TCM buffer (50 mM Tris-HCl [pH 7.4] containing 5 mM CaCl₂ and 5 mM MgCl₂). Membranes were isolated, and protein concentrations were measured by a modification of the Lowry method. For spectral analysis, membranes (8 mg) were resuspended in 50% (vol/vol) glycerol and analyzed in an SLM-Aminco DW 2000 spectrophotometer. Samples were reduced with a few grains of sodium dithionite in the presence or absence of KCN, and difference spectra at 77K were recorded. The c-type cytochrome spectra were measured after membranes were precipitated with 0.01 N HCl in acetone as described by Goodhew et al. (22). Heme peroxidase staining was performed to detect c-type cytochrome bands. Membranes (3 mg) were washed with trichloroacetic acid (10% [vol/vol]) and separated by SDS-PAGE. SDS treatment removed noncovalently bound hemes, so that only c-type cytochrome was revealed after heme peroxidase staining. Protein blotting and detection were performed with the Amersham (Piscataway, N.J.) enhanced chemiluminescence Western blotting detection reagents.

Oxidase activities were determined as described by Flores-Encarnacion et al. (16) with various substrates at the following final concentrations: 3 mM NADH, 10 mM glucose, 10 mM ethanol, 10 mM acetaldehyde, 10 mM ascorbate plus 2 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or 10 mM ascorbate plus 1.5 mM 2,3,5,6-tetrachloro-1,4-benzenediol (THQ). The oxidase activities were measured polarographically with a Clark oxygen electrode in 2 ml of 50 mM potassium phosphate buffer (pH 7.4 or 6.0) at 30°C. Dehydrogenase activities were measured spectrophotometrically with TMPD as the alternative electron acceptor, as described by Flores-Encarnacion et al. (16).

DNA manipulations, sequencing, and analysis. The DNA region flanking the Tn5 integration was sequenced using the primer 5'-CGTTCAGGACGCTAC-3', complementary to bases 17 to 34 within the Tn5 inverted repeat. Sequencing was performed with an ABI automated sequencer (model 373A) by using a PRISM Ready Reaction Dye Deoxy terminator cycle sequencing kit (Perkin-Elmer, Boston, Mass.; DNA sequencing facility, University of Arizona). DNA sequences were identified by using the BLAST server of the National Center for Biotechnology Information accessed over the Internet. Computer-assisted sequence analysis was performed using programs of the University of Wisconsin package version 10.0 Genetics Computer Group software.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of IAA by wild-type strain PAl5. In an attempt to elucidate the IAA biosynthetic pathway(s) in G. diazotrophicus, IAA and possible intermediates were analyzed by HPLC and TLC. The HPLC elution profiles of acidic ethyl acetate extracts contained two minor peaks and three major peaks: two early-eluting unknown substances at 9 and 11 min, ILA, IAA, and indole-3-carboxylic acid (ICA) (Fig. 1). The sharp peak in the beginning of the chromatogram is an internal standard. TLC of ethyl acetate extracts from G. diazotrophicus cultures showed IAA, ILA, and TOL, but no ICA could be detected by this method (data not shown). ILA and TOL are intermediates of the IpyA pathway. ICA is a decarboxylated, relatively inactive form of IAA. Therefore, the results indicated that G. diazotrophicus produces IAA through the IpyA pathway. Quantification of the indole derivatives was achieved by measuring the peak area of the HPLC chromatogram, and yields of 5 to 7 µg of IAA/ml were found in the cell-free supernatants of stationary cultures of G. diazotrophicus PAl5.

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FIG. 1. Reversed-phase HPLC chromatogram of the culture fluid of G. diazotrophicus wild-type PAl5 (a) and IAA– mutant strain MAd22 (b) after 4 days of growth. The cultures were grown in minimal medium (LGI) supplemented with 100 µg of Trp/ml. AU, absorption unit.

Isolation and genetic analysis of a mutant deficient in IAA biosynthesis. Various approaches to clone an indole-3-pyruvic acid decarboxylase gene (ipdC gene), a key enzyme in the IpyA pathway, were not successful. In order to isolate genes encoding enzymes involved in IAA biosynthesis, a different genetic approach was adopted using the transposon Tn5. Following Tn5 mutagenesis, 2,500 exconjugants were analyzed for IAA production as described in Materials and Methods. Three mutant candidates with decreased IAA and one IAA-overproducing mutant candidate were further characterized by HPLC and TLC (data not shown). Among IAA^– mutant candidates, MAd10 was further characterized, because MAd10 excreted only 6% of the amount of IAA produced by the parent strain in liquid culture and it is neither a Trp auxotroph nor a Nif^– strain (data not shown). The Tn5-linked region of MAd10 was cloned and sequenced; it encoded gene products with a high similarity to several antibiotic synthetase enzymes from Bacillus subtilis, Streptomyces sp., and Pseudomonas sp. (27, 29, 39). This result suggested that the IAA^– phenotype of MAd10 was not linked to the Tn5 insertion site. When the region containing the Tn5 and flanking DNA from MAd10 was inserted into the chromosome of wild-type strain PAl5, the resulting strain was IAA⁺. This also indicated that the Tn5 insertion in the antibiotic synthetase region in MAd10 was not linked to the IAA^– phenotype.

Complementation of the mutant MAd10. To determine where the mutation causing the IAA^– phenotype was located on the chromosome, a pLAFR3 cosmid library of G. diazotrophicus was introduced into MAd10 (46). Two cosmids complementing the IAA^– phenotype of MAd10 were isolated after screening 2,000 tetracycline-resistant transconjugants using the colorimetric method described earlier. These cosmids had overlapping restriction sites, which indicated that they probably contained the same gene or genes complementing decreased IAA production in MAd10. The region from one of the cosmids, pSL10, was subcloned to determine the smallest fragment that complemented the IAA^– phenotype. A 5.6-kb HindIII/EcoRI fragment (pSL12) successfully complemented the IAA^– phenotype, restoring wild-type IAA levels (Table 2).

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TABLE 2. Indole compounds produced by Ccm– mutants of G. diazotrophicus in a liquid medium

Sequence analysis. To determine the nucleotide sequence, pSL12 was subcloned to smaller fragments and each was sequenced. Examination of the sequence revealed five contiguous open reading frames (ORFs) (Fig. 2), the deduced products of which had high similarity to proteins involved in cytochrome c biogenesis (ccm genes [cytochrome c maturation genes]) in E. coli (37 to 48% identity) or cyc genes in Bradyrhizobium japonicum (38 to 52% identity). The ccm operon sequence is reported under GenBank accession number AY456185. Products of the ccmABCDEFGH genes in E. coli are required for the formation of holocytochrome c, the products being subunits of an ABC transporter (CcmABC), a heme chaperone (CcmE), a putative cytochrome c heme lyase (CcmEF), a postulated redox system (CcmGH), and a membrane protein that stabilizes the complex (CcmD) (reviewed in references 26, 48, and 49). In B. japonicum these genes are named cyc and are arranged somewhat differently than the ccm genes in E. coli and now in G. diazotrophicus (data not shown).

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FIG. 2. Physical and restriction maps of G. diazotrophicus ccm operon and ccm mutants. (Top) The 5.6-kb G. diazotrophicus genomic fragment harboring the ccm operon, with restriction sites indicated. B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; M, MscI; Sp, SphI; St, StuI. (Bottom) A series of ccm mutants in which ccm genes were disrupted by deletion or insertion of a streptomycin () cassette.

Characterization of the ccm mutants. To confirm that one or more of the ccm genes are responsible for the IAA^– phenotype, several mutant strains were generated by inserting (Sm^r) cassette and/or by deleting a portion of the genes to yield the strains MAd20, MAd21, and MAd22 (Fig. 2). Mutagenesis results indicated that a mutation in any of the ccmCEF genes results in the IAA^– phenotype.

IAA production by both ccm mutants and the wild type was investigated by HPLC analysis. The ccm mutants produced a barely detectable amount of IAA (4 to 6% of the wild type) after 4 days of growth. The detectable IpyA pathway intermediate, ILA, was also reduced significantly compared with wild type (Fig. 1; Table 2). The mobilization of a plasmid (pSL12) containing the functional ccm operon restored the ability to produce IAA in the three new mutants (Table 2). This indicated that a defect in cytochrome c production is responsible for the IAA mutant phenotype.

The cytochrome c content of G. diazotrophicus cells was characterized with membranes obtained from cells as described by Flores-Encarnacion et al. (16). Noncovalently bound hemes were removed by washing membranes with 0.01 N HCl in acetone to determine cytochrome c content without the spectral interference of b-type cytochromes. The dithionite-reduced minus persulfate-oxidized spectra (77K) of acidic acetone-treated membranes from wild type and ccm mutants were determined (Fig. 3B). The spectra of wild-type membranes revealed the characteristic peaks at 417, 520, and 549.5 nm originating from the c-type cytochromes, while these peaks were absent or significantly reduced in the ccm mutants (Fig. 3B). Different spectra produced by cyanide treatment of the reduced preparation revealed the presence of an a-type cytochrome in both ccm mutants and the wild type, which was accompanied by a large enhancement of the signals at 589 nm. Additionally, b-type cytochromes were retained in ccm mutant membranes, as suggested by shoulders at 430, 529, and 560 nm in reduced plus CN^– minus oxidized spectra (Fig. 3A).

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FIG. 3. Difference spectra at 77K for c-type cytochromes associated with membranes of G. diazotrophicus PAl5 and its ccm mutants (MAd10, MAd20, MAd21, and MAd22). (A) Reduced plus KCN minus oxidized spectra. Samples were reduced with dithionite in the presence of 1.0 mM KCN and oxidized with air. (B) Dithionite-reduced minus persulfate-oxidized spectra of acidic acetone-treated membranes. An 800-µg aliquot of protein was used in each sample.

To analyze the ccm mutant phenotypes in more detail, membrane fractions of aerobically grown wild-type and mutant cells were analyzed on SDS-PAGE for the presence of heme-associated peroxidase activity (heme staining). In the wild-type cells, four c-type cytochromes with apparent molecular masses of 67, 56, 52, and 45 kDa were detected (Fig. 4). No membrane-bound c-type cytochromes were detected in membranes prepared from the ccm mutants.

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FIG. 4. Membrane-bound c-type cytochromes of G. diazotrophicus PAl5 and its ccm mutants detected by heme staining after SDS-PAGE. Each lane contains membrane protein samples. Lane a, PAl5 (200 µg); lanes b to e, ccm mutants MAd10, MAd20, MAd21, and MAd22 (1 mg each); lane f, horse heart cytochrome c (10 µg).

Since in many organisms cytochrome c proteins are involved in respiration, oxidase, and dehydrogenase activities, these activities of the wild-type and mutant strains were measured polarographically with a Clark oxygen electrode and spectrophotometrically with TMPD as an electron acceptor. In decreasing order, glucose, NADH, acetaldehyde, ascorbate plus THQ, ethanol, ascorbate plus TMPD, and lactate were the best substrates for the oxidase activities associated with G. diazotrophicus membranes (Table 3). In the case of ccm mutants, no ethanol, acetaldehyde, and lactate oxidase activities were detected; however, similar glucose and NADH oxidase activities were retained. The dehydrogenase activities of ccm mutants were significantly decreased when acetaldehyde, ethanol, or lactate was used as substrate, but the activities with glucose or NADH were similar to those of the wild type (Table 4).

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TABLE 3. Oxidase activities associated with membranes of G. diazotrophicus

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TABLE 4. Dehydrogenase activities associated with membranes of G. diazotrophicus

Top Abstract Introduction Materials and Methods Results Discussion References

 
Improved plant growth via plant-associated bacterial nitrogen fixation has been reported in many plant-microbe interactions (reviewed in references 38 and 45). One example of this beneficial symbiotic relationship is that between sugarcane and G. diazotrophicus. G. diazotrophicus may provide biologically fixed nitrogen as well as a significant amount of bacterial IAA. In this work, we isolated genes that are involved in IAA production.

Based on the presence of intermediates, it was hypothesized that G. diazotrophicus synthesizes IAA through the IpyA pathway (7, 17). Our work also suggested that G. diazotrophicus produces IAA through the IpyA pathway. Two IpyA intermediates, ILA and TOL, were detected in the stationary-phase liquid culture (Fig. 1). Other intermediates, IpyA and IAAld, were not detected with the solvent and conditions used in this experiment, possibly due to an inherent instability of IpyA and IAAld (14). However, the present study did not clarify whether ILA and TOL are directly involved in the IAA metabolism of this bacterium. Metabolic studies with isotopically labeled substrates are necessary to confirm this result. The presence of ICA on the HPLC chromatogram indicated that IAA catabolism is associated with oxidative decarboxylation processes. This pathway has been reported in Rhizobium phaseoli and Rhizobium trifolii (5, 14). Decarboxylated forms of IAA such as ICA are thought to be regulators of endogenous IAA levels in microorganisms (8, 24).

The mutation responsible for the reduced IAA production was located in the cytochrome c biogenesis genes (ccm genes). Cytochrome c protein is an electron carrier with a relatively wide distribution among prokaryotes. In contrast to other cytochromes, cytochrome c enzymes carry a prosthetic heme group covalently attached to the apoprotein through thioester bonds between the vinyl groups of heme and the cysteine sulfurs of a CXXCH peptide motif (1, 2, 47, 48). Biogenesis of c-type cytochromes in many gram-negative bacteria is a complex process, requiring cytochrome c maturation proteins (ccm gene products) and the disulfide bond-oxidizing and disulfide bond-reducing proteins DsbA, DsbB, DsbD, and TrxA (15, 47). The amino acid sequence analysis of the G. diazotrophicus ccm operon revealed that the individual proteins have conserved motifs, transmembrane helices, and active sites that exist in other bacteria (data not shown). There is an orf10 encoding a protein of 60 amino acids between ccmC and ccmE that has no similarity to any known protein. It is possible that this ORF functions as a CcmD protein, although the sequence is so divergent that it could not be recognized as a ccmD gene.

To test the involvement of G. diazotrophicus ccm genes in cytochrome c maturation, several ccm mutant strains were constructed and analyzed. None of the membrane-bound c-type cytochromes was detected in these mutant strains; on the other hand, the ccm mutants had no effect on the biogenesis of a-type or b-type cytochromes (Fig. 3 and 4). Likewise, it has been reported that the maturation of the membrane-bound or soluble cytochrome c, but not a- or b-type cytochromes, is completely abolished in the ccm mutants of other bacteria (31).

Measurement of oxidase and dehydrogenase activities also confirmed that ccm mutants of G. diazotrophicus resulted in loss of functional cytochrome c. All ccm mutants are devoid of ethanol, acetaldehyde and lactate dehydrogenases, and oxidase activities, since these dehydrogenases contain subunits bearing cytochrome c, with molecular masses ranging from 45 to 82 kDa (3, 28).

It has been reported that ccmC or ccmG mutants of Paracoccus denitrificans have a much slower growth rate on rich media, probably due to the lack of protection against oxidized thiol compounds in periplasmic proteins (31). Reduced disulfide bonds provide protection against the oxidized compounds, facilitated by CcmC and CcmG. The growth rates of wild-type G. diazotrophicus and the ccm mutants in both minimal and rich media were the same (data not shown), unlike that observed in P. denitrificans.

There has been renewed interest in cytochrome c as a result of the discovery that programmed cell death in eukaryotes requires the release of cytochrome c from mitochondria (23, 37). Additionally, in some prokaryotes, a defect in the biogenesis of cytochrome c leads to a dramatic increase in synthesis and excretion of heme biosynthetic intermediates (10, 21). Other reported phenotypes of cytochrome c mutants are loss of copper resistance (51) and pyoverdine production (19) in Pseudomonas fluorescens, defects in nitrogen fixation in B. japonicum and R. phaseoli (36, 43), and reduced intracellular infection in Legionella pneumophila (50), as well as defects in high-affinity iron acquisition in Rhizobium leguminosarum and P. fluorescens (9, 52). However, there are no reports describing the relationship between IAA production and cytochrome c in the literature. The involvement of cytochrome c in IAA synthesis reported here may represent another function. Mutation in the ccmCEF region resulted in a dramatic decrease of IAA production. It could be that a cytochrome c is involved in the IAA biosynthetic pathway in G. diazotrophicus. There are no known intermediates of IAA accumulated in the ccm mutants, which suggests that the decreased IAA production is not due to defects in the known IAA biosynthetic pathways. The possibility of the presence of different IAA biosynthetic pathways may explain the relationship between the ccm genes and IAA production.

Another possible explanation is the involvement of a cytochrome c protein in the regulation of IAA production. It has been shown that the level of IAA is important in determining whether it will have beneficial influences or pathogenic effects. High concentrations of IAA can be detrimental to plant growth, since it results in the inhibition of root growth and enlargement of plant cells, causing plant tumors (33). Therefore, it is reasonable to propose that IAA production is tightly regulated. It has been demonstrated that transcription of Pseudomonas putida ipdC is regulated by tryptophan and a stationary-phase sigma factor, RpoS (32). As mentioned above, ccm mutants synthesize very little IAA and no intermediates accumulate, suggesting that cytochrome c may have a role in the regulation of IAA biosynthesis.

In summary, the ccm gene products are required for cytochrome c biogenesis as well as IAA biosynthesis in G. diazotrophicus. Biochemical studies of the enzymes involved in the IAA biosynthetic pathway are required to reveal whether cytochrome c is necessary for the function of any individual enzyme. Sugarcane plant growth experiments using an IAA^– ccm mutant indicated that while the colonization and growth of the ccm mutant inside sugarcane plants was similar to that of wild-type G. diazotrophicus, the mutant did not promote plant growth (S. Lee et al., submitted for publication). These results are consistent with the idea that IAA production is a factor in this beneficial plant-microbe association and support further investigation of this hypothesis.

 
We thank the National Science Foundation (IBN-9728184) and the Universidad Nacional Autonoma de Mexico (PAPIIT-IN215801) for funding this research.

 
* Corresponding author. Mailing address: Division of Plant Pathology and Microbiology, Department of Plant Sciences, P.O. Box 210036, Forbes 204, University of Arizona, Tucson, AZ 85721. Phone: (520) 621-9835. Fax: (520) 621-9290. E-mail: ckennedy{at}u.arizona.edu.

Present address: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, August 2004, p. 5384-5391, Vol. 186, No. 16
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.16.5384-5391.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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