The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping

The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche.     

How Do Bacteria Avoid High Oxygen Concentrations?

Bacteria, such as Escherichia coli and Azospirillum brasilense, avoid microenvironments with elevated oxygen concentrations, not by sensing reactive oxygen derivatives, but by sensing a metabolic down-shift that results from elevated oxygen levels. A novel protein, Aer, and the chemotaxis serine receptor, Tsr, have recently been identified as transducers for aerotaxis which monitor internal energy levels in the bacteria.     

Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense

A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [Δ(cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.     

NH4+-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH₄⁺. Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH₄⁺. The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O₂) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH₄Cl. Both mutants failed to incorporate [¹⁴C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10⁷ cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a ¹⁵N₂-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of ¹⁵N inside root and shoot material than the wild type did. The results of our nitrogen balance and ¹⁵N enrichment studies indicated that NH₄⁺-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Microaerophilic Cooperation of Reductive and Oxidative Pathways Allows Maximal Photosynthetic Membrane Biosynthesis in Rhodospirillum rubrum

The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions). R. rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose. Employing a unique partial O₂ pressure (pO₂) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect. A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions. Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose. Fructose was utilized via the Embden-Meyerhof-Parnas pathway. High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures. No glucose-6-phosphate dehydrogenase activity was detected under any conditions. We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions. The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions. Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions. We propose a model by which the primary “switching” of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R. rubrum.     

Oxygen taxis and proton motive force in Azospirillum brasilense.

The microaerophilic nitrogen-fixing bacterium Azospirillum brasilense formed a sharply defined band in a spatial gradient of oxygen. As a result of aerotaxis, the bacteria were attracted to a specific low concentration of oxygen (3 to 5 microM). Bacteria swimming away from the aerotactic band were repelled by the higher or lower concentration of oxygen that they encountered and returned to the band. This behavior was confirmed by using temporal gradients of oxygen. The cellular energy level in A. brasilense, monitored by measuring the proton motive force, was maximal at 3 to 5 microM oxygen. The proton motive force was lower at oxygen concentrations that were higher or lower than the preferred oxygen concentration. Bacteria swimming toward the aerotactic band would experience an increase in the proton motive force, and bacteria swimming away from the band would experience a decrease in the proton motive force. It is proposed that the change in the proton motive force is the signal that regulates positive and negative aerotaxis. The preferred oxygen concentration for aerotaxis was similar to the preferred oxygen concentration for nitrogen fixation. Aerotaxis is an important adaptive behavioral response that can guide these free-living diazotrophs to the optimal niche for nitrogen fixation in the rhizosphere.     

Magneto-aerotaxis in marine coccoid bacteria.

Magnetotactic cocci swim persistently along local magnetic field lines in a preferred direction that corresponds to downward migration along geomagnetic field lines. Recently, high cell concentrations of magnetotactic cocci have been found in the water columns of chemically stratified, marine and brackish habitats, and not always in the sediments, as would be expected for persistent, downward-migrating bacteria. Here we report that cells of a pure culture of a marine magnetotactic coccus, designated strain MC-1, formed microaerophilic bands in capillary tubes and used aerotaxis to migrate to a preferred oxygen concentration in an oxygen gradient. Cells were able to swim in either direction along the local magnetic field and used magnetotaxis in conjunction with aerotaxis, i.e., magnetically assisted aerotaxis, or magneto-aerotaxis, to more efficiently migrate to and maintain position at their preferred oxygen concentration. Cells of strain MC-1 had a novel, aerotactic sensory mechanism that appeared to function as a two-way switch, rather than the temporal sensory mechanism used by other bacteria, including Magnetospirillum megnetotacticum, in aerotaxis. The cells also exhibited a response to short-wavelength light (< or = 500 nm), which caused them to swim persistently parallel to the magnetic field during illumination.     

Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation

Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed on their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, it was shown that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR-p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.     

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.     

Responses of Azospirillum brasilense to Nitrogen Deficiency and to Wheat Lectin: A Diffuse Reflectance Infrared Fourier Transform (DRIFT) Spectroscopic Study

For the rhizobacterium Azospirillum brasilense, the optimal nutritional range of C:N ratios corresponds to the presence of malate (ca. 3 to 5 g l⁻¹ of its sodium salt) and ammonium (ca. 0.5 to 3 g l⁻¹ of NH₄Cl) as preferred carbon and nitrogen sources, respectively. This microaerophilic aerotactic bacterium is known to have a narrow optimal oxygen concentration range of ca. 3 to 5 μM, which is 1.2% to 2% of oxygen solubility in air-saturated water under normal conditions. In this work, the effects of stress conditions (bound-nitrogen deficiency related to a high C:N ratio in the medium; excess of oxygen) on aerobically grown A. brasilense Sp245, a native wheat-associated endophyte, were investigated in the absence and presence of wheat germ agglutinin (WGA, plant stress protein and a molecular host-plant signal for the bacterium) using FTIR spectroscopy of whole cells in the diffuse reflectance mode (DRIFT). The nutritional stress resulted in the appearance of prominent spectroscopic signs of poly-3-hydroxybutyrate (PHB) accumulation in the bacterial cells; in addition, splitting of the amide I band related to bacterial cellular proteins indicated some stress-induced alterations in their secondary structure components. Similar structural changes were observed in the presence of nanomolar WGA both in stressed A. brasilense cells and under normal nutritional conditions. Comparative analysis of the data obtained and the relevant literature data indicated that the stress conditions applied (which resulted in the accumulation of PHB involved in stress tolerance) and/or the presence of nanomolar concentrations of WGA induced synthesis of bacterial cell-surface (glyco)proteins rich in β-structures, that could be represented by hemagglutinin and/or porin.     

Aerotactic response of Azospirillum brasilense.

Five strains of Azospirillum brasilense and two of Azospirillum spp., from Israel, responded to self-created and preformed oxygen gradients by forming aerotactic bands in capillary tubes and actively moving toward a specific zone with low dissolved oxygen. Increasing the oxygen concentration in capillaries containing phosphate buffer increased the number of attracted bacteria and decreased band velocity. High O2 concentrations and H2O2 temporarily repulsed the bacteria, causing the formation of a bacterial arc around the capillary mouth. There was no band formation under anaerobic conditions, although the bacteria remained highly motile. Exogenous energy sources were unnecessary for aerotaxis in Azospirillum spp. The addition of oxidizable substrates to the capillary slightly enhanced aerotaxis, possibly by accelerating O2 consumption. Aerotactic band formation was affected by pH, bacterial concentration and age, incubation time, and respiratory inhibitors, but not by the lack of combined nitrogen in the growth medium. It is proposed that aerotaxis plays a role in the capacity of Azospirillum spp. to reach an environment suitable for N2 fixation.     

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA ⁻ strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.     

Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism.     

Optimization of biofloc production in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> (MTCC-125) and evaluation of its adherence with the roots of certain crops

The phenomenon of flocculation in Azospirillum brasilense (MTCC-125) was studied under different combinations of carbon and nitrogen sources. Fructose and Potassium nitrate at a pH of 6.4 in the cultural medium favour a higher bio-floc production. The biofloc was studied for root adhesion and its survival efficiency in the rhizoplane and rhizosphere of certain crops such as sorghum and sunflower under dryland condition. It has been demonstrated that the flocculated cultures of Azospirillum were found to have maximum adhesion to the root surface and higher survival rate in the rhizoplane and rhizosphere under different moisture stressed conditions as compared to the log phase cells of Azospirillum.     

Characterization of the ntrBC genes of Azospirillam brasilense Sp7: Their involvement in the regulation of nitrogenase synthesis and activity

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif⁺. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.     

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO₂ fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO₂ fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.     

Metabolic Analysis of Adaptation to Short-Term Changes in Culture Conditions of the Marine Diatom Thalassiosira pseudonana

This report describes the metabolic and lipidomic profiling of 97 low-molecular weight compounds from the primary metabolism and 124 lipid compounds of the diatom Thalassiosira pseudonana. The metabolic profiles were created for diatoms perturbed for 24 hours with four different treatments: (I) removal of nitrogen, (II) lower iron concentration, (III) addition of sea salt, (IV) addition of carbonate to their growth media. Our results show that as early as 24 hours after nitrogen depletion significant qualitative and quantitative change in lipid composition as well as in the primary metabolism of Thalassiosira pseudonana occurs. So we can observe the accumulation of several storage lipids, namely triacylglycerides, and TCA cycle intermediates, of which citric acid increases more than 10-fold. These changes are positively correlated with expression of TCA enzymes genes. Next to the TCA cycle intermediates and storage lipid changes, we have observed decrease in N-containing lipids and primary metabolites such as amino acids. As a measure of counteracting nitrogen starvation, we have observed elevated expression levels of nitrogen uptake and amino acid biosynthetic genes. This indicates that diatoms can fast and efficiently adapt to changing environment by altering the metabolic fluxes and metabolite abundances. Especially, the accumulation of proline and the decrease of dimethylsulfoniopropionate suggest that the proline is the main osmoprotectant for the diatom in nitrogen rich conditions.     

Tactic responses to oxygen in the phototrophic bacterium Rhodobacter sphaeroides WS8N

The temporal and spatial behavior of a number of mutants of the photosynthetic, facultative anaerobe Rhodobacter sphaeroides to both step changes and to gradients of oxygen was analyzed. Wild-type cells, grown under a range of conditions, showed microaerophilic behavior, accumulating in a 1.3-mm band about 1.3 mm from the meniscus of capillaries. Evidence suggests this is the result of two signaling pathways. The strength of any response depended on the growth and incubation conditions. Deletion of either the complete chemosensory operons 1 and 2 plus the response regulator genes cheY(4) and cheY(5) or cheA(2) alone led to the loss of all aerotactic responses, although the cells still swam normally. The Prr system of R. sphaeroides responds to electron flow through the alternative high-affinity cytochrome oxidase, cbb(3), controlling expression of a wide range of metabolic pathways. Mutants with deletions of either the complete Prr operon or the histidine kinase, PrrB, accumulated up to the meniscus but still formed a thick band 1.3 mm from the aerobic interface. This indicates that the negative aerotactic response to high oxygen levels depends on PrrB, but the mutant cells still retain the positive response. Tethered PrrB(-) cells also showed no response to a step-down in oxygen concentration, although those with deletions of the whole operon showed some response. In gradients of oxygen where the concentration was reduced at 0.4 micro M/s, tethered wild-type cells showed two different phases of response, with an increase in stopping frequency when the oxygen concentration fell from 80 to 50% dissolved oxygen and a decrease in stopping at 50 to 20% dissolved oxygen, with cells returning to their normal stopping frequency in 0% oxygen. PrrB and CheA(2) mutants showed no response, while PrrCBA mutants still showed some response.     

Microaerophilic cooperation of reductive and oxidative pathways allows maximal photosynthetic membrane biosynthesis in Rhodospirillum rubrum

The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions). R. rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose. Employing a unique partial O(2) pressure (pO(2)) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect. A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions. Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose. Fructose was utilized via the Embden-Meyerhof-Parnas pathway. High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures. No glucose-6-phosphate dehydrogenase activity was detected under any conditions. We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions. The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions. Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions. We propose a model by which the primary "switching" of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R. rubrum.