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     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

A glycine betaine transport system in Aphanothece halophytica and other glycine betaine-synthesising cyanobacteria

Uptake of exogenous ¹⁴C-glycine betaine has been followed in the cyanobacterium Aphanothece halophytica and other species able to synthesise glycine betaine in response to osmotic stress. At 1 mmol dm^–3 uptake was rapid (flux rate=29.50 nmol m^–2 s^–1), equilibrating at an internal concentration of 120 mmol dm^–3 within 30 min. This rapid uptake, coupled with high internal accumulation, was characteristic of glycine betaine-synthesising cyanobacteria only. The ¹⁴C-glycine betaine transported was not catabolised. Kinetic studies indicated a Michaelis-Menten type relationship (K _m=2.0 mol dm^–3, V _max=45 nmol min^–1 mm^–3 cell volume), with a pH optimum of 8.0–8.5. Darkness dramatically decreased the flux rate. Higher ¹⁴C-glycine betaine levels occurred in cells growth in medium of elevated osmotic strength, and glycine betaine uptake was sensitive to changes in external salinity. A relationship between Na⁺ availability and glycine betaine uptake was observed, with >80 mmol dm^–3 Na⁺ required for optimal stimulation of uptake in seawater-grown cells. Severe hyperosmotic stress (1000 mmol dm^–3 NaCl) reduced the rate of glycine betaine uptake but increased internal glycine betaine concentration at equilibrium. Hypo-osmotic stress caused a decline in the internal glycine betaine concentration due to an increased rate of loss, indicating that the efflux system was also sensitive to ambient salinity changes. It is envisaged that this active transport system may be an adaptive mechanism in halophilic glycine betaine-synthesising cyanobacteria.     

Betaine status in wheat in relation to nitrogen stress and to transient salinity stress

Summary Glycine betaine is readily accumulated in wheat (Triticum aestivum cv. Inia) shoots during periods of salinity stress. The ability of the plant to utilize betaine as a source of nitrogen remains unresolved. We, therefore, conducted solution culture experiments in a greenhouse to test the hypothesis that betaine is degraded in wheat shoots under conditions of severe nitrogen deficiency. Betaine concentrations increased in continuously salt stressed plants for only 17 days after salinity was imposed. After this period, concentrations (dry weight basis) decreased steadily until plants died 32 days later. Decreases in betaine concentration were also observed in treatments where salinity stress was removed. The rate of decrease in concentration was greatest in the N-free treatment. These decreases in betaine concentration were the result of dilution by plant growth. Betaine contents (mol shoot^–1) remained unchanged after removal of substrate nitrate. Therefore our results support the hypothesis that betaine is a stable end product of metabolism.     

Effect of amaranth on the growth of Rhizobium and Bradyrhizobium strains

The growth rate of different strains of Bradyrhizobium and Rhizobium was studied in media containing amaranth seed meal instead of yeast extract. Results obtained in erlenmeyer flasks and stirred fermenters show that both Bradyrhizobium japonicum strains E109, E110, 5019, 587 and Rhizobium melilotistrains B36, B323, B399, Lq₂₂, Lq₄₂, Lq₅₁ and U₃₂₂, grow satisfactorily in amaranth seed meal medium. Cell count obtained for the strains tested was greater than 4 × 10¹⁰ viable cells.ml^–1. Amaranth seed meal (4 g.l^–1) is a suitable component for culture media that can be used instead of yeast extract.     

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     

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.     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni²⁺ (MIC, 75 M) and showed concentration-dependent Ni²⁺ uptake in a wide concentration range (50–500 M). Ni²⁺ uptake up to a certain threshold limit also increased thiol content (66 nmol mg^–1 protein), proline content (10.85 nmol mg^–1 protein) and urease specific activity (500 nmol min^–1 mg^–1 protein) maximum corresponding to 100 M Ni²⁺ as the external concentration or 151 nmol Ni²⁺ mg^–1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni²⁺ concentration (25 M). Higher intracellular Ni²⁺ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni²⁺ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO₃, 100 mM) to the crude extract. The induction of thiol or proline by Ni²⁺, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Use of the gusA gene marker in a competition study of the Rhizobium strains nodulating the common bean (Phaseolus vulgaris) in Senegal soils

Indigenous rhizobial population is among the factors which influence increased crop yield through inoculation with elite strains. In this study, we compared in greenhouse conditions the competitiveness of Rhizobium strain ISRA 355 for nodulation of the common bean (Phaseolus vulgaris) cultivated in different unsterile Senegal soils in terms of pH, N and C contents. The strain ISRA 355 produced a stable GUS+ transconjugant which was used for competition with indigenous soil rhizobia in six localities. At Bayakh, the transconjugant ISRA 355gusA was less competitive than the indigenous rhizobial strains, whereas in the other localities, it was more competitive since it occupied more than 90% of the nodules. Thus the Rhizobium strain ISRA 355 should be used for successfully inoculating the common bean in Senegal soils.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Use of a promoter-specific probe to identify two loci from the Rhizobium meliloti nodulation regulon

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.     

Growth of Rhizobium sp. in the presence of catechol

The ability of Rhizobium sp., isolated from Lablab purpureus to survive in soil containing a phenolic derivative catechol was investigated. It survived for 9 months in soil containing catechol. In synthetic medium, Rhizobium sp. utilized catechol up to 10 mM as sole carbon source and catechol 1, 2-dioxygenase was present in the catechol grown cells. In the presence of organic acids and sugars, catechol was co-metabolized; but catechol 1, 2-dioxygenase induction was inhibited. The ability of Rhizobium sp. to utilize various phenolic substances provides potential advantage to overcome the phenolic toxicants present in soils.     

Ecology of Hup+ Rhizobium strains of cow pea miscellany: native frequency and competence

Rhizobium strains nodulating summer legumes cow pea [Vigna unguiculata (L.)], green gram [V. radiata (L.) (Wilczek)], black gram [V. mungo (L.) (Hepper)] and cluster bean [Cyamopsis tetragonoloba (L.) (Taub)] and a winter legume chick pea [Cicer arietinum (L.)] were surveyed in the Northern Plains of India and screened for hydrogenase activity to determine distribution of Hup character in the native ecosystem. It was observed that 56% of the Rhizobium strains of summer legumes were Hup⁺ whereas that of the winter legume, chick pea, were all Hup^-. Ex planta acetylene reduction activity was observed in most of the Hup⁺ but not in the Hup^- strains of any of the host species. In summer legume, mixed inoculation of Hup⁺ and Hup^- strains, under sterilized as well as unsterilized soil conditions, showed that the host species were predominantly nodulated with Hup⁺ strain.     

Chemotaxis by Rhizobium meliloti

Summary Chemotaxis by Rhizobium meliloti strain Ve 26 has been studied and conditions required for chemotaxis have been defined, using the Adler capillary assay technique. Several sugars and amino-acids were shown to be attractants with varying effectiveness for this organism: sugars are weak attractants (except gluconate) and amino-acids are good attractants (except unpolar amino-acids).     

Host-specificity mutants of Rhizobium meliloti have additive effects in situ on initiation of alfalfa nodules

Pairs of Rhizobium meliloti nod mutants were co-inoculated onto alfalfa (Medicago saliva L.) roots to determine whether one nod mutant could correct, in situ, for defects in nodule initiation of another nod mutant. None of the Tn5 or nod deletion mutants were able to help each other form nodules when co-inoculated together in the absence of the wild-type. However, as previously observed, individual nod mutants significantly increased nodule initiation by low dosages of co-inoculated wild-type cells. Thus, nod mutants do produce certain signal substances or other factors which overcome limits to nodule initiation by the wild-type. When pairs of nod mutants were co-inoculated together with the wild-type, the stimulation of nodulation provided by individual nodABC mutants was not additive. However, clearly additive or synergistic stimulation was observed between pairs of mutants with a defective host-specificity gene (nodE, nodF, or nodH). Each pair of host-specificity mutants stimulated first nodule formation to nearly the maximum levels obtainable with high dosages of the wild-type. Mutant bacteria were recovered from only about 10% of these nodules, whereas the co-inoculated wild-type was present in all these nodules and substantially outnumbered mutant bacteria in nodules occupied by both. Thus, these mutant co-inoculants appeared to help their parent in situ even though they could not help each other. Sterile culture filtrates from wild-type cells stimulated nodule initiation by low dosages of the wild-type, but only when a host-specificity mutant was also present. The results from our studies seem consistent with the possibility that pairs of host-specificity mutants are able to help the wild-type initiate nodule formation by sustained production of complementary signals required for induction of symbiotic host responses.     

Identification of structural nif genes in an iron-oxidising moderate thermophile

The effect of 500 mM NaCl on the growth, and phosphatase production of a Citrobacter sp. was investigated. Although growth was retarded, phosphatase production was enhanced by 50%. Relief from osmotic stress using the osmoprotectant glycine betaine gave normal growth, but phosphatase activity was reduced. The Citrobacter sp. ceased to grow following a shift to anaerobic conditions, but anaerobically-incubated cells continued to produce phosphatase after a transient lag.     

Quorum-sensing in Rhizobium

Quorum-sensing signals are found in many species of legume-nodulating rhizobia. In a well-characterized strain of R. leguminosarum biovar viciae, a variety of autoinducers are synthesised, and all have been identified as N-acyl-homoserine lactones. One of these N-acyl-homoserine lactones, is N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone, previously known as small bacteriocin, which inhibits the growth of several R. leguminosarum strains. The cinRI locus is responsible for the production of small bacteriocin. CinR induces cinI in response to the AHL made by CinI, thus forming a positive autoregulatory induction loop. A complex cascade of quorum-sensing loops was characterized, in which the cinIR locus appears to be the master control for three other AHL-dependent quorum-sensing control systems. These systems include the raiI/raiR, traI/triR and rhiI/rhiR. Other rhizobial strains appear to share some of these quorum sensing loci, but not all loci are found in all strains. Small bacteriocin along with the other N-acyl-homoserine lactones produced by these three AHL-based control systems regulate (i) growth inhibition of sensitive strains, (ii) transfer of the symbiotic plasmid pRL1JI, and (iii) expression of the rhizosphere-expressed (rhi) genes that influence nodulation. Some of the genes regulated by these systems have been identified. While the functions of some, such as the trb operon regulated by triR are clear, several of the regulated genes have no homologues of known function. It is anticipated that several other genes regulated by these systems have yet to be identified. Therefore, despite the regulation of one of the most complex quorum-sensing cascade being understood, several of the functions regulated by the quorum-sensing genes remain to be elucidated. This revised version was published online in August 2006 with corrections to the Cover Date.