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Abstract:  Rhizobium "hedysari" HCNT1 and Sinorhizobium meliloti 41 were investigated and compared for their ability to shift from a typical aerobic, growth-supporting metabolism to O2–limiting, low-energy-expending, basal activities. Such metabolic conversion leads bacteria to stop reproduction although allows them to survive. Once anaerobic, both rhizobia started to consume their internal energy budget and most of the cells remained metabolically active for a long time, as revealed by microscope-based analyses. However, although R. "hedysari" HCNT1 also maintained the same number of culturable cells, S. meliloti 41 started to reduce this number almost immediately when anaerobic incubation took place. In the presence of NO2– the ability of R. "hedysari" HCNT1 to restore normal growth reduced drastically. A mutant strain, previously obtained by inactivation of the gene encoding nitrite reductase (nirK), did not burn up internal adenosine triphosphate when exposed to the same O2–limiting conditions in the presence of NO2–. This finding indicated that NO2–-reduction activity in the wild type strain, HCNT1, results in a decrease in cell energy content and culturability. Therefore, R. "hedysari" HCNT1 and S. meliloti 41 follow different paths to reduce the internal energy pool towards the so-called viable-not-culturable state that can be reached within a relatively large interval of internal energy charge, depending upon the bacterial strain.

Ex planta, bacteroids of the sulla-symbiont Rhizobium “hedysari” strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 µM nitrite transiently impeded O2 uptake in bacteroids, which resumed consumption of O2 when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. “hedysari” strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.

Rhizobium trifolii, R. leguminosarum, andR. “hedysarum”, grownex planta under anoxic conditions in a chemically defined medium, evolve N2O from NO3−, NO2−, and (NH4)2NO3. The amount of nitrous oxide formed after 96 hours is about 0.2μM×mg−1 cells d.w. Large availability of organic matter enhances the production of N2O from nitrate by free-livingR. trifolii in peat/sand mixtures. Denitrification of the above species andR. meliloti was detected also in planta. Nitrous oxide production increases almost linearly from 10–45μM×mg−1 nodules d.w. when nitrogen-fixing plants are exposed to increasing concentrations of nitrate (1–12μM).

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.

Three cDNAs encoding the β chain of Cap Z have been isolated from λgt11 chicken libraries screened with antibodies. The coding region, which is identical among the cDNAs, contains 831 basepairs encoding a protein with 277 amino acid residues and Mr= 31,352. The predicted protein sequence contains eight regions that match perfectly the NH2-terminal sequence of eight peptides isolated from muscle Cap Z β. The amino acid compositions of the protein predicted from the cDNA sequence and Cap Z β are similar. Comparison of the cDNA sequence and the predicted protein sequence with those of other actin-binding proteins and all sequences in the GenBank and NBRF Protein databases shows no remarkable similarities.

We investigated the binding properties of the type I insulin-like growth factor (IGF) receptor expressed in NIH-3T3 fibroblasts transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and 17 pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous growth factors bind to immunologically distinct epitopes on the type I receptor.

There are numerous poorly known and unexploited rhizobia-legume symbioses in arid ecosystems. These symbiotic associations have a considerable potential as sources for food, fodder, and energy. Expanded acquisition of information is needed on rhizobial strain specificities, salt and drought resistance, and on inoculation needs and applicable technologies for arid lands. Development of appropriate technologies for maximal protein recovery and for production of legume biomass as an energy source should be implemented. An important aspect is the utilization of molecular genetics for the development of economically improved legume symbioses.

Seed inoculation is widely used in agriculture, especially for grain legumes. However, it may be suitable for forage legumes too, particularly for the introduction of a new crop or for soil reclamation in marginal areas. This is the case of the present study, aimed at evaluating some strains of Rhizobium sp. (Rhizobium “hedy-sari “) to inoculate Hedysarum coronarium plants used for reclamation of marginal areas in Tuscany.Strain CC1335, able to produce growth inhibitors in vitro, was able to nodulate the roots both in pot and in all the field inoculation trials, while strain RH19st3 did not enter root system by any inoculation techniques employed. Strains HCNT1 and HCNA, showing detectable competition activity in vitro, produced good nodulation only by using calcium sulphate with the peat-arabic gum inoculant. The buffering effect of lime played evidently a determinant role in the performance of the inoculant.The results reported here suggest that improved inoculation methods will make possible the utilization of highly nitrogen fixing strains even if unable to compete with indigenous ones. Moreover, the introduction of highly competitive strains, which might interfere with soil microbiota, can be avoided.

A sandy soil amended with different forms and amounts of fertilizer nitrogen (urea, ammonium sulphate and potassium nitrate) was investigated in model experiments for N2O emission, which may be evolved during both oxidation of ammonia to nitrate and anaerobic respiration of nitrate. Since C2H2 inhibits both nitrification and the reduction of N2O to N2 during denitrification, the amount of N2O evolved in the presence and absence of C2H2 represents the nitrogen released through nitrification and denitrification.

Rhizobium “hedysari” strain HCNT 1 rapidly reduced nitrite to N2O, only slowly reduced nitrate to nitrite and did not exhibit nitrous oxide reductase activity. Nitrite reduction in this rhizobium strain may be a detoxification mechanism for conversion of nitrite, which inhibits O2 uptake, to non-toxic N2O. Concentrations of nitrite as small as 3 µM diminished O2 uptake in whole cells. The bacterium did not couple energy conservation with nitrate or nitrite reduction. Cells neither grew anaerobically at the expense of these nitrogen oxides nor translocated protons during reduction of nitrite. Induction of nitrite reductase activity was not a response to the presence of nitrate or nitrite, but occurred instead when the O2 concentration in culture atmospheres fell to <16.5% of air saturation. Sensitivity of cytochrome o, which is synthesized only in cells grown under O2-limited conditions, may account for the toxicity of nitrite in strain HCNT 1.

cDNA encoding the alpha chain of Cap Z has been isolated by screening a lambda gt11 library with affinity-purified antibodies. A single cDNA insert (designated CE2) of 2153 base pairs (bp) contains an open reading frame of 836 bp, which is incomplete at its 5' end. The technique of "rapid amplification of cDNA ends" has been used to extend the 5' end of this open reading frame to a potential transcription initiation site that is preceded by 320 bp of an apparently untranslated region. The protein predicted by the resulting nucleotide sequence has a Mr of 32,960 and contains four regions that show close homology with four alpha-chymotryptic digestion fragments of the alpha chain. The amino acid composition of the alpha chain of Cap Z and the predicted protein are also similar. Northern blot analysis of whole chicken embryos shows two mRNA species of 1.9 and 2.4 kilobases, respectively, that hybridize with CE2. Three potential polyadenylylation signals in two regions of CE2 460 bp apart are identified, suggesting that the two messages may result from the use of alternative polyadenylylation sites. Comparison of the sequence data with that of other known actin-capping and severing proteins shows no significant homologies, suggesting that Cap Z may be a member of a unique group of capping, nonsevering proteins.

Most denitrifying bacteria reduce nitrate to the inert gases nitrous oxide or nitrogen. A remarkable exception to this is Rhizobium sullae strain HCNT1, which catalyses only a single step in the denitrification pathway, the reduction of nitrite to the reactive molecule nitric oxide. Further study demonstrated that HCNT1 does not encode the genes for NO reductase. Prolonged incubation of HCNT1 under anoxic conditions revealed that the cells had reduced culturability but not viability when nitrite was present. This may indicate an adaptation to anoxic conditions to provide resistance to environmental stresses. A closely related strain of R. sullae, strain CC1335, which is unable to denitrify, was found to lose culturability but not viability irrespective of the presence of nitrite. When the gene for nitrite reductase was mobilized into CC1335, this increased culturability with or without nitrite. These results indicate that the presence of nitrite reductase can influence the long-term survival of R. sullae strains and may provide an explanation as to why HCNT1 possesses this unusual truncation of its denitrification electron transport chain.

Polyacrylamide gel electrophoresis profiles of various cell surface lipopolysaccharides (LPS) obtained from fast-growing strains and plasmidless mutant strains of Rhizobium ‘hedysari’, R. leguminosarum and from slow-growing Bradyrhizobium japonicum strains have been compared. Correlations were obtained by using serological techniques. Two of the fast-growing strains and one slow-growing strain employed showed several LPS with slow mobility and a few others characterized by fast mobility in the gel. Other fast-growing rhizobium strains and all the other slow-growing bradyrhizobia tested had bands widely distributed in the gels. Location of bands, in these rhizobia, did not correlate with rapidity of growth of the strains. Induced antibiotic resistance did not affect LPS composition for either the fast- or slow-growing rhizobia. The LPS composition was apparently affected by the loss of plasmids. It appears to be associated with the lack of some bands in the region ranging from 30–50 kDa. The LPS composition was also affected by the mutation in the fast mobility region. The mutant strain lacked a low molecular mass band compared with the parental strain. [1H]-NMR spectra of LPS extracts from the latter strain also revealed a signal that is missing from the spectrum of the LPS of mutant strain.