A newly recognized protozoan human parasite, Cyclospora has been incriminated as the cause of prolonged diarrhea. Five patients had episodes of diarrhea accompanied by nausea, weight loss, and/or low-grade fever for 10-45 days. Multiple fecal samples fixed in sodium acetate-acetic acid-formalin contained spherical organisms measuring 8-10 microns in diameter; a modified concentration technique was used to detect them. The sediment was examined by direct microscopy and autofluorescence, and the identification was confirmed by acid-fast stain. All patients had visited either Mexico or Thailand. The presence of Cyclospora organisms in these patients shows that these can be etiologic agents of traveler's diarrhea in both immunocompetent and immunocompromised hosts. Fecal specimens from patients with unexplained diarrhea should be routinely examined for their presence.

Spores of Bacillus cereus mutants selected for slow response to germinants and sensitivity to lysozyme were found to be deficient in coat, but were heat-resistant and contained the same quantity of dipicolinic acid as the wild-type. While the average coat protein content of the spores was 25% of the wild-type, many spores were coatless with large whorls of coat deposited in the cytoplasm. These coat deposits were isolated in Renografin gradients and found to cross-react immunologically with wild-type coat. The proteins extractable from these deposits were virtually identical to those extracted from wild-type spores. The morphology of the coat deposits was very similar to coats of wild-type spores, but with a deficiency of the outermost cross-patched layer. The sites of formation and deposition were altered. Since the mutant reverted to a phenotype identical to the parental strain with a frequency consistent with an initial point mutation, apparently a single defect resulted in alteration of the deposition of the spore coats on to the outer forespore membrane. Despite this defect, mutant cells were able to synthesize and process spore coat precursors into an array of morphological layers very similar to the wild-type. There are apparently distinct morphogenetic pathways for the formation of the spore body and coat layers.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38°C). The spores also germinated slowly when produced at 350C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 350C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 103 plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 109 PFU/ml at 37 C and 7 x 108 PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.Preliminary investigations of five temperature-sensitive (ts) sporulation mutants of Bacillus cereus T indicated that a sporadically induced lytic agent was present in the culture supernatant fluids of all five mutants. Through its ability to form discrete plaques on lawns of sensitive bacteria and through observation of the particles under the electron microscope, we were able to identify the lytic agent as a bacteriophage.The present paper describes the isolation and preliminary characterization of the ts mutants, some physical and biological properties of the phage which distinguish it from similar phages previously studied (1, 6, 16, 17; Goldberg and Gollakata, Bacteriol. Proc. p. 47, 1960; Gollakata and Halvorson, Bacteriol. Proc. p. 86, 1960), and some aspects of the phage-host cell relationship in the mutants and in the parent strain. MATERIALS AND METHODSBacterial strains and cultivation. The strain from which our mutants were derived was a trypto-' Journal Article no. 6184 from the Michigan Agricultural Experiment Station.phan auxotroph of B. cereus T, isolated in our laboratory. The tryptophan requirement was a convenient marker for verifying the origin of the sporulation mutants. B. cereus NRRL569 and B. cereus ATCC6464 UM4, which are sensitive to bacteriophage CP-53, and B. cereus ATCC6464, which carries phage CP-53, were obtained from Curtis B. Thorne (University of Massachusetts, Amherst). Stocks of all bacterial strains were stored in 60% glycerol buffered with 0.1 M sodium phosphate (pH 7.0) at -20 C. These stocks were transferred at 6-month intervals. The stock spore suspensions, from which inoculations were made routinely, were grown either in G medium (8)...