Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads

Abstract

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 ...

Rippling Is a Predatory Behavior in Myxococcus xanthus

“…Since rippling pattern formation occurs similarly when M. xanthus cells are in the presence of dead prey as it does with live prey, we felt that it was prudent to examine the role of developmental autolysis in connection with developmental rippling. There is contradictory evidence as to whether or not cell lysis is an essential component of the developmental program, and we considered that part of this discrepancy could be strain dependent (18,22). We analyzed the autolytic rates of DZ2 and DK1622 cells that had been resuspended in MMC buffer and incubated in 12-well plates at 32°C.…”

Role of autocide AMI in development of Myxococcus xanthus

“…The first proposed mechanism is based on the observed lytic effect of low concentrations of autocides and model fatty acids on vegetative and starving M. xanthus cells (6,29,30). The observation that a substantial proportion of a developing population of M. xanthus cells undergo lysis (22,32) led to the notion that autocides affect development by initiating this observed developmental lysis, thus releasing cellular components necessary for development to proceed. Accordingly, rescue of development would be observed in mutants which were incapable of sufficient lysis to initiate development on their own.…”

“…In addition, most of the isobranched fatty acids tested displayed lytic activity but no detectable spore rescue activity. It should be noted that the lysis activity of lipids was measured only on vegetative cultures; demonstration of developmental lysis has been reported in microbeads (22), but the procedure is complex and does not lend itself to routine testing of lytic compounds.…”

Flow cytometric analysis of entire microbial colonies

“…For example, the interaction between cells held together at close proximity may be studied (7). In particular such interactions between two different strains co-entrapped in the same bead can serve as a bioassay to detect products of one strain that inhibit or enhance the growth of another strain.…”

“…The ability to perform such studies would be enhanced significantly if such colonies were made amenable to studies by flow cytometry. Weaver et al (6,11,12) and Nir et al (3)(4)(5)7,8) have suggested that this can be done by entrapping the cells within small spheres in a way that would allow the cells to multiply and form a microcolony that is fully contained within the spheres. Methods for performing this entrapment within highly uniform spheres of the size range amenable to flow cytometric analysis have been developed (3).…”