Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble.     

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.     

Cloning of genes responsible for acetic acid resistance in Acetobacter aceti.

Five acetic acid-sensitive mutants of Acetobacter aceti subsp. aceti no. 1023 were isolated by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Three recombinant plasmids that complemented the mutations were isolated from a gene bank of the chromosome DNA of the parental strain constructed in Escherichia coli by using cosmid vector pMVC1. One of these plasmids (pAR1611), carrying about a 30-kilobase-pair (kb) fragment that conferred acetic acid resistance to all five mutants, was further analyzed. Subcloning experiments indicated that a 8.3-kb fragment was sufficient to complement all five mutations. To identify the mutation loci and genes involved in acetic acid resistance, insertional inactivation was performed by insertion of the kanamycin resistance gene derived from E. coli plasmid pACYC177 into the cloned 8.3-kb fragment and successive integration into the chromosome of the parental strain. The results suggested that three genes, designated aarA, aarB, and aarC, were responsible for expression of acetic acid resistance. Gene products of these genes were detected by means of overproduction in E. coli by use of the lac promoter. The amino acid sequence of the aarA gene product deduced from the nucleotide sequence was significantly similar to those of the citrate synthases (CSs) of E. coli and other bacteria. The A. aceti mutants defective in the aarA gene were found to lack CS activity, which was restored by introduction of a plasmid containing the aarA gene. A mutation in the CS gene of E. coli was also complemented by the aarA gene. These results indicate that aarA is the CS gene.     

The plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus

Abstract Alcaligenes eutrophus strain H16 harbors a 450 kilobase pairs (kb) conjugative plasmid which codes for the ability of the organism to grow lithoautotrophically on hydrogen and carbon dioxide (reviewed in [1]). The genes for hydrogen oxidation, designated hox , are clustered on plasmid pHG1 in a DNA region of approximately 100-kb in size ([2], Fig. 1). The hox genes and their organization have been analyzed by isolation of Hox-deficient mutants, by complementation analysis, by cloning of hox genes, identification of hox -encoded polypeptides and, most recently, by DNA sequencing. The hox cluster is flunked by the two structural gene regions, hoxS and hoxP ; it contains a regulatory locus, hoxC , and additional genes like hoxN and hoxM whose products play a role in the formation of catalytically active hydrogenase proteins. Of four indigenous 1.3-kb insertion elements, two copies of IS491 map in the hox gene cluster. These elements may be involved in rearrangements and deletions which occur particularly frequently in this region of the megaplasmid (Schwartz, Kortlüke and Friedrich, unpublished).     

Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.

Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.     

Increased Nitrogenase-Dependent H(2) Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H(2) photoproduction. However, a mutation in a structural hup gene did not result in maximum H(2) production rates, indicating that the capacity to recycle H(2) was not completely lost. Highest H(2) production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H(2) recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.     

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Cloning and characterization of elastase genes from Pseudomonas aeruginosa.

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.     

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.     

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli.

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.     

Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.     

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.     

Characterization of two genes (hupD and hupE) required for hydrogenase activity in Azotobacter chroococcum

In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity. Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis. A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL. Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase. The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product. The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively. A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.     

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.     

Isolation of genes required for hydrogenase synthesis in Escherichia coli

A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate. From E. coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain. Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated. The former plasmid restored activity in strain AK23 while the latter did not. The smallest active DNA fragment in plasmid pAK23C was 0.9 kb. This gene is designated hydE. Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression. Strain AK23 was also devoid of formate hydrogenlyase and formate dehydrogenase activities and these activities were restored by some of the plasmids. Hydrogenase and formate-related activities in strain AK23 were restored by growth of cells in a high concentration of nickel. Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa.     

Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase

The poly-beta-hydroxybutyrate biosynthetic thiolase gene from Zoogloea ramigera was used as a hybridization probe to screen restriction digests of Alcaligenes eutrophus H16 DNA. Specific hybridization signals were obtained and two fragments (a 2.3-kilobase PstI fragment and a 15-kilobase EcoRI fragment) cloned in the Escherichia coli vector pUC8 (plasmids pAeT3/pAeT10 and pAeT29, respectively). Biochemical analysis of lysates of E. coli cells containing each plasmid identified significant levels of beta-ketothiolase and acetoacetyl-CoA reductase enzyme activities in lysates of E. coli cells containing plasmids pAeT10 or pAeT29. Nucleotide sequence analysis of the pAeT10 insert identified two open reading frames which encode polypeptides of Mr = 40,500 and Mr = 26,300 corresponding to the structural genes for beta-ketothiolase (phbA) and acetoacetyl-CoA reductase (phbB), respectively. Amino acid sequence homologies between the two bacterial and two mammalian thiolases are discussed with respect to the chain length specificity exhibited by the different thiolase enzymes.     

The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa.

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.