Stable Transformation of the ActinobacteriaFrankiaspp.

ABSTRACTA stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genusFrankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into differentFrankiastrains by a filter mating withEscherichia colistrain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced intoFrankia casuarinaestrain CcI3 at a frequency of 4.0 × 10−3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced intoF. casuarinaestrain CcI3 and otherFrankiastrains at frequencies ranging from 10−2to 10−4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerantFrankiasp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitiveF. casuarinaestrain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed inF. casuarinaestrain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression inFrankiaspp., overcoming a major hurdle in the field. This step in the development of genetic tools inFrankiaspp. will open up new avenues for research on actinorhizal symbiosis.IMPORTANCEThe absence of genetic tools forFrankiaresearch has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids intoFrankiaspp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.

Contrasted Reactivity to Oxygen Tensions inFrankiasp. Strain CcI3 throughout Nitrogen Fixation and Assimilation

Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria. Although nitrogenase is oxygen and reactive oxygen species-labile, oxygen tension is required to sustain respiration. In the nitrogen-fixingFrankia, various strategies have been developed through evolution to control the respiration and nitrogen-fixation balance. Here, we assessed the effect of different oxygen tensions onFrankiasp. strain CcI3 growth, vesicle production, and gene expression under different oxygen tensions. Both biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen-deficient conditions. The mRNA levels for the nitrogenase structural genes (nifHDK) were high under hypoxic and hyperoxic conditions compared to oxic conditions. The mRNA level for the hopanoid biosynthesis genes (sqhC andhpnC) was also elevated under hyperoxic conditions suggesting an increase in the vesicle envelope. Under nitrogen-deficient conditions, thehup2 mRNA levels increased with hyperoxic environment, whilehup1 mRNA levels remained relatively constant. Taken together, these results indicate thatFrankiaprotects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection.

Casuarina Root Exudates Alter the Physiology, Surface Properties, and Plant Infectivity of Frankia sp. Strain CcI3

ABSTRACTThe actinomycete genusFrankiaforms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions betweenFrankiaand host plants in the rhizosphere. Root exudates are important inRhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences inFrankiaphysiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses.Casuarina cunninghamianaroot exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested onFrankiasp. strain CcI3. Root exudates increased the growth yield ofFrankiain the presence of a carbon source, butFrankiawas unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal “curling” inFrankiacells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes.Frankiacells preexposed toC. cunninghamianaroot exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants andFrankiain the rhizosphere.

Diminished Exoproteome of Frankia spp. in Culture and Symbiosis

ABSTRACT Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N2 fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

Truncated hemoglobins in Frankia CcI3: effects of nitrogen source, oxygen concentration, and nitric oxide

Frankia strain CcI3 produces 2 truncated hemoglobins, HbN and HbO. Using ion-exchange chromatography, we characterized the expression of the relative amounts of HbN and HbO in −N (nitrogen-fixing) cultures and +N (nitrogen-supplemented) cultures. The −N cultures maintained an approximately constant ratio of HbO to HbN throughout the life of the culture, with HbO constituting 80%–85% of the total hemoglobin produced. In contrast, in +N cultures, HbN was observed to increase over time and HbO decreased. Total hemoglobin as a fraction of total protein was approximately constant throughout the growth phase in −N cultures, while it decreased somewhat in +N cultures. Subjecting −N cultures to a NO generator resulted in increased production of HbN, relative to the controls. Nitrite accumulated in +N cultures, but not in −N cultures. This suggests that the greater amount of HbN in +N cultures might be due to NO produced by the reduction of nitrite. The effects of O2 concentration were determined in +N cultures. Cultures grown in 1% O2 produced about 4 times more HbO than cultures grown in 20% O2. Overall, these results provide evidence for a role of HbN in NO oxidation and for a role of HbO in adaptation to low oxygen concentrations.

Nitric Oxide and Oxygen Regulate Truncated Hemoglobin Gene Expression in Frankia Strain CcI3

ABSTRACT The Frankia genome contains two truncated hemoglobin genes (hboN and hboO) whose functions remain to be determined. Nitric oxide (NO) generated by the addition of 400 μM SNAP (S-nitroso-N-acetylpenicillamine) caused a 10-fold increase in hboN gene expression but had no effect on hboO expression. The addition of the NO scavenger, carboxy-PT10, reduced the effect of SNAP. hboO gene expression increased under low-oxygen conditions, while hboN expression was unaffected. These results suggest that HboN may function in protection from nitrosative stress and that HboO may act as an oxygen transport molecule for increased respiration in hypoxic environments.