The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

A novel class of ammonium assimilation mutants of the photosynthetic bacterium Rhodobacter capsulatus

Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH ₄ ⁺ -supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed.     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

The contribution of Rhizobium meliloti to soil denitrification

The ability of Rhizobium meliloti cells to denitrify in soils under several conditions was tested. All the strains tested were able to remove large amounts of N-NO₃ ^- from soils. Both water filled pore space above 36% and temperatures above 20°C greatly increased nitrogen losses. However, even with optimal conditions for denitrification and the highest rhizobial populations found in agricultural soils, the contribution of Rhizobium to the total denitrification was virtually negligible as compared to other soil microorganisms.To whom correspondence should be addressed     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Rhizobium meliloti distribution in the soil following alfalfa inoculation

Summary Alfalfa seeds, inoculated with an antibiotic-resistantRhizobium meliloti strain, were planted in three replicated field plots at Clayton, N.C. Core samples were taken three times in the next year at 0, 10, and 20 cm from the edge of each plot. Soil subsamples were taken from within each core sample at 0, 6, 12, and 18 cm depths. The numbers of the inoculum Rhizobium strain in each soil subsample were determined by inoculation of alfalfa plants with diluted soil samples. In general the distribution of rhizobia showed some movement outward and downward in the soil. Lower counts were obtained at the surface during summer. The Rhizobium persistence pattern in the soil differed in the three plots which is consistent with the variability in Rhizobium numbers often observed in established alfalfa stands. Cooperative investigation of the United States Department of Agriculture, Science and Education Administration, Agricultural Research and the North Carolina Agricultural Research Service, Raleigh, North Carolina. Paper No. 6818 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh.     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Stability and transmissibility of the cryptic plasmids of Rhizobium meliloti GR4

Rhizobium meliloti strain GR4 harbours two cryptic plasmids sharing extensive regions of homology between them and with other non-symbiotic plasmids of different strains of R. meliloti. They both are very stable showing a segregation rate of less than 0.1% loss per generation. pRmeGR4a (115 MDa) is a self-transmissible plasmid at a variable frequency to other species, and it is also responsible for promoting, at low frequency, the contransfer of pRmeGR4b (140 MDa), the other cryptic plasmid of the strain. A 4.8 kb PstI fragment of pRmeGR4a, responsible for the high stability in cis of this plasmid, has been isolated and several recombinant plasmids have been constructed showing different segregation rates in the strains used in this study. Their stabilities can be considerably improved by insertion of the stabilization mrs/par region of RK2.     

The response ofRhizobium meliloti to L-methionine DL-sulphoximine

A strain ofRhizobium meliloti has been shown to be capable of growth in the presence of methionine sulphoximine concentrations at least two orders of magnitude higher than that required for the complete inhibition of glutamine synthetase activity. Neither the specific growth rate, nor the nutritional requirements of the organism were affected by methionine sulphoximine in the medium.Rhizobium meliloti appeared to assimilate ammoniavia the glutamate dehydrogenase pathway during growth in the presence of methionine sulphoximine. This suggests thatRhizobium meliloti may have some regulatory mechanism controlling ammonia assimilation that is not present in other enterobacteria possessing similar enzymatic machinery     

Ammonium assimilation in Proteus vulgaris,Bacillus pasteurii,and Sporosarcina ureae

No active uptake of ammonium was detected in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae, which indicates that these bacteria depend on the passive diffusion of ammonia across the cell membrane. In P. vulgaris the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and glutamate dehydrogenase (GDH) were present, and these enzymes exhibited high affinities for ammonium. In B. pasteurii and S. ureae, however, no GS activity was detected, and GOGAT activity was only present in S. ureae. GDH enzymes were present in these two organisms, but showed only low affinity for ammonium, with apparent K _m-values of 55.2 mM in B. pasteurii and 36.7 mM in S. ureae, repectively. These observations explain why P. vulgaris is able to grow at neutral pH and low ammonium concentration (2 mM), while B. pasteurii and S. ureae require high ammonium concentration (40 mM) and alkaline pH for growth.Non-standard abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GT glutamyl transferase - MA methylammonium - NB nutrient broth - YE yeast extract - NA nocotinic acid     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Denitrifying ability of thirteen Rhizobium meliloti strains

The denitrifying ability of thirteen strains of Rhizobium meliloti was tested. Most of the strains were able to reduce nitrate to nitrous oxide or dinitrogen. However, they failed to use nitrate as electron acceptor for ATP generation or growth at low oxygen tensions. Under micro-aerobic conditions, free-living cells of R. meliloti 102-F-51 strain exhibited a constitutive nitrate reductase activity independent of the presence of nitrate. On the other hand, nitrite reductase activity was dependent not only on low levels of oxygen but also on the presence of a high nitrate concentration in the medium. Denitrification activity proceeded immediately once a threshold level of nitrite was accumulated in the medium or in cells incubated with 1mM nitrite. However, a lag period was required when cells were incubated with nitrate.     

Identification of salt- and drought-tolerant Rhizobium meliloti L. strains

The first set of experiments identified sodium chloride (NaCl) tolerance of 92 accessions of Rhizobium meliloti L. from various rhizobia collections and arid and saline areas of the Intermountain West. Accessions were incubated in salinized (0, 176, 352, 528, 616, 704 or 792 m M) yeast extract mannitol (YEM) medium. Growth was measured by turbidity at 420 nm after 3 d in culture. Rhizobial strains were classified by their growth response at an optical density (OD) of 704 m M; Groups One and Two did not exceed 0.10 and 0.33, respectively. Forty three different rhizobial strains were identified as salt-sensitive and 49 as salt-tolerant at 704 m M NaCl. None grew in a saline solution of 792 m M NaCl.The second set of experiments investigated the drought tolerance of R. meliloti accessions that exhibited differential salt tolerance. Fifteen salt-sensitive and 15 salt-tolerant strains of R. meliloti from the first experiment were exposed to simulated drought stress by adding polyethylene glycol 6,000 (PEG-6,000) to the YEM medium at concentrations of 0, –0.4, –0.8 or –1.0 MPa. Rhizobium strains were incubated for 10 days at 25°C and growth turbidity was measured at 420nm. Growth turbidity of the 30 accessions ranged from 100% at –0.4 MPa to 0% at –1.0 MPa. With one exception, strains that were more drought-tolerant (at –1.0 MPa) were also more salt-tolerant (616 m M). However, some of the more salt-tolerant strains at 616 m M were not the more drought-tolerant stains at –1.0 MPa. These salt-and drought-tolerant Rhizobium accessions are excellent models to study the mechanism(s) of such resistance, and to elucidate the role of genetics of NaCl and drought tolerance.     

Choline derivatives increase two different acid phosphatases in Rhizobium meliloti and Pseudomonas aeruginosa

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole carbon and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by EDTA-lysozyme treatment. The R. meliloti acid phosphatase activity of crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence of 5 mM choline, betaine, trimethylammonium or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. Among several phosphoesters tested only pyridoxal-5-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) of crude extracts obtained from bacteria grown in the presence of serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected when alpha-naphthylphosphate or pyridoxal-5-phosphate were used as substrates. In conclusion, although the coline metabolites are capable of increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.Abbreviations p-NPP p-nitrophenyl phosphate - PLP pyridoxal-5-phosphate - PMP pyridoxamine-5-phosphate Recipient of a Fellowship from the CONICORMember of the SAPIU-CONICETCareer Member of the CONICET     

The relationship between the state of adenylylation of glutamine synthetase I and the export of ammonium in free-living,N2-fixing Rhizobium

The relationship between ammonium assimilation and ammonium export has been studied in free-living, N₂-fixing Rhizobium sp. 32H1. After 55 to 67 h of microaerobic growth under a gas phase of 0.2% O₂ – 1.0% CO₂ – 98.8% Ar high levels of nitrogenase were observed concomitant with a slightly adenylylated glutamine synthetase (GSI) and some glutamine synthetase (GSII) activity. However, after growth of 89 h, or longer, GSI became adenylylated and the level of GSII had decreased. When the gas phase was shifted to 0.2% O₂ – 1.0% CO₂ – 98.8% N₂, a lag was observed before ammonium export could be detected in the 55 to 67 h cultures. No lag in ammonium export was observed in the cultures previously grown for 89 h. The onset of ammonium export in the 55 to 67 h cultures was found to correlate with the adenylylation state of GSI. There appeared to be no correlation between the level of GSII and the export of ammonium. Neither an increase in the adenylylation level of GSI nor ammonium export was observed when the 55 to 67 h cultures were maintained under the Ar gas mixture.Abbreviations GOGAT Glutamate synthase - GS glutamine synthetase - BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] - CTAB cetyltrimethylammonium bromide - MES [2-(N-morpholino)-ethane sulfonic acid]     

Nitrogen assimilation in the facultative methylotroph Hyphomicrobium X

A survey of the possible nitrogen assimilation pathways in Hyphomicrobium X showed that when the nitrogen source was satisfied by ammonium sulphate or methylamine and the supply was in excess, NADPH-dependent glutamate dehydrogenase was used to assimilate nitrogen. When the nitrogen supply was limited the cells expressed high levels of glutamine synthetase and NADH-dependent glutamine:2-oxoglutamate aminotransferase activity whilst the activity of the glutamate dehydrogenase was lower. When nitrate was the N-source, the glutamine synthetase/glutamine oxoglutamate aminotransferase pathway was utilised irrespective of the nitrogen concentration in the medium. Evidence was obtained to suggest that the glutamine synthetase activity was regulated by adenylylation/deadenylylation. Carbon-limited chemostat cultures showed low glutamine synthetase activity levels but the synthesis of the enzyme was derepressed when the cultures became N-limited.     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Enhanced biosynthesis of polyhydroxyalkanoates in a mutant strain of Rhizobium meliloti

Strains of Rhizobium spp. isolated from leguminous plants and standard strains accumulated 27% to 57% polyhydroxyalkanoate (PHA) of their cell biomass. Among these cultures, one strain of Rhizobium meliloti synthesized 10–30% more PHA than others and contained 3% hydroxyvalerate (HV) when grown on sucrose as carbon substrate. The occurrence of hydroxybutyrate (HB) and HV was confirmed by GC and ¹H NMR analysis. Treatment of the culture with 4-N-piperidinobutyl-2-chlorophenoxazine resulted in a mutant which synthesized upto 69%, PHA of the cell biomass with an improved yield of 11 to 47% under different carbon and nitrogen ratios, compared to the parent strain.