Vol. 130, No. 3
JOUIRAL OF BACTRzIOLOGY, June 1977, p. 1199-1205 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Effect of Macromolecular Synthesis on the Coordinate Morphogenesis of Polar Surface Structures in Caulobacter crescentus AKIO FUKUDA* AND YOSHIMI OKADA Department ofBiophysics and Biochemistry, Faculty of Sciences, University of Tokyo, Hongo,
Tokyo 113, Japan Received for publication 17 March 1977
The Caulobacter polar surface structures (flagella, pili, and the deoxyribonucleic acid phage OCbK receptors), which are expressed at proximal sites of swarmer cells in a coordinate manner (Shapiro, Annu. Rev. Microbiol., 30:377407, 1976) could be blocked by a single mutation. The mutant C. crescentus CB13 ple-801 did not form these surface structures when grown at 3500. Upon shift down to 2500, the mutant cells initiated the formation of the surface structures. When mitomycin C was added to the mutant culture upon shift down from 35 to 2500, 4CbK receptor formation was inhibited to a minimal level. Rifampin and chloramphenicol completely inhibited 4>CbK receptor formation when added to the mutant culture upon shift down. Deoxyribonucleic acid as well as ribonucleic acid and protein synthesis seem to be required for the formation of qCbK receptors. Penicillin V also inhibited 4CbK receptor formation, indicating the involvement of cell wall synthesis. When the mutant CB13 ple-801 cells were shifted down briefly from 35 to 2500 and then shifted up to 3500, flagella and 0CbK receptors were formed even at 3500 to different extents depending on how long the cells were incubated at 2500. This formation of the surface structures at 3500 was inhibited by rifampin. From these results, it appears that translation, assembly, or localization processes for the formation ofthe surface structures are not temperature sensitive at 3500 in the pleiotropic mutant CB13 ple-801. The syntheses of deoxyribonucleic acid and the cell wall do not appear to be temperature sensitive either, since the mutant grows normally at 3500. It is suggested that there exists a regulatory step that commits the cells to initiate the synthesis of requisite ribonucleic acid for the formation of the polar surface structures.
Cellular structures that are useful for the study of Caulobacter cell differentiation are a single flagellum, pili, and deoxyribonucleic acid (DNA) phage receptors. These are all polar surface structures that are under temporal control and appear at a similar defined time in the Caulobacter cell cycle at proximal sites in the swarmer cell (8). The morphogenesis of these polar surface structures is manifested in cell motility and susceptibility to phages and can be estimated by assaying specific phage-adsorbing capacities of the cells (2, 3, 8). The pili are ribonucleic acid (RNA) phage receptors (7), and active flagella serve as attachment sites for certain flagellotropic DNA phages (2, 5). The nature of DNA phage receptors is not yet known. It has been shown previously that pleiotropic single mutations affect cell motility and susceptibility to phage infection (3, 4). A single muta-
tion simultaneously blocks the formation of flagella, pili, and DNA phage receptors in C. crescentus CB13, whereas other mutations block the formation of some of these surface structures. A large number of phenotypic mutants have been isolated and classified into several groups with respect to cell motility and susceptibility to phage infection. From the analysis of these mutants, it is suggested that there exists an ordered sequence in the coordinate morphogenesis of the Caulobacter surface structures at the polar cell sites (3). Mutants in group I are those that become nonmotile and simultaneously resistant to both DNA and RNA phages (3). Temperature-sensitive mutants in this group were isolated and partially characterized previously (3). These mutants offer an opportunity to investigate mechanisms for the coordinate morphogenesis of the cell surface structures. We describe in
1199
1200
FUKUDA AND OKADA
J. BACTERIOL.
this paper the effects of macromolecular synthesis on the formation of the polar surface structures in C. crescentus CB13 and discuss possible regulatory sites.
tus CB13 cells
were grown fresh in PYE medium to optical density at 660 nm (OD660) of 0.1 (4.4 x 108 cells per ml) at temperatures indicated. Chloramphenicol was added to 0.5 to 1.0 ml of CB13 culture
an
at a final concentration of 20 ,ug/ml, and 32P-labeled was added to achieve a multiplicity above 12.0. At the concentration of chloramphenicol used, the cells remained motile for 1 h without growth, and at a multiplicity above 12.0, linearity existed between 32P-labeled 4CbK or 4Cp34 adsorbed to the
phage
MATERIALS AND METHODS Bacteria, phages, and growth conditions. C. crescentus CB13Bla wild type and CB13 ple-801,
a
tem-
perature-sensitive pleiotropic mutant, and its spontaneous revertants, CB13 ple-801 R1, CB13 ple801R2, and CB13 ple-801R3 (3), were used. DNA phage 4CbK (1) and flagellotropic DNA phage 4Cp34 (2) were described previously. Bacteria and phages were routinely grown and propagated in peptone-yeast extract (PYE) nutrient broth (6). Phage stocks of high titer were prepared as described previously (2). Preparation of 32P-labeled phages. For the preparation of 32P-labeled 4CbK or qCp34, [32P]phosphate (2 mCi, carrier-free, Japan Isotope Association, Tokyo) was added to 50 ml of CB13 wild-type culture (1.3 x 108 cells per ml) upon phage infection at a multiplicity of 0.03. The infected culture was incubated at 30°C for 24 h with reciprocal shaking for phage propagation. The lysates were centrifuged at 8,000 rpm for 10 min to remove cell debris. The supernatant fluid was then centrifuged at 15,000 rpm and 4°C for 60 min, and the sediment was suspended in 1 ml of PYE medium. After removal of remaining cell debris by low-speed centrifugation (6,000 rpm, 5 min), the phage suspension was sedimented through a linear 30-ml gradient from 5 to 20% sucrose in PYE medium at 20,000 rpm and 4°C for 30 min in a Hitachi RPS25 rotor. The peak fractions of phage infectivity were pooled and filtered through a double layer of one membrane filter disk (HAWP, 0.45-,um pore size, 2.4-cm diameter; Millipore Corp., Bedford, Mass.) and one Whatman glass filter paper disk (GF/B, 2.4-cm diameter; Whatman, Inc., Clifton, N.J.). The titer and radioactivity of such a phage preparation were routinely 2.0 x 1011 plaque-forming units (PFU)/ ml, 8.0 x 10- 32p cpm/PFU, for qbCbK and 4.0 x 1010 PFU/ml, 3.0 x 10-5 32p cpm/PFU, for 0Cp34. Assay of 32P-labeled phage adsorption. C. crescew,
cells and the cell concentration. The cell-phage mixwas then incubated for 15 min at 25°C with gentle shaking. The adsorption of 32P-labeled 4OCbK or OCp34 usually reached a plateau within 10 min of incubation. The mixture was filtered onto a Whatman GF/B filter paper disk (2.4-cm diameter) by gentle aspiration, washed six times with 5-ml portions of PYE medium, dried at 80°C for 30 min, and counted in toluene base scintillation fluid. Chemicals. Chloramphenicol, mitomycin C, and rifampin were purchased from Sigma Chemical Co., St. Louis, Mo. ture
RESULTS
Formation of flagella and phage receptors in the temperature-sensitive
mutant CB13
ple-801 grown at different temperatures. A temperature-sensitive mutant, C. crescentus CB13 ple-801, becomes nonmotile and simultaneously resistant to both DNA and RNA phages when grown at a nonpermissive temperature (35°0) (3). To estimate the formation of flagella and phage receptors in CB13 ple-801 grown at different temperatures, phage-adsorbing capacities of the cells were assayed by the use of 32P-labeled DNA phage OCbK and flagellotropic DNA phage OCp34. The mutant CB13 ple-801 did not adsorb 32P-labeled 4CbK and 4Cp34 when grown at 350C (Table 1). The mutant cells possessed phage-adsorbing capacities when grown at a lower temperature. However, the formation of flagella, defined here as 32p_
labeled OCp34-adsorbing capacity, and of ¢CbK
receptors appeared to be substantially reduced
TABLE 1. Phage adsorption to C. crescentus CB13 wild type and temperature-sensitive pleiotropic mutant CB13 ple-801 and its revertants, grown at different temperatures Phagea
Strain
4CbK 30'C
oCp34
25°C 35°C 25°C 300C 35°C CB13 wild type 100 83.1 65.5 100 100.1 64.9 CB13 ple-801 44.1 37.6 0 80.1 35.4 2.7 CB13 ple-801R1 82.6 63.0 99.9 72.6 CB13ple-801R2 91.1 50.3 103.5 62.6 CB13 ple-801R3 103.5 36.8 101.1 52.3 a 32P-labeled phages adsorbed to cells are presented as a percentage of those on CB13 wild type grown at 250C. One hundred percent 4CbK adsorption was 10,884 32P cpm/ml at an OD66o of 0.1, and 100% 0Cp34 was 10,344 32p cpm/ml of an ODm of 0.1.
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SURFACE STRUCTURE MORPHOGENESIS IN CAULOBACTER
in the CB13 ple-801 cells, even when grown at 250C. All the spontaneous revertants isolated from CB13 ple-801 regained the phage-adsorbing capacities, together with cell motility, irrespective of growth temperatures. As reported previously (3), the mutant CB13 ple-801 cells did not possess pili when grown at a nonpermissive temperature. These results support the previous notion (3, 8) that a single mutation pleiotropically affects the formation of flagella and phage receptors. The question now arises whether or not the lack of phage-adsorbing capacities in CB13 ple801 grown at a nonpermissive temperature is attributable to enhanced temperature sensitivity of phage receptors. The strains CB13 wild type and CB13 ple-801 were grown at 250C in PYE medium. After addition of chloramphenicol at a concentration of 20 ,ug/ml to inhibit further synthesis of phage receptors, the CB13 wild-type and CB13 ple-801 cells were shifted up to 350C, and 32P-labeled OCbK-adsorbing capacities were assayed at intervals. The 4CbKadsorbing capacity of the wild-type cells decreased exponentially at 350C by single-hit kinetics but were stable at 250C (Fig. 1). This result indicates that the wild-type OCbK receptors are already temperature sensitive at 350C and agrees with the reduced level of 4CbK adsorption to the wild-type cells grown at 350C (Table 1). Similarly, the flagellotropic OCp340
30
TIME (min ) 60 90
120
.
A00 50 10-
10=
5-
FIG. 1. Temperature sensitivity of 4CbK receptors in the wild-type CB13 and the mutant CB13 ple-801. Wild-type CB13 and the temperature-sensitive pleiotropic mutant CB13 ple-801 cells were freshly grown at 25°C to an ODfo of 0.1 (4.4 x 108 cells per ml) in PYE medium and mixed with chloramphenicol at a final concentration of 20 pg/ml. One-half portion of each culture was incubated at 25°C, and the other half was shifted up to 35°C. At intervals, portions (1.0 ml) were removed for the assay of 32P-labeled 4CbK-adsorbing capacities as described in the text. One hundred percent 4CbK adsorption was 7,610 32p cpmlml at an OD60 of 0.1 for the mutant CB13 ple801. Symbols: 0, CB13 wild type, 35°C; *, CB13 ple801, 35°C; CB13 wild type, 25°C; *, CB13 ple5,
801, 25°C.
1201
adsorbing capacity of the wild-type cells was temperature sensitive at 350C. The 4CbK-adsorbing capacity of the temperature-sensitive mutant CB13 ple-801 decreased at 350C similarly to that of the wild-type strain (Fig. 1). There was no enhancement of temperature sensitivity in 4)CbK adsorption to the mutant cells. The XCbK receptors of the mutant cells were also stable at 250C. The results with OCp34 were similar. These results indicate that the flagella and phage receptors, themselves, are not temperature sensitive, but rather that their formation is, in the pleiotropic mutant CB13 ple-801 at a nonpermissive temperature. Effect of macromolecular synthesis on surface structure formation. The cell cycle of the temperature-sensitive mutant CB13 ple-801 proceeds normally at 350C, as does that of the wild-type strain, with respect to cell growth, gross cell morphology, and asymmetric stalk formation (3). This mutant thus offers an opportunity to examine the effect of macromolecular synthesis on the formation of the polar surface structures upon shift down to a permissive temperature. The mutant CB13 ple-801 was freshly grown at 350C in PYE medium to an OD6. Of 0.1 (4.41 x 108 cells per ml) and shifted down to 250C. Mitomycin C, rifampin, or chloramphenicol was added to the culture at various times after shift down and, at intervals, portions (1.0 ml) of the mutant culture were removed for assay of 32P-labeled OCbK-adsorbing capacity (4CbK receptors). Without addition of macromolecular inhibitors, 4CbK receptors began to be formed at 15 min after shift down, and thereafter the specific activity (32P counts per minute per milliliter at an OD660 of 0.1) of 4CbK receptors increased continuously for at least 180 min (Fig. 2). Similarly, flagella formation (32P-labeled 4Cp34 adsorption) and cell motility (as observed under a phase-contrast microscope) began to appear at 30 min after shift down. When a DNA inhibitor, mitomycin C, was added upon shift down (at 0 min) at a concentration of 5 ug/ml, the formation of ¢CbK receptors was inhibited to a minimal level (Fig. 2). When the drug was added later, at 20 or 60 min after shift down, the already initiated formation of CbK receptors continued for an additional 80 min and plateaued. Similar amounts of ¢CbK receptors were formed, irrespective of the addition time of the drug at 20 or 60 min after shift down. These results indicate that DNA synthesis is necessary for the formation of the surface structures. In similar experiments, when an RNA inhibitor, rifampin, was added upon shift down (at 0
1202
J. BACTERIOL.
FUKUDA AND OKADA
4 C x
U, 0
3
0 c
2 N, C
E
U1 E-
CN4 m
TIME( min )
FIG. 2. Effects of macromolecular inhibitors on the formation of 4CbK receptors. Temperature-sensitive pleiotropic mutant CB13 ple-801 cells were freshly grown in PYE medium to an OD,O of 0.1 (4.4 x 108 cells per ml) at 35°C and shifted down to 25°C. The temperature of the culture decreased to 25°C within 2 min. After shift down, mitomycin C (MC, 5 Mg/ml), rifampin (RIF, 4 pg/ml), or chloramphenicol (CM, 20 pg/mi) was added to the culture at 0, 20, and 60 min. At intervals after drug addition, portions (1.0 ml) were removed for the assay of 32P-labeled 4CbK-adsorbing capacities as described in the text. Symbols: 0, No drug addition; *, drug addition, 0 min; *, drug addition, 20 min; 0, drug addition, 60 min.
min) at a concentration of 4 ,Ig/ml, the formation of OCbK receptors was completely inhibited. However, appreciable amounts of OCbK receptors were formed after rifampin addition at 20 or 60 min after shift down. This formation of OCbK receptors after drug addition continued for about 80 min before it leveled off. It appears that de novo RNA synthesis after shift down is required for the formation of oCbK receptors. The residual formation of OCbK receptors for 80 min in the presence of rifampin might indicate that, once synthesized, requisite RNA is rather stable. When a protein inhibitor, chloramphenicol, was added upon shift down (at 0 min) at a concentration of 20 ,ug/ml, the formation of 4CbK receptors was completely inhibited, as in the case of rifampin addition. Unlike rifampin addition, there was no residual receptor formation when the drug was added at 20 min after shift down. Upon drug addition at 60 min after shift down, however, residual formation of 4CbK receptors was observed, indicating that some receptors could be formed in the absence of protein synthesis once the cells were incubated long enough at a low temperature (25°C). The effects of macromolecular synthesis described above suggest that (i) de novo RNA and protein syntheses are required for the formation of the surface structures upon shift down in
the temperature-sensitive pleiotropic mutant CB13 ple-801, and (ii) DNA synthesis is also a prerequisite for the cells to efficiently form the surface structures. Surface structure formation in the mutant CB13 ple-801 upon temperature shift down and subsequent shift up. Temperature shift down and subsequent shift up experiments were carried out to examine a possible control step for the morphogenesis of the polar surface structures. The temperature-sensitive mutant CB13 ple-801 was grown freshly at 35°C in PYE medium to the early log phase (OD660, 0.1, or 4.41 x 108 cells per ml). The culture was shifted down to 250C and, after incubation for different times, shifted up again to 35°C. 32P-labeled 4CbK- and 4Cp34-adsorbing capacities were assayed in the meantime. OCbK receptors were formed even at 350C, once the cells were briefly exposed to 250C (Fig. 3). When the cells were shifted up to 350C at 4 min after shift down to 25°C, 4CbK receptors were immediately formed, in contrast with the later formation (15 min) when cells were continuously incubated at 250C. This difference should reflect the difference in cellular activity at 250C (generation time, 215 min) and at 350C (generation time, 150 min) of the mutant in PYE medium. The extent of 4CbK receptor formation in
SURFACE STRUCTURE MORPHOGENESIS IN CA ULOBACTER
VOL. 130, 1977
1203
o 0
E
C)
C? "I
cU
ci
.E O ci)
E
2 ,,,
a-
E~ -C
Q-
-o
30
60
90 0 30 TIME (min )
60
90
FIG. 3. Formation ofpolar surface structures in the temperature-sensitive pleiotropic mutant CB13 ple-801 temperature shift down and subsequent shift up. Mutant CB13 ple-801 cells were freshly grown in PYE medium to an OD6N of 0.1 (4.4 x 108 cells per ml) at 35°C, divided into three portions, and shifted down to 25°C. (a and b) One portion ofthe culture was incubated at 25°C (0), and the other two portions were shifted up to 35°C at 4 min (a) and 11 min (0) after shift down for O4CbK adsorption and at 10 min (*) and 20 min (0) for flagellotropic 4Cp34 adsorption. At intervals during shift down and subsequent shift up, samples (1.0 ml) were removed for the assay of 32P-labeled phage-adsorbing capacities. Specific phage-adsorbing capacities are presented as 32p counts per minute per milliliter at an OD660 of 0.1. (c and d) In separate experiments, the mutant CB13 ple-801 (at an OD6,. of 0.1 in PYE medium) was shifted up to 35°C at 20 min after shift down to upon
25°C. Rifampin (RIF, 4 pg/ml) was added to one portion of the culture upon shift up. 32P-labeled phageadsorbing capacities were assayed as above. Specific phage-adsorbing capacities are presented as 32p counts per minute per milliliter at an OD660 of 0.1 . Symbols: 0, No drug addition, 25°C; 0, no drug addition, shifted up to 35°C; *, drug addition, shifted up to 35°C.
early times after shift up was dependent on how long the cells were incubated at 25°C before shift up to 35°C. This 4CbK receptor formation after shift up to 35°C was inhibited by rifampin (Fig. 3). Therefore, it appears that requisite RNA for 4CbK receptor formation can be synthesized at 35°C. 4CbK receptor formation after shift up to 35°C continued for about 25 min. CbK receptor activity then decreased gradually when the cells were continously incubated at 35°C. This decrease of 4CbK receptor activity might be attributed to the inherent temperature sensitivity of 4CbK receptors (Fig. 1) and to the transition of swarmer cells to stalked cells, during which 4CbK receptors are lost from the cells (8).
Quite similar results were obtained with flagella formation in temperature shift down and subsequent shift up experiments (Fig. 3). Flagella formation after shift up to 35°C was also inhibited by rifampin. The results described above indicate that DNA, RNA, and protein syntheses and assembly for the formation of the polar surface structures are not temperature sensitive in the mutant CB13 ple-801. Effect of penicillin V on the formation of the surface structures. The cell wall is involved directly or indirectly in the formation of cell surface structures. To examine the effect of cell wall synthesis on the formation of the Caulobacter surface structures, penicillin V was added to CB13 ple-801 culture at a concentra-
1204 FUKUDA AND OKADA tion of 800 ug/ml upon shift down from 35 to 250C, and 4CbK receptor formation was assayed by 32P-labeled 4)CbK adsorption to the cells. Penicillin V inhibited the formation of 4CbK receptors (Fig. 4). There was no flagella formation, either, in the presence of penicillin V. Although an aberrant effect of penicillin addition on the cells is not rigorously excluded, the above results suggest that the formation of the Caulobacter surface structures requires cell wall synthesis. DISCUSSION In this paper we have described the effects of macromolecular synthesis on the formation of the Caulobacter polar surface structures (flagella and phage receptors). As shown in previous and the present experiments (3; Table 1) the formation of the C.'crescentus CB13 polar surface structures is regulated by a common mechanism. A single mutation simultaneously blocks the formation of flagella, pili (RNA phage receptors; 7), and DNA phage receptors. The temperature-sensitive pleiotropic mutant C. crescentus CB13 ple-801 grows normally at either 25 or 350C, as does the wild-type strain. At 350C this mutant is unable to form flagella, pili, and DNA phage 4CbK receptors, but upon shift down .to 250C it initiates the formation of these surface structures (Fig. 2 and 3). Thus, effects of macromolecular synthesis on the surface structure formation in the mutant could be
E 2 E C
7 -7
CL
005
n
0
30 60 TIME (min
90
0
o
FIG. 4. Effect of penicillin V on 4CbK receptor formation. Mutant CB13 ple-801 cells were grown fresh in PYE medium at 350C to an ODma of 0.095 (4.2 108 cells per ml), divided into two portions, and shifted down to 25°C. Upon shift down (0 min), penicillin V was added to one portion of the culture at a concentration of 800 pg/ml. At intervals, portions (1.0 ml) were removed for the assay of 32P-labeled oCbK-adsorbing capacities. ODm values of the culture were also measured in parallel. Specific phageadsorbing capacities are presented as 32P counts per minute per milliliter at an ODsw of 0.1. Symbols: 0, 32p counts per minute, no drug addition; o, 32p_ counts per minute, drug addition; 0, OD6n, no drug addition; OD680, drug addition. x
0,
J. BACTERIOL.
conveniently examined in temperature shift experiments by the use of macromolecular inhibitors. In addition, the extent of surface structure formation could be quantitated by assaying specific phage adsorption. When DNA synthesis was inhibited, the surface structure formation was also inhibited to a great extent. This was typically shown by assaying 4CbK receptor formation in the CB13 ple-801 cells upon shift down from 35 to 250C in the presence of mitomycin C (Fig. 2). It is not clear how DNA synthesis is related to the surface structure formation that occurs at a defined time in the cell cycle (8). Presumably, only those cells that enter the critical period at a permissive temperature become committed to form the surface structures, and this process requires DNA synthesis. Rifampin completely inhibited the surface structure formation when added to the CB13 ple-801 culture upon temperature shift down (Fig. 2). Once the mutant CB13 ple-801 cells were incubated briefly at 250C and then shifted up to 350C, the surface structure formation occurred, which was again inhibited by rifampin (Fig. 3). These results indicate that the synthesis of requisite RNA per se is not temperature sensitive at 350C. It then seems likely that there exists a regulatory step that commits the cells to initiate the synthesis of requisite RNA for surface structure formation. As so far examined, there was no stimulatory effect of dibutyryl cyclic adenosine 3',5'-monophosphate or dibutyryl cyclic guanosine 3',5'-monophosphate on the formation of CbK receptors and flagella in CB13 ple-801 grown at 350C in PYE medium or in Casamino Acids-supplemented minimal medium (A. Fukuda, unpublished data). Chloramphenicol completely inhibited the surface structure formation when added early (within about 20 min) after shift down from 35 to 250C (Fig. 2), indicating that de novo protein synthesis is essential. When the mutant CB13 ple-801 cells were incubated at 250C for a longer time (60 min), precursors seemed to be synthesized sufficiently for a residual formation of 4CbK receptors in the presence of chloramphenicol (Fig. 2). As described above, the surface structures were formed in CB13 ple-801 even at 350C, once the cells were incubated briefly at 250C before subsequent shift up (Fig. 3). This result seems to exclude the possibility that, among conceivable regulatory steps for surface structure formation, translation, assembly, or localization is temperature sensitive in CB13 ple-801 at 350C. The syntheses of DNA and cell wall, which also appear to be involved in surface structure formation (Fig. 2 and 4), should not
VOL. 130, 1977
SURFACE STRUCTURE MORPHOGENESIS IN CAULOBACTER.
be temperature sensitive either, since the tant grows normally at 35°C.
mu-
ACKNOWLEDGMENT This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan. LITERATURE CITED 1. Agabain-Keshishian, N., and L. Shapiro. 1970. Stalked bacteria: properties of deoxyribonucleic acid bacteriophage 46CbK. J. Virol. 5:795-800. 2. Fukuda, A., K. Miyakawa, H. Iba, and Y. Okada. 1976. A flagellotropic bacteriophage and flagella formation in Caulobacter. Virology 71:583-592. 3. Fukuda, A., K. Miyakawa, H. Iida, and Y. Okada. 1976. Regulation of polar surface structure in Caulto-
4.
5.
6. 7.
8.
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bacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors. Mol. Gen. Genet. 149:167-173. Fukuda, A., K. Miyakawa, and Y. Okada.1974. A pleiotropic mutation in Caulobacter crescentus that affects the flagellar formation and susceptibility to phage infection. Proc. Jpn. Acad. 50:839-842. Jollick, J. D., and B. L. Wright. 1974. A flagella specific bacteriophage for Caulobacter. J. Gen. Virol. 22:197205. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. Schmidt, J. M. 1966. Observations on the adsorption of Caulobacter bacteriophages containing ribonucleic acid. J. Gen. Microbiol. 45:347-353. Shapiro, L. 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377-407.
JOUIRAL OF BACTRzIOLOGY, June 1977, p. 1199-1205 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Effect of Macromolecular Synthesis on the Coordinate Morphogenesis of Polar Surface Structures in Caulobacter crescentus AKIO FUKUDA* AND YOSHIMI OKADA Department ofBiophysics and Biochemistry, Faculty of Sciences, University of Tokyo, Hongo,
Tokyo 113, Japan Received for publication 17 March 1977
The Caulobacter polar surface structures (flagella, pili, and the deoxyribonucleic acid phage OCbK receptors), which are expressed at proximal sites of swarmer cells in a coordinate manner (Shapiro, Annu. Rev. Microbiol., 30:377407, 1976) could be blocked by a single mutation. The mutant C. crescentus CB13 ple-801 did not form these surface structures when grown at 3500. Upon shift down to 2500, the mutant cells initiated the formation of the surface structures. When mitomycin C was added to the mutant culture upon shift down from 35 to 2500, 4CbK receptor formation was inhibited to a minimal level. Rifampin and chloramphenicol completely inhibited 4>CbK receptor formation when added to the mutant culture upon shift down. Deoxyribonucleic acid as well as ribonucleic acid and protein synthesis seem to be required for the formation of qCbK receptors. Penicillin V also inhibited 4CbK receptor formation, indicating the involvement of cell wall synthesis. When the mutant CB13 ple-801 cells were shifted down briefly from 35 to 2500 and then shifted up to 3500, flagella and 0CbK receptors were formed even at 3500 to different extents depending on how long the cells were incubated at 2500. This formation of the surface structures at 3500 was inhibited by rifampin. From these results, it appears that translation, assembly, or localization processes for the formation ofthe surface structures are not temperature sensitive at 3500 in the pleiotropic mutant CB13 ple-801. The syntheses of deoxyribonucleic acid and the cell wall do not appear to be temperature sensitive either, since the mutant grows normally at 3500. It is suggested that there exists a regulatory step that commits the cells to initiate the synthesis of requisite ribonucleic acid for the formation of the polar surface structures.
Cellular structures that are useful for the study of Caulobacter cell differentiation are a single flagellum, pili, and deoxyribonucleic acid (DNA) phage receptors. These are all polar surface structures that are under temporal control and appear at a similar defined time in the Caulobacter cell cycle at proximal sites in the swarmer cell (8). The morphogenesis of these polar surface structures is manifested in cell motility and susceptibility to phages and can be estimated by assaying specific phage-adsorbing capacities of the cells (2, 3, 8). The pili are ribonucleic acid (RNA) phage receptors (7), and active flagella serve as attachment sites for certain flagellotropic DNA phages (2, 5). The nature of DNA phage receptors is not yet known. It has been shown previously that pleiotropic single mutations affect cell motility and susceptibility to phage infection (3, 4). A single muta-
tion simultaneously blocks the formation of flagella, pili, and DNA phage receptors in C. crescentus CB13, whereas other mutations block the formation of some of these surface structures. A large number of phenotypic mutants have been isolated and classified into several groups with respect to cell motility and susceptibility to phage infection. From the analysis of these mutants, it is suggested that there exists an ordered sequence in the coordinate morphogenesis of the Caulobacter surface structures at the polar cell sites (3). Mutants in group I are those that become nonmotile and simultaneously resistant to both DNA and RNA phages (3). Temperature-sensitive mutants in this group were isolated and partially characterized previously (3). These mutants offer an opportunity to investigate mechanisms for the coordinate morphogenesis of the cell surface structures. We describe in
1199
1200
FUKUDA AND OKADA
J. BACTERIOL.
this paper the effects of macromolecular synthesis on the formation of the polar surface structures in C. crescentus CB13 and discuss possible regulatory sites.
tus CB13 cells
were grown fresh in PYE medium to optical density at 660 nm (OD660) of 0.1 (4.4 x 108 cells per ml) at temperatures indicated. Chloramphenicol was added to 0.5 to 1.0 ml of CB13 culture
an
at a final concentration of 20 ,ug/ml, and 32P-labeled was added to achieve a multiplicity above 12.0. At the concentration of chloramphenicol used, the cells remained motile for 1 h without growth, and at a multiplicity above 12.0, linearity existed between 32P-labeled 4CbK or 4Cp34 adsorbed to the
phage
MATERIALS AND METHODS Bacteria, phages, and growth conditions. C. crescentus CB13Bla wild type and CB13 ple-801,
a
tem-
perature-sensitive pleiotropic mutant, and its spontaneous revertants, CB13 ple-801 R1, CB13 ple801R2, and CB13 ple-801R3 (3), were used. DNA phage 4CbK (1) and flagellotropic DNA phage 4Cp34 (2) were described previously. Bacteria and phages were routinely grown and propagated in peptone-yeast extract (PYE) nutrient broth (6). Phage stocks of high titer were prepared as described previously (2). Preparation of 32P-labeled phages. For the preparation of 32P-labeled 4CbK or qCp34, [32P]phosphate (2 mCi, carrier-free, Japan Isotope Association, Tokyo) was added to 50 ml of CB13 wild-type culture (1.3 x 108 cells per ml) upon phage infection at a multiplicity of 0.03. The infected culture was incubated at 30°C for 24 h with reciprocal shaking for phage propagation. The lysates were centrifuged at 8,000 rpm for 10 min to remove cell debris. The supernatant fluid was then centrifuged at 15,000 rpm and 4°C for 60 min, and the sediment was suspended in 1 ml of PYE medium. After removal of remaining cell debris by low-speed centrifugation (6,000 rpm, 5 min), the phage suspension was sedimented through a linear 30-ml gradient from 5 to 20% sucrose in PYE medium at 20,000 rpm and 4°C for 30 min in a Hitachi RPS25 rotor. The peak fractions of phage infectivity were pooled and filtered through a double layer of one membrane filter disk (HAWP, 0.45-,um pore size, 2.4-cm diameter; Millipore Corp., Bedford, Mass.) and one Whatman glass filter paper disk (GF/B, 2.4-cm diameter; Whatman, Inc., Clifton, N.J.). The titer and radioactivity of such a phage preparation were routinely 2.0 x 1011 plaque-forming units (PFU)/ ml, 8.0 x 10- 32p cpm/PFU, for qbCbK and 4.0 x 1010 PFU/ml, 3.0 x 10-5 32p cpm/PFU, for 0Cp34. Assay of 32P-labeled phage adsorption. C. crescew,
cells and the cell concentration. The cell-phage mixwas then incubated for 15 min at 25°C with gentle shaking. The adsorption of 32P-labeled 4OCbK or OCp34 usually reached a plateau within 10 min of incubation. The mixture was filtered onto a Whatman GF/B filter paper disk (2.4-cm diameter) by gentle aspiration, washed six times with 5-ml portions of PYE medium, dried at 80°C for 30 min, and counted in toluene base scintillation fluid. Chemicals. Chloramphenicol, mitomycin C, and rifampin were purchased from Sigma Chemical Co., St. Louis, Mo. ture
RESULTS
Formation of flagella and phage receptors in the temperature-sensitive
mutant CB13
ple-801 grown at different temperatures. A temperature-sensitive mutant, C. crescentus CB13 ple-801, becomes nonmotile and simultaneously resistant to both DNA and RNA phages when grown at a nonpermissive temperature (35°0) (3). To estimate the formation of flagella and phage receptors in CB13 ple-801 grown at different temperatures, phage-adsorbing capacities of the cells were assayed by the use of 32P-labeled DNA phage OCbK and flagellotropic DNA phage OCp34. The mutant CB13 ple-801 did not adsorb 32P-labeled 4CbK and 4Cp34 when grown at 350C (Table 1). The mutant cells possessed phage-adsorbing capacities when grown at a lower temperature. However, the formation of flagella, defined here as 32p_
labeled OCp34-adsorbing capacity, and of ¢CbK
receptors appeared to be substantially reduced
TABLE 1. Phage adsorption to C. crescentus CB13 wild type and temperature-sensitive pleiotropic mutant CB13 ple-801 and its revertants, grown at different temperatures Phagea
Strain
4CbK 30'C
oCp34
25°C 35°C 25°C 300C 35°C CB13 wild type 100 83.1 65.5 100 100.1 64.9 CB13 ple-801 44.1 37.6 0 80.1 35.4 2.7 CB13 ple-801R1 82.6 63.0 99.9 72.6 CB13ple-801R2 91.1 50.3 103.5 62.6 CB13 ple-801R3 103.5 36.8 101.1 52.3 a 32P-labeled phages adsorbed to cells are presented as a percentage of those on CB13 wild type grown at 250C. One hundred percent 4CbK adsorption was 10,884 32P cpm/ml at an OD66o of 0.1, and 100% 0Cp34 was 10,344 32p cpm/ml of an ODm of 0.1.
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in the CB13 ple-801 cells, even when grown at 250C. All the spontaneous revertants isolated from CB13 ple-801 regained the phage-adsorbing capacities, together with cell motility, irrespective of growth temperatures. As reported previously (3), the mutant CB13 ple-801 cells did not possess pili when grown at a nonpermissive temperature. These results support the previous notion (3, 8) that a single mutation pleiotropically affects the formation of flagella and phage receptors. The question now arises whether or not the lack of phage-adsorbing capacities in CB13 ple801 grown at a nonpermissive temperature is attributable to enhanced temperature sensitivity of phage receptors. The strains CB13 wild type and CB13 ple-801 were grown at 250C in PYE medium. After addition of chloramphenicol at a concentration of 20 ,ug/ml to inhibit further synthesis of phage receptors, the CB13 wild-type and CB13 ple-801 cells were shifted up to 350C, and 32P-labeled OCbK-adsorbing capacities were assayed at intervals. The 4CbKadsorbing capacity of the wild-type cells decreased exponentially at 350C by single-hit kinetics but were stable at 250C (Fig. 1). This result indicates that the wild-type OCbK receptors are already temperature sensitive at 350C and agrees with the reduced level of 4CbK adsorption to the wild-type cells grown at 350C (Table 1). Similarly, the flagellotropic OCp340
30
TIME (min ) 60 90
120
.
A00 50 10-
10=
5-
FIG. 1. Temperature sensitivity of 4CbK receptors in the wild-type CB13 and the mutant CB13 ple-801. Wild-type CB13 and the temperature-sensitive pleiotropic mutant CB13 ple-801 cells were freshly grown at 25°C to an ODfo of 0.1 (4.4 x 108 cells per ml) in PYE medium and mixed with chloramphenicol at a final concentration of 20 pg/ml. One-half portion of each culture was incubated at 25°C, and the other half was shifted up to 35°C. At intervals, portions (1.0 ml) were removed for the assay of 32P-labeled 4CbK-adsorbing capacities as described in the text. One hundred percent 4CbK adsorption was 7,610 32p cpmlml at an OD60 of 0.1 for the mutant CB13 ple801. Symbols: 0, CB13 wild type, 35°C; *, CB13 ple801, 35°C; CB13 wild type, 25°C; *, CB13 ple5,
801, 25°C.
1201
adsorbing capacity of the wild-type cells was temperature sensitive at 350C. The 4CbK-adsorbing capacity of the temperature-sensitive mutant CB13 ple-801 decreased at 350C similarly to that of the wild-type strain (Fig. 1). There was no enhancement of temperature sensitivity in 4)CbK adsorption to the mutant cells. The XCbK receptors of the mutant cells were also stable at 250C. The results with OCp34 were similar. These results indicate that the flagella and phage receptors, themselves, are not temperature sensitive, but rather that their formation is, in the pleiotropic mutant CB13 ple-801 at a nonpermissive temperature. Effect of macromolecular synthesis on surface structure formation. The cell cycle of the temperature-sensitive mutant CB13 ple-801 proceeds normally at 350C, as does that of the wild-type strain, with respect to cell growth, gross cell morphology, and asymmetric stalk formation (3). This mutant thus offers an opportunity to examine the effect of macromolecular synthesis on the formation of the polar surface structures upon shift down to a permissive temperature. The mutant CB13 ple-801 was freshly grown at 350C in PYE medium to an OD6. Of 0.1 (4.41 x 108 cells per ml) and shifted down to 250C. Mitomycin C, rifampin, or chloramphenicol was added to the culture at various times after shift down and, at intervals, portions (1.0 ml) of the mutant culture were removed for assay of 32P-labeled OCbK-adsorbing capacity (4CbK receptors). Without addition of macromolecular inhibitors, 4CbK receptors began to be formed at 15 min after shift down, and thereafter the specific activity (32P counts per minute per milliliter at an OD660 of 0.1) of 4CbK receptors increased continuously for at least 180 min (Fig. 2). Similarly, flagella formation (32P-labeled 4Cp34 adsorption) and cell motility (as observed under a phase-contrast microscope) began to appear at 30 min after shift down. When a DNA inhibitor, mitomycin C, was added upon shift down (at 0 min) at a concentration of 5 ug/ml, the formation of ¢CbK receptors was inhibited to a minimal level (Fig. 2). When the drug was added later, at 20 or 60 min after shift down, the already initiated formation of CbK receptors continued for an additional 80 min and plateaued. Similar amounts of ¢CbK receptors were formed, irrespective of the addition time of the drug at 20 or 60 min after shift down. These results indicate that DNA synthesis is necessary for the formation of the surface structures. In similar experiments, when an RNA inhibitor, rifampin, was added upon shift down (at 0
1202
J. BACTERIOL.
FUKUDA AND OKADA
4 C x
U, 0
3
0 c
2 N, C
E
U1 E-
CN4 m
TIME( min )
FIG. 2. Effects of macromolecular inhibitors on the formation of 4CbK receptors. Temperature-sensitive pleiotropic mutant CB13 ple-801 cells were freshly grown in PYE medium to an OD,O of 0.1 (4.4 x 108 cells per ml) at 35°C and shifted down to 25°C. The temperature of the culture decreased to 25°C within 2 min. After shift down, mitomycin C (MC, 5 Mg/ml), rifampin (RIF, 4 pg/ml), or chloramphenicol (CM, 20 pg/mi) was added to the culture at 0, 20, and 60 min. At intervals after drug addition, portions (1.0 ml) were removed for the assay of 32P-labeled 4CbK-adsorbing capacities as described in the text. Symbols: 0, No drug addition; *, drug addition, 0 min; *, drug addition, 20 min; 0, drug addition, 60 min.
min) at a concentration of 4 ,Ig/ml, the formation of OCbK receptors was completely inhibited. However, appreciable amounts of OCbK receptors were formed after rifampin addition at 20 or 60 min after shift down. This formation of OCbK receptors after drug addition continued for about 80 min before it leveled off. It appears that de novo RNA synthesis after shift down is required for the formation of oCbK receptors. The residual formation of OCbK receptors for 80 min in the presence of rifampin might indicate that, once synthesized, requisite RNA is rather stable. When a protein inhibitor, chloramphenicol, was added upon shift down (at 0 min) at a concentration of 20 ,ug/ml, the formation of 4CbK receptors was completely inhibited, as in the case of rifampin addition. Unlike rifampin addition, there was no residual receptor formation when the drug was added at 20 min after shift down. Upon drug addition at 60 min after shift down, however, residual formation of 4CbK receptors was observed, indicating that some receptors could be formed in the absence of protein synthesis once the cells were incubated long enough at a low temperature (25°C). The effects of macromolecular synthesis described above suggest that (i) de novo RNA and protein syntheses are required for the formation of the surface structures upon shift down in
the temperature-sensitive pleiotropic mutant CB13 ple-801, and (ii) DNA synthesis is also a prerequisite for the cells to efficiently form the surface structures. Surface structure formation in the mutant CB13 ple-801 upon temperature shift down and subsequent shift up. Temperature shift down and subsequent shift up experiments were carried out to examine a possible control step for the morphogenesis of the polar surface structures. The temperature-sensitive mutant CB13 ple-801 was grown freshly at 35°C in PYE medium to the early log phase (OD660, 0.1, or 4.41 x 108 cells per ml). The culture was shifted down to 250C and, after incubation for different times, shifted up again to 35°C. 32P-labeled 4CbK- and 4Cp34-adsorbing capacities were assayed in the meantime. OCbK receptors were formed even at 350C, once the cells were briefly exposed to 250C (Fig. 3). When the cells were shifted up to 350C at 4 min after shift down to 25°C, 4CbK receptors were immediately formed, in contrast with the later formation (15 min) when cells were continuously incubated at 250C. This difference should reflect the difference in cellular activity at 250C (generation time, 215 min) and at 350C (generation time, 150 min) of the mutant in PYE medium. The extent of 4CbK receptor formation in
SURFACE STRUCTURE MORPHOGENESIS IN CA ULOBACTER
VOL. 130, 1977
1203
o 0
E
C)
C? "I
cU
ci
.E O ci)
E
2 ,,,
a-
E~ -C
Q-
-o
30
60
90 0 30 TIME (min )
60
90
FIG. 3. Formation ofpolar surface structures in the temperature-sensitive pleiotropic mutant CB13 ple-801 temperature shift down and subsequent shift up. Mutant CB13 ple-801 cells were freshly grown in PYE medium to an OD6N of 0.1 (4.4 x 108 cells per ml) at 35°C, divided into three portions, and shifted down to 25°C. (a and b) One portion ofthe culture was incubated at 25°C (0), and the other two portions were shifted up to 35°C at 4 min (a) and 11 min (0) after shift down for O4CbK adsorption and at 10 min (*) and 20 min (0) for flagellotropic 4Cp34 adsorption. At intervals during shift down and subsequent shift up, samples (1.0 ml) were removed for the assay of 32P-labeled phage-adsorbing capacities. Specific phage-adsorbing capacities are presented as 32p counts per minute per milliliter at an OD660 of 0.1. (c and d) In separate experiments, the mutant CB13 ple-801 (at an OD6,. of 0.1 in PYE medium) was shifted up to 35°C at 20 min after shift down to upon
25°C. Rifampin (RIF, 4 pg/ml) was added to one portion of the culture upon shift up. 32P-labeled phageadsorbing capacities were assayed as above. Specific phage-adsorbing capacities are presented as 32p counts per minute per milliliter at an OD660 of 0.1 . Symbols: 0, No drug addition, 25°C; 0, no drug addition, shifted up to 35°C; *, drug addition, shifted up to 35°C.
early times after shift up was dependent on how long the cells were incubated at 25°C before shift up to 35°C. This 4CbK receptor formation after shift up to 35°C was inhibited by rifampin (Fig. 3). Therefore, it appears that requisite RNA for 4CbK receptor formation can be synthesized at 35°C. 4CbK receptor formation after shift up to 35°C continued for about 25 min. CbK receptor activity then decreased gradually when the cells were continously incubated at 35°C. This decrease of 4CbK receptor activity might be attributed to the inherent temperature sensitivity of 4CbK receptors (Fig. 1) and to the transition of swarmer cells to stalked cells, during which 4CbK receptors are lost from the cells (8).
Quite similar results were obtained with flagella formation in temperature shift down and subsequent shift up experiments (Fig. 3). Flagella formation after shift up to 35°C was also inhibited by rifampin. The results described above indicate that DNA, RNA, and protein syntheses and assembly for the formation of the polar surface structures are not temperature sensitive in the mutant CB13 ple-801. Effect of penicillin V on the formation of the surface structures. The cell wall is involved directly or indirectly in the formation of cell surface structures. To examine the effect of cell wall synthesis on the formation of the Caulobacter surface structures, penicillin V was added to CB13 ple-801 culture at a concentra-
1204 FUKUDA AND OKADA tion of 800 ug/ml upon shift down from 35 to 250C, and 4CbK receptor formation was assayed by 32P-labeled 4)CbK adsorption to the cells. Penicillin V inhibited the formation of 4CbK receptors (Fig. 4). There was no flagella formation, either, in the presence of penicillin V. Although an aberrant effect of penicillin addition on the cells is not rigorously excluded, the above results suggest that the formation of the Caulobacter surface structures requires cell wall synthesis. DISCUSSION In this paper we have described the effects of macromolecular synthesis on the formation of the Caulobacter polar surface structures (flagella and phage receptors). As shown in previous and the present experiments (3; Table 1) the formation of the C.'crescentus CB13 polar surface structures is regulated by a common mechanism. A single mutation simultaneously blocks the formation of flagella, pili (RNA phage receptors; 7), and DNA phage receptors. The temperature-sensitive pleiotropic mutant C. crescentus CB13 ple-801 grows normally at either 25 or 350C, as does the wild-type strain. At 350C this mutant is unable to form flagella, pili, and DNA phage 4CbK receptors, but upon shift down .to 250C it initiates the formation of these surface structures (Fig. 2 and 3). Thus, effects of macromolecular synthesis on the surface structure formation in the mutant could be
E 2 E C
7 -7
CL
005
n
0
30 60 TIME (min
90
0
o
FIG. 4. Effect of penicillin V on 4CbK receptor formation. Mutant CB13 ple-801 cells were grown fresh in PYE medium at 350C to an ODma of 0.095 (4.2 108 cells per ml), divided into two portions, and shifted down to 25°C. Upon shift down (0 min), penicillin V was added to one portion of the culture at a concentration of 800 pg/ml. At intervals, portions (1.0 ml) were removed for the assay of 32P-labeled oCbK-adsorbing capacities. ODm values of the culture were also measured in parallel. Specific phageadsorbing capacities are presented as 32P counts per minute per milliliter at an ODsw of 0.1. Symbols: 0, 32p counts per minute, no drug addition; o, 32p_ counts per minute, drug addition; 0, OD6n, no drug addition; OD680, drug addition. x
0,
J. BACTERIOL.
conveniently examined in temperature shift experiments by the use of macromolecular inhibitors. In addition, the extent of surface structure formation could be quantitated by assaying specific phage adsorption. When DNA synthesis was inhibited, the surface structure formation was also inhibited to a great extent. This was typically shown by assaying 4CbK receptor formation in the CB13 ple-801 cells upon shift down from 35 to 250C in the presence of mitomycin C (Fig. 2). It is not clear how DNA synthesis is related to the surface structure formation that occurs at a defined time in the cell cycle (8). Presumably, only those cells that enter the critical period at a permissive temperature become committed to form the surface structures, and this process requires DNA synthesis. Rifampin completely inhibited the surface structure formation when added to the CB13 ple-801 culture upon temperature shift down (Fig. 2). Once the mutant CB13 ple-801 cells were incubated briefly at 250C and then shifted up to 350C, the surface structure formation occurred, which was again inhibited by rifampin (Fig. 3). These results indicate that the synthesis of requisite RNA per se is not temperature sensitive at 350C. It then seems likely that there exists a regulatory step that commits the cells to initiate the synthesis of requisite RNA for surface structure formation. As so far examined, there was no stimulatory effect of dibutyryl cyclic adenosine 3',5'-monophosphate or dibutyryl cyclic guanosine 3',5'-monophosphate on the formation of CbK receptors and flagella in CB13 ple-801 grown at 350C in PYE medium or in Casamino Acids-supplemented minimal medium (A. Fukuda, unpublished data). Chloramphenicol completely inhibited the surface structure formation when added early (within about 20 min) after shift down from 35 to 250C (Fig. 2), indicating that de novo protein synthesis is essential. When the mutant CB13 ple-801 cells were incubated at 250C for a longer time (60 min), precursors seemed to be synthesized sufficiently for a residual formation of 4CbK receptors in the presence of chloramphenicol (Fig. 2). As described above, the surface structures were formed in CB13 ple-801 even at 350C, once the cells were incubated briefly at 250C before subsequent shift up (Fig. 3). This result seems to exclude the possibility that, among conceivable regulatory steps for surface structure formation, translation, assembly, or localization is temperature sensitive in CB13 ple-801 at 350C. The syntheses of DNA and cell wall, which also appear to be involved in surface structure formation (Fig. 2 and 4), should not
VOL. 130, 1977
SURFACE STRUCTURE MORPHOGENESIS IN CAULOBACTER.
be temperature sensitive either, since the tant grows normally at 35°C.
mu-
ACKNOWLEDGMENT This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan. LITERATURE CITED 1. Agabain-Keshishian, N., and L. Shapiro. 1970. Stalked bacteria: properties of deoxyribonucleic acid bacteriophage 46CbK. J. Virol. 5:795-800. 2. Fukuda, A., K. Miyakawa, H. Iba, and Y. Okada. 1976. A flagellotropic bacteriophage and flagella formation in Caulobacter. Virology 71:583-592. 3. Fukuda, A., K. Miyakawa, H. Iida, and Y. Okada. 1976. Regulation of polar surface structure in Caulto-
4.
5.
6. 7.
8.
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bacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors. Mol. Gen. Genet. 149:167-173. Fukuda, A., K. Miyakawa, and Y. Okada.1974. A pleiotropic mutation in Caulobacter crescentus that affects the flagellar formation and susceptibility to phage infection. Proc. Jpn. Acad. 50:839-842. Jollick, J. D., and B. L. Wright. 1974. A flagella specific bacteriophage for Caulobacter. J. Gen. Virol. 22:197205. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. Schmidt, J. M. 1966. Observations on the adsorption of Caulobacter bacteriophages containing ribonucleic acid. J. Gen. Microbiol. 45:347-353. Shapiro, L. 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377-407.
