Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131.

Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from a XhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cure A. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain of Anabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication in Anabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged from after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation to A. variabilis, where they appeared to recombine with pRDS1.

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra.

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.

Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana.

The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of NH+4 from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa] in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 microM O2. In contrast, respiration in NH+4-grown cells was saturated by O2 between 8 and 10 microM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in NH+4-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.

Oxygen protection of nitrogenase in Frankia sp. HFPArI3.

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O2 uptake and acetylene reduction rates in response to substrate concentration. O2 of NH4+-grown cells showed an apparent KmO2 of approximately 1 microM O2. In N2-fixing cultures a second Km O2 of about 215 microM O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent Km for acetylene was more than 10-fold greater than reported in vitro values. These data were interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica.

The mechanism of O(2) protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O(2) inhibition of nitrogenase activity correlated with the O(2) tension of the medium in which heterocyst formation was induced. O(2) resistance also correlated with the apparent K(m) for acetylene, indicating that O(2) tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O(2) that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O(2) sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O(2) but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O(2) diffusion barrier and active respiration by the heterocyst.