The unusual sensitivity of a Pythium ultimum isolate to three inhibitors of prokaryote protein synthesis [tetracycline (TET), chloramphenicol (CAM), erythromycin (ERY)] was investigated. TET inhibited growth in shake culture by 40 yo at 10 ,ug ml-I and 99 yo at 100 pg ml-l within 5 h (one doubling period). CAM inhibited growth by 40 yo at 500 pg ml-l and 70°,, at I mg ml-l. Cycloheximide (CHI), a eukaryote protein synthesis inhibitor, blocked growth completely at 1 pg ml-l. These agents reduced incorporation of leucine and phenylalanine into protein by 40 to 50 yo (100 pg TET ml-l, 500 pg CAM ml-l) or 93 0;(1 pg CHI ml-l) within 20 min. This rapid inhibitory effect could not be attributed to impaired mitochondrial energy generation. TET inhibition increased to 90 yo by 60 min, but CAM inhibition did not change for 5 h. TET inhibited both the transport and protein synthetic (cytoplasmic) components of amino acid incorporation, but CAM impaired only the transport activity. CHI blocked protein synthesis, but did not inhibit transport detectably. In contrast to these results, growth inhibition by ERY did not increase with dose (4004 inhibition at 10 to 500 pg ml-l), and ERY at 500 pg ml-l inhibited neither amino acid transport nor protein synthesis in 5 h. The results indicate that the sensitivity of P. ultimum to these prokaryote inhibitors reflects unusual permeability properties, which account for impaired transport (CAM, TET) and allow access of the drugs to sensitive intracellular sites, the cytoplasmic (TET) and mitochondrial (ERY) protein synthetic systems.
A method is described for growing uniform mycelial pretreated for 10 min with a variety of inhibitors known to suspensions of Pvthium ultimum and Phytophthora impair specific cytoplasmic and mitochondrial functions. tnegasperma var. sojae in a defined medium in shake culture. The cultures were maintained easily, and after about 12 hr Dry weight doubling times of these fungi were 5 to 6 hr and from the seeding of fresh cultures they were ready for use. The about 12 hr, respectively. Incorporation of radioactive rapid growth and the ease and reproducibility of sampling leucine, adenine, glycerol, and acetate into proteins, nucleic make possible short-term manipulations of small amounts of acids, and lipids by 3-ml cultures could be measured easily cell material as well as the rapid production of large amounts with 10-min pulse periods. These synthetic activities were of uniform, actively growing mycelium. blocked in a predictable fashion if the cell samples were The utility of liquid culture methods for physiological In our work with an isolate of Pvthium ultimum Trow, studies of filamentous fungi often is restricted by one or one of the species used by Tolmsoff (9), we found the more factors (5, 8). Preparation of test cultures from a establishment of such cultures to be possible but timemycelial mat or tight mycelial clumps leads to sampling consuming and their maintenance was especially difficult difficulties owing to nonuniformity of culture material. In at high cell densities. Further, we were unable to adapt a addition, many fungi grow slowly in liquid culture. Phytophthora megasperma Drechs. var. sojae Hildb. Although slow growth and cell heterogeneity may be isolate to such hyphal suspension growth owing to overcome to some extent by seeding media with hyphal difficulty in satisfactorily fragmenting the mycelium fragments and mechanically shaking the cultures, many without extensive destruction. Even though Tolmsoff (9) fungi still exhibit extensive mycelial aggregation when did not study Phytophthora spp., he noted that the grown in shake culture. blending conditions and transfer interval were critical and In particular, these problems are common with that they varied with the organism. In this report we Pythium spp. and Phytophthora spp. Many of the latter describe a shake culture method which is applicable to a species typically grow very slowly, and in shake culture fast-growing P. ultimum isolate and a slow-growing P. hyphal seed pieces of both groups of fungi give rise to megasperma var. sojae isolate, that provides for rapid tight pellets of mycelium. Information regarding some of growth in a defined medium, ease of culture production the unusual features of these fungi, such as their and maintenance, near-uniformity of culture material, sensitivity to antibacterial drugs (3), is limited, in part sampling ease with good reproducibility, and rapid assay because their growth habit precludes certain types of of metabolic activities. A brief report of part of this work experimentation. The results of sev...
Hybridization of [3H]polyuridylic acid to RNA isolated from Botryodiplodia theobromae pycnidiospores yielded an estimate of about 6.25 x 10(5) polyadenylate-containing RNA (poly A(+) RNA) molecules per dormant spore. The number increased about fourfold by the time of germ tube emergence at 3 h. The average size of this presumed mRNA was about 4.1 x 10(5) daltons (1275 nucleotides), with an average polyadenylate segment length of 26 nucleotides. Neither of these values changed significantly during germination. The earliest detectable (first 30 min of germination) de novo synthesized mRNA's were rapidly incorporated into polyribosomes. This newly synthesized, presumably functional, mRNA was composed of both poly A(+) RNA and polyadenylate-lacking RNA. The average sizes of the two polyribosomal mRNA subpopulations and the total poly A(+) RNA population were identical.
Portion of a dissertation submitted in partial fulfillment of the requirements for the Ph. D. degree, University of Kentucky.
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