Expression Of The Direct Glucose Oxidative Pathway In Acetobacter Diazotrophicus

CINDEFI, Facultad de Cs. Exactas (UNLP)-CONICET, Argentina

A. diazotrophicus oxidizes glucose extracellularly to gluconate via a PQQ-linked glucose dehydrogenase (GDH). This is considered the main route for glucose catabolism in this bacterium. Moreover, aldose oxidation through GDH allows N2-fixing A. diazotrophicus to direct the electron flux through a more efficient branch of its respiratory chain generating extra utilizable energy (Luna et al. 2000). In this study the expression of GDH by A. diazotrophicus PAL3 under different environmental conditions was tested.

A. diazotrophicus PAL 3 was grown fixing N2 in continuous cultures at different glucose concentrations. GDH activity increased with the concentration of glucose in the medium. Growth yield was maximum for 10 g/L of glucose and decreased for concentrations above 20 g/L. Cultures with 10 and 20 g/L glucose were C-limited whereas at higher sugar concentrations cultures were under carbon-excess conditions. Nevertheless glucose was almost entirely consumed. Carbon-excess cultures accumulated gluconate and ketogluconates. This is a typical overflow metabolism behavior. It is commonly observed that bacteria under carbon-excess excrete partially oxidized intermediates, capsular material and protein. In the case of A. diazotrophicus, although some polysaccharides and protein could be detected, gluconate was the main overflow product. Therefore, within the plant, where this organism lives in a sugar rich environment (likely at very low growth rates), A. diazotrophicus seems to be able to oxidize glucose at high rates provided that oxygen is not limiting. The extracellular glucose oxidation yields biologically utilizable energy that can be used for N2-fixation and, probably could also function as a mechanism of respiratory protection of nitrogenase.

The pH of the culture medium had a profound influence on the steady state biomass concentration and growth yield. The range of optimum pH was between 5.5 and 6.0 and yields decreased towards acidic or alkaline pHs. The culture became unstable and washed-out (no growth) at pH values over 7.7. These data are in accordance with those obtained in batch cultures: at an initial pH of 3.5, biomass yield was half of that obtained at 5.5 and no growth was observed at 7.5. At these pH value no GDH activity could be detected. Since this enzyme is on the inner membrane oriented towards the periplasm, the pH of the growth environment could influence either its synthesis or activity, as already reported for other microorganisms. To study the pH-dependence of GDH activity and synthesis, cells grown at different pHs were incubated with glucose at the optimum pH (6.0) and at the culture pH, and the gluconate production rates examined. At pHs ranging from 4.5 to 6.5 no significant differences between both activities were measured. But at pH values distant from the optimum, and particularly under relative alkaline environments, although GDH is actively synthesized (gluconate production at pH 6.0 was high), very low activities could be detected at the culture pHs. From these results it could be concluded, in accordance with previous speculations (Luna et al. 2000), that glucose metabolism in A. diazotrophicus proceeds mainly via GDH and, under conditions where extracellular glucose oxidation is somehow impeded, growth is profoundly affected. In accordance with this proposal a gdh mutant of A. diazotrophicus PAL 3 could be grown in glucose containing media (indicating that the direct oxidative pathway is not the only route for glucose catabolism in this bacterium), but biomass yields were significantly lower than those obtained with the parental strain under N2-fixation. References

Luna MF, Mignone CF, Boiardi JL (2000) Appl. Microbiol. Biotechnol. 54, 564-569 Partly supported by ANPCyT (PICT 97 No. 1196)

INVOLVEMENT OF CYTOCHROME c IN THE BIOSYNTHESIS OF IAA IN GLUCONACETOBACTER DIAZOTROPHICUS AND ASSESSMENT OF A ROLE FOR IAA PRODUCTION IN SUGARCANE GROWTH ENHANCEMENT

S. Lee1, E. Pierson1, E. Escamilla2, C. Kennedy1

department of Plant Pathology, University of Arizona, Tucson, AZ 85721, USA

2Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico

Gluconacetobacter diazotrophicus, an endophyte of sugarcane, is beneficial to sugarcane growth possibly by two mechanisms, one dependent, and one not, on nitrogen fixation by the bacterial partner (Sevilla et al. 2001). To test the hypothesis that IAA production is a factor leading to sugarcane growth enhancement (Fuentes-Ramirez et al. 1993), genes known to be involved in IAA biosynthesis in other organisms were sought by PCR and complementation strategies. These were not successful. Screening of Tn5 mutants of G. diazotrophicus strain PA15 led to the isolation of strain MAdlO, which produced very little IAA (-6% of wild-type levels). The mutation which led to decreased IAA production was not associated with the insertion site of Tn5 in MAdlO, determined by cloning Tn5 and flanking regions in a suicide vector and reinsertion of this DNA into the G. diazotrophicus genome. To determine the site of the mutation leading to decreased levels of IAA in MAdlO, a pLAFR3 library carrying G. diazotrophicus DNA inserts was transferred by conjugation into MAdlO, followed by screening of transconjugants for IAA production. In two IAA+ transconjugants, the cosmids isolated shared identical regions in the insert fragments; analysis by subcloning, complementation, and DNA sequencing, indicated that the mutation in MAdlO was located in the ccmC gene, involved in cytochrome c maturation. The ccm operon was sequenced and found to encode Ccm proteins of -50% identity to those of the corresponding operon in Bradyrhizobium japonicum. Insertion of kan cassettes into the G. diazotrophisuc ccm genes cloned on suicide vectors, followed by their reintroduction into the G. diazotrophicus genome, led to the construction of several ccm mutants. The mutations in ccmC, ccmD, or ccmE genes led to the IAA" phenotype, and each produced -4-6% levels of IAA compared to strain PA15. Mobilization of the entire ccm operon into these mutants restored IAA production. Therefore cytochrome c is likely to be an essential component of an IAA biosynthetic enzyme in G. diazotrophicus.

Spectral analysis of cytochrome c, heme-associated peroxidase activities, and membrane-associated respiratory activities in the wild-type and mutant strains showed that the Ccm proteins of G. diazotrophicus are involved in cytochrome c biogenesis. Growth on several media and ability to fix nitrogen were not influenced in the ccm mutant strains; the only phenotype observed was a decrease in IAA production. The effect of a ccmC mutation on plant growth enhancement was examined, either singly or in combination with a mutation in nifD. Regardless of nitrogen supply, plants inoculated with wild-type PA15 were larger than uninoculated plants. Plants inoculated with a ccmC mutant, a nifD mutant, or a ccmC-nifD double mutant were no larger than uninoculated plants. These results are consistent with the hypothesis that both nitrogen fixation and IAA production are factors that allow G. diazotrophicus to benefit plant growth, and that a threshold of input by either factor must be attained before growth enhancement is achieved.

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