Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose.

Pseudomonas fluorescens HV37a inhibited growth of the fungus Pythium ultimum on potato dextrose agar (PDA). An antibiotic activity produced under these conditions was fractionated and partially characterized. Extracts prepared from the PDA on which HV37a was grown revealed a single peak of antibiotic activity on thin-layer chromatograms. Similar extracts were prepared from mutants of HV37a. Their analysis indicated that the antibiotic observed in thin-layer chromatograms was responsible for fungal inhibition observed on PDA. The production of the PDA antibiotic required the presence of glucose, whereas two other antibiotic activities were produced only on potato agar without added glucose. Two mutants (denoted AfuIa and AfuIb) previously characterized as deficient in fungal inhibition on PDA showed altered regulation of the production of all three antibiotics in response to glucose. These mutants were also deficient in glucose dehydrogenase. Mutants isolated as deficient in glucose dehydrogenase were also deficient in fungal inhibition and were grouped into two classes on the basis of complementation analysis with an AfuI cosmid. Glucose regulation of antibiotic biosynthesis therefore involves at least two components and requires glucose dehydrogenase.

Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad.

Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition.

A diffusion assay for detection and quantitation of methyl-esterified proteins on polyacrylamide gels.

The methyl esterification of bacterial and mammalian proteins is a subject of increasing interest and effort. Such studies in intact cells typically involve the use of [methyl-3H]methionine which is taken up and incorporated into S-adenosyl-L-methionine, the methyl donor. The level of methylation, however, is much less than the incorporation of labeled methionine directly into protein. A diffusion assay which distinguishes [3H]methionine from the base-labile [3H]methyl esters is described here. The ester linkage is hydrolyzed at high pH to release [3H]methanol from the sample which diffuses into an adjacent pool of scintillation fluid. The assay is contained in a scintillation vial which can be counted directly.

Replacement and amplification of bacterial genes with sequences altered in vitro.

An efficient method for the replacement of chromosomal DNA by segments altered in vitro has been developed for bacteria. The method requires (i) a recombinant plasmid with a ColE1-like replicon and (ii) a strain defective in DNA polymerase I (polA), which is unable to replicate the plasmid extrachromosomally. This method is of general use since there are a number of suitable vectors and polA strains are available in both Escherichia coli and Salmonella typhimurium, the two most widely studied bacterial species. Using the method, we have constructed two chromosomal deletions in the chemotaxis gene region of S. typhimurium. In addition, plasmid sequences integrated into the chromosome have been amplified up to 30-fold by varying the concentration of ampicillin or tetracycline in the growth medium.