A consolidated analysis of the physiologic and molecular responses induced under acid stress in the legume-symbiont model-soil bacterium Sinorhizobium meliloti.

Abiotic stresses in general and extracellular acidity in particular disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0-6.1. Under such stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a higher aerobic-respiration rate in the acid-stressed rhizobia. Multivariate analysis of global metabolome data served to unequivocally correlate specific-metabolite profiles with the extracellular pH, showing that at low pH the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Further analyses should be focused on the time course of the observed changes, its associated intracellular signaling, and on the comparison with the changes that operate during the sub lethal acid-adaptive response (ATR) in rhizobia.

Colonization of sorghum and wheat by seed inoculation with Gluconacetobacter diazotrophicus.

Colonization of sorghum and wheat after seed inoculation with Gluconacetobacter diazotrophicus strains PAL 5 and UAP 5541/pRGS561 (containing the marker gene gusA) was studied by colony counting and microscopic observation of plant tissues. Inoculum levels as low as 10(2) CFU per seed were enough for root colonization and further spreading in aerial tissues. Rhizoplane colonization was around 7 log CFU g(-1) (fresh weight). G. diazotrophicus was found inside sorghum and wheat roots with populations higher than 5 log CFU g(-1) (fresh weight). Stem colonization remained stable for 30 days post inoculation with endophyte concentrations from 4 to 5 log CFU g(-1) (fresh weight) (in both plants). Population in leaves decreased continuously being undetectable after 17 days post inoculation.

Periplasmic PQQ-dependent glucose oxidation in free-living and symbiotic rhizobia.

The expression of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) of Rhizobium tropici CIAT899 and Sinorhizobium meliloti RCR2011 was investigated under different nutrient-limiting conditions in continuous cultures, under different conditions of phosphate availability, and in S. meliloti bacteroids. The presence of free PQQ in alfalfa root exudates has also been assayed. It was shown that apo-GDH or holoenzyme was actively synthesized by these rhizobia, with the concomitant production of gluconate from glucose, under certain environmental conditions. GDH activity was also detected in bacteroids from alfalfa root nodules inoculated with either S. meliloti RCR2011 or 102F34. It was also shown that free PQQ was present in root exudates of alfalfa, but its production is ascribed to the activity of Erwinia sp., a normal contaminant of these seeds.

The carbon source influences the energetic efficiency of the respiratory chain of N2-fixing Acetobacter diazotrophicus.

Acetobacter diazotrophicus is a diazotrophic bacterium that colonizes sugarcane tissues. Glucose is oxidized to gluconate in the periplasm prior to uptake and metabolism. A membrane-bound glucose dehydrogenase quinoenzyme [which contains pyrroloquinoline quinone (PQQ) as the prosthetic group] is involved in that oxidation. Gluconate is oxidized further via the hexose monophosphate pathway and tricarboxylic acid cycle. A. diazotrophicus PAL3 was grown in a chemostat with atmospheric nitrogen as the sole N source provided that the dissolved oxygen was maintained at 1.0-2.0% air saturation. The biomass yields of A. diazotrophicus growing with glucose or gluconate with fixed N were very low compared with other heterotrophic bacteria. The biomass yields under N-fixing conditions were more than 30% less than with ammonium as the N source using gluconate as the carbon source but, surprisingly, were only about 14% less with glucose. The following scheme for the metabolism of A. diazotrophicus through the different pathways emerged: (1) the respiratory chain of this organism had a different efficiency of ATP production in the respiratory chain (P:O ratio) under different culture conditions; and (2) N fixation was one (but not the sole) condition under which a higher P:O ratio was observed. The other condition appears to be the expression of an active PQQ-linked glucose dehydrogenase.

Role of bean lectins inRhizobium phaseoli-Phaseolus vulgaris interactions. Some properties of lectins from two bean cultivars.

Lectins from twoPhaseolus vulgaris L. cultivars were isolated and purified by salt fractionation, affinity chromatography and gel permeation chromatography. The cultivars used were: 'alubia', with a low-nodulating ability, and Bat 76 with a good symbiotic aptitude. Differences in properties of the two lectins were noted: 'alubia' lectin gave only one peak with haemagglutinating activity following gel permeation chromatography while Bat 76 yielded two active peaks, although both lectins had several bands of about 30 kDa following gel electrophoresis, Bat 76 lecting had three bands of about 50 kDa which were not present in 'alubia' and red kidney bean lectins. Peptide-mapping, by limited proteolysis and two dimensional gel electrophoresis, also showed differences between the lectins which are therefore judged to be different.

The role of magnesium and calcium ions in the glucose dehydrogenase activity of Klebsiella pneumoniae NCTC 418.

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 microM CaCl2 in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl2 was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg2+ and Ca2+ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca2+ ions could replace Mg2+ ions. The high GDH activity which has been found in cells grown under Mg2(+)-limited conditions in the presence of higher concentrations of Ca2+ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.