Precision control of ammonium release in Azotobacter vinelandii.

The capture and reduction of atmospheric dinitrogen gas to ammonium can be accomplished through the enzyme nitrogenase in a process known as biological nitrogen fixation (BNF), by a class of microbes known as diazotrophs. The diazotroph Azotobacter vinelandii is a model organism for the study of aerobic nitrogen fixation, and in recent years has been promoted as a potential producer of biofertilizers. Prior reports have demonstrated the potential to partially deregulate BNF in A. vinelandii, resulting in accumulation and extracellular release of ammonium. In many cases, deregulation requires the introduction of transgenic genes or elements to yield the desired phenotype, and the long-term stability of these strains has been reported to be somewhat problematic. In this work, we constructed two strains of A. vinelandii where regulation can be precisely controlled without the addition of any foreign genes or genetic markers. Regulation is maintained through native promoters found in A. vinelandii that can be induced through the addition of extraneous galactose. These strains result in varied degrees of regulation of BNF, and as a result, the release of extracellular ammonium is controlled in a precise, and galactose concentration-dependent manner. In addition, these strains yield high biomass levels, similar to the wild-type A. vinelandii strain and are further able to produce high percentages of the bioplastic polyhydroxybutyrate.

A deoxyviolacein-based transposon insertion vector for pigmented tracer studies.

Pigments provide a simple means to rapidly visually ascertain the quantities or presence of specific microbes in a complex community. The selection of pigment-producing colonies that are simple to differentiate from common colony phenotypes provides a high degree of certainty for the identity of pigment-tagged strains. Successful employment of pigment production is dependent on various intrinsic factors related to proper levels of gene expression and pigment production that are not always easy to predict and vary within each microbe. We have constructed a simple transposon system that incorporates the genes for the production of deoxyviolacein, a pigment produced from intracellular reserves of the amino acid tryptophan, to randomly insert these genes throughout the genome. This tool allows the user to select from many thousands of potential sites throughout a bacterial genome for an ideal location to generate the desired amount of pigment. We have applied this system to a small selection of endophytes and other model bacteria to differentiate these strains from complex communities and confirm their presence after several weeks in natural environments. We provide two examples of applications using the pigments to trace strains following introduction into plant tissues or to produce a reporter strain for extracellular nitrogen compound sensing. We recognize that this tool could have far broader utility in other applications and microbes, and describe the methodology for use by the greater scientific community.

Enhanced extracellular ammonium release in the plant endophyte Gluconacetobacter diazotrophicus through genome editing.

IMPORTANCE: Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus. Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe.

Gluconacetobacter diazotrophicus Gene Fitness during Diazotrophic Growth.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.