Search

Abstract 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.

Abstract 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.

ABSTRACT The plant growth-promoting bacterium Gluconacetobacter diazotrophicus was originally discovered in association with sugarcane plants as an endophyte. As a member of the small class of organisms defined as diazotrophs, G. diazotrophicus is capable of fixing nitrogen from the atmosphere and could serve an important role in minimizing the requirements for nitrogen from industrial-derived fertilizers. In addition to sugarcane, G. diazotrophicus is capable of forming endophyte associations with a variety of other important crops. It has been reported that this microbe requires micro-aerobic conditions to effectively fix nitrogen gas from the atmosphere through the enzyme nitrogenase, making it slightly more difficult to study the diazotrophic lifestyle in the laboratory. The ability of the strain to reside within the plant during growth means that any extracellular nitrogen released by this microbe would immediately become available to the plant host. For this reason, it is an ideal target for development as an improved biofertilizer strain. In this work, we constructed strains of G. diazotrophicus that result in enhanced ammonium release, as measured by growing with a closely associated algal strain under micro-aerobic conditions, and by further quantifying ammonium concentrations accumulated under micro-aerobic and aerobic growth. 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.