Genes encoding a fructose-1,6-bisphosphate aldolase and a fructose-1,6-bisphosphatase are present within the gene cluster for mimosine degradation in Rhizobium sp. strain TAL1145

Rhizobium sp. strain TAL1145 can catabolize mimosine, a toxic amino acid produced by the tree-legume leucaena. The mid and pyd genes involved in mimosine degradation in TAL1145 are located in two clusters within a 25-kb region in the chromosome, which was cloned in plasmid pUHR263. A 5.5-kb EcoRI fragment, located between the mid and pyd genes in pUHR263, was characterized by sequencing and transposon-insertion mutagenesis and six open reading frames (ORF) were identified. Based on high homologies with other known proteins and conserved signature domains, ORF1 and ORF2 were identified as fba and fbp genes, encoding fructose-1,6-bisphosphate aldolase (FBA) and fructose-1,6-bisphosphatase (FBP), respectively. The fba mutant showed a slightly reduced growth rate compared to TAL1145 while the fbp mutant did not show any growth defects. Both mutants could catabolize mimosine and formed normal nitrogen-fixing nodules on leucaena, suggesting that these genes are not involved in mimosine degradation and symbiosis.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

midD-encoded ‘rhizomimosinase’ from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K _m, V _max and K _cat of MidD were 1.27 × 10^?4 mol, 4.96 × 10^?5 mol s^?1 mg^?1, and 2,256.05 s^?1, respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5′-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including l-tyrosine, l-tryptophan and l-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C–N lyase.     

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Characterization of a Rhizobium etli chromosomal gene required for nodule development on Phaseolus vulgaris L.

A chromosomal gene, required for nodule development on Phaseolus bean, was characterized from Rhizobium etli strain TAL182. MLC640 is a Tn5 insertion mutant of TAL182 which shows decreased motility in soft TY agar and is defective in nodule development. The site of Tn5 insertion in MLC640 mapped to a 3.6-kb EcoRI chromosomal fragment. The 3.6-kb fragment was subcloned from the cosmid pUHR80 which complemented MLC640. Further subcloning and site-directed Tn5 mutagenesis localized the gene for nodule development to a 1.7-kb region within the 3.6-kb EcoRI fragment. Southern hybridization using the 3.6-kb EcoRI fragment as the probe against genomic DNA of several Rhizobium spp. indicated that this gene is conserved in different rhizobia.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gimore 402, Honolulu, Hawaii 96822. USA;     

Effects of Growth Mode and Pyruvate Carboxylase on Succinic Acid Production by Metabolically Engineered Strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Anaplerotic Function of Phosphoenolpyruvate Carboxylase in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> USDA110

In bacteria, anaplerotic carbon fixation necessary for growth on carbon sources that are metabolized to three-carbon intermediates is provided by the activity of pyruvate carboxylase (PYC) and/or phosphoenolpyruvate carboxylase (PPC). In contrast to other rhizobia, which encode only one of these enzymes in their genomes, Bradyrhizobium japonicum USDA110 encodes both. Streptavidin-HRP western blot analysis of B. japonicum extracts demonstrated the presence of a biotin-containing protein whose molecular mass was indistinguishable from those of PYCs produced by Sinorhizobium meliloti and Rhizobium etli. Sequence analysis of the possible B. japonicum PYC revealed the lack of a pyruvate binding site as well as other characteristics indicating that the enzyme is non-functional, and PPC activity, but not PYC activity, was detectible in extracts prepared from strain USDA110. A B. japonicum cosmid genomic library was used to clone the ppc by functional complementation of S. meliloti pyc mutant RmF991. S. meliloti RmF991-carrying plasmids containing the B. japonicum ppc regained the ability to grow with glucose as a carbon source and produced PPC activity. The cloned ppc gene was inactivated by insertion mutagenesis and recombined into the USDA110 genome. The resulting ppc mutant was essentially devoid of PPC activity and grew poorly with glucose as carbon source in comparison to the wild-type strain. These data indicate that B. japonicum utilizes PPC, and not PYC, as an anaplerotic enzyme for growth on carbon sources metabolized to three-carbon intermediates.     

pyd genes of Rhizobium sp. strain TAL1145 are required for degradation of 3-hydroxy-4-pyridone, an aromatic intermediate in mimosine metabolism

Rhizobium sp. strain TAL1145 degrades the Leucaena toxin mimosine and its degradation product 3-hydroxy-4-pyridone (HP). The aim of this investigation is to characterize the Rhizobium genes for HP degradation and transport. These genes were localized by subcloning and mutagenesis on a previously isolated cosmid, pUHR263, containing mid genes of TAL1145 required for mimosine degradation. Two structural genes, pydA and pydB, encoding a metacleavage dioxygenase and a hydrolase, respectively, are required for degradation of HP, and three genes, pydC, pydD, and pydE, encoding proteins of an ABC transporter, are involved in the uptake of HP by TAL1145. These genes are induced by HP, although both pydA and pydB show low levels of expression without HP. pydA and pydB are cotranscribed, while pydC, pydD, and pydE are each transcribed from separate promoters. PydA and PydB show no homology with other dioxygenases and hydrolases in Sinorhizobium meliloti, Mesorhizobium loti, and Bradyrhizobium japonicum. Among various root nodule bacteria, the ability to degrade mimosine or HP is unique to some Leucaena-nodulating Rhizobium strains.     

Exopolysaccharide-deficient mutants of Astragali rhizobia are symbiotically effective on Astragalus sinicus, an indeterminate nodulating host

Five exopolysaccharide-deficient mutants were isolated after rhizobial strain 107 was subjected to transposon Tn5 mutagenesis. The amount of EPS produced by the mutants was dramatically decreased to between 3% and 6% of wild-type level. All mutants carried a singel copy of Tn5. Two mutants (NA3 and NA10) were complemented by the R. meliloti exoA gene and the functionally equivalent exoD gene of Rhizobium sp. strain NGR234. Two other mutants (NA7 and NA8) were complemented by the R. meliloti exoB gene and the functionally equivalent NGR234 exoC gene. The remaining mutant (NA11) was not complemented by any exo genes of R. meliloti or Rhizobium NGR234. All mutants induced normal nitrogen-fixing nodules on Astragalus sinicus, an indeterminate nodulating host.     

C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance ¹³C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The -alanine level increases further upon incubation with the non-permissive substrate -glucose. -Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with -glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on -fructose and -glutamate, alanine also accumulates within 3 h upon transfer to -glucose.     

Plasmid content of salt stress-tolerant <Emphasis Type="Italic">Rhizobium</Emphasis> strains from Egyptian soils nodulating common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Plasmid profile analysis is useful to characterize Rhizobium strains within the same species. Among the 16 Rhizobium strains examined, 14 had distinct plasmid profiles. The size of plasmids ranged from 40 to 650 kb, and three plasmids of 650, 510 and 390 kb were common to several strains. Plasmid analysis revealed that Rhizobium etli contained a mega-plasmid, similar in size to Rhizobium tropici. All the salt-tolerant strains examined had a plasmid of 250 kb, except for strain EBRI 29. This suggests that this plasmid may play an important adaptive role under salt stress conditions.     

Isolation of Insertion Sequence ISRLdTAL1145-1 from a Rhizobium sp. (Leucaena diversifolia) and Distribution of Homologous Sequences Identifying Cross-Inoculation Group Relationships

Insertion sequence (IS) element ISRLdTAL1145-1 from Rhizobium sp. (Leucaena diversifolia) strain TAL 1145 was entrapped in the sacB gene of the positive selection vector pUCD800 by insertional inactivation. A hybridization probe prepared from the whole 2.5-kb element was used to determine the distribution of homologous sequences in a diverse collection of 135 Rhizobium and Bradyrhizobium strains. The IS probe hybridized strongly to Southern blots of genomic DNAs from 10 rhizobial strains that nodulate both Phaseolus vulgaris (beans) and Leucaena leucocephala (leguminous trees), 1 Rhizobium sp. that nodulates Leucaena spp., 9 R. meliloti (alfalfa) strains, 4 Rhizobium spp. that nodulate Sophora chrysophylla (leguminous trees), and 1 nonnodulating bacterium associated with the nodules of Pithecellobium dulce from the Leucaena cross-inoculation group, producing distinguishing IS patterns for each strain. Hybridization analysis revealed that ISRLdTAL1145-1 was strongly homologous with and closely related to a previously isolated element, ISRm USDA1024-1 from R. meliloti, while restriction enzyme analysis found structural similarities and differences between the two IS homologs. Two internal segments of these IS elements were used to construct hybridization probes of 1.2 kb and 380 bp that delineate a structural similarity and a difference, respectively, of the two IS homologs. The internal segment probes were used to analyze the structures of homologous IS elements in other strains. Five types of structural variation in homolog IS elements were found. The predominate IS structural type naturally occurring in a strain can reasonably identify the strain's cross-inoculation group relationships. Three IS structural types were found in Rhizobium species that nodulate beans and Leucaena species, one of which included the designated type IIB strain of R. tropici (CIAT 899). Weak homology to the whole IS probe, but not with the internal segments, was found with two Bradyrhizobium japonicum strains. The taxonomic and ecological implications of the distribution of ISRLdTAL1145-1 are discussed.     

Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilize it as a source of carbon and nitrogen

Growth of most Rhizobium strains is inhibited by mimosine, a toxin found in large quantities in the seeds, foliage and roots of plants of the genera Leucaena and Mimosa. Some Leucaena-nodulating strains of Rhizobium can degrade mimosine (Mid⁺) and are less inhibited by mimosine in the growth medium than the mimosine-nondegrading (Mid^-) strains. Ten Mid⁺ strains were identified that did not degrade 3-hydroxy-4-pyridone (HP), a toxic intermediate of mimosine degradation. However, mimosine was completely degraded by these strains and HP was not accumulated in the cells when these strains were grown in a medium containing mimosine as the sole source of carbon and nitrogen. The mimosine-degrading ability of rhizobia is not essential for nodulation of Leucaena species, but it provides growth advantages to Rhizobium strains that can utilize mimosine, and it suppresses the growth of other strains that are sensitive to this toxin.     

Symbiotic Characteristics and Rhizobium Requirements of a Leucaena leucocephala × Leucaena diversifolia Hybrid and Its Parental Genotypes

In 56-day-old plants, Leucaena leucocephala and its hybrid with L. diversifolia showed 100% more total N than did L. diversifolia. Significant (P < 0.01) host-inoculation interaction in total N was 14.4% of the total phenotypic variation. The most effective and competitive Rhizobium sp. for the leucaenas was TAL 1145. Three-strain mixed inoculation was inferior to TAL 1145 alone.     

Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.