Nutrient-induced spore germination of a Bacillus amyloliquefaciens biocontrol agent on wheat spikes.

AIMS: In this study, we investigated the feasibility of applying nutrient germinants to plant surfaces to stimulate the spore germination of the plant disease biocontrol agent Bacillus amyloliquefaciens strain TrigoCor. METHODS AND RESULTS: Using the terbium chloride assay and phase-contrast microscopy, we screened potential germinants of TrigoCor spores and found that a combination of d-glucose, d-fructose and potassium chloride (GFK), in addition to either l-asparagine (Asn-GFK) or l-alanine (Ala-GFK), induced maximal levels of TrigoCor spore germination in vitro. The germinant mixture Asn-GFK was also able to significantly stimulate Bacillus spore germination on wheat surfaces. CONCLUSIONS: The successful in vivo stimulation of Bacillus spore germination suggests that nutrient-induced spore germination on plant surfaces is a feasible strategy for improving Bacillus biocontrol. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the challenges of applying Bacillus biological control agents to aboveground plant parts is that Bacillus cells transition to a metabolically dormant spore state on plant surfaces, making them unable to prevent subsequent pathogen attacks. This study demonstrates that using nutrients to stimulate Bacillus spore germination in vivo is a promising option for improving disease control and should be pursued further.

Reduced flux through the purine biosynthetic pathway results in an increased requirement for coenzyme A in thiamine synthesis in Salmonella enterica serovar typhimurium.

Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in Salmonella enterica serovar Typhimurium. Prior work showed that panE mutants (panE encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of panE mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth.

The panE gene, encoding ketopantoate reductase, maps at 10 minutes and is allelic to apbA in Salmonella typhimurium.

In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The apbA gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858-4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene panE. Although previous reports placed panE at 87 min on the Escherichia coli chromosome, we show herein that apbA and panE are allelic and map to 10 min on both the S. typhimurium and E. coli chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.

ApbA, the ketopantoate reductase enzyme of Salmonella typhimurium is required for the synthesis of thiamine via the alternative pyrimidine biosynthetic pathway.

The apbA gene of Salmonella typhimurium was shown to encode ketopantoic acid reductase. ApbA was purified from crude cell-free extracts to greater than 95% homogeneity after two chromatographic steps. N-terminal amino acid sequencing (first 15 amino acids) and Western blot analysis confirmed the isolated protein was ApbA. The functional protein was a monomer with a molecular mass of 31.1 kDa. Optimal reaction conditions for the reduction of ketopantoic acid were established at a pH of 6.25, and a temperature of 42 degreesC. The preferred electron source was NADPH, and the apparent Km constants of the enzyme for NADPH and ketopantoic acid were determined to be 0.776 +/- 0.09 mM and 0.742 +/- 0.01 mM, respectively. The homogeneous enzyme had a specific activity of 64.3.

Differential reflectance spectrophotometry-I High-reflectance method for determination of micro amounts of substances resolved on thin plates.

The basic principles of differential high-reflectance spectroscopy are discussed from the standpoint of the determination of substances resolved on chromatoplates. Results obtained with the use of two systems, nickel dimethylglyoximate or copper neocuproinate adsorbed on cellulose, are used as illustrations. A graphical method for selecting the optimum concentration range for analysis and for determining the maximum accuracy attainable is also outlined. When contrasted with the conventional method of measuring reflectance, the technique promises substantially increased accuracy over a wider concentration range and seems particularly suited to the analysis of trace amounts of material.

Selection of the optimum concentration range for reflectance spectrophotometric analysis.

Two graphical methods for selecting the optimum range and determining the maximum accuracy of reflectance spectrophotometric analysis are discussed. Results obtained with the use of two systems (Rhodamine B, which absorbs in the visible region of the spectrum, and aspirin, which absorbs in the ultraviolet region, both adsorbed on silica gel) are employed to illustrate how the methods might be employed in practice. Experimental results are contrasted with those expected by application of the Kubelka-Munk equation. The results indicate that the minimum relative error in concentration to be expected in reflectance spectrophotometric analysis is about 6% per 1 % reading error, and that the optimum range for analysis can be arrived at after plotting the reflectance data for either of the two systems discussed, whether the system conforms to the Kubelka-Munk equation or not.