Diazotroph mutants designed using metabolic engineering to excrete surplus ammonium were used to enhance nitrogen fixation and plant growth, as the levels of nitrogen fixation attained with diazotrophs are insufficient for the plant’s needs. In this study, wild-type (A1501) and engineered ammonium-excreting (1568/pVA3) strains of nitrogen-fixing Pseudomonas stutzeri strains were tested in vitro based on plant growth-promoting traits, such as phosphate solubilization ability, indole acetic acid (IAA) production and nitrogenase activities, as well as ammonium excretion as affected by mannitol-mediated osmotic stress. The maize plant growth-promoting effect of the A1501 and 1568/pVA3 strains was evaluated in pots and in the field, and the ¹⁵N-dilution technique was employed to assess the proportion of plant nitrogen derived from nitrogen fixation. The results demonstrate that the 1568/pVA3 strain displayed higher IAA production and nitrogenase activity than A1501 and released significant quantities of ammonium. After 50 days, in all of the conditions assayed, maize inoculated with 1568/pVA3 accumulated more plant biomass (3.3% on average) and fixed N (39.4% on average) than plants inoculated with A1501. In the field experiment, the grain yield of maize was enhanced by 5.6% or 5.9% due to the inoculation of seeds with 1568/pVA3 in the absence or presence of exogenous N fertilizer, respectively. Therefore, the engineered P. stutzeri strain tested in the greenhouse and field was shown to perform better than the wild-type strain with respect to maize growth parameters and biologically fixed nitrogen.

Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and β-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.

Background/Objectives: Bacillus velezensis has recently received increased attention as a potential biological agent because of its broad-spectrum antagonistic capacity against harmful bacteria and fungi. This study aims to thoroughly analyze the genomic characteristics of B. velezensis BRI3, thereby providing theoretical groundwork for the agronomic utilization of this strain. Methods: In this work, we evaluated the beneficial traits of the newly isolated strain B. velezensis BRI3 via in vitro experiments, whole-genome sequencing, functional annotation, and comparative genomic analysis. Results: B. velezensis BRI3 exhibits broad-spectrum antifungal activity against various soilborne pathogens, displays inhibitory effects comparable to those of the type strain FZB42, and exhibits particularly effective antagonism against Sclerotinia sclerotiorum (Lib.) de Bary. Whole-genome sequencing and assembly revealed that the genome of BRI3 contains one chromosome and two plasmids, which carry a large amount of genetic information. Moreover, 13 biosynthetic gene clusters (BGCs) involved in the biosynthesis of secondary metabolites were predicted within the BRI3 genome. Among these, two unique BGCs (cluster 11 and cluster 13), which were not previously reported in the genomes of other strains and could potentially encode novel metabolic products, were identified. The results of the comparative genomic analysis demonstrated the genomic structural conservation and genetic homogeneity of BRI3. Conclusions: The unique characteristics and genomic data provide insights into the potential application of BRI3 as a biocontrol and probiotic agent.