Vol. 125, No. 3 Printed in U.S.A.
JouRNAL OF BACTURIOLOGY, Mar. 1976, P. 968-974 Copyright C 1976 American Society for Microbiology
Requirement for Pantothenate for Filament Formation by Erwinia carotovora MARY M. GRULA*
AND
E. A. GRULA
School of Biological Sciences, Oklahoma State University, Stillwater, Oklahoma 74074
Received for publication 25 November 1975
Pantothenate is required for the formation of filaments by Erwinia carotoThis has been demonstrated for the following division-inhibiting agents: Dserine, D-cycloserine, penicillin, vancomycin, fluoride ion, and ultraviolet light. D-Serine inhibits pantothenate synthesis in an ammonia-glucose or an ammonia-pyruvate medium; therefore, it is necessary to add pantothenate to obtain filament formation in these media, using n-serine as the division-inhibiting agent. Under conditions in which pantothenate synthesis is not inhibited by the agent producing filaments, the need for it for filamentation was shown by the use of salicylate, an inhibitor of endogenous pantothenate synthesis. Evidence is presented that the production offilaments is a specific response to pantothenate, rather than a nonspecific growth stimulation. vora.
Although numerous studies on the mechanism of the formation of filaments by rodshaped bacteria have been done, the phenomenon is not yet understood, nor can an adequate generalization be made about the conditions inducing filamentation. The diversity of the chemical nature of agents causing filamentation has been noted (5, 16). Some agents that induce our strain of Erwinia carotovora to form filaments are: certain D-amino acids, various forms of penicillin, including penicillanic acid, vancomycin, n-cycloserine, and ultraviolet light (8, 9). Most of these agents inhibit synthesis of the mucopeptide component of the cell envelope of Erwinia (10); however, decreased wall synthesis itself is an insufficient condition for the formation of filaments (10). Evidence for specific nutritional requirements for filament formation by Erwinia has been presented (13). The key observation that led to the finding that pantothenate is required was the failure of D-serine to promote production of filamentous cells if an ammonium salt was the sole source of nitrogen. Addition of catalytic amounts of pantothenate to such a medium results in the formation of very long cells. In media with other nitrogen sources, the presence of D-serine leads to the production of long cells without added pantothenate. This paper is a report of experiments designed to test whether filaments obtained by adding pantothenate result from nonspecific growth stimulation or because a specific nutritional requirement for the development of long cells is supplied. Also reported are certain re-
sults concerned with a possible mechanism of pantothenate action. MATERIALS AND METHODS Bacterial strain. In previous publications (9, 11, 12) we designated the strain employed as Erwinia species, since it did not correspond exactly with any species despribed in the 7th edition of Bergey's Manual of Determinative Bacteriology. On the basis of a number of anaerogenic sugar fermentations, H2S production, reduction of nitrate to nitrite, and certain other characteristics, we stated that it resembled E. aroideae most closely; however, its optimum growth temperature was well below the 37 C optimum of E. aroideae. The taxonomy of the genus Erwinia has received much attention in recent years. Starr and Chatterjee (17) have stated that the original decision to lump all gram-negative peritrichate plant pathogens in one genus (Erwinia) has had a disastrous effect on the development of knowledge about these organisms. Dye (6) and Graham (7), who have studied hundreds of isolates from various parts of the world, have established a set of criteria for species and varieties of species of Erwinia (the soft rot group) that has been generally accepted and incorporated into the 8th edition of Bergey's Manual (2). An important observation of Dye (6) is that, since gas production is highly variable and cannot be correlated with other characters, it is not a property that is useful taxonomically. Graham (7) and Dye (6) have concluded that there should be only one species of the soft rot group, viz., E. carotovora. Dye (6) recognized E. carotovora and four other varieties: atroseptica, chrysanthemi, rhapontici, and cypripedii. On the bases of their criteria and others in Bergey's Manual, 8th edition, our strain is E. carotovora 968
VOL. 125, 1976
PANTOTHENATE AND FILAMENT FORMATION
var. carotovora, except that its optimum growth temperature is between 25 and 30 C. Maintenance of the strain, methods ofgrowing in chemically defined media, and methods of determining growth and cell length have been described (8, 9). Measurement of filaments. Cell length is reported here as an approximated average (arithmetic mean) value, obtained from cell length distributions according to the following formula: [(percentage in interval) x (midpoint of interval)]/100. Intervals chosen were (,um) 50. The final figure for average length is an approximation, differing from the true average by a quantity that depends on the variability and on the distribution of lengths. In all cases, differences in average length reported as due to some external variable were always far greater than any deviation of calculated length from the true arithmetic mean. Pantothenate uptake. Pantothenate (sterilized by filtration) was added as the sodium salt of -pantothenic acid (obtained from Schwarz/Mann or Sigma Chemical Co.). Experiments with radioactive ,-alanine or pantothenate were carried out using [1-_4C].8-alanine or pantothenate labeled in the carboxyl group of the ,alanine moiety in the medium at a level of 0.01 ,uCi/ ml. Enough nonradioactive compound was added to produce a final molar concentration of 10-4 (a8-alanine) or 106 (pantothenate). Cells were extracted for coenzyme A (CoA) at pH 2.1 by the method of Das and Toennies (4). The residue, whose radioactivity is considered due to acyl carrier protein, was digested in 0.5 ml of 1.0 N NaOH at 34 to 35 C before counting. Counting techniques were those of Das and Toennies (3), using Bray (1) scintillation fluid. The chemically defined medium of Howard-Flanders et al. (14) was used for experiments with Escherichia coli strain B. Cultures were grown at 35 C with reciprocal shaking in 4-ml volumes in 16-mm test tubes. Cultures were examined for length of cells and amount of growth in the same manner as Erwinia cultures.
969
growth enough to allow the cells to elongate. However, the following observations indicate that this is not the case. (i) With either pyru-
vate or mannose as a carbon source, added pantothenate results in marked cell elongation with very little apparent stimulation of growth. (ii) Addition of pantothenate during early exponential growth results in significant elongation before any visible turbidity is produced. (iii) A negative correlation exists between the extent of overcoming of growth inhibition by pantothenate and the degree of elongation produced. Data illustrating this, obtained by the use of high and low concentrations of NH4+ ion, are given in Fig. 1. Since growth (increase of cell mass) is essential for filament formation, the lack of filaments in ammonia media containing n-serine could result simply from a lack of growth. To test this possibility, we used the plate count as an indicator of growth. Cultures were grown in NH4Cl-pyruvate-D-serine medium. Addition of pantothenate will allow development of filaments up to 100 ,m, with only a minimal increment in cell mass (as indicated by optical density), over the culture without added pantothenate. Data from a typical experiment are given in Table 1. Although growth was strongly inhibited by D-serine, it was by no means completely prevented. In the culture without pantothenate, two doublings of the viable count had occurred at 12 h and three had occurred at 16 h. The smaller increase in the plate count in the culture with pantothenate at 12 h, coupled with no increase after another 4 h, is probably related to an increase in average length of the colonyforming unit. The latter value is unknown, but this does not invalidate the fact that an increase of cell mass did occur in both cultures. Without pantothenate apparently normal cell RESULTS AND DISCUSSION division took place, whereas with it division D-Serine, which promotes production of fila- seemed to be nearly completely inhibited. mentous forms of E. carotovora in aspartic acid To obtain further evidence that the elongatmedia, does not do so in media with NH,Cl ing effect of pantothenate was not simply a (0.075 M) as the sole nitrogen source. Yet it is nonspecific reflection of increased growth, we highly toxic in ammonia media, indicating that determined the degree of elongation of Erwinia it has access to certain critical metabolic reac- cells, in the presence of n-serine, under conditions within the cell. Cell elongation has not tions of nutritional deficiency other than pantobeen observed in these media at any time up to thenate. If a pantothenate deficiency severe 24 h, with concentrations of -serine ranging enough to markedly limit growth would prefrom 0.4 to 2 mM. Lowering the concentration vent the formation of filaments, simply because of NH4Cl to 0.015 M results in the production of not enough growth had occurred, other nutria few (1% or less) cells from 5 to 15 um in tional deficiencies should produce similar relength. Addition of pantothenate partially sults. E. carotovora does not have an absolute reovercomes growth inhibition by D-serine and results in formation of filaments. The obvious quirement for any vitamin, amino acid, purine, inference is that pantothenate simply increases or pyrimidine (12). Thus, the nutritional defi-
970
J. BACTERIOL.
GRULA AND GRULA .9 .8 .7 .6
.
.5 .4 z
Ul 0
4
4
0
0
CONTROL
J
D-SERINE + PANTOTHENATE
.2
-SERINE I
_
.1
12
11
13
12
14 15 HOURS
13
HOURS C. to
a90
90
U
17
18
19
20
0.
s
0
70
cc
60
60 Z 50
40
.j
16
E 80 2
80 ao 70 (2
U
0-
D-SERINE + PANTOTHENATE
30
2
50
i
40
Ul U
D-SERINE + PANTOTHENATE
30
CU
40
4 20 c
20 1U0
D-SERINE
CONTROL
i
< 10
*
i ,=,,D=-SERINE
11
12
13
14 15 HOURS
16
17
18
19
20
11
12
13
15 14 HOURS
16
17
u
18
CONTROL
19
20
FIG. 1. Growth and cell length of E. carotovora in ammonia-glucose medium, as influenced by D-serine and D-serine plus pantothenate. (A) Growth with 0.075 M NH4Cl; (B) growth with 0.015 M NH4CI; (C) cell length with 0.075 M NH4CI; (D) cell length with 0.015 M NH4Cl. D-Serine concentration was 12 x 10-3 M, and pantothenate was 10-6 M.
TABLE 1. Effect of added pantothenate on growth and cell length in ammonia-pyruvate mediuma
Average length of cells or filaments (m)
.OD'
Time after inoculation (h)
0 12 16 21
No pantothenate
0.006 0.008 0.018
With pantothenate
No pantothenate
With pan-
0.00 0.015 0.022
2 3.3 3.2 2
2 20 46 51
Plate countc (Colony-forming units/ml)
No pantothenate
With pantothen-
3.7x107 15 x 107 30 x 107
4.2x107 12 x 107 12 x 107
tothenate
ate
NH4Cl, 0.015 M; pyruvate, 8 mM; D-serine, 1.3 mM; sodium pantothenate, 0.002 mM. b OD, Optical density. c Obtained by averaging the counts of duplicate plates at each of two dilutions. Plating medium was Trypticase soy agar. a
ciencies we were limited to comprised nitrogen (NH4Cl or aspartic acid), carbon energy source, magnesium ion, sulfate ion, and the mineral mixture of our basal medium. Media containing D-serine were prepared with graded limiting amounts of each of the above nutrients, with the compositon otherwise unchanged. Growth and cell length in the critical ranges were observed. Results are given in Table 2. A deficiency of magnesium sulfate is easy to demonstrate. Growth in MgSO4-deficient media to
which D-serine had been added (at 13 h when control cultures would be in exponential growth) was very low; there was no turbidity. Yet the cells were very long; in the case of the medium with no added MgSO4, 30% were in the range of 50 to 100 ,um. With every nutritional deficiency examined, D-serine resulted in very long cells in situations wherein growth was strongly limited. Similar results utilizing other basal media are presented in Table 3. It is evident that a culture
PANTOTHENATE AND FILAMENT FORMATION
VOL. 125, 1976
TABLE 2. Effect of certain nutritional deficiencies on cell length of E. carotovora in the presence of D-serinea
GrwhAverage
Required nutrient concentration as Growth a fraction of control
cell
nof
length
55 47
5.0 4.0
60 50
20.0 21.0
75 38 5
23.0 19.0 49.0
84 74
33
35.0 46.0 71.0
77 28 5
67.0 52.0 70.0
normnal)
(gLm)
Nitrogen (NH4+)' No added pantothenate 1/66
1/0oo With 10-6 M pantothenate 1/66
1/1oo
MgSO4C
1/2 1/3
None added
Aspartic acid (with glucose)
1/2 1/4
1/1o Aspartic acid (without glucose)
1/2
1/4
1/1o D-Serine was present in all media at a concentration of 10-3 M. b NH4Cl was the sole source of N. Glucose was the carbon source. c Basal medium was asparate-glucose. The requirement for MgSO4 is largely for the SO42- ion; since no other source of Mg2+ was added, it was assumed that the Mg2+ requirement was met by the minimal level of MgSO4. a
limited in growth by a low concentration of a specific nutrient may contain very long filaments. The very small increases in turbidity accompanying an elongation that is the equivalent of three or four cell divisions may be accounted for by the lower light-scattering power per unit mass of filaments. Pantothenate, if it actually is necessary for filament formation, should be required in all media. However, this is not always readily observed; in aspartic acid media, for example, filaments are induced by D-serine without the addition of pantothenate. The synthesis of pantothenate by E. carotovora may be conveniently blocked in any medium, including those with exogenous aspartic acid, by sodium salicylate. Maas (15), using a wild-type E. coli, demonstrated that salicylate
interfered with pantoate, and hence pantothenate, synthesis. At one time we felt that if salicylate should act similarly in the metabolism of our strain of Erwinia, it might mimic the action
971
of r-serine, i.e., induce cells to form filaments. However, 10-4 M salicylate resulted in the formation of very short cells, rather than filaments (11). When salicylate was combined with 1)-serine, a synergistic toxicity occurred, and the formation of filaments was almost wholly prevented. Microbial assays indicated that the synthesis of pantothenate by E. carotovora was decreased in the presence of salicylate by 70% (11). Further evidence that salicylate produced a pantothenate deficiency was the fact that 10-6 M pantothenate completely overcame growth inhibition by salicylate, and, if a division-inhibiting agent was also present, formation of filaments occurred (11). Salicylate prevented elongation of Erwinia cells with every division-inhibiting agent with which it was tested. This included the six namino acids that inhibit division (7), D-cycloserine, penicillin, vancomycin, fluoride ion, and ultraviolet light. In all cases, supplementation with pantothenate resulted in very long cells. Data are given in Table 4. Pantothenate can be replaced in these situations by pantoic acid, ketopantoic acid, or aketoisovaleric acid but not by a-methyl pantothenate or /8-alanine. Salicylate added to preformed filaments had no effect on their length. We interpret its action in preventing filament formation solely on the basis of its inhibition of pantothenate synthesis. It should be emphasized that, by itself, pantothenate has no ability to elongate cells beyond their normal length; the presence of a division-inhibiting agent, such as D-serine or one of the cell wall synthesis-inhibiting antibiotics, is required. Tests using E. coli. To determine whether the pantothenate requirement for filament formation observed with E. carotovora might have TABLE 3. Filament formation accompanied by minimal growth Medium
Aspartateglucose
NH4+-glucosepantothenate
Modification Trace minerals omitted NH4+ concentration l/so of control None
Average Growth length (Ism) 100 0.01 0.015
75
80 0.00 Aspartatemalate 100 0.005 10-" M pantoAspartatethenate added malate 90 0.006 None Aspartate at 14 were observed nm. 540 Cultures a Optical density at to 16 h. In aspartate-glucose and NH4+-glucose-pantothenate, D-serine was 10-' M; in the other media, it was 1.3 x
10-3 M.
972
J. BACTERIOL.
GRULA AND GRULA
wider applicability, we utilized E. coli strain B. Crystal violet (18) was chosen as a divisioninhibiting agent since D-serine does not mediate filament formation in E. coli B (E. Grula, unpublished data). Results are given in Table 5. Although not as dramatic as with E. carotovora, changes in cell length occurred under conditions that suggest a critical level of pantothenate or a derivative is necessary for crystal violet to mediate filament formation in E. coli B.
Possible mode of action of pantothenate. Since pantothenate or a derivative appears to be required for cell elongation mediated by a variety of inhibitors (as opposed to a general growth requirement), it is of interest to inquire as to its mode of action.
TABLE 4.
That pantothenate is acting physiologically, instead of in some nonspecific, nonenzymatic way, is indicated by the fact that co-methyl pantothenate (an analogue that might replace pantothenate in a nonenzymatic reaction) would not replace pantothenate in any situation where the deficient factor was pantoic acid. If (3-alanine was the limiting factor, cu-methyl pantothenate stimulated growth and filament formation to some extent, probably because of a partial hydrolysis yielding (8-alanine. Failure of co-methyl pantothenate to replace pantothenate shows that pantothenate action is not associated with the stimulation of division by pantoyl lactone (9); in the latter case, &omethyl pantoyl lactone is more active on a molar basis than pantoyl lactone.
Cell lengths of E. carotovora as affected by certain division-inhibiting agents and added salicylate and pantothenate Cell length
Medium
Division-inhibiting agent
Glucose-aspartate
r-Serine (10-3 M) Fluoride ion (4 x 10-3 M) Penicillin (3 ,ug/ml) Vancomycin (10 ,ug/ml) UV lightb D-Cycloserine (10-6 M) Penicillin D-Cycloserinec
Glucose-ammonia
No further ad- Salicylate (10-5 M) ditions
20-50 20-300 50-300 3-50 10-50 20-100 10-100 2-5
.'
2-3 2-3 3-10 2-3 3-5 2-3 1-2
(Am)a Salicylate + pantothenate (10-6 M) 20-50 20-300 20-300 3-50 10-50 10-50 10-50 10-50
(Pantothenate only) 50-300
3-5 100-300 Penicillin Malate-ammonia Range including 90% of cells or filaments. b Cells were exposed to ultraviolet (UV) light and cultured after exposure as reported previously (9). c Failure of elongation in the presence of D-cycloserine in glucose-ammonia medium indicates a possibility of inhibition of pantothenate synthesis. Addition of pantothenate resulted in an increase in optical density from 0.02 to 0.30 (16 h of incubation). a
TABLE 5. Cell length of E. coli B as influenced by crystal violet, salicylate, and pantothenate in a chemically defined medium Cell length distributione Growtha 20-50 1-2 2-5 5-10 10-20
Additive(s)
100 None 0.77 100 0.30 Salicylate (0.0012 M) 100 0.25 Salicylate (0.0018 M) 30 20 20 0.55 Crystal violet (2 gg/ml) 40 15 25 0.54 Crystal violet (4 jug/ml) 9.9 0.1% 90 0.39 Crystal violet (2 ,ug/ml) + salicylate (0.0018 M) 3 30 15 50 0.57 Crystal violet (2 ,Ag/ml) + salicylate (0.0018 M) + pantothenate (10-6 M) a Optical density at 540 nm. Cultures were observed at 18 h. b Percentage of cells in each length range. Lengths expressed in micrometers. c Some filaments up to 300 in length were present.
jsm
20 20 2
>50
10
10o
to 100 j.m
VOL. 125, 1976
PANTOTHENATE AND FILAMENT FORMATION
Pantoyl taurine would not replace pantothenate in the stimulation of filament formation. It inhibited growth in media in which pantothenate synthesis was limiting; this inhibition was partially relieved by adding pantothenate at a fourfold lower concentration. It was believed desirable to determine the relative amounts of CoA and acyl carrier protein (ACP) in short and in filamentous cells, information that should be useful in analyzing the mechanism of pantothenate action. Das and Toennies (4) found, with Streptococcus faecalis, that in the presence of an excess of precursors during exponential growth the cellular content of CoA and ACP remained constant and in a molar ratio of 4 to 1. Under conditions of stress, ACP was maintained at the expense of CoA. We have compared the CoA and ACP contents of normal and filamentous cells using several media in which -serine was the division-inhibiting agent. Initial experiments were done by adding n-serine and labeled 18-alanine at time zero, in which the effect of n-serine on overall synthesis of CoA and ACP would be shown. Cells were harvested during exponential growth. In these tests, the CoA/ACP ratio of -serine-grown cells was lower than that of the control cells (Table 6). The ACP content of D-serine-grown cells per unit dry weight was increased by about 50% and the CoA content was decreased by 25 to 30%. If -serine was added during early exponential growth, the CoA/ACP ratio continued to be lower in -serine-grown cells, but the difference between these and the control cells was less than that observed using the first approach. A third type of experiment was done to determine whether the diminished CoA levels could be attributed to loss by leakage. Cells were grown to the early exponential phase in a medium with [1-'4C],8-alanine. They were removed from the medium, washed, and placed in fresh unlabeled medium, with and without n-serine. No appreciably greater leakage of radioactive material from cells was observed in the presence of -serine than in its absence. Therefore, it is likely that the decrease in the CoA/ACP ratio observed in n-serine-grown cells is real and not an artifact resulting from greater leakage of CoA. The fact that certain physiological indicators of CoA deficiency occur in -serine-grown cells, including the accumulation of keto acids (13), is consistent with a decreased CoA content. It is premature to speculate on the significance of this alteration in CoA or the CoA/ACP ratio. Some type of association of filament formation with a change in the pattern of fatty
973
TABLE 6. CoAIACP ratios of filaments and of normal cells ofE. carotovora ~~Average length CoA/ACP cell Time of
aditioonf
Basal medium
ratio of DserineP Con- D-Ser(h) trol ine
NH4+-glucose
#-alanine
NH4+-glucose
(_Lm) Con- D-Sertrol
ine
0
5.2
2.7
5
30
12
5.6
4.8
3
20
#-alanine
30 0 4.7 2.5 2 NH4+-glucose pantothenate 10 14 5.3 4.7 3 NH4+-glucose pantothenate 12 0 5.8 2.7 2 NH4+-pyruvate 8-alanine a 0 h is time of inoculation. Cultures were harvested at 16 h, at which time cell length was determined.
acid synthesis is a possibility, but so far no conclusive evidence that such an association exists has been obtained. LITERATURE CITED 1. Bray, G. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279-285. 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore. 3. Das, D. N., and G. Toennies. 1968. A versatile buffered scintillation system. Anal. Biochem. 23:26-34. 4. Das, D. N., and G. Toennies. 1969. Relations between coenzyme A and presumptive acyl carrier protein in different conditions of streptococcal growth. J. Bacteriol. 98:898-902. 5. Duguid, J. P., and J. F. Wilkinson. 1961. Environmentally induced changes in bacterial morphology. Symp. Soc. Gen. Microbiol. 11:69-99. 6. Dye, D. W. 1969. A taxonomic study of the genus Erwinia. II. The 'carotovora' group. N. Z. J. Sci. 12:8197. 7. Graham, D. C. 1972. Identification of soft rot coliform bacteria, p. 273-279. In H. P. Maas Gustermus (ed.), Proceedings of the Third International Conference on Plant Pathogenic Bacteria. Center for Agricultural Publishing and Documentation, Wagenigen, The
Netherlands. 8. Grula, E. A. 1960. Cell division in a species ofErwinia. II. Inhibition of division by 1-amino acids. J. Bacteriol. 80:375-385. 9. Grula, E. A., and M. M. Grula. 1962. Cell division in a species of Erwinia. II. Reversal of inhibition of cell division caused by D-amino acids, penicillin, and ultraviolet light. J. Bacteriol. 83:981-989. 10. Grula, E. A., and M. M. Grula. 1964. Cell division in a species of Erwinia. VII. Amino sugar content ofdividing and non-dividing cells. Biochem. Biophys. Res. Commun. 17:341-346. 11. Grula, M. M., and E. A. Grula. 1962. Cell division in a species of Erwinia. IV. Metabolic blocks in pantothenate biosynthesis and their relationship to inhibition of cell division. J. Bacteriol. 83:989-997. 12. Grula, M. M., R. W. Smith, C. F. Parham, and E. A.
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GRULA AND GRULA
Grula. 1968. Cell division in a species of Erwinia. XI. Some aspects of the carbon and nitrogen nutrition of Erwinia species. Can. J. Microbiol. 14:1217-1224. 13. Grula, M. M., R. W. Smith, C. F. Parham, and E. A. Grula. 1968. Cell division in a species ofErwinia. XI. A study of nutritional influences in D-serine inhibition of growth of Erwinia sp., and of certain specific sites of D-serine action. Can. J. Microbiol. 14:12251238. 14. Howard-Flanders, P., E. Simson, and L. Theriot. 1964. A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics 49:237-246.
J. BACTERIOL. 15. Mas, W. K. 1952. Pantothenate studies. II. Evidence from mutants for interference by salicylate with pantoate synthesis. J. Bacteriol. 63:227-232. 16. Sister, M., and M. Schaechter. 1974. Control of cell division in bacteria. Bacteriol. Rev. 38:199-221. 17. Starr, M. P., and A. K. Chatterjee. 1972. The genus Erwinia: enterobacteria pathogenic to plants and ani-
mals. Annu. Rev. Microbiol. 26:389-426. 18. Walker, J. R., N. A. Shafiq, and R. G. Allen. 1971. Bacterial cell division regulation: physiological effects of crystal violet onEscherichia coli lon+ and lon strains. J. Bacteriol. 108:1296-1303.
JouRNAL OF BACTURIOLOGY, Mar. 1976, P. 968-974 Copyright C 1976 American Society for Microbiology
Requirement for Pantothenate for Filament Formation by Erwinia carotovora MARY M. GRULA*
AND
E. A. GRULA
School of Biological Sciences, Oklahoma State University, Stillwater, Oklahoma 74074
Received for publication 25 November 1975
Pantothenate is required for the formation of filaments by Erwinia carotoThis has been demonstrated for the following division-inhibiting agents: Dserine, D-cycloserine, penicillin, vancomycin, fluoride ion, and ultraviolet light. D-Serine inhibits pantothenate synthesis in an ammonia-glucose or an ammonia-pyruvate medium; therefore, it is necessary to add pantothenate to obtain filament formation in these media, using n-serine as the division-inhibiting agent. Under conditions in which pantothenate synthesis is not inhibited by the agent producing filaments, the need for it for filamentation was shown by the use of salicylate, an inhibitor of endogenous pantothenate synthesis. Evidence is presented that the production offilaments is a specific response to pantothenate, rather than a nonspecific growth stimulation. vora.
Although numerous studies on the mechanism of the formation of filaments by rodshaped bacteria have been done, the phenomenon is not yet understood, nor can an adequate generalization be made about the conditions inducing filamentation. The diversity of the chemical nature of agents causing filamentation has been noted (5, 16). Some agents that induce our strain of Erwinia carotovora to form filaments are: certain D-amino acids, various forms of penicillin, including penicillanic acid, vancomycin, n-cycloserine, and ultraviolet light (8, 9). Most of these agents inhibit synthesis of the mucopeptide component of the cell envelope of Erwinia (10); however, decreased wall synthesis itself is an insufficient condition for the formation of filaments (10). Evidence for specific nutritional requirements for filament formation by Erwinia has been presented (13). The key observation that led to the finding that pantothenate is required was the failure of D-serine to promote production of filamentous cells if an ammonium salt was the sole source of nitrogen. Addition of catalytic amounts of pantothenate to such a medium results in the formation of very long cells. In media with other nitrogen sources, the presence of D-serine leads to the production of long cells without added pantothenate. This paper is a report of experiments designed to test whether filaments obtained by adding pantothenate result from nonspecific growth stimulation or because a specific nutritional requirement for the development of long cells is supplied. Also reported are certain re-
sults concerned with a possible mechanism of pantothenate action. MATERIALS AND METHODS Bacterial strain. In previous publications (9, 11, 12) we designated the strain employed as Erwinia species, since it did not correspond exactly with any species despribed in the 7th edition of Bergey's Manual of Determinative Bacteriology. On the basis of a number of anaerogenic sugar fermentations, H2S production, reduction of nitrate to nitrite, and certain other characteristics, we stated that it resembled E. aroideae most closely; however, its optimum growth temperature was well below the 37 C optimum of E. aroideae. The taxonomy of the genus Erwinia has received much attention in recent years. Starr and Chatterjee (17) have stated that the original decision to lump all gram-negative peritrichate plant pathogens in one genus (Erwinia) has had a disastrous effect on the development of knowledge about these organisms. Dye (6) and Graham (7), who have studied hundreds of isolates from various parts of the world, have established a set of criteria for species and varieties of species of Erwinia (the soft rot group) that has been generally accepted and incorporated into the 8th edition of Bergey's Manual (2). An important observation of Dye (6) is that, since gas production is highly variable and cannot be correlated with other characters, it is not a property that is useful taxonomically. Graham (7) and Dye (6) have concluded that there should be only one species of the soft rot group, viz., E. carotovora. Dye (6) recognized E. carotovora and four other varieties: atroseptica, chrysanthemi, rhapontici, and cypripedii. On the bases of their criteria and others in Bergey's Manual, 8th edition, our strain is E. carotovora 968
VOL. 125, 1976
PANTOTHENATE AND FILAMENT FORMATION
var. carotovora, except that its optimum growth temperature is between 25 and 30 C. Maintenance of the strain, methods ofgrowing in chemically defined media, and methods of determining growth and cell length have been described (8, 9). Measurement of filaments. Cell length is reported here as an approximated average (arithmetic mean) value, obtained from cell length distributions according to the following formula: [(percentage in interval) x (midpoint of interval)]/100. Intervals chosen were (,um) 50. The final figure for average length is an approximation, differing from the true average by a quantity that depends on the variability and on the distribution of lengths. In all cases, differences in average length reported as due to some external variable were always far greater than any deviation of calculated length from the true arithmetic mean. Pantothenate uptake. Pantothenate (sterilized by filtration) was added as the sodium salt of -pantothenic acid (obtained from Schwarz/Mann or Sigma Chemical Co.). Experiments with radioactive ,-alanine or pantothenate were carried out using [1-_4C].8-alanine or pantothenate labeled in the carboxyl group of the ,alanine moiety in the medium at a level of 0.01 ,uCi/ ml. Enough nonradioactive compound was added to produce a final molar concentration of 10-4 (a8-alanine) or 106 (pantothenate). Cells were extracted for coenzyme A (CoA) at pH 2.1 by the method of Das and Toennies (4). The residue, whose radioactivity is considered due to acyl carrier protein, was digested in 0.5 ml of 1.0 N NaOH at 34 to 35 C before counting. Counting techniques were those of Das and Toennies (3), using Bray (1) scintillation fluid. The chemically defined medium of Howard-Flanders et al. (14) was used for experiments with Escherichia coli strain B. Cultures were grown at 35 C with reciprocal shaking in 4-ml volumes in 16-mm test tubes. Cultures were examined for length of cells and amount of growth in the same manner as Erwinia cultures.
969
growth enough to allow the cells to elongate. However, the following observations indicate that this is not the case. (i) With either pyru-
vate or mannose as a carbon source, added pantothenate results in marked cell elongation with very little apparent stimulation of growth. (ii) Addition of pantothenate during early exponential growth results in significant elongation before any visible turbidity is produced. (iii) A negative correlation exists between the extent of overcoming of growth inhibition by pantothenate and the degree of elongation produced. Data illustrating this, obtained by the use of high and low concentrations of NH4+ ion, are given in Fig. 1. Since growth (increase of cell mass) is essential for filament formation, the lack of filaments in ammonia media containing n-serine could result simply from a lack of growth. To test this possibility, we used the plate count as an indicator of growth. Cultures were grown in NH4Cl-pyruvate-D-serine medium. Addition of pantothenate will allow development of filaments up to 100 ,m, with only a minimal increment in cell mass (as indicated by optical density), over the culture without added pantothenate. Data from a typical experiment are given in Table 1. Although growth was strongly inhibited by D-serine, it was by no means completely prevented. In the culture without pantothenate, two doublings of the viable count had occurred at 12 h and three had occurred at 16 h. The smaller increase in the plate count in the culture with pantothenate at 12 h, coupled with no increase after another 4 h, is probably related to an increase in average length of the colonyforming unit. The latter value is unknown, but this does not invalidate the fact that an increase of cell mass did occur in both cultures. Without pantothenate apparently normal cell RESULTS AND DISCUSSION division took place, whereas with it division D-Serine, which promotes production of fila- seemed to be nearly completely inhibited. mentous forms of E. carotovora in aspartic acid To obtain further evidence that the elongatmedia, does not do so in media with NH,Cl ing effect of pantothenate was not simply a (0.075 M) as the sole nitrogen source. Yet it is nonspecific reflection of increased growth, we highly toxic in ammonia media, indicating that determined the degree of elongation of Erwinia it has access to certain critical metabolic reac- cells, in the presence of n-serine, under conditions within the cell. Cell elongation has not tions of nutritional deficiency other than pantobeen observed in these media at any time up to thenate. If a pantothenate deficiency severe 24 h, with concentrations of -serine ranging enough to markedly limit growth would prefrom 0.4 to 2 mM. Lowering the concentration vent the formation of filaments, simply because of NH4Cl to 0.015 M results in the production of not enough growth had occurred, other nutria few (1% or less) cells from 5 to 15 um in tional deficiencies should produce similar relength. Addition of pantothenate partially sults. E. carotovora does not have an absolute reovercomes growth inhibition by D-serine and results in formation of filaments. The obvious quirement for any vitamin, amino acid, purine, inference is that pantothenate simply increases or pyrimidine (12). Thus, the nutritional defi-
970
J. BACTERIOL.
GRULA AND GRULA .9 .8 .7 .6
.
.5 .4 z
Ul 0
4
4
0
0
CONTROL
J
D-SERINE + PANTOTHENATE
.2
-SERINE I
_
.1
12
11
13
12
14 15 HOURS
13
HOURS C. to
a90
90
U
17
18
19
20
0.
s
0
70
cc
60
60 Z 50
40
.j
16
E 80 2
80 ao 70 (2
U
0-
D-SERINE + PANTOTHENATE
30
2
50
i
40
Ul U
D-SERINE + PANTOTHENATE
30
CU
40
4 20 c
20 1U0
D-SERINE
CONTROL
i
< 10
*
i ,=,,D=-SERINE
11
12
13
14 15 HOURS
16
17
18
19
20
11
12
13
15 14 HOURS
16
17
u
18
CONTROL
19
20
FIG. 1. Growth and cell length of E. carotovora in ammonia-glucose medium, as influenced by D-serine and D-serine plus pantothenate. (A) Growth with 0.075 M NH4Cl; (B) growth with 0.015 M NH4CI; (C) cell length with 0.075 M NH4CI; (D) cell length with 0.015 M NH4Cl. D-Serine concentration was 12 x 10-3 M, and pantothenate was 10-6 M.
TABLE 1. Effect of added pantothenate on growth and cell length in ammonia-pyruvate mediuma
Average length of cells or filaments (m)
.OD'
Time after inoculation (h)
0 12 16 21
No pantothenate
0.006 0.008 0.018
With pantothenate
No pantothenate
With pan-
0.00 0.015 0.022
2 3.3 3.2 2
2 20 46 51
Plate countc (Colony-forming units/ml)
No pantothenate
With pantothen-
3.7x107 15 x 107 30 x 107
4.2x107 12 x 107 12 x 107
tothenate
ate
NH4Cl, 0.015 M; pyruvate, 8 mM; D-serine, 1.3 mM; sodium pantothenate, 0.002 mM. b OD, Optical density. c Obtained by averaging the counts of duplicate plates at each of two dilutions. Plating medium was Trypticase soy agar. a
ciencies we were limited to comprised nitrogen (NH4Cl or aspartic acid), carbon energy source, magnesium ion, sulfate ion, and the mineral mixture of our basal medium. Media containing D-serine were prepared with graded limiting amounts of each of the above nutrients, with the compositon otherwise unchanged. Growth and cell length in the critical ranges were observed. Results are given in Table 2. A deficiency of magnesium sulfate is easy to demonstrate. Growth in MgSO4-deficient media to
which D-serine had been added (at 13 h when control cultures would be in exponential growth) was very low; there was no turbidity. Yet the cells were very long; in the case of the medium with no added MgSO4, 30% were in the range of 50 to 100 ,um. With every nutritional deficiency examined, D-serine resulted in very long cells in situations wherein growth was strongly limited. Similar results utilizing other basal media are presented in Table 3. It is evident that a culture
PANTOTHENATE AND FILAMENT FORMATION
VOL. 125, 1976
TABLE 2. Effect of certain nutritional deficiencies on cell length of E. carotovora in the presence of D-serinea
GrwhAverage
Required nutrient concentration as Growth a fraction of control
cell
nof
length
55 47
5.0 4.0
60 50
20.0 21.0
75 38 5
23.0 19.0 49.0
84 74
33
35.0 46.0 71.0
77 28 5
67.0 52.0 70.0
normnal)
(gLm)
Nitrogen (NH4+)' No added pantothenate 1/66
1/0oo With 10-6 M pantothenate 1/66
1/1oo
MgSO4C
1/2 1/3
None added
Aspartic acid (with glucose)
1/2 1/4
1/1o Aspartic acid (without glucose)
1/2
1/4
1/1o D-Serine was present in all media at a concentration of 10-3 M. b NH4Cl was the sole source of N. Glucose was the carbon source. c Basal medium was asparate-glucose. The requirement for MgSO4 is largely for the SO42- ion; since no other source of Mg2+ was added, it was assumed that the Mg2+ requirement was met by the minimal level of MgSO4. a
limited in growth by a low concentration of a specific nutrient may contain very long filaments. The very small increases in turbidity accompanying an elongation that is the equivalent of three or four cell divisions may be accounted for by the lower light-scattering power per unit mass of filaments. Pantothenate, if it actually is necessary for filament formation, should be required in all media. However, this is not always readily observed; in aspartic acid media, for example, filaments are induced by D-serine without the addition of pantothenate. The synthesis of pantothenate by E. carotovora may be conveniently blocked in any medium, including those with exogenous aspartic acid, by sodium salicylate. Maas (15), using a wild-type E. coli, demonstrated that salicylate
interfered with pantoate, and hence pantothenate, synthesis. At one time we felt that if salicylate should act similarly in the metabolism of our strain of Erwinia, it might mimic the action
971
of r-serine, i.e., induce cells to form filaments. However, 10-4 M salicylate resulted in the formation of very short cells, rather than filaments (11). When salicylate was combined with 1)-serine, a synergistic toxicity occurred, and the formation of filaments was almost wholly prevented. Microbial assays indicated that the synthesis of pantothenate by E. carotovora was decreased in the presence of salicylate by 70% (11). Further evidence that salicylate produced a pantothenate deficiency was the fact that 10-6 M pantothenate completely overcame growth inhibition by salicylate, and, if a division-inhibiting agent was also present, formation of filaments occurred (11). Salicylate prevented elongation of Erwinia cells with every division-inhibiting agent with which it was tested. This included the six namino acids that inhibit division (7), D-cycloserine, penicillin, vancomycin, fluoride ion, and ultraviolet light. In all cases, supplementation with pantothenate resulted in very long cells. Data are given in Table 4. Pantothenate can be replaced in these situations by pantoic acid, ketopantoic acid, or aketoisovaleric acid but not by a-methyl pantothenate or /8-alanine. Salicylate added to preformed filaments had no effect on their length. We interpret its action in preventing filament formation solely on the basis of its inhibition of pantothenate synthesis. It should be emphasized that, by itself, pantothenate has no ability to elongate cells beyond their normal length; the presence of a division-inhibiting agent, such as D-serine or one of the cell wall synthesis-inhibiting antibiotics, is required. Tests using E. coli. To determine whether the pantothenate requirement for filament formation observed with E. carotovora might have TABLE 3. Filament formation accompanied by minimal growth Medium
Aspartateglucose
NH4+-glucosepantothenate
Modification Trace minerals omitted NH4+ concentration l/so of control None
Average Growth length (Ism) 100 0.01 0.015
75
80 0.00 Aspartatemalate 100 0.005 10-" M pantoAspartatethenate added malate 90 0.006 None Aspartate at 14 were observed nm. 540 Cultures a Optical density at to 16 h. In aspartate-glucose and NH4+-glucose-pantothenate, D-serine was 10-' M; in the other media, it was 1.3 x
10-3 M.
972
J. BACTERIOL.
GRULA AND GRULA
wider applicability, we utilized E. coli strain B. Crystal violet (18) was chosen as a divisioninhibiting agent since D-serine does not mediate filament formation in E. coli B (E. Grula, unpublished data). Results are given in Table 5. Although not as dramatic as with E. carotovora, changes in cell length occurred under conditions that suggest a critical level of pantothenate or a derivative is necessary for crystal violet to mediate filament formation in E. coli B.
Possible mode of action of pantothenate. Since pantothenate or a derivative appears to be required for cell elongation mediated by a variety of inhibitors (as opposed to a general growth requirement), it is of interest to inquire as to its mode of action.
TABLE 4.
That pantothenate is acting physiologically, instead of in some nonspecific, nonenzymatic way, is indicated by the fact that co-methyl pantothenate (an analogue that might replace pantothenate in a nonenzymatic reaction) would not replace pantothenate in any situation where the deficient factor was pantoic acid. If (3-alanine was the limiting factor, cu-methyl pantothenate stimulated growth and filament formation to some extent, probably because of a partial hydrolysis yielding (8-alanine. Failure of co-methyl pantothenate to replace pantothenate shows that pantothenate action is not associated with the stimulation of division by pantoyl lactone (9); in the latter case, &omethyl pantoyl lactone is more active on a molar basis than pantoyl lactone.
Cell lengths of E. carotovora as affected by certain division-inhibiting agents and added salicylate and pantothenate Cell length
Medium
Division-inhibiting agent
Glucose-aspartate
r-Serine (10-3 M) Fluoride ion (4 x 10-3 M) Penicillin (3 ,ug/ml) Vancomycin (10 ,ug/ml) UV lightb D-Cycloserine (10-6 M) Penicillin D-Cycloserinec
Glucose-ammonia
No further ad- Salicylate (10-5 M) ditions
20-50 20-300 50-300 3-50 10-50 20-100 10-100 2-5
.'
2-3 2-3 3-10 2-3 3-5 2-3 1-2
(Am)a Salicylate + pantothenate (10-6 M) 20-50 20-300 20-300 3-50 10-50 10-50 10-50 10-50
(Pantothenate only) 50-300
3-5 100-300 Penicillin Malate-ammonia Range including 90% of cells or filaments. b Cells were exposed to ultraviolet (UV) light and cultured after exposure as reported previously (9). c Failure of elongation in the presence of D-cycloserine in glucose-ammonia medium indicates a possibility of inhibition of pantothenate synthesis. Addition of pantothenate resulted in an increase in optical density from 0.02 to 0.30 (16 h of incubation). a
TABLE 5. Cell length of E. coli B as influenced by crystal violet, salicylate, and pantothenate in a chemically defined medium Cell length distributione Growtha 20-50 1-2 2-5 5-10 10-20
Additive(s)
100 None 0.77 100 0.30 Salicylate (0.0012 M) 100 0.25 Salicylate (0.0018 M) 30 20 20 0.55 Crystal violet (2 gg/ml) 40 15 25 0.54 Crystal violet (4 jug/ml) 9.9 0.1% 90 0.39 Crystal violet (2 ,ug/ml) + salicylate (0.0018 M) 3 30 15 50 0.57 Crystal violet (2 ,Ag/ml) + salicylate (0.0018 M) + pantothenate (10-6 M) a Optical density at 540 nm. Cultures were observed at 18 h. b Percentage of cells in each length range. Lengths expressed in micrometers. c Some filaments up to 300 in length were present.
jsm
20 20 2
>50
10
10o
to 100 j.m
VOL. 125, 1976
PANTOTHENATE AND FILAMENT FORMATION
Pantoyl taurine would not replace pantothenate in the stimulation of filament formation. It inhibited growth in media in which pantothenate synthesis was limiting; this inhibition was partially relieved by adding pantothenate at a fourfold lower concentration. It was believed desirable to determine the relative amounts of CoA and acyl carrier protein (ACP) in short and in filamentous cells, information that should be useful in analyzing the mechanism of pantothenate action. Das and Toennies (4) found, with Streptococcus faecalis, that in the presence of an excess of precursors during exponential growth the cellular content of CoA and ACP remained constant and in a molar ratio of 4 to 1. Under conditions of stress, ACP was maintained at the expense of CoA. We have compared the CoA and ACP contents of normal and filamentous cells using several media in which -serine was the division-inhibiting agent. Initial experiments were done by adding n-serine and labeled 18-alanine at time zero, in which the effect of n-serine on overall synthesis of CoA and ACP would be shown. Cells were harvested during exponential growth. In these tests, the CoA/ACP ratio of -serine-grown cells was lower than that of the control cells (Table 6). The ACP content of D-serine-grown cells per unit dry weight was increased by about 50% and the CoA content was decreased by 25 to 30%. If -serine was added during early exponential growth, the CoA/ACP ratio continued to be lower in -serine-grown cells, but the difference between these and the control cells was less than that observed using the first approach. A third type of experiment was done to determine whether the diminished CoA levels could be attributed to loss by leakage. Cells were grown to the early exponential phase in a medium with [1-'4C],8-alanine. They were removed from the medium, washed, and placed in fresh unlabeled medium, with and without n-serine. No appreciably greater leakage of radioactive material from cells was observed in the presence of -serine than in its absence. Therefore, it is likely that the decrease in the CoA/ACP ratio observed in n-serine-grown cells is real and not an artifact resulting from greater leakage of CoA. The fact that certain physiological indicators of CoA deficiency occur in -serine-grown cells, including the accumulation of keto acids (13), is consistent with a decreased CoA content. It is premature to speculate on the significance of this alteration in CoA or the CoA/ACP ratio. Some type of association of filament formation with a change in the pattern of fatty
973
TABLE 6. CoAIACP ratios of filaments and of normal cells ofE. carotovora ~~Average length CoA/ACP cell Time of
aditioonf
Basal medium
ratio of DserineP Con- D-Ser(h) trol ine
NH4+-glucose
#-alanine
NH4+-glucose
(_Lm) Con- D-Sertrol
ine
0
5.2
2.7
5
30
12
5.6
4.8
3
20
#-alanine
30 0 4.7 2.5 2 NH4+-glucose pantothenate 10 14 5.3 4.7 3 NH4+-glucose pantothenate 12 0 5.8 2.7 2 NH4+-pyruvate 8-alanine a 0 h is time of inoculation. Cultures were harvested at 16 h, at which time cell length was determined.
acid synthesis is a possibility, but so far no conclusive evidence that such an association exists has been obtained. LITERATURE CITED 1. Bray, G. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279-285. 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore. 3. Das, D. N., and G. Toennies. 1968. A versatile buffered scintillation system. Anal. Biochem. 23:26-34. 4. Das, D. N., and G. Toennies. 1969. Relations between coenzyme A and presumptive acyl carrier protein in different conditions of streptococcal growth. J. Bacteriol. 98:898-902. 5. Duguid, J. P., and J. F. Wilkinson. 1961. Environmentally induced changes in bacterial morphology. Symp. Soc. Gen. Microbiol. 11:69-99. 6. Dye, D. W. 1969. A taxonomic study of the genus Erwinia. II. The 'carotovora' group. N. Z. J. Sci. 12:8197. 7. Graham, D. C. 1972. Identification of soft rot coliform bacteria, p. 273-279. In H. P. Maas Gustermus (ed.), Proceedings of the Third International Conference on Plant Pathogenic Bacteria. Center for Agricultural Publishing and Documentation, Wagenigen, The
Netherlands. 8. Grula, E. A. 1960. Cell division in a species ofErwinia. II. Inhibition of division by 1-amino acids. J. Bacteriol. 80:375-385. 9. Grula, E. A., and M. M. Grula. 1962. Cell division in a species of Erwinia. II. Reversal of inhibition of cell division caused by D-amino acids, penicillin, and ultraviolet light. J. Bacteriol. 83:981-989. 10. Grula, E. A., and M. M. Grula. 1964. Cell division in a species of Erwinia. VII. Amino sugar content ofdividing and non-dividing cells. Biochem. Biophys. Res. Commun. 17:341-346. 11. Grula, M. M., and E. A. Grula. 1962. Cell division in a species of Erwinia. IV. Metabolic blocks in pantothenate biosynthesis and their relationship to inhibition of cell division. J. Bacteriol. 83:989-997. 12. Grula, M. M., R. W. Smith, C. F. Parham, and E. A.
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GRULA AND GRULA
Grula. 1968. Cell division in a species of Erwinia. XI. Some aspects of the carbon and nitrogen nutrition of Erwinia species. Can. J. Microbiol. 14:1217-1224. 13. Grula, M. M., R. W. Smith, C. F. Parham, and E. A. Grula. 1968. Cell division in a species ofErwinia. XI. A study of nutritional influences in D-serine inhibition of growth of Erwinia sp., and of certain specific sites of D-serine action. Can. J. Microbiol. 14:12251238. 14. Howard-Flanders, P., E. Simson, and L. Theriot. 1964. A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics 49:237-246.
J. BACTERIOL. 15. Mas, W. K. 1952. Pantothenate studies. II. Evidence from mutants for interference by salicylate with pantoate synthesis. J. Bacteriol. 63:227-232. 16. Sister, M., and M. Schaechter. 1974. Control of cell division in bacteria. Bacteriol. Rev. 38:199-221. 17. Starr, M. P., and A. K. Chatterjee. 1972. The genus Erwinia: enterobacteria pathogenic to plants and ani-
mals. Annu. Rev. Microbiol. 26:389-426. 18. Walker, J. R., N. A. Shafiq, and R. G. Allen. 1971. Bacterial cell division regulation: physiological effects of crystal violet onEscherichia coli lon+ and lon strains. J. Bacteriol. 108:1296-1303.
