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Journal of Bacteriology, July 2001, p. 4389-4392, Vol. 183, No. 14
Department of Molecular Biology and
Microbiology, Tufts University School of Medicine, Boston,
Massachusetts 02111,1 and Laboratory for
Microbiology, Department of Biology, Philipps University Marburg,
D-35032 Marburg, Germany2
Received 20 February 2001/Accepted 25 April 2001
The complete Bacillus subtilis genome contains four
genes (proG, proH, proI, and comER) with
the potential to encode The pathway of proline synthesis
from glutamate, the most common mechanism of proline biosynthesis,
comprises three enzymatic steps (Fig. 1).
The corresponding genes of Escherichia coli, proB, proA, and proC, encode
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4389-4392.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Multiple Genes for the Last Step of Proline
Biosynthesis in Bacillus subtilis
ABSTRACT
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Abstract
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References
1-pyrroline-5-carboxylate
reductase, a proline biosynthetic enzyme. Simultaneous defects in three
of these genes (proG, proH, and proI)
were required to confer proline auxotrophy, indicating that the
products of these genes are mostly interchangeable with respect to the
last step in proline biosynthesis.
TEXT
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-glutamyl kinase,
-glutamyl phosphate reductase, and
1-pyrroline-5-carboxylate (P5C) reductase,
respectively (21). The proBA-dependent pathway
of proline synthesis was shown to function also in Bacillus
subtilis; mutations within the proBA locus cause
auxotrophy for proline (8, 29). While B. subtilis has a single proA-like gene, a second
proB-like gene, proJ of the proHJ
locus (B. R. Belitsky and A. L. Sonenshein, GenBank accession
number AF006720) has been found. In a manner unique to this bacterium,
either ProB-like enzyme can provide enough -glutamyl kinase activity
to support growth in the absence of exogenous proline (unpublished
results). Apparently, previously described mutations to auxotrophy in
the proBA locus either affect proA or are
proB alleles that are polar on proA expression.
No proC mutant of B. subtilis has been described,
and four genes have the potential to encode ProC-like proteins with P5C
reductase activity: proH (also called orf257 and
proC), comER (also called comED),
proI (also called yqjO), and ykeA
(here renamed proG) (1, 14, 20). The four genes
are located at 172.3°, 225.5°, 211.2°, and 116.1° on the
chromosomal map (http://genolist.pasteur.fr/SubtiList [26]) and code
for proteins of 271, 273, 278, and 272 amino acids, respectively (the
originally reported coding region of proH [1, 20] was
extended by resequencing the proH 3' end [GenBank accession number AF006720]). ProH and ProI are 42% identical to each other and
up to 35% identical to many other P5C reductases from bacteria,
archaea, and eukaryotes. ProG and ComER have more limited similarity to
other P5C reductases and to each other. The functions of the four
B. subtilis genes are not known. In this work we sought to
identify the gene(s) responsible for the last step of proline biosynthesis.
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FIG. 1.
Pathways of proline biosynthesis in B.
subtilis. The major pathway of proline synthesis from glutamate
is shown as a descending series of reactions. Some proline is also
apparently synthesized from glutamate via ornithine (an intermediate in
arginine synthesis) by the action of the RocD product (unpublished
results). Since no ortholog of E. coli ArgE protein is
present in B. subtilis, conversion of
N-acetylglutamic -semialdehyde (an intermediate in
arginine synthesis) to -glutamic semialdehyde (21)
apparently does not occur. Details of the anabolic pathway from
glutamate to ornithine and the catabolic reaction from -glutamic
semialdehyde to glutamate have been omitted. The catabolism of
citrulline includes its conversion to ornithine (unpublished data) but
has not been characterized further. Ornithine cyclodeaminase (dashed
line) is not present in B. subtilis. The
proG and proI genes have been previously
known as ykeA and yqjO, respectively.
Construction and properties of a proG
(ykeA) null mutant.
To create pBB1081, the 1.56-kb
PvuII-EcoRI fragment from pCM103
(23) containing most of the proG gene and the
5' end of the dppA gene was cloned between the
PstI (blunt-ended) and EcoRI sites of pJPM1, a
derivative of pBS (Stratagene) containing a chloramphenicol resistance
marker (27). Methods for plasmid isolation, agarose gel
electrophoresis, use of restriction and DNA modification enzymes, DNA
ligation, PCR, Southern hybridization with digoxigenin-labeled DNA
probes, and electroporation of E. coli JM107 or DH5
cells
were as described by Sambrook et al. (30). DNA and protein
sequences were analyzed using the DNA Strider (22) or
BLAST (2) programs. A deletion-insertion mutation within
the proG gene was created by replacing the 0.10-kb PstI-SacI fragment of pBB1081 with a 1.43-kb
PstI-SacI ble cassette determining
resistance to phleomycin, excised from pJPM136 (6). The
orientation of the ble gene in the resulting
proG::ble plasmid, pBB1082,
coincides with that of the proG gene. pBB1082 was introduced into B. subtilis SMY, and phleomycin-resistant,
chloramphenicol-sensitive transformants, arising from double-crossover
homologous recombination events, were selected. Growth of B. subtilis cells, transformation by chromosomal or plasmid DNA, and
isolation of chromosomal DNA were as described previously
(6). The replacement of the chromosomal proG gene by the
proG::ble allele in strain BB1951
was confirmed by comparing sizes of the PCR products from the wild-type
and mutant proG chromosomal loci. Strain BB1951
(proG::ble) had the growth
characteristics of a wild-type strain in the presence and absence of proline.
Construction and properties of a proH null
mutant.
The 0.27-kb EcoRI-PstI 3'-end
fragment of the proH gene was subcloned in several steps
from pLS23-17 (7) between the EcoRI and
PstI sites of pBB544, a derivative of pBluescript SK(
)
(Stratagene) containing a neomycin resistance marker (5).
The resulting plasmid, pBB575, was integrated via a single-crossover
recombination event into the chromosome of B. subtilis
strain SMY at the proH locus. To clone DNA adjacent to the
site of integration of pBB575, the chromosomal DNA of the resulting
strain was digested with HindIII, self-ligated, and
introduced by electroporation into E. coli cells. The
isolated plasmid, pBB576, had a 1.32-kb insert of chromosomal DNA
carrying most of proH. A deletion-insertion mutation within
the proH gene was created by replacing the 0.55-kb BclI-EcoRI fragment of pBB576 with a 1.9-kb
BamHI-EcoRI tet cassette, excised from
pBEST307 (17). The orientation of the tet gene in the resulting plasmid, pBB734, coincides with that of the
proH gene. Strain BB286
(proH::tet) was constructed as
described above for strain BB734, using pBB734 and selecting for
tetracycline-resistant, neomycin-sensitive transformants. Strain
BB286 (proH::tet) had the growth
characteristics of a wild-type strain in the presence and absence of proline.
Construction and properties of a proI
(yqjO) null mutant.
The 1.85-kb 'yqjP proI
yqjN' chromosomal region was amplified by PCR using custom
synthesized oligonucleotides as primers. To create pJS18, the internal
1.62-kb ClaI-NsiI fragment of the PCR product,
including the entire proI gene and the flanking regions of
the yqjP and yqjN genes, was cloned in
pBluescript SK() (Stratagene), cleaved with ClaI and
PstI. For construction of pJS20
(proI::spc), the 0.25-kb
BclI-StuI fragment of pJS18 that is internal to
proI was replaced with the 1.3-kb
BamHI-XbaI (filled-in) fragment, excised
from plasmid pRMK65 (18), which contains the
spc gene. The orientation of the spc gene in this
construction is opposite to that of the proI gene. Strain
JSB9 (proI::spc) was isolated by
transformation of strain JH642 (trpC2 pheA1) (obtained from J. Hoch) with BamHI-linearized DNA of pJS20.
Spectinomycin-resistant transformants were selected, and the
correct double-crossover integration event was verified by Southern
hybridization using the SalI-NotI
fragment of pJS18 as a probe. Strain JSB9
(proI::spc) had the growth
characteristics of a wild-type strain in the presence and absence of proline.
Construction and properties of multiple mutants.
comER mutants were constructed previously and shown to be
prototrophic (14, 16). Strains containing all possible
combinations of two or three mutations in the proG,
proH, proI, and comER genes and the corresponding
quadruple mutant were constructed by transformation of strain SMY with
chromosomal DNAs from appropriate mutants. The Pro phenotype of some of
the double and triple mutants is shown in Table
1. The growth rate in glucose-ammonia
medium of any of the double mutants was identical to the growth rate of a wild-type strain. No contribution of the comER gene to the
cells' ability to grow without proline was detected for any of the
mutants. The proG proH proI triple mutant required proline
for growth, demonstrating that the P5C reductase enzymes are in fact
required for proline synthesis in B. subtilis as in other
organisms but that this function can be taken over by any one of three
proteins, ProG, ProH, or ProI. In some experiments the proG
proI mutant exhibited a long lag period before initiating growth
in minimal medium without proline. This effect probably reflects the
need for proH to be induced in order to provide enough
enzyme to support proline synthesis. We cannot exclude the possibility
that spontaneous mutations which elevate expression of proH
accumulate in the culture of the proG proI mutant.
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Role of proC-like genes in proline generation
through the arginase pathway.
In addition to the anabolic pathway
of proline synthesis from glutamate, two catabolic pathways can lead to
proline from ornithine; neither of these pathways requires the first
two steps of proline synthesis from glutamate (Fig. 1). B. subtilis does not contain any gene that could code for ornithine
cyclodeaminase (cyclase) (20, 28, 31) but has a
well-characterized arginase pathway for arginine degradation
(11). In that pathway, ornithine aminotransferase, the
product of the rocD gene, generates -glutamic
semialdehyde, a substrate of P5C reductase, from ornithine (Fig. 1)
(4, 13). B. subtilis cells are able to utilize
extracellular ornithine or the related amino acids arginine and
citrulline as sources of proline (8), and they failed to
do so in a rocD mutant, indicating that the arginase pathway
is essential for proline generation under these conditions (Fig. 1).
Formation of proline from ornithine, arginine or citrulline was also
dependent on the presence of proG, proH, or
proI, the same genes that can support proline synthesis from
glutamate. The proG proI double mutant, whose only P5C
reductase is encoded by proH, again had a small growth
defect under these conditions (Table 1). To ensure that no proline was
derived from glutamate, which can be formed from ornithine and related
amino acids, we introduced a proB::cat
mutation into our strains and confirmed the requirement for either
proG, proH, or proI (Table 1). Thus,
proG, proH, or proI is essential for
proline formation, both through the glutamate pathway and through the
arginase pathway.
Possible roles of multiple P5C reductases. Participation of at least three P5C reductase isoenzymes in the last step of proline synthesis is unique to B. subtilis among characterized organisms and may reflect specialized functions or regulation or both. Multiple genes with potential to code for P5C reductase isoenzymes have been detected in the genomes of the gram-positive bacteria Bacillus halodurans (32), Bacillus anthracis (http://www.tigr.org), Enterococcus faecalis (http://www.tigr.org), and Clostridium difficile (http://www.sanger.ac.uk) and the gram-negative bacterium Pseudomonas putida (http://www.tigr.org), but their functions have not been verified.
Though we could not detect a unique role for ProG, ProH, or ProI in proline synthesis from either glutamate or arginine, it is possible that such a role exists under some physiological conditions. Transcription of the proBA and proI genes is increased during proline limitation (unpublished data) and seems to be regulated by a termination-antitermination control mechanism, the T-box system (15); both proBA and proI contain 18-bp T-box-like sequences with the predicted proline specifier codons CCU and CCC (9). Coordinate induction of proI and proBA by proline limitation suggests that ProI is the major P5C reductase under such conditions. The proHJ locus, encoding enzymes for the first and the last steps of proline synthesis, is induced by high concentrations of salt (unpublished data), in keeping with the role of proline as the major endogenously produced osmoprotectant in B. subtilis (19, 25, 33). Finally, multiple P5C reductase isoenzymes may be involved in removal of excess P5C, which was reported to be toxic in Aspergillus nidulans (3) and in human cells (24) and has also been shown to be toxic for B. subtilis cells (unpublished data). The role of the comER gene remains unknown (16). comER itself and its unusual overlapping, divergent orientation with respect to that of the comEA-EB-EC operon (14) are conserved in B. halodurans, B. anthracis, and Bacillus stearothermophilus, i.e., all Bacillus species for which sequencing information is available. comER expression decreases in stationary phase of growth in competence medium (14); no effect of comER mutations on cell competence was observed in earlier work (14, 16). The comER gene is at least partially under sporulation control, and its putative
E-dependent promoter
has been identified (10). We could not detect any effect
of comER mutations on sporulation efficiency in nutrient broth medium or in minimal medium either without proline or in the
presence of a limiting amount of proline when the proG proH proI mutant was used.
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ACKNOWLEDGMENTS |
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We are grateful to D. Dubnau for a gift of strains.
This work was supported by U.S. Public Health Service grant GM36718, the Deutsche Forschungsgemeinschaft (SFB-395 and Graduiertenkolleg Proteinfunktion auf atomarer Ebene), and the Fonds der Chemischen Industrie.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6762. Fax: (617) 636-0337. E-mail: address: bbelit02{at}tufts.edu.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Ahn, K. S., and R. G. Wake. 1991. Variations and coding features of the sequence spanning the replication terminus of Bacillus subtilis 168 and W23 chromosomes. Gene 98:107-112[CrossRef][Medline]. |
| 2. |
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402 |
| 3. | Arst, H. N., S. A. Jones, and C. R. Bailey. 1981. A method for the selection of deletion mutations in the L-proline catabolism gene cluster of Aspergillus nidulans. Genet. Res. 38:171-195[Medline]. |
| 4. |
Baumberg, S., and C. R. Harwood.
1979.
Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis.
J. Bacteriol.
137:189-196 |
| 5. |
Belitsky, B. R.,
M. C. Gustafsson,
A. L. Sonenshein, and C. Von Wachenfeldt.
1997.
An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport.
J. Bacteriol.
179:5448-5457 |
| 6. |
Belitsky, B. R., and A. L. Sonenshein.
1998.
Role and regulation of Bacillus subtilis glutamate dehydrogenase genes.
J. Bacteriol.
180:6298-6305 |
| 7. |
Bohannon, D. E.,
M. S. Rosenkrantz, and A. L. Sonenshein.
1985.
Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source.
J. Bacteriol.
163:957-964 |
| 8. |
Buxton, R. S.
1980.
Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage.
J. Gen. Virol.
46:427-437 |
| 9. | Chopin, A., V. Biaudet, and S. D. Ehrlich. 1998. Analysis of the Bacillus subtilis genome sequence reveals nine new T-box leaders. Mol. Microbiol. 29:662-664[CrossRef][Medline]. |
| 10. |
Fawcett, P.,
P. Eichenberger,
R. Losick, and P. Youngman.
2000.
The transcriptional profile of early to middle sporulation in Bacillus subtilis.
Proc. Natl. Acad. Sci. USA
97:8063-8068 |
| 11. | Fisher, S. H. 1993. Utilization of amino acids and other nitrogen-containing compounds, p. 221-228. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, D.C. |
| 12. |
Fouet, A., and A. L. Sonenshein.
1990.
A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis.
J. Bacteriol.
172:835-844 |
| 13. | Gardan, R., G. Rapoport, and M. Debarbouille. 1995. Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. J. Mol. Biol. 249:843-856[CrossRef][Medline]. |
| 14. | Hahn, J., G. Inamine, Y. Kozlov, and D. Dubnau. 1993. Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA. Mol. Microbiol. 10:99-111[Medline]. |
| 15. | Henkin, T. M. 1994. tRNA-directed transcription antitermination. Mol. Microbiol. 13:381-387[CrossRef][Medline]. |
| 16. |
Inamine, G. S., and D. Dubnau.
1995.
ComEA, a Bacillus subtilis integral membrane protein required for genetic transformation, is needed for both DNA binding and transport.
J. Bacteriol.
177:3045-3051 |
| 17. | Itaya, M. 1992. Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome. Biosci. Biotechnol. Biochem. 56:685-686[Medline]. |
| 18. | Kappes, R. M., B. Kempf, S. Kneip, J. Boch, J. Gade, J. Meier-Wagner, and E. Bremer. 1999. Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis. Mol. Microbiol. 32:203-216[CrossRef][Medline]. |
| 19. | Kempf, B., and E. Bremer. 1998. Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolarity environments. Arch. Microbiol. 170:319-330[CrossRef][Medline]. |
| 20. | Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256[CrossRef][Medline]. |
| 21. | Leisinger, T. 1996. Biosynthesis of proline, p. 434-441. In F. C. Neidhardt, et al. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C. |
| 22. |
Marck, C.
1988.
`DNA Strider': a `C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
Nucleic Acids Res.
16:1829-1836 |
| 23. | Mathiopoulos, C., J. P. Mueller, F. J. Slack, C. G. Murphy, S. Patankar, G. Bukusoglu, and A. L. Sonenshein. 1991. A Bacillus subtilis dipeptide transport system expressed early during sporulation. Mol. Microbiol. 5:1903-1913[CrossRef][Medline]. |
| 24. |
Maxwell, S. A., and G. E. Davis.
2000.
Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines.
Proc. Natl. Acad. Sci. USA
97:13009-13014 |
| 25. | Measures, J. C. 1975. Role of amino acids in osmoregulation of non-halophilic bacteria. Nature 257:398-400[CrossRef][Medline]. |
| 26. | Moszer, I. 1998. The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis. FEBS Lett. 430:28-36[CrossRef][Medline]. |
| 27. |
Mueller, J. P.,
G. Bukusoglu, and A. L. Sonenshein.
1992.
Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system.
J. Bacteriol.
174:4361-4373 |
| 28. |
Muth, W. L., and R. N. Costilow.
1974.
Ornithine cyclase (deaminating). II. Properties of the homogeneous enzyme.
J. Biol. Chem.
249:7457-7462 |
| 29. |
Ogura, M.,
M. Kawata-Mukai,
M. Itaya,
K. Takio, and T. Tanaka.
1994.
Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis.
J. Bacteriol.
176:5673-5680 |
| 30. | Sambrook, J., E. F. Fritsch, and T. J. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 31. | Sans, N., U. Schindler, and J. Schroder. 1988. Ornithine cyclodeaminase from Ti plasmid C58: DNA sequence, enzyme properties and regulation of activity by arginine. Eur. J. Biochem. 173:123-130[Medline]. |
| 32. |
Takami, H.,
K. Nakasone,
Y. Takaki,
G. Maeno,
R. Sasaki,
N. Masui,
F. Fuji,
C. Hirama,
Y. Nakamura,
N. Ogasawara,
S. Kuhara, and K. Horikoshi.
2000.
Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis.
Nucleic Acids Res.
28:4317-4331 |
| 33. |
Whatmore, A. M.,
J. A. Chudek, and R. H. Reed.
1990.
The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis.
J. Gen. Microbiol.
136:2527-2535 |
Journal of Bacteriology, July 2001, p. 4389-4392, Vol. 183, No. 14
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4389-4392.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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