Relatedness among Rhizobium and Agrobacterium species determined by three methods of nucleic acid hybridization.

Deoxyribonucleic acid (DNA) was isolated from 20 strains of Rhizobium and Agrobacterium and from one strain of Serratia marcescens; the guanine plus cytosine content of each DNA sample was determined by thermal denaturation. Radioactive DNA was isolated from three reference strains following the uptake of [2-(14)C]thymidine in the presence of deoxyadenosine. Ribonucleic acid (RNA) polymerase was used to synthesize radioactive RNA on DNA templates from the three reference strains. Radioactive DNA and RNA from the three reference strains were each hybridized with filter-bound DNA from all of the 21 test strains in 6 x SSC (standard saline citrate) and 50% formamide at 43 C for 40 hr. DNA/DNA relatedness was also determined by spectrophotometric measurement of the rates of association of single-stranded DNA. The order of relatedness between strains was similar by each method. Overall standard deviations for the DNA/DNA and DNA/RNA membrane filter techniques were +/-0.87 and +/-1.03%, respectively; that for the spectrophotometric technique was +/-4.11%. The DNA/DNA membrane technique gave higher absolute values of hybridization than did the DNA/RNA technique. R. leguminosarum and R. trifolii could not be distinguished from each other by these techniques. These results also indicated close relationships between R. lupini and R. japonicum, and (with less certainty) between R. meliloti and R. phaseoli. Of all the rhizobia tested against the A. tumefaciens 371 reference strain, the R. japonicum strains were the most unrelated. The three Agrobacterium strains used were as related to the R. lupini and R. leguminosarum references as were several rhizobium strains.

Temperature-sensitive mutants of Saccharomyces cerevisiae variable in the methionine content of their protein.

Temperature-sensitive mutants were derived from Saccharomyces cerevisiae Y5alpha by ethyl methane sulfonate mutagenesis, in a search for mutants that would produce methionine-rich protein at the nonpermissive temperature. A total of 132 mutant strains were selected which showed adequate growth on minimal medium at 25 degrees C but little or no growth on the same medium supplemented with a high concentration (2 mg/ml) of l-methionine at 37 degrees C. Several of these mutants were found to increase the proportion of methionine in their protein to much higher levels than that of the wild-type parent after a temperature shift from 25 to 37 degrees C. Two strains, 476 and 438, which were temperature sensitive only in the presence of methionine, produced cellular protein with methionine contents as high as 3.6 and 4.3%, respectively, when incubated in the presence of methionine. The former strain contained 2.5% methionine even when incubated at 37 degrees C in the absence of methionine. Wild strain Y5alpha, on the other hand, had 1.75% methionine under all conditions tested. Most temperature-sensitive mutants isolated had the same methionine content as the wild strain. It is concluded that the proportion of a specific amino acid, such as methionine, in S. cerevisiae protein can be altered by culturing certain temperature-sensitive mutants at an elevated temperature.

Sequential cold-sensitive mutations in Aspergillus fumigatus.

Mutants of thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 degrees C were sought. Cold-sensitive mutants were enriched from progeny spores of gamma-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through chessecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 degrees C, respectively. Mutants unable to grow at 37 degrees C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 degrees C was isolated and mutations further increasing the cold sensitivity by increments of 3-5 degrees C were found to occur. Mutants completely unable to grow at 37 degrees C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 degrees C and higher. Each mutant produced revertants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.

Methods for the determination of deoxyribonucleic Acid homologies in streptomyces.

Variations of the membrane filter technique for deoxyribonucleic acid (DNA) hybridizations were studied with respect to Streptomyces species. At the temperatures required for specific hybridization of DNA with the high melting temperature (T(m)) characteristic of Streptomyces, large amounts (up to 97%) of filter-bound DNA became eluted, in all reaction mixtures studied, within 21 hr. In most solutions this leaching was increased by the presence of sheared denatured DNA. Incubation of DNA-loaded filters in a solution of 50% formamide containing 6x standard saline citrate, at 48 C for 40 hr, was judged to be the best set of conditions tested based on relatively good retention of immobilized DNA, very low hybridization with unrelated DNA of a similarly high T(m) (from Sarcina lutea), and the formation of complexes similar in thermal stability to the native DNA. The expression of results as sheared DNA bound in relation to long-chain DNA retained is recommended when a high concentration of sheared DNA relative to immobilized DNA is used.

High-temperature production of protein-enriched feed from cassava by fungi.

A simple, nonaseptic, low-cast process for the conversion of cassava, a starchy tropical root crop, into microbial protein for use as animal feed was sought. Screening tests culminated in the isolation of a thermotolerant, amylase-producing mold, designated I-21, which was identified as Aspergillus fumigatus. The optimum pH for protein synthesis was 3-5, but the optimum temperature was less than the desired temperature (larger than or equal to 45 C) required for a nonaseptic fermentation. A. fumigatus I-21 and its asporogenous mutant I-21A grew equally well in a medium prepared from whole cassava roots with a mean protein doubling time at 45 C and pH 3.5 of 3.5 h. In batch culture, approximately 4% carbohydrate, supplied as whole cassava, could be feremented in 20 h, giving a final yield of 24 g of dry product, containing 36.9% crude protein, per liter. The conversion of carbohydrate used to crude protein was 22.1%. When determined as amino acids, the protein content of the product, which contained cassava bark and other unfermented residues, was 27.1%. With urea as the nitrogen source, no pH control was necessary. Preliminary data indicated that medium prepared from whole cassava roots was inhibitory to the mold unless the cassava pulp was heated to 70 C immediately after being ground. Heating to 70 C was required to gelatinize the starch and permit its complete utilization.

Sequential cord-sensitive mutations in Aspergillus fumigatus. III. Mechanism of cold sensitivity.

The mechanism of cold sensitivity of Aspergillus fumigatus ON5, a 37 degrees C-sensitive mutant derived from A. fumigatus I-21 (ATCC 32722) by five sequential mutations, was investigated. The rate of in vivo protein synthesis by ON5 was not affected for 2 h following a shift from 45 to 34 degrees C, but the rate of in vivo RNA synthesis dropped almost immediately. The RNA polymerases 2 h following a shift from 45 to 34 degrees C, but the rate of in vivo RNA synthesis dropped almost immediately. The RNA polymerases of ON5 possessed wild-type activity in vitro at a nonpermissive temperature (34 degrees C) indicating that the reduction in the rate of in vivo RNA synthesis did not result from cold sensitivity in transcription, but was possibly a result of rapid feedback inhibition of transcription. Mutant ON5 was not able to produce ribosomes at a nonpermissive temperature as evidenced by the fact that no 3H-labelled amino acids were incorporated into the monosome, large ribosomal subunit, or small ribosomal subunit at 34 degrees C. Ribosomal subunit assembly or ribosomal RNA processing appears, therefore, to be the cold-sensitive cellular function in ON5.

Mutants of Saccharomyces cerevisiae and Candida utilis with increased susceptibility to digestive enzymes.

Mutants of Candida utilis and a haploid strain of Saccharomyces cerevisiae were isolated, after ultraviolet light mutagenesis, which had increased sensitivities to snail gut enzymes (ses). Three of the five S. cerevisiae mutants tested had increased sensitivities to porcine pepsin, all were more susceptible to a sequential treatment with pepsin, lipase, peptidase, and trypsin, four were sensitive to osmotic shock, and two had increased glucan/mannan ratios in their cell walls. All combinations of mutants showed positive complementation in heterozygous diploids, although complementation between one pair, which had the same phenotype, was incomplete, indicating that four to five different cistrons were involved. All mutations were found to be recessive. Haploid strains bearing pairs of ses mutations were not markedly more sensitive to mammalian digestive enzymes than strains with single mutations. Rat-feeding experiments with three mutants and the parental strains indicated that the protein was efficiently utilized in all cases. Net protein ratios for the two mutants of S. cerevisiae tested were slightly higher than that for their parent, but the differences were of marginal significance.

Production of Microbial Biomass Protein from Potato Processing Wastes by Cephalosporium eichhorniae.

The use of Cephalosporium eichhorniae 152 (ATCC 38255) (reclassified as Acremonium alabamense; see Addendum in Proof), a thermophilic, acidophilic, amylolytic fungus, for the conversion of potato processing wastes into microbial protein for use as animal feed was studied. The fungus was not inhibited by alpha-solanine or beta-2-chaconine, antimicrobial compounds in potatoes, or by morpholine or cyclohexylamine (additives to steam used in the peeling process) at levels likely to be encountered in this substrate. Mixed effluent from holding tanks at a potato-processing plant contained about 10 bacteria per ml and inhibited fungal growth. The fungus grew well on fresh potato wastes containing up to 5% total carbohydrate and utilized both starch and protein at 45 degrees C and pH 3.75. On potato homogenate medium containing 2% carbohydrate (about 14% fresh potato) supplemented with monoammonium phosphate (0.506 g/liter) and ferric iron (0.1 g/liter), with pH control (at 3.75) and additional nitrogen supplied by the automatic addition of ammonium hydroxide, typical yields were 0.61 g (dry weight) of product and 0.3 g of crude protein per g of carbohydrate supplied. An aerobic, spore-forming bacterium, related to Bacillus brevis, commonly contaminated nonsterilized batch cultures but was destroyed by heating for 15 min at 100 degrees C.

Sequential cold-sensitive mutations in Aspergillus fumigatus. II. Analysis by the parasexual cycle.

From Aspergillus fumigatus I-21 (ATCC 32722), which grows at temperatures from 12 to 50 degrees C, three multistep, independently derived, cold-sensitive mutants unable to grow at 37 degrees C or below (Cs-37) were obtained by sequential exposure to ethylmethane sulfonate (strain AT2) or N-methyl-N'-nitro-N-nitrosoguanidine (AT1 and AT3). These mutants and ON5, a five-step Cs-37 mutant, were marked by mutations affecting spore color and nutritional requirements and crossed in four combinations by classical parasexual means. The heterokaryons demonstrated partial complementation with respect to auxotrophic requirements (suboptimal growth on minimal medium) and cold sensitivity (growth at 37 degrees C but not at 25 degrees C). Most presumed diploids, formed by exposure of the heterokaryons to d-camphor vapors, showed complete complementation but were unstable, as demonstrated by variations in spore sizes and markedly different ratios of segregant classes derived from different clones. Analysis of the segregants of the diploids or aneuploids, induced by Benomyl, indicated that multiple genes were responsible for cold sensitivity in each Cs-37 mutant, since segregants with various levels of cold sensitivity were obtained. The higher than predicted frequency of reversion to temperatures two or more steps back in the sequence of cold sensitivity mutations suggested that these genes or their products interacted. No Cs-37 segregant yielding a consistently lower frequency of revertants than the original mutants was obtained.

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae.

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45 degrees to 47 degrees C, maximum protein yield at 45 degrees C, and no growth at 25 degrees C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45 degrees C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10 to 10 per animal) injected by various routes into normal and gamma-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.