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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.

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.

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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.

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]-linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N(2) or NH(3)). Its synthesis was stimulated by conditions of high energetic demand (i.e., N(2)-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N(2)-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)-linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.

The ability to solubilize insoluble inorganic pho- sphate compounds by Gluconacetobacter diazotrophicus was studied using different cul-ture approaches. Qualitative plate assays using tricalcium phosphate as the sole P-source showed that G. diazotrophicus produced solu-bilization only when aldoses were used as the C-source. Extracellular aldose oxidation via a pyrroloquinoline quinone-linked glucose dehy-drogenase (PQQ-GDH) is the main pathway for glucose metabolism in G. diazotrophicus. In batch cultures with 5 g l-1 of hydroxyapatite as the P-source and glucose as the C-source, more than 98% of insoluble P was solubilized. No solubilization was observed neither using glyc-erol nor culturing a PQQ-GDH mutant of G. di-azotrophicus. Solubilizaton was not affected by adding 100 mmol l-1 of MES buffer. Continuous cultures of G. diazotrophicus showed significant activities of PQQ-GDH either under C or P limi-tation. An intense acidification in the root envi-ronment of tomato and wheat seedlings inocu-lated with a G. diazotrophicus PAL5 was ob-served. Seedlings inoculated with a PQQ-GDH mutant strain of G. diazotrophicus showed no acidification. Our results suggest that G. di-azotrophicus is an excellent candidate to be used as biofertilizer because in addition to the already described plant growth-promoting abili-ties of this organism, it shows a significant mineral phosphate solubilization capacity

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.