A genomic library of the asexual pathogenic yeast Candida albicans was constructed in the S. cerevisiae vector YEp13. The library contains a representation of the entire genome with a probability of 99%. The expression of the genes of C. albicans in S. cerevisiae was examined and two mutations his3-1 and trp1-289 of S. cerevisiae were complemented by the cloned genes of C. albicans. The hybridization data indicates that the plasmids complementing the mutations of S. cerevisiae contain sequences from C. albicans.
We removed by recombinant deoxyribonucleic acid (DNA) techniques a small DNA segment from within a cloned gene (the 0.4 kb gene) in which transcription in under sporulation control in Bacillus subtilis. These deletion mutation was introduced into the B. subtilis chromosome by transformation with cloned DNA. Competent cells bearing a mutation (tms-26) that is closely linked to the 0.4 kb gene were transformed with linearized plasmid DNA containing the truncated 0.4 kb gene and the wild-type allele of the tms locus. Selection for Tms+ transformants yielded oligosporogenous mutants of unusually dark-brown colony pigmentation. This phenotype was caused by a mutation which mapped at or very near the site of the 0.4 kg gene deletion, whose presence and position in chromosomal DNA was confirmed by Southern hybridization analysis. Phase-contrast microscopy and electron microscopy showed that the mutation, which we designated as spoVG, impaired sporulation at about the fifth stage; bacteria harboring the spoVG mutation proceeded normally through stage IV of development but frequently lysed thereafter, apparently as a result of disintegration of an immature spore cortex. This identifies the 0.4 kb gene (or DNA in its immediate vicinity) as a new sporulation locus and shows that its product functions at a late stage in development.
A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 ,ug of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.Myxococcus xanthus, the most extensively studied member of the Myxobacterales, is a gram-negative, gliding bacterium, characterized by a complex, multicellular development program (33). Development of M. xanthus, which culminates in the building of spore-filled fruiting bodies, is contingent upon the fulfillment of several conditions: (i) a minimal cell density, (ii) a solid substratum to support the aggregating cells, and (iii) nutritional downshift of exponentially growing cells (25). Successful completion of the developmental process depends upon the proper functioning of many genes. Some of these genes are directly involved in the intercellular signaling necessary for coordinating specific multicellular activities (16); others affect development by controlling the motility (11, 12) or cohesiveness (27) of the cells.Autocides are substances produced by vegetative M. xanthus cells which are capable of inducing lysis in the producing and related strains (29). The two major autocides, referred to as AMI and AMV, have been isolated and chemically characterized (6, 30). Autocide AMV is phosphatidylethanolamine (PE), the major component of the inner and outer cell membranes of M. xanthus. Autocide AMI, a mixture of saturated, monounsaturated, and polyunsaturated fatty acids, appears to be an enzymatic breakdown product of AMV and its active lytic moiety. In addition to its autolytic activity, autocide AMI has recently been shown to be capable of accelerating aggregation and sporulation in the wild-type strain and of rescuing aggregation and sporulation in dsg development mutan...
When suspended in a liquid starvation medium, exponentially growing Myxococcus xanthus sporulated within 3 days. These myxospores were similar to spores developed within fruiting bodies, as determined by electron microscopy and the production of spore-specific protein S. This liquid sporulation system may be useful as a means of preparing large quantities of myxospores incubation, approximately 15% of the initial number of DK1622 cells had become mature, resistant spores and had aggregated into large, macroscopic clumps. When M. xanthus DK5057, a mutant incapable of fruiting-body formation and sporulation under classic developmental conditions (10), was prepared in the manner described above, fewer than 50 spores per ml were observed, although the vegetative cells remained viable under these conditions for at least 6 days (data not shown).The rapid decrease in turbidity of the DK1622 culture may be due to lysis of some proportion of the cells, to a change in turbidity due to the formation of macroscopic clumps, or to some interplay of the two processes. The culture containing DK5057 cells gradually decreased in turbidity, although no macroscopic clumping was observed. Cell-cell interactions play an important role in the life-style of M. xanthus (7,13,20), and it is interesting that, in the absence of a solid surface, the MCM sporulating cells seek an alternative form of multicellular cooperation in order to carry out an altered form of the developmental program. A similar effect was noted by Burchard (3). Vegetative cells of a nondispersed growing mutant formed large clumps in 1CT growth medium; when transferred to starvation buffer, these cells differentiated into spores shaped like fruiting-body spores within these clumps.In order to compare spores produced in MCM buffer with fruiting-body and glycerol-induced spores, thin sections of spores were prepared for electron microscopic analysis by the procedure of Inouye et al. (11). Both MCM spores and fruiting-body spores displayed thick, multilayered extracellular capsules which were absent in glycerol-induced spore samples (Fig. 2).The presence of protein S further demonstrated that MCM spores resembled fruiting-body spores and differed from glycerol-induced spores. Protein S is a spore-specific protein whose synthesis is turned on early in development (12) and which is subsequently assembled onto the surface of the spores. Rinsed and sonicated 7-day spore samples at ca. 3 x 107/ml were incubated for 60 min at 37°C in the presence of an equal volume of a 12-mg/ml stock solution of rabbit anti-protein S immunoglobulin G (IgG) prepared in 0.15 M NaCl; 10 mM CaCl2 was added to prevent removal of protein S from the spores by NaCl (11). Samples were centrifuged at 15,000 rpm (Mikroliter; Hettich, Tuttlingen, Federal Republic of Germany) for 2 min, rinsed once, and suspended in 0.15 M NaCl-10 mM CaCl2 at 10 times the original volume. A second incubation was performed at 37°C for 30 min in the presence of 1/10 volume fluorescein isothiocyanate-conjugated goat anti-ra...
Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pill on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A- S+ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A+ S- mutants, i.e., strains capable of movement by the A-motility system only, and (iii) a retardation of streaming speed in the wild-type strain (A+ S+). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pill. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.
Vegetative cells of Myxococcus xanthus were immobilized in 25-jLm-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus. Multicellular development and differentiation comprise a complex set of processes that have been studied in many systems. Among the simplest organisms capable of the cell communication and interaction necessary to carry out such a sequence of events are the members of the order Myxobacterales, of which Myxococcus xanthus is the most extensively studied (11,15).In this species, development into heat-resistant spores proceeds within macroscopic fruiting bodies. Initiation of these events depends upon the 'fulfillment of several prerequisites: (i) a solid surface must be available to support the developing fruiting bodies, (ii) a downshift in the availability of certain nutrients must occur, and (iii) the cell culture must have achieved a critical density prior to starvation (14).Close to 15 years ago, Wireman and Dworkin reported that the development of M. xanthus was accompanied by cell lysis (18, 19). This developmental lysis has played an important role as a marker of development (see, e.g., reference 17). However, an easily reproducible methodology for measuring this lysis has been difficult to develop. In an effort to examine developmental lysis by using new methods, at least one group has obtained results which have led to a reevaluation of the very existence of developmental lysis (9). The present study presents evidence for a strong correlation between lysis and sporulation in M. xanthus and indicates that both of these phenomena are cell density dependent, as has been shown for fruiting-body development (4, 16).A recently developed technique (R. Nir, R. Lamed, and E. Sahar, manuscript in preparation) was used for studying various aspects of the myxobacterial life ...
Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo-mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo-mutant classes, most dramatically in the dsg mutants.Myxobacteria are unique in the procaryotic world, displaying several atypical characteristics: (i) gliding motility, (ii) communal aspects during the entire life cycle, and (iii) a developmental program under starvation conditions culminating in the production of spore-bearing fruiting bodies (for a review, see reference 13).In the 1970s, it was reported that an integral part of the developmental program is massive cell lysis (19,20). This finding has been corroborated over the years by various investigators (e.g., see references 5, 7, and 15). It has been suggested that the lysed cells serve as signals or nutrients or both for the developing cells. Recently, however, the same laboratory which originally demonstrated this lysis has succeeded in interrupting lysis (by the use of a specific monoclonal antibody) without interfering with the developmental process (M. Dworkin, personal communication). Apparently, fruiting body formation, sporulation, and cell lysis interplay in complex ways which are only now beginning to be appreciated.Myxococcus xanthus produces at least five substances, termed autocides, which induce lysis in cultures of the producing and closely related strains (16). One of the two major autocides, AMI, has been shown to be a mixture of saturated and unsaturated free fatty acids (17). The other major autocide, AMV, is phosphatidylethanolamine (2). The autolytic activity of AMV depends on it being degraded to AMI by phospholipases. All strains of M. xanthus tested were sensitive to 1 ,ug of AMI per ml. The observations that developing cells undergo autolysis and that M. xanthus cells are peculiarly sensitive to lysis by the autocides led us to the hypothesis that these autocides play a role in developmental lysis.One category of sporulation-deficient (Spo-) mutants, those defective in cell-cell signaling, has been grouped into four classes (4, 11). This classification is based on the ability of two different mutants to complement each other extracelluarly, thereby forming fruiting bodies and spores. Two mutants belonging to the same class are incapable of this phenotypic complementation, whereas two mutants capable of complementation will, by definition, be assign...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.