JOURNAL OF BACrERIOLOGY, Mar. 1975, p. 1200-1202 Copyright i 1975 American Society for Microbiology
Vol. 121, No. 3 Printed in U.S.A.
NOTES Minicell Yield and Cell Division Suppression in Bacillus subtilis Mutants NEIL H. MENDELSON* AND SHEILA I. COYNE Department of Microbiology and Medical Technology, and Graduate Committee Arizona, Tucson, Arizona 85721
on
Genetics, University of
Received for publication 24 December 1974
Minicell yield is determined by the probability of a minicell-producing division and the relationship of growth to division in Bacillus subtilis mutants.
We recently discovered that minicell-producing cells of the div IV-A1 mutant grow as long filaments primarily because the cells are suppressed in general division ability compared with wild-type cells (2). In the present communication we examine the relationship of growth to division in another minicell-producing mutant, div IV-BJ. Our findings indicate that the div IV-Bl mutant is less suppressed in division ability than the div IV-AJ mutant and consequently minicell-producing cells of div IV-BJ are not as long as those found in div IV-A1 cultures. We have also explored the possibility of estimating minicell yields on the basis of division ability and minicell division frequency. All data were obtained from clones during the initial stages of outgrowth after spore germination. Detailed methods have been described previously (1, 4). Both mutants were studied in a common genetic background (strain CU403). Table 1 indicates the relationship of division ability to clone growth found in the analysis of 51 div IV-BJ clones. The large standard deviation of clone lengths at each stage of division was similar to that found in the div IV-Al mutant and represented individual clone differences in division ability. The equation included in Table 1 represents the overall division ability of div IV-B1 clones relative to growth. Similar equations have previously been derived for wild-type CU403 and for the div IV-Al mutant
TABLE 1. Relationship of clone length to cell division in CU403 diu IV-BJa
lStandard deviation of Avg clone clone length length (mm) clone in In clone measured measured ~(pm) No. of No. .o
No. of No. of
0 1 2 3 4 5 6 7 8 9 10 11 12 1:3
42 53 31 20 25 30 13 13 7 16 8 6 2 3 3 4 3
14 16 19
DE,,
32.0 59.1 88.8 85.4 97.1 114.0 143.1 143.5 127.7 172.2 212.6 269.5 264.0 319.6 271.6 342.5 468.3
17.0 26.3 49.3 26.4 30.7 32.3 86.5 53.6 17.8 66.4 50.9 139.2 57.9 68.0 122.7 90.5 74.8
(sm - 27.04 20.8 /
Clones were grown from spores on Trypticase soy agar plus 20 Mg of thymine per ml in microscope growth chambers as previously described (1). ' Obtained from regression line derived from plot of division number versus clone length. DE, Division
equivalents.
(2).
We calculated the number of division equiva- mined for the wild-type CU403 strain [2].1 The lents expressed in div IV-B1 and found that, in percentage of division equivalents expressed 51 clones examined, there were 1,359.0 actual was 38.75. divisions present. The number of divisions exFluid-grown populations of div IV-B1 were pected based on the equation derived from also suppressed in division ability (Table 3). To wild-type clones was 3,506.59. {The equation achieve the first division during clone outDEwt = [(,m - 17.6) V8.8 was previously deter- growth, div IV-B1 clones must grow nearly 2.5 1200
VOL. 121, 1975
NOTES
TABLE 2. Cell length relationships of wild-type and div IV-BI populations during initial outgrowth from spores in fluid culturesa Genotype
Standard Meanlength ength deviation No. of clones Mean of at first measured length at first division (sam) division (im)
div IV-Bl + div IV-BI
76 97
8.06 20.12
1.87 4.04
a Spores were germinated and grown in Trypticase soy broth plus 20 gg of thymine per ml at 30 C. Samples were fixed in formaldehyde and photographed from wet mounts using a phase contrast microscope. Negatives were projected with a photographic enlarger and drawn with pencil, and contour lengths were measured from the drawings with a wheel-type map-measuring instrument. The ratio of mean lengths at first division was 20.12/8.06 = 2.49.
times as long as wild-type clones. Since minicell production is not present before the first division in these clones, minicell production cannot be the cause of excess length of minicell-producing cells in comparison with the length of wild-type cells. The yield of minicells produced by mutants may be calculated as shown in Table 4. Two parameters must be known: (i) either the division ability equation or the percentage of division equivalents expressed relative to wildtype cells; and (ii) P, the probability of a minicell-producing division. The div IV-Bl and div
1201
IV-Al mutants had complementing differences in the values of P and division suppression (Table 2). As a result, the expected yields of minicells were nearly identical. A note of caution should be mentioned at this point. To extend such calculations to fluid cultures at different growth stages requires the knowledge that the parameters obtained from early outgrowth clones are valid for the conditions of growth being considered. Although we have previously observed that the division suppression of div IV-A1 is maintained at later stages of clone outgrowth (2), we have also found that the P values of div IV-Bl fluid-grown populations vary with growth stage (3). It is important, therefore, to obtain data for determination of P values and division ability under the conditions for which predictions are desired. It appears from these and our previous studies that three phenotypic aspects of minicellproducing mutants may be identified: (i) division site location; (ii) frequency of minicell-producing divisions; and (iii) division ability. Since all are ramifications of a single mutation (either div IV-A1 or div IV-Bl), we are searching for a common mechanism that may regulate these diverse aspects of cell division. Genome location and the manner in which genomes segregate during clone development in minicell-producing mutants are currently being investigated, for the absence of deoxyribonucleic acid in minicells indicates a likely relationship of these parameters to cell division.
TABLE 3. Minicell yield calculations Genotype
Determination
diu IV-BI
DEa vs. clone length ........
Probability of a minicell-producing division ........ Total percentage of DE expressed relative to wild type .................. No. of minicells expected per clone of 100 Mm length Ae .... ........ ......... B' ................... a
2704)8
div IV-AI
DEA
=
Mm-44.6)4
0.31d
0.39c
38.75
25.27c
1.08 1.12
Wild-type
DEwt =
88
100
0.8183 0.9224
DE, Division equivalents.
5See Table 1.
From reference (2). From reference (1). e Determined by solving appropriate line 1 equation using ,m = 100 and multiplying answer by P value from line 2. t Determined by solving wild-type equation from line 1 using Mm = 100 and multiplying answer by appropriate percentage of division equivalents expressed (line 3) and by P value (line 2). d
1202
NOTES
We wish to thank W. T. Starmer for advice on statistics. This work was supported by Public Health Service research grant GM-18735 and career development award K04 GM 70555, both from the National Institute of General Medical Sciences to N. H. M. S. I. C. is the recipient of HEA Title IX-B Fellowship (formerly NDEA Title IV).
LITERATURE CITED 1. Coyne, S. I., and N. H. Mendelson. 1974. Clonal analysis of cell division in the Bacillus subtilis div IV-BI mini-
J. BACTERIOL.
cell-producing mutant. J. Bacteriol. 118:15-20. 2. Mendelson, N. H. 1975. Cell division suppression in the Bacillus subtilis div IV-Al minicell-producing mutant. J. Bacteriol. 121:1166-1172. 3. Mendelson, N. H., and S. L. Keener. 1974. Physiological influences on cell division patterns in the div IV-Bl minicell-producing mutant of Bacillus subtilis. J. Bacteriol. 118:1190-1191. 4. Reeve, J. N., N. H. Mendelson, S. I. Coyne, L. L. Hallock, and R. M. Cole. 1973. Minicells of Bacillus subtilis. J. Bacteriol. 114:860-873.
Vol. 121, No. 3 Printed in U.S.A.
NOTES Minicell Yield and Cell Division Suppression in Bacillus subtilis Mutants NEIL H. MENDELSON* AND SHEILA I. COYNE Department of Microbiology and Medical Technology, and Graduate Committee Arizona, Tucson, Arizona 85721
on
Genetics, University of
Received for publication 24 December 1974
Minicell yield is determined by the probability of a minicell-producing division and the relationship of growth to division in Bacillus subtilis mutants.
We recently discovered that minicell-producing cells of the div IV-A1 mutant grow as long filaments primarily because the cells are suppressed in general division ability compared with wild-type cells (2). In the present communication we examine the relationship of growth to division in another minicell-producing mutant, div IV-BJ. Our findings indicate that the div IV-Bl mutant is less suppressed in division ability than the div IV-AJ mutant and consequently minicell-producing cells of div IV-BJ are not as long as those found in div IV-A1 cultures. We have also explored the possibility of estimating minicell yields on the basis of division ability and minicell division frequency. All data were obtained from clones during the initial stages of outgrowth after spore germination. Detailed methods have been described previously (1, 4). Both mutants were studied in a common genetic background (strain CU403). Table 1 indicates the relationship of division ability to clone growth found in the analysis of 51 div IV-BJ clones. The large standard deviation of clone lengths at each stage of division was similar to that found in the div IV-Al mutant and represented individual clone differences in division ability. The equation included in Table 1 represents the overall division ability of div IV-B1 clones relative to growth. Similar equations have previously been derived for wild-type CU403 and for the div IV-Al mutant
TABLE 1. Relationship of clone length to cell division in CU403 diu IV-BJa
lStandard deviation of Avg clone clone length length (mm) clone in In clone measured measured ~(pm) No. of No. .o
No. of No. of
0 1 2 3 4 5 6 7 8 9 10 11 12 1:3
42 53 31 20 25 30 13 13 7 16 8 6 2 3 3 4 3
14 16 19
DE,,
32.0 59.1 88.8 85.4 97.1 114.0 143.1 143.5 127.7 172.2 212.6 269.5 264.0 319.6 271.6 342.5 468.3
17.0 26.3 49.3 26.4 30.7 32.3 86.5 53.6 17.8 66.4 50.9 139.2 57.9 68.0 122.7 90.5 74.8
(sm - 27.04 20.8 /
Clones were grown from spores on Trypticase soy agar plus 20 Mg of thymine per ml in microscope growth chambers as previously described (1). ' Obtained from regression line derived from plot of division number versus clone length. DE, Division
equivalents.
(2).
We calculated the number of division equiva- mined for the wild-type CU403 strain [2].1 The lents expressed in div IV-B1 and found that, in percentage of division equivalents expressed 51 clones examined, there were 1,359.0 actual was 38.75. divisions present. The number of divisions exFluid-grown populations of div IV-B1 were pected based on the equation derived from also suppressed in division ability (Table 3). To wild-type clones was 3,506.59. {The equation achieve the first division during clone outDEwt = [(,m - 17.6) V8.8 was previously deter- growth, div IV-B1 clones must grow nearly 2.5 1200
VOL. 121, 1975
NOTES
TABLE 2. Cell length relationships of wild-type and div IV-BI populations during initial outgrowth from spores in fluid culturesa Genotype
Standard Meanlength ength deviation No. of clones Mean of at first measured length at first division (sam) division (im)
div IV-Bl + div IV-BI
76 97
8.06 20.12
1.87 4.04
a Spores were germinated and grown in Trypticase soy broth plus 20 gg of thymine per ml at 30 C. Samples were fixed in formaldehyde and photographed from wet mounts using a phase contrast microscope. Negatives were projected with a photographic enlarger and drawn with pencil, and contour lengths were measured from the drawings with a wheel-type map-measuring instrument. The ratio of mean lengths at first division was 20.12/8.06 = 2.49.
times as long as wild-type clones. Since minicell production is not present before the first division in these clones, minicell production cannot be the cause of excess length of minicell-producing cells in comparison with the length of wild-type cells. The yield of minicells produced by mutants may be calculated as shown in Table 4. Two parameters must be known: (i) either the division ability equation or the percentage of division equivalents expressed relative to wildtype cells; and (ii) P, the probability of a minicell-producing division. The div IV-Bl and div
1201
IV-Al mutants had complementing differences in the values of P and division suppression (Table 2). As a result, the expected yields of minicells were nearly identical. A note of caution should be mentioned at this point. To extend such calculations to fluid cultures at different growth stages requires the knowledge that the parameters obtained from early outgrowth clones are valid for the conditions of growth being considered. Although we have previously observed that the division suppression of div IV-A1 is maintained at later stages of clone outgrowth (2), we have also found that the P values of div IV-Bl fluid-grown populations vary with growth stage (3). It is important, therefore, to obtain data for determination of P values and division ability under the conditions for which predictions are desired. It appears from these and our previous studies that three phenotypic aspects of minicellproducing mutants may be identified: (i) division site location; (ii) frequency of minicell-producing divisions; and (iii) division ability. Since all are ramifications of a single mutation (either div IV-A1 or div IV-Bl), we are searching for a common mechanism that may regulate these diverse aspects of cell division. Genome location and the manner in which genomes segregate during clone development in minicell-producing mutants are currently being investigated, for the absence of deoxyribonucleic acid in minicells indicates a likely relationship of these parameters to cell division.
TABLE 3. Minicell yield calculations Genotype
Determination
diu IV-BI
DEa vs. clone length ........
Probability of a minicell-producing division ........ Total percentage of DE expressed relative to wild type .................. No. of minicells expected per clone of 100 Mm length Ae .... ........ ......... B' ................... a
2704)8
div IV-AI
DEA
=
Mm-44.6)4
0.31d
0.39c
38.75
25.27c
1.08 1.12
Wild-type
DEwt =
88
100
0.8183 0.9224
DE, Division equivalents.
5See Table 1.
From reference (2). From reference (1). e Determined by solving appropriate line 1 equation using ,m = 100 and multiplying answer by P value from line 2. t Determined by solving wild-type equation from line 1 using Mm = 100 and multiplying answer by appropriate percentage of division equivalents expressed (line 3) and by P value (line 2). d
1202
NOTES
We wish to thank W. T. Starmer for advice on statistics. This work was supported by Public Health Service research grant GM-18735 and career development award K04 GM 70555, both from the National Institute of General Medical Sciences to N. H. M. S. I. C. is the recipient of HEA Title IX-B Fellowship (formerly NDEA Title IV).
LITERATURE CITED 1. Coyne, S. I., and N. H. Mendelson. 1974. Clonal analysis of cell division in the Bacillus subtilis div IV-BI mini-
J. BACTERIOL.
cell-producing mutant. J. Bacteriol. 118:15-20. 2. Mendelson, N. H. 1975. Cell division suppression in the Bacillus subtilis div IV-Al minicell-producing mutant. J. Bacteriol. 121:1166-1172. 3. Mendelson, N. H., and S. L. Keener. 1974. Physiological influences on cell division patterns in the div IV-Bl minicell-producing mutant of Bacillus subtilis. J. Bacteriol. 118:1190-1191. 4. Reeve, J. N., N. H. Mendelson, S. I. Coyne, L. L. Hallock, and R. M. Cole. 1973. Minicells of Bacillus subtilis. J. Bacteriol. 114:860-873.
