Kenneth B. Raper

Cell aggregation in Dictyostelium discoideum.

Abstract The capacity of small populations of myxamebas of Dictyostelium discoideum to aggregate, i.e., to form pseudoplasmodia, was investigated under contrasting cultural and environmental conditions, including: (1) populations developed in situ on washed or purified agar where growth of myxamebas was restricted by limiting the nutrient (bacteria), and (2) populations of pregrown myxamebas that were harvested, washed, and deposited in known numbers at predetermined densities upon washed or purified agar varying in composition and prepared in different ways. The investigations centered upon the type culture of Dictyostelium discoideum (strain NC-4) and certain substrains derived from it, but included also nine other isolations of this species unrelated to NC-4 in point of origin. Aggregative capacity was found to be similar in all these when small populations were grown and tested under like conditions. The conditions under which tests of aggregative capacity were made profoundly influenced the results obtained. Factors of primary importance to aggregation seemed to include the following: (1) the existence of a critical relationship between the number of myxamebas and their density per unit area of substrate; (2) the necessity for cells to remain within reasonable proximity over a period of several hours subsequent to nutrient depletion in undisturbed cultures or after deposition on agar of pregrown populations in minute drops of water or saline; (3) the removal by washing from normal agar of certain unidentified substances that permit spreading of implanted drops and accentuate cell dispersal; and (4) the optimal reconstitution and/or preparation of the agar gel. The ability of a small population of 100–150 myxamebas, or fewer, to aggregate was found to be independent of any special type of cell recognizable by the writers. Rather this capacity is thought to result from physiological changes emergent within the population as a whole following nutrient depletion, on the one hand, or implantation of a pregrown population on the other. Unusually large myxamebas, believed to represent the so-called “I-cells” reported in existing literature, were occasionally observed; but such did not occur in any reasonably definite ratio in our cultures, and their diverse characteristics seemed to suggest no common origin. Finally, their total absence from the great majority of our test populations fails to support the notion that they represent a unique cell type of special significance to pseudoplasmodium formation in Dictyostelium discoideum .

The microbiological production of gibberellins A and X.

Abstract A method is described for the production of crude crystalline gibberellin in yields of about 12 g. per 160 gal. of culture liquor. This product is a mixture of the known gibberellin A ([α]D +36 °) and a new gibberellin (C19H22O6) with a rotation of [α]D +92 °.

Preservation of Molds by the Lyophil Process

Subsequent to the establishment of the four Regional Research Laboratories by the Department of Agriculture, a large collection of industrially important microorganisms was assembled at the Northern Regional Research Laboratory as an integral part of the research program of the Fermentation Division. From the outset it was recognized that this collection could be of the greatest possible value only if variation in the organisms was kept at an absolute minimum. It was likewise recognized that the routine labor involved in propagating individual cultures should be reduced as much as possible in order that a large and varied collection could be maintained. The accomplishment of both objectives through preservation of cultures by some type of vacuum desiccation from the frozen state appeared as a real possibility. The experience of numerous bacteriologists (Shackell, 1909; Hammer, 1911; Rogers, 1914; Swift, 1921, 1937; Brown, 1932; Elser, Thomas, and Steffen, 1935; and Flosdorf and Mudd, 1935, 1938) during the past quarter of a century left little doubt that this method could be successfully used for the preservation of bacterial cultures contained in the collection. Some published reports (Rogers, 1914; Elser, Thomas and Steffen, 1935) and other work then known to be in progress (Wickerham and Andreasen, 1942) likewise indicated that the method could probably be successfully applied to the yeasts. Regarding the molds, however, there were few and fragmentary reports, and these were generally not too

Spore germination inDictyostelium discoideum: Trehalase and the requirement for protein synthesis

Abstract Protein synthesis occurs during spore germination of Dictyostelium discoideum . The use of amino acid analogs, cycloheximide, and puromycin demonstrated that the emergence stage of germination required this synthesis for completion. The reversible inhibition of trehalase formation demonstrated that protein synthesis was probably required for the synthesis of new proteins as well as for protein turnover during germination. Not all enzymes needed for spore germination appear to be made de novo during the process itself. It was hypothesized that glucose served as the energy source for the early stages of germination whereas trehalose served for the later stages. The possibility of a link between trehalase activity and the autoinhibitor(s) of spore germination was discussed.

Synoptic key to Aspergillus nidulans group species and related Emericella species

A synoptic key for identification of 30 taxa in the Aspergillus nidulans-Emericella assemblage is presented. The key uses definitive and readily observable features : conidiophore length, width of conidial columns, hulle cell distribution and colour in mass, ascospore morphology, location of cleistothecia in relation to hulle cells, and, for certain taxa, colony appearance with specified growth conditions. Brief descriptions are included as an aid to comparison and to guide the reader to detailed technical descriptions.

Induction of fruiting in two aggregateless mutants of Dictyostelium discoideum.

Abstract Five general groups of morphogenetically aberrant mutants of Dictyostelium discoideum were isolated. Each group of mutants was characterized either by the absence of any fruiting structures or by the formation of abnormal fructifications. Among these developmental mutants were two aggregateless isolates, Agg−-1 and Agg−-2, that could be induced to form normal sorocarps under certain conditions. Sorocarps of the normal D. discoideum type were formed when growing myxamoebae from either of these mutants were allowed to come in contact with myxamoebae of the other mutants, wild-type D. discoideum, D. purpureum, or D. mucoroides. No sorocarps were formed when myxamoebae of Agg−-1 and Agg−-2 were paired. These two aggregateless mutants, while incapable of aggregating or fruiting when cultivated singly with Escherichia coli B/r on a glucose-salts medium, formed normal fruiting structures after being freed of what appeared to be a product of bacterial growth. The spores produced by Agg−-1 and Agg−-2 myxamoebae again gave rise to aggregateless clones of the original parental types.

Control of sorocarp size in the cellular slime mold Dictyostelium discoideum

Abstract Factors that control the size of sorocarps in Dictyostelium discoideum were investigated. Artificially crowding large populations of myxamoebae of the wild type, NC-4(S2), prior to culmination did not increase the average size of the developing fruiting bodies compared to controls grown and permitted to fruit on the surface of conventional plate cultures. It was concluded that there exists a critical mass of cells above which complete intercellular integration cannot be maintained; loss of such integration within a cell population leads to the formation of two or more sorocarps. Because of this critical mass, sorocarps do not exceed a certain size even in the presence of an abnormally large number of myxamoebae. Certain petite mutants, derived from the wild type, develop only small sorocarps in both conventional plate cultures and artificially crowded populations. Other mutants form fruiting bodies similar to the wild type in such crowded populations although they remain petite in plate cultures of the usual type. By employing a method for measuring directly the size of the critical mass, it could be shown that the petites of the second group had a critical mass almost identical to that of the wild type; in contrast, the critical mass of the first group was reduced under all conditions. It was shown, therefore, that the size of the critical mass is under genetical control and may be altered by mutation. In crowded populations of myxamoebae the critical mass of the strain or mutant is responsible in limiting the size of the sorocarps. In sparse populations, on the other hand, the size of the aggregation territory and the density of the myxamoebae per unit area assume determining roles.

Acytostelium leptosomum: A Unique Cellular Slime Mould with an Acellular Stalk

SUMMARY: A new genus and species of the Acrasieae is described, and for this the binomial Acytostelium leptosomum is applied in recognition of its acellular sorophores and the diminutive proportions of its sorocarps. This slime mould has been isolated upon four occasions from soil and forest litter collected from deciduous forests within the United States. The vegetative stage of A. leptosomum, as that of the other cellular slime moulds of this Class, consists of the independent growth of myxamoebae which feed upon bacterial cells. The fruiting stage likewise duplicates that of other members of the Acrasieae in its early manifestations; but it differs markedly from these in its terminal phase, i.e. the construction of the mature fructification, or sorocarp. A. leptosomum is at present unique among these slime moulds in its capacity to produce a sorocarp with a non-cellular stalk. The morphogenetic process whereby this is accomplished is not thoroughly understood; nevertheless, a possible explanation of how the stalk may be formed without the expenditure of any cells is offered.

THE INFLUENCE OF LIGHT ON THE TIME OF CELL AGGREGATION IN THE DICTYOSTELIACEAE

The time at which aggregation begins in the Dictyosteliaceae was found to be influenced by the conditions of light to which the myxamoebae were exposed during the vegetative and preaggregative stages. The myxamoebae were usually pregrown in the dark on Escherichia coli or Aerobacter aerogenes, harvested, washed by centrifugation, resuspended, deposited on non-nutrient agar, and incubated under varying regimens of light and darkness. Myxamoebae of the single strains of Dictyostelium mucoroides, D. purpureum, Polysphondylium violaceum and P. pallidum included in the tests, and one strain of D. discoideum (Acr-12), aggregated earliest if exposed to constant light. Other strains of D. discoideum, including the type, NC-4, and substrains derived from it, aggregated optimally after an initial dark period of 6 to 8 hours followed by about 4 hours of light. When these latter strains were grown in light rather than in darkness, the initial dark period required for early aggregation was reduced to 4 hours. When lig...

The fine structure of macrocyst germination in Dictyostelium mucoroides.

Abstract The ultrastructure of macrocysts during germination is described from OsO 4 fixed material. The process begins with the splitting of the tertiary wall into two parts. This is followed by the cleavage of the protoplast into large uninucleate pro-amoebae. As these proamoebae become less electron dense most of the endocyte fragments are digested. Simultaneously the secondary wall and the outer part of the tertiary wall break, leaving only the thin inner part of the tertiary wall intact. The pro-amoebae divide several times to produce the myxamoebae that escape during germination by breaking through the inner layer of the tertiary wall. These myxamoebae multiply or form sorocarps or macrocysts, depending on conditions.