A Quantitative Assay to Study Cell Movement in the Myxobacteria

jf. Cell Sci. 25, 173-178 (i977) 173 Printed in Great Britain

A QUANTITATIVE ASSAY TO STUDY CELL MOVEMENT IN THE MYXOBACTERIA

JOSEPH LONSKI, RONALD HEROMIN AND DAVID INGRAHAM Department of Biology, Bucknell University, Lezvisburg, Pennsylvania, U.S.A.

SUMMARY A simple quantitative assay has been developed to test the rate of cell movement of myxobacteria. The assay employs an agar surface and at no time are the cells cultured in a liquid environment. Isolation of a rate-increasing substance(s) from fruiting Myxococcus xartthus is reported. The understanding of the aggregative process in these bacteria will be aided by characterization of the chemotactic system.

INTRODUCTION The myxobacteria represent a unique group of procaryotes which interact with each other throughout their life cycle. These interactions play a role both in the swarming and feeding process, as well as in fruiting body formation. The general properties of the myxobacteria have been reviewed several times (Dworkin, 1966; 1972). A distinctive feature of the myxobacteria is that when cells on the surface of a solid medium are deprived of specific nutrients, they shift from growth to development and begin to migrate, by means of a gliding motility, into aggregation centres (Dworkin, 1963). A number of investigations (Lev, 1954; Jennings, 1961; McVittie & Zahler, 1962; Fleugel, 1963) have suggested that this aggregation is in response to a chemotactic substance but this has not yet been carefully examined. Having aggregated, the cells then construct a macroscopic fruiting body. For Myxococcus xanthus this is a simple mound of myxospores encased in a slime sheath. This research was initiated to aid in the search for the mechanism of aggregation of these procaryotes. In order to understand the mechanism it will be necessary to know the chemical identity of the chemotactic agent. We describe here a quantitative bioassay which does allow for the first time an analysis of the response of myxobacteria to various chemical substances in a controlled environment. The problem was approached by preparing stable solutions from the area of active fruiting body formation by M. xanthus on agar. It was observed that in the presence of these solutions starved myxobacteria moved more rapidly on an agar surface, and this could be quantified, since the speed of the myxobacteria was found to be proportional to the concentration of the extracted preparation. As was the case for the early work on aggregation in the cellular slime moulds (Bonner, Kelso & Gilmor, 1966), it is not known whether the chemical solution contains both an orientation factor and a separate rate-increasing substance, or whether both occur. This problem could be

12 CEL 25 174 J- Lonski, R. Heromin and D. Ingraham approached by using time-lapse photomicrography, as Reichenbach (1965) has done, to follow individual cells. The object of our assay is to record the speed of the myxobacteria in the presence of a test solution. If the bacteria are placed in contact with the text substance in a concentrated group, they will move outward from the dense centre. If their progress is measured at 2 points in time, the average rate of outward movement may be computed.

MATERIALS AND METHODS The method, which is similar to that developed for the cellular slime moulds (Bonner et al. 1966), is as follows: 1 ml of the test solution is carefully mixed with 1 ml of hot 3 % Bacto agar containing o-2 % MgSO4 (on a slide-warming tray kept at 60 °C) in a small plastic Petri dish (50 x 12 mm - Falcon No. 1006 - with tight-fitting lid). After the agar has solidified the covers are removed from the dishes for 3 min to allow excess moisture on the surface to evaporate. The myxobacteria used for the test must be prepared very carefully. Myxococais xanthus, FB (ATCC no. 25232), is continuously maintained in our laboratory as M. xanthus, JL-i, using the following methods. The cells are allowed to grow vegetatively as confluent growth for 7 days on Casitone agar plates (CT) (Dworkin, 1962). The cells are then transferred to amino acid fruiting agar plates (AAF) (Dworkin, 1963) for 4 days, where they aggregate and form fruiting bodies. The transfers are made by adding 4 ml of sterile distilled water to a donor plate and then 1 ml of the cell suspension is drawn up into a sterile pipette and cri-ml drops are placed on an acceptor plate. Sterile plastic Petri dishes (100 x 15 mm) are used. Continuous transfers from CT to AAF to CT have insured that the myxobacteria do not lose their ability to undergo their normal life cycle. Dworkin (1962) reports that M. xanthus VC lost the ability to form either myxospores or fruiting bodies after 100 serial liquid transfers. Cultures are grown at 30 °C in the dark. Stock cultures can be maintained by transferring cells from CT to phosphate-buffered agar (15% Bacto agar; 010% MgSO4; and 001 M (K2HPO4-KH2PO4) and kept in a re- frigerator at 2 °C for 4 weeks. The bacteria for the actual assay are taken from 7-day-old cultures, well into stationary phase, growing on CT plates as confluent growth. Approximately 3 ml of sterile distilled water are added to a plate and the cell suspension aseptically pipetted to circles of cellulose dialysing membrane (wall thickness 00889 mm (0-0043 m-))> roughly 6 mm in diameter. The circles are cut from a sheet of membrane using a paper punch and then boiled in EDTA solution (io~3 M) for 20 min, rinsed several times with distilled water and then autoclaved. The circles are then placed on agar in Petri dishes (100 x 15 mm) containing 1-5 % Bacto agar and o-i % MgSO4 before pipetting the cells. The bacteria are allowed to incubate on the circles for 20-24 h at 30 °C, so as to sensitize the cells to starvation but not initiate fruiting-body formation. The circles with the sensitized bacteria are removed with forceps and placed on one of the test Petri dishes, bacteria side up, 3 circles being placed in each dish. All of the procedures utilize aseptic technique and are carried out in a laminar flow clean air station. The test plates are incubated in a 30 °C incubator, dark, for approximately 24 h. During this time the bacteria will have crossed the edge of the cellulose circle on to the agar. With a needle holder, 6 grooves 2 mm in width are scratched on the bottom of the plastic dish at a tangent to the cellophane circles. Then with the use of an ocular micrometer in a dissecting microscope the distances between a line scratched near the edge of the cellophane circle and 4 streams of cells furthest out are measured and averaged, for each of the 6 grooves. The same procedure is repeated after 8 h and thus it is possible to have rates of outward movement of the bacteria expressed in terms of mm h"1. At the time of the second test readings well defined aggregation streams are usually present on the agar control plates and mature fruiting bodies are found after 4 days. Cell movement and myxobacteria 175

RESULTS Over the last 2 years we have found the test to be very consistent. Data from 130 control plates during this period show a mean rate of 0-054 mm h"1 (standard devi- ation o-on). In order to give some idea of the consistency of this test, 4 different sets of data have been chosen at random from the past 2 years and are presented in Table 1.

Table 1. Representative data to demonstrate consistency of method

Increase of experi- Experi- mental Control, mental, over 1 1 Date mm h" Mean S.D. mm h" Mean S.D. control, %

0056 0-132 0-049 0-126 0-051 Aug. 1975 O-I2I 0-054 OI25 0054 OIl8 0048 0-052 0-004 O-I22 0-008 J35 0-050 0050 0160 0-046 0-164 Nov. 1975 0-049 0-168 0-047 0-171 0052 0-049 0159 0163 0-007 233 0-048 0-179 0050 0-183 Dec. 1975 0-054 0-187 0-052 0196 0059 0-192 0-056 0-053 0005 0-181 0188 0-007 254 0-057 0-189 0-060 0-194 0-060 0-191 Feb. 1976 0-056 0199 0052 0202 0059 0057 0004 0188 0-194 0-006 240 Four experiments and their controls picked at random. Each column of 6 figures represents the 6 marked sides of the 3 membrane circles on one Petri dish. Each figure is the mean of the 4 fastest streams of cells at about 20 h subtracted from the 4 fastest at 8 h later. The standard deviations are of the 6 means (of 4 streams each).

A number of variables were examined while developing this test. The age of the cells, either starved or non-starved, did not affect the control rates of movement. However, vegetative cells (taken directly from CT agar plates and placed on cellophane circles) do not respond to the isolated rate-increasing substance(s) described in this paper. The length of time between the initial reading and the final reading was also treated as a variable. Data collected from control plates using 2, 4, 8, 10, or 20 h indicate that 176 J. Lonski, R. Heromin and D. Ingraham the mean rates of movement were nearly the same but with times less than 6 h the mean range was larger. This is probably due to the error involved in discerning shorter distances of cell movement between initial and final readings. Rates of movement are unaffected by light (fluorescent lamps, cool white, 500- 800 ft. c. (5-28 x io3 —8-45 x io3 lm m~2)) and gravity. Cells move at the same rate in this test on Petri dishes incubated right side up, inverted, and vertically. The tempera- ture optimum for this test is approximately 30 °C. These findings confirm the report of Burchard (1974) on M. xanthus, IBy, a gliding strain. Measurements of rates of cell movement as a function of agar concentration were also recorded. These indicate that varying the agar concentration (1-3%) had no effect on the mean rates of movement. However, agar concentrations below 0-5 % and above 3-5% were not satisfactory. Burchard (1974) has reported that the maximum rate of colony expansion of M. xanthus was always observed on 1-1*5 % agar; minimal rates were observed at 0-5 and 3% agar. Colony expansion of gliding strains is attributable to active movement as well as an increase in cell mass when nutrients are available. Burchard (1974) observed that colony expansion occurs on 1 % purified agar containing only salts and EDTA and explained this by the fact that agar itself contains 0-3% protein by weight. The cells, which produce extracellular proteases, presumably metabolize this protein. We have not measured the cell mass during the rate test but have repeatedly made the observa- tion that with an increase in time few cells are found near the membrane circles and a distinct outer ring of cells is present when the final rate test readings are taken. Controls have not shown this behaviour. Whereas cells growing under vegetative conditions maintain cell densities in the entire area of the expanding circle, other evidence for gliding motility, rather than colony expansion due to an increase in cell mass, comes from an analysis of the rates of movement of starved cells (0-05 to 0*19 mm h"1), which are faster than can be accounted for by growth and division of these 7-/(m-long cells with generation time of 5-6 h on agar. Burchard (1974) presented similar evidence. Another variable investigated was cell density. The result was identical with all of the cell densities examined. At low cell densities (2 x io5 cells ml"1) it was more diffi- cult to follow cell movements but the rates of movement were all the same. Generally, we worked with cellulose circles covered with a uniform layer of cells at a density of 2 x io8 cells ml"1. Solutions which showed an ability to increase the rates of cell movement were obtained using the following procedure. The bacteria were grown vegetatively on CT agar plates by depositing four o-i-ml drops of cell suspension from AAF plates on the agar surface. After 7 days of vegetative growth a plate was washed free of cells with 2 ml of sterile distilled water. One millilitre of this cell suspension was deposited as drops of liquid in Petri dishes (100 x 15 mm) containing 1*5% agar and o-i % MgSO4. Two days later, during fruiting-body formation, the plate was washed free of cells with 4 ml of sterile distilled water and the collected cell suspension centrifuged immediately in a Sorvall refrigerated centrifuge at 20000 g. The supernatant was re- tained and immediately autoclaved for 20 min. The pellet material was also found to Cell movement and myxobacteria 177 increase the rate of cell movement, but will not be considered here. Work on the analysis of both the supernatant and pellet fraction is in progress. The amount of liquid recovered from one AG agar plate was used as 1 x concentration of test substance. This could be diluted with sterile distilled water or concentrated by placing the fluid in a vessel suspended in a boiling water bath. Our test was found to be quantitative when the test solution was diluted in a series of experiments and it was found that the rate of movement of the myxobacteria was reduced. A series of 1 in 5 dilutions over a 20-fold range were run using the membrane circle assay method and in 8 separate experiments it was shown that a 5-fold increase in concentration produces a i^-fold increase in the rate of movement. The plain agar controls move at about 0-05 mm h"1 and with very active solutions it is possible to obtain speeds over 0-19 mm h"1 All rate-increasing activity is lost if the substance (from either the supernatant or pellet) is not autoclaved or boiled immediately after centrifugation. This suggests that, as with the chemotactic system of some of the cellular slime moulds (Chang, 1968), an enzyme may be present which specifically destroys the substance. However, at present we have no evidence for such a mechanism. We suggest that the bacteria are generating a gradient in our assay by utilizing or degrading the test substance. It is possible that active test material is penetrating the cellophane membrane. McVittie & Zahler (1962) present results showing that centres of aggregation of M. xanthus lying below a cellulose dialysing membrane excrete a compound capable of passing through the membrane and initiating the formation of an aggregate (which later becomes a fruiting body) on the top of the membrane. Chemical substances, either shown or suspected to have chemotactic activity for the cellular slime moulds, were tested using our bioassay and were not found to increase the rates of cell movement of M. xanthus. These chemicals included cyclic AMP, 5'-AMP, ADP and folic acid. All 4 chemicals were tested separately in the concentration range of io~3 to io~6 M. This work was supported by a grant from Research Corporation.

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