Previous Article | Next Article 
Journal of Bacteriology, January 2000, p. 504-507, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Proteolysis of the McpA Chemoreceptor Does Not
Require the Caulobacter Major Chemotaxis Operon
Jeng-Wen
Tsai and
M. R. K.
Alley*
Department of Biochemistry, Imperial College
of Science, Technology and Medicine, London SW7 2AY, United Kingdom
Received 30 July 1999/Accepted 14 October 1999
 |
ABSTRACT |
The degradation of the McpA chemoreceptor in Caulobacter
crescentus accompanies the swarmer cell to the stalked-cell
differentiation event. To further analyze the requirements for its
degradation, we have constructed a series of strains that have
deletions in the mcpA gene and in the mcpA
chemotaxis operon. Internal deletions of the mcpA gene
demonstrate that the highly conserved domain (signalling unit) and the
methylation domains are not required for cell cycle-regulated
proteolysis. The deletion of the chemotaxis operon, which is absolutely
required for chemotaxis and McpA chemoreceptor methylation, has no
effect on McpA proteolysis.
| |
TEXT |
Proteolysis is an important
mechanism in both cell differentiation and progression through the cell
cycle. In Caulobacter crescentus, swarmer cells must undergo
an obligate differentiation event to become sessile stalked cells
before they can start to grow and divide. The differentiation of the
motile swarmer cell to nonmotile stalked cell is accompanied by the
proteolysis of key proteins involved in motility (3, 5, 7, 10,
17). The proteolysis of the McpA chemoreceptor (3) is
accompanied by a dramatic decrease in methylesterase and
methyltransferase activities (7). Unlike swarmer cells, the
newly formed stalked cells are capable of initiating DNA replication.
Once DNA replication is initiated, the stalked cells grow and will
progress through the cell cycle to develop into predivisional cells,
where the components required for motility are synthesized and targeted to the portion of the cell that will form the swarmer cell. The chemoreceptor McpA is specifically localized to the flagellated pole of
the predivisional cell (2). The ability to localize chemoreceptors to the poles of the cell is not restricted to
C. crescentus, as it has been shown to occur in
Escherichia coli (15) and is likely to be a
universal phenomenon found in other prokaryotes (8). Unlike
in other prokaryotes, the chemoreceptors in C. crescentus
are degraded during its life cycle; this proteolysis event plays an
important part in the asymmetric distribution of the polarly localized
chemoreceptors (3).
The CheR and CheB binding site, a pentapeptide (4, 20), is
conserved in McpA and is located at the extreme C terminus of
chemoreceptors. McpA is a methylated chemoreceptor (1), suggesting that CheR and CheB bind to the C terminus. The extreme C
terminus of McpA has been shown to be required for its degradation (3). Therefore, one explanation for the observation that
C-terminal deletions were no longer degraded is that they were not able
to form complexes with CheR and the other proteins of the chemotaxis machinery. The methylesterase (CheB) and methyltransferase (CheR) activities are lost (7) about the same time as McpA is
degraded, suggesting that there is some coordinate regulation of the
chemotaxis machinery at the level of proteolysis. Because of the
coincidental loss of the methylesterase, methyltransferase, and McpA,
we wanted to determine what role the methylation domains (Fig.
1A) play in the degradation of McpA. We
constructed internal deletions of the mcpA gene in order to
avoid perturbation of the C terminus, as this has been already shown to
be required for proteolysis (3). The construction of
mcpA internal deletions will aid in determining whether the
highly conserved signalling domain (HCD) is required for McpA
proteolysis and also map the N-terminal extent of the putative
degradation signal.
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 1.
Cell cycle immunoblots of mcpA internal
deletions. (A) mcpA internal deletions. The pale grey boxes
denote the extent of the McpA protein present in the various
chromosomal deletions of the mcpA gene in the named strains
(Table 1). The transmembrane domains are denoted by TM1 and TM2 and are
colored in grey. The methylation domains (checkered) are named after
the K1- and R1-methylated peptides shown in the E. coli Tsr
chemoreceptor (11). The HCD (striped box) is the most highly
conserved domain in all chemoreceptors (13); it is involved
in signalling. The gaps denote the extent of the deletions in the
mcpA gene in the named C. crescentus strains. The
black box at the C terminus of McpA is the CheR-CheB binding site (R/B)
(4, 20). (B) Cell cycle immunoblots. Strains MRKA208, which
is the wild-type strain in these experiments, MRKA21, MRKA25, MRKA26,
and MRKA24 were synchronized by Percoll (Pharmacia) density
centrifugation. Samples were taken at the times (minutes) indicated
during the 90-min cell cycle. The same amount of cells was loaded in
each lane. The cell extracts were subjected to electrophoresis on a
sodium dodecyl sulfate-8% polyacrylamide gel and transferred to
nitrocellulose (19); the primary antiserum was to McpA, and
the secondary antiserum was anti-rabbit conjugated to horseradish
peroxidase.
|
|
The first deletion strain, MRKA21 (Table
1), was constructed by conjugating the
suicide plasmid pWA12
AS (Table 2) into MRKA208 by using the helper plasmid pRK600 in MT607 (Table 1). The
kanamycin sucrose-sensitive transconjugants were then spread on 3%
sucrose plates to obtain excision of the kanamycin-resistant plasmid
from the chromosome. All sucrose-resistant colonies were subjected to
Southern and immunoblot analyses, and one positive colony was named
MRKA21. All subsequent internal deletions were obtained with this
methodology. The MRKA21 strain had most of the HCD deleted from the
mcpA gene on the chromosome (Fig. 1A). The deletion of the
HCD in McpA did not prevent its proteolysis (Fig. 1B), and the
degradation pattern was not significantly different from the pattern
observed with the wild-type strain MRKA208 (Fig. 1B). Deletion of part
of the HCD and the intervening sequence to the K1 methylation domain
resulted in strain MRKA25 (Fig. 1A), which still degraded McpA (Fig.
1B). Therefore, we deleted the sequence coding for the K1 methylation
domain and most of the HCD in mcpA to create strain MRKA26,
as shown in Fig. 1A. Strain MRKA26 still degraded McpA (Fig. 1B). The
absence of an effect on McpA degradation in the various K1 and HCD
deletion derivatives of McpA suggested that a much larger deletion
should be created. Hence, we deleted the entire K1 methylation domain
and HCD signalling unit (Fig. 1A). The resulting deletion strain MRKA24
still degraded McpA (Fig. 1B). The only conserved cytoplasmic domain
remaining that is known to interact with other chemotaxis proteins in
the McpA deletion present in strain MRKA24 (Fig. 1A) was the C-terminal CheR-CheB binding site. Because C-terminal deletions that removed this
CheR-CheB binding site from McpA have been shown to be stable (3), we wanted to determine whether CheR and CheB are
required for McpA degradation. Because cheR and
cheB reside in an operon with cheA and
cheW (Fig. 2A), we decided to
delete the entire operon to test whether any of the chemotaxis genes
present in this operon are required for McpA degradation. Therefore, we
created strain MRKA580; the che17 deletion removes the
mcpA operon promoter and every gene in the mcpA
operon except cheE (Fig. 2A). Since the mcpA
operon promoter is deleted (Fig. 2A), the only gene left intact
(cheE) will not be expressed. The deletion of the
cagA gene will have no phenotypic effect because there is a
second copy of the cagA gene in the C. crescentus
genome, and deletion of the first cagA gene has no
observable phenotype (M. R. K. Alley, unpublished data). The
che17 chemotaxis deletion strain MRKA580 produces very
small swarms in semisolid media (Fig. 2B). The swarmer cells do not
reverse their swimming direction, and predivisional cells swim with
their stalks in front, which is the predominant direction of swimming
(12). The che17 deletion removes the mcpA gene, so we introduced the mcpA gene on a
plasmid, pRMCP4, by triparental mating with MT607. The plasmid pRMCP4
is a low-copy-number plasmid (two to five copies per cell) of the
IncP-1 incompatibility group. Since the number of copies of the
mcpA gene on pRMCP4 are above the normal level found in the
wild-type strain MRKA208, we also introduced pRMCP4 into MRKA208 as a
control for any plasmid copy number effects. As shown in the immunoblot
in Fig. 3A, the wild-type strain makes
slightly more McpA than the che17 deletion strain
MRKA580, which is probably due to the extra copy present on the
chromosome of MRKA208. The MRKA580 strain is not able to methylate its
McpA chemoreceptors (Fig. 3B), further demonstrating that the MRKA580
strain is defective in chemotaxis. No methylation of McpA was observed
in extracts from the MRKA580 strain, even after a 1-month exposure to
X-ray film. Therefore, the cheR gene in the
mcpA operon is essential for McpA methylation. The wild-type strain bearing pRMCP4 degrades McpA (Fig. 3C), and therefore, there are
no effects on McpA degradation due to the increase in levels of McpA or
the presence of the IncP-1 plasmid. McpA is degraded in the
che17 deletion strain MRKA580, suggesting that the genes
in the mcpA operon are not required for McpA proteolysis and
that McpA does not need a functional chemotaxis system for its
degradation. Furthermore, if CheR methyltransferase and CheB methylesterase are not required for McpA proteolysis, we can postulate that the CheR-CheB binding site is involved only in McpA methylation; therefore, we can exclude its requirement in McpA degradation.
View larger version (73K):
[in this window]
[in a new window]
|
FIG. 2.
The che17 deletion strain MRKA580 is
defective in chemotaxis. (A) The grey boxes represent the open reading
frames for the named genes (AJ006687). The extent of the deletion in
che17 strains is shown by the black box. (B) A yeast
extract swarm agar (0.005% yeast extract, 0.5 mM MgSO4,
0.5 mM CaCl2, 0.15% Bacto agar) plate after 48 h
inoculated with equal amounts of MRKA208 (wild type [WT]) and MRKA580
(indicated by the no. 17) strains bearing the plasmid pRMCP4.
|
|
View larger version (69K):
[in this window]
[in a new window]
|
FIG. 3.
McpA methylation and cell cycle degradation in a
mcpA operon deletion. (A) Immunoblot of strains MRKA208
(wild type [WT]) and MRKA580 (indicated by the no. 17) using McpA
antisera. The arrow indicates the McpA protein band. (B)
Immunoprecipitation of McpA in cell extracts from chloramphenicol
pretreated cells labelled with [3H]methylmethionine
(17). Equal numbers of counts per minute were
immunoprecipitated. The cell extracts were from strains MRKA208 (WT)
and MRKA580 (no. 17). (C) McpA cell cycle immunoblots of strains
MRKA208 (WT) and MRKA580 (no. 17) bearing the plasmid pRMCP4. The
numbers above the panel are the time points (minutes) at which the
samples were taken.
|
|
The chemoreceptors are modified by methylation by CheR and CheB and
form large functional complexes with CheA and CheW (6, 14).
The possibility that these proteins might be involved in the stability
of the McpA chemoreceptor is derived from the fact that stability of
other proteins can be altered by protein complex formation. The
methylesterase (CheB) and methyltransferase (CheR) activities are lost
during the cell cycle (7), suggesting that the proteolysis
of the chemotaxis machinery might be coordinately regulated. One
possible way to coordinate the proteolysis of the chemotaxis machinery
is to target the entire complex for degradation. In this study, we show
that deletion of the K1 and HCD domains of McpA do not prevent its
degradation (Fig. 1). When the mcpA operon was deleted, McpA
was not methylated and there was no effect on the degradation of McpA.
Therefore, we can assume that none of the genes in the mcpA
operon are required for McpA degradation. The combined data from the
internal and operon deletions would suggest that McpA is the target for
its protease. Thus, McpA and the other chemoreceptors might be required
for the degradation of the methylesterase and methyltransferase. To
test this hypothesis, we are presently generating antisera to CheR and CheB.
| |
ACKNOWLEDGMENTS |
This study was funded by a Wellcome Trust project grant (#044761)
to M.R.K.A.
M.R.K.A. is a Royal Society Research Fellow.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Imperial College of Science, Technology and Medicine,
London SW7 2AY, United Kingdom. Phone: 44-171-594 5304. Fax: 44-171-594 5207. E-mail: d.alley{at}ic.ac.uk.
| |
REFERENCES |
| 1.
|
Alley, M. R. K.,
S. L. Gomes,
W. Alexander, and L. Shapiro.
1991.
Genetic analysis of a temporally transcribed chemotaxis gene cluster in Caulobacter crescentus.
Genetics
129:333-341[Abstract].
|
| 2.
|
Alley, M. R. K.,
J. R. Maddock, and L. Shapiro.
1992.
Polar localization of a bacterial chemoreceptor.
Genes Dev.
6:825-836[Abstract/Free Full Text].
|
| 3.
|
Alley, M. R. K.,
J. R. Maddock, and L. Shapiro.
1993.
Requirement of the carboxyl terminus of a bacterial chemoreceptor for its targeted proteolysis.
Science
259:1754-1757[Abstract/Free Full Text].
|
| 4.
|
Barnakov, A. N.,
L. A. Barnakova, and G. L. Hazelbauer.
1999.
Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation.
Proc. Natl. Acad. Sci. USA
96:10667-10672[Abstract/Free Full Text].
|
| 5.
|
Domian, I. J.,
K. C. Quon, and L. Shapiro.
1997.
Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle.
Cell
90:415-424[CrossRef][Medline].
|
| 6.
|
Gegner, J. A.,
D. R. Graham,
A. F. Roth, and F. W. Dahlquist.
1992.
Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway.
Cell
70:975-982[CrossRef][Medline].
|
| 7.
|
Gomes, S. L., and L. Shapiro.
1984.
Differential expression and positioning of chemotaxis methylation proteins in Caulobacter.
J. Mol. Biol.
178:551-568[CrossRef][Medline].
|
| 8.
|
Harrison, D. M.,
J. Skidmore,
J. P. Armitage, and J. R. Maddock.
1999.
Localization and environmental regulation of MCP-like proteins in Rhodobacter sphaeroides.
Mol. Microbiol.
31:885-892[CrossRef][Medline].
|
| 9.
|
Hynes, M. F.,
J. Quandt,
M. P. O'Connell, and A. Puhler.
1989.
Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene.
Gene
78:111-120[CrossRef][Medline].
|
| 10.
|
Jenal, U., and L. Shapiro.
1996.
Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell.
EMBO J.
15:2393-2406[Medline].
|
| 11.
|
Kehry, M. R.,
M. W. Bond,
M. W. Hunkapiller, and F. W. Dahlquist.
1983.
Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product.
Proc. Natl. Acad. Sci. USA
80:3599-3603[Abstract/Free Full Text].
|
| 12.
|
Koyasu, S., and Y. Shirakihara.
1984.
Caulobacter crescentus flagellar filament has a right-handed helical form.
J. Mol. Biol.
173:125-130[CrossRef][Medline].
|
| 13.
|
Le Moual, H., and D. E. Koshland, Jr.
1996.
Molecular evolution of the C-terminal cytoplasmic domain of a superfamily of bacterial receptors involved in taxis.
J. Mol. Biol.
261:568-585[CrossRef][Medline].
|
| 14.
|
Liu, Y.,
M. Levit,
R. Lurz,
M. G. Surette, and J. B. Stock.
1997.
Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.
EMBO J.
16:7231-7240[CrossRef][Medline].
|
| 15.
|
Maddock, J. R., and L. Shapiro.
1993.
Polar location of the chemoreceptor complex in the Escherichia coli cell.
Science
259:1717-1723[Abstract/Free Full Text].
|
| 16.
|
Sharma, S. B., and E. R. Signer.
1990.
Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in planta revealed by transposon Tn5-gusA.
Genes Dev.
4:344-356[Abstract/Free Full Text].
|
| 17.
|
Shaw, P.,
S. L. Gomes,
K. Sweeney,
B. Ely, and L. Shapiro.
1983.
Methylation involved in chemotaxis is regulated during Caulobacter differentiation.
Proc. Natl. Acad. Sci. USA
80:5261-5265.
|
| 18.
|
Spratt, B. G.,
P. J. Hedge,
S. te Heesen,
A. Edelman, and J. K. Broome-Smith.
1986.
Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9.
Gene
41:337-342[CrossRef][Medline].
|
| 19.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 20.
|
Wu, J.,
J. Li,
G. Li,
D. G. Long, and R. M. Weis.
1996.
The receptor binding site for the methyltransferase of bacterial chemotaxis is distinct from the sites of methylation.
Biochemistry
35:4984-4993[CrossRef][Medline].
|
Journal of Bacteriology, January 2000, p. 504-507, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
da Rocha, R. P., de Miranda Paquola, A. C., do Valle Marques, M., Menck, C. F. M., Galhardo, R. S.
(2008). Characterization of the SOS Regulon of Caulobacter crescentus. J. Bacteriol.
190: 1209-1218
[Abstract]
[Full Text]
-
Alvarez-Martinez, C. E., Baldini, R. L., Gomes, S. L.
(2006). A Caulobacter crescentus Extracytoplasmic Function Sigma Factor Mediating the Response to Oxidative Stress in Stationary Phase.. J. Bacteriol.
188: 1835-1846
[Abstract]
[Full Text]
-
Galhardo, R. S., Rocha, R. P., Marques, M. V., Menck, C. F. M.
(2005). An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus. Nucleic Acids Res
33: 2603-2614
[Abstract]
[Full Text]
-
Apidianakis, Y., Mindrinos, M. N., Xiao, W., Lau, G. W., Baldini, R. L., Davis, R. W., Rahme, L. G.
(2005). Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression. Proc. Natl. Acad. Sci. USA
102: 2573-2578
[Abstract]
[Full Text]
-
Potocka, I., Thein, M., Osteras, M., Jenal, U., Alley, M. R. K.
(2002). Degradation of a Caulobacter Soluble Cytoplasmic Chemoreceptor Is ClpX Dependent. J. Bacteriol.
184: 6635-6641
[Abstract]
[Full Text]
-
West, L., Yang, D., Stephens, C.
(2002). Use of the Caulobacter crescentus Genome Sequence To Develop a Method for Systematic Genetic Mapping. J. Bacteriol.
184: 2155-2166
[Abstract]
[Full Text]
-
Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H.
(2002). Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. Proc. Natl. Acad. Sci. USA
99: 4632-4637
[Abstract]
[Full Text]
-
Tsai, J.-W., Alley, M. R. K.
(2001). Proteolysis of the Caulobacter McpA Chemoreceptor Is Cell Cycle Regulated by a ClpX-Dependent Pathway. J. Bacteriol.
183: 5001-5007
[Abstract]
[Full Text]
-
Jones, S. E., Ferguson, N. L., Alley, M. R. K.
(2001). New members of the ctrA regulon: the major chemotaxis operon in Caulobacter is CtrA dependent. Microbiology
147: 949-958
[Abstract]
[Full Text]
-
Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., Heidelberg, J. F., Alley, M. R. K., Ohta, N., Maddock, J. R., Potocka, I., Nelson, W. C., Newton, A., Stephens, C., Phadke, N. D., Ely, B., DeBoy, R. T., Dodson, R. J., Durkin, A. S., Gwinn, M. L., Haft, D. H., Kolonay, J. F., Smit, J., Craven, M. B., Khouri, H., Shetty, J., Berry, K., Utterback, T., Tran, K., Wolf, A., Vamathevan, J., Ermolaeva, M., White, O., Salzberg, S. L., Venter, J. C., Shapiro, L., Fraser, C. M.
(2001). Complete genome sequence of Caulobacter crescentus. Proc. Natl. Acad. Sci. USA
10.1073/pnas.061029298v1
[Abstract]
[Full Text]
-
Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., Heidelberg, J. F., Alley, M. R. K., Ohta, N., Maddock, J. R., Potocka, I., Nelson, W. C., Newton, A., Stephens, C., Phadke, N. D., Ely, B., DeBoy, R. T., Dodson, R. J., Durkin, A. S., Gwinn, M. L., Haft, D. H., Kolonay, J. F., Smit, J., Craven, M. B., Khouri, H., Shetty, J., Berry, K., Utterback, T., Tran, K., Wolf, A., Vamathevan, J., Ermolaeva, M., White, O., Salzberg, S. L., Venter, J. C., Shapiro, L., Fraser, C. M.
(2001). Complete genome sequence of Caulobacter crescentus. Proc. Natl. Acad. Sci. USA
98: 4136-4141
[Abstract]
[Full Text]
Top
Abstract
Text
