Relationship of the major constituents of the Neurospora crassa cell wall to wild-type and colonial morphology. J. Bacteriol. 90:1073-1081. 1965.-The relationship of cell wall to morphology in Neurospora crassa was studied by correlating the levels of structural polymers of the cell wall with wild-type and colonial morphology. The cell wall of N. crassa contains at least four major complexes: a peptide-polysaccharide complex; two glucose polymers, one of which was found to be a laminarinlike,8-1,3-glucan; and, lastly, chitin. The levels of one or more of these structural polymers are consistently altered in single-gene mutants with colonial growth, and in sorbose-induced colonial growth. The proportions of these polymers, particularly of the peptide-polysaccharide complex and the f3-1,3-glucan, appear to be important to morphology.
The distribution and localization of structural polymers in the cell wall of Neurospora crassa has been studied by selective removal and light and electron microscope examination. Observations with the light microscope indicated that each polymer by itself can provide structural integrity to the cell wall. Examination by electron microscopy showed that the cell wall consists of an outer layer of thick fibrils, identified chemically as a glucan-peptidegalactosamine complex, and an inner layer made up of f-1,3 glucan and thin fibrils of chitin.
Human peripheral blood mononuclear cell proliferation induced by Mycobacterium leprae could be inhibited by the suppressor factor in the lysate of the macrophages of lepromatous leprosy patients. Macrophages from normal subjects and tuberculoid patients did not show production of a suppressor factor. Inhibition occurred only when the factor was present in the initial stages of lymphocyte culture. The factor is heat stable and nondialyzable. Proliferation induced by some mycobacteria and concanavalin A could also be blocked by the factor. Interestingly, blastogenic response by a few other antigens and phytohemagglutinin could not be inhibited by the suppressor factor. Mononuclear cells pretreated with such lysate from lepromatous macrophages for 24 h could induce suppressive activity in the cells in vitro in an autologous system. Treatment of these cells with carbonyl iron after the induction phase, to remove phagocytic cells, did not abolish their suppressive activity. The lepromatous macrophage lysate also generated suppressive activity in a T-lymphocyte-enriched population of normal subjects. These studies are interpreted to indicate that immunosuppression in lepromatous patients is produced by both macrophages and T lymphocytes. The exact phase in which either of these cells acts as a suppressor may be different. Specific suppression by macrophages to M. leprae can be an early event, and nonspecific suppression by T lymphocytes may be a later event in the course of lepromatous leprosy.
The cell wall of Neurospora crassa contains bound enzymes that can digest its structural polymers. These enzymes are not present at the same levels at all stages of growth. The levels of these autolytic enzymes vary and generally show some relationship to the process of branching. These enzymes were removed from the cell wall by f3-mercaptoethanol extraction and were tested for activity against isolated cell wall fractions. Such studies, as well as autolytic studies, showed that enzymes acting on the protein portion of the cell wall (proteases) are more prominent than enzymes that act on the glucan portion (glucanases) of the cell wall. Comparative studies between the wild type and a spreading colonial mutant spco-J showed that earlier and higher frequency of branching in spco-J was correlated with a greater amount of these enzymes bound to the cell walls. It is concluded from these observations that autolytic enzymes acting on the protein and glucan portion of the cell walls occur as wall-bound and participate in the process of branching in Neurospora.The important role of the cell wall and the relative ratios of cell wall polymers in the control of morphology in Neurospora crassa have been clearly demonstrated (3, 6). This relationship was established by studying the cell wall polymers of the wild type and several single-gene-controlled morphological mutants of the fungus. Among the morphological mutants, those that grow compactly and tightly, referred to as "colonial," have higher frequencies of branches per unit length of mycelia than the wild-type strain (3). This might lead to the compactness in growth that appears to distinguish it from the wild type. Thus the process of branching becomes very important and biochemical processes behind the morphological variation due to branching are of interest. It was suggested that changes in the relative ratios of cell wall polymers (6), especially those that clearly add to the structural integrity of the cell wall (7), can result in alteration of branching leading to morphological variation. Studies carried out to determine the presence of enzymes bound to the wall and the activity of these enzymes during the growth of the fungus are reported in this paper. Such enzymes might play a part in the process of branching in the fungus. This study was made after observing the presence of an enzyme in the extract of Neurospora with activity toward ,B-1, 3 glucan of the cell wall. MATERIALS AND METHODSThe strains of N. crassa used were Perkins wild type (PA) and a spreading colonial mutant, spco-1 . Conidia were obtained from cultures of the above strains, grown regularly in 2% agar medium containing 2% sucrose and minimal salt solution. The conidial suspension in water was filtered through glass wool to remove mycelial debris and agar pieces. The conidia in the suspension were counted, and up to 3 X 1010 conidia were inoculated in 150 ml of 2% sucroseminimal salt medium and grown on a rotary shaker for various periods. After each period, the culture was filtered through a mem...
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