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Journal of Bacteriology, December 1998, p. 6511-6518, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Department of Bacteriology, University of
Wisconsin
Madison, Madison, Wisconsin 53706-1567
Received 16 July 1998/Accepted 8 October 1998
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Expression of the prpBCDE operon of Salmonella typhimurium LT2 required (i) the synthesis of propionyl-coenzyme A (CoA) by the PrpE protein or the acetyl-CoA-synthesizing systems of the cell and (ii) the synthesis of 2-methylcitrate from propionyl-CoA and oxaloacetate by the PrpC protein. We propose that either 2-methylcitrate or a derivative of it signals the presence of propionate in the environment. This as yet unidentified signal is thought to serve as a coregulator of the activity of PrpR, the member of the sigma-54 family of transcriptional activators needed for activation of prpBCDE transcription. The CobB protein was also required for expression of the prpBCDE operon, but its role is less well understood. Expression of the prpBCDE operon in cobB mutants was restored to wild-type levels upon induction of the propanediol utilization (pdu) operon by 1,2-propanediol. This effect did not require catabolism of 1,2-propanediol, suggesting that a Pdu protein, not a catabolite of 1,2-propanediol, was responsible for the observed effect. We explain the existence of these redundant functions in terms of metabolic pathway integration. In an environment with 1,2-propanediol as the sole carbon and energy source, expression of the prpBCDE operon is ensured by the Pdu protein that has CobB-like activity. Since synthesis of this Pdu protein depends on the availability of 1,2-propanediol, the cell solves the problem faced in an environment devoid of 1,2-propanediol where propionate is the sole carbon and energy source by having cobB located outside of the pdu operon and its expression independent of 1,2-propanediol. At present, it is unclear how the CobB and Pdu proteins affect prpBCDE expression.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Understanding metabolic pathway integration is a central issue in cell physiology. Learning more about this important aspect of cell function requires that we uncover and dissect the strategies used by the cell to ensure the coordinated and timely synthesis or degradation of metabolites.
In the recent past, the catabolism of propionic acid in
Salmonella typhimurium LT2 (11, 13, 34) and
Escherichia coli (9, 32) has been investigated.
The cluster of genes required for the catabolism of propionate in these
bacteria was first identified in and characterized for S. typhimurium LT2 (11, 13), with the closely related gene
cluster in E. coli being reported as part of the genome
project of this bacterium (3). These genes are referred to
as the prp genes and are located in the 8.5-centisome region
of the chromosome. These genes constitute a locus comprised of five
genes organized in two transcriptional units (Fig.
1). One of these units contains the
prpR gene, which encodes a putative member of the sigma-54
(RpoN) family of transcriptional activators (27). PrpR
activity is required for the catabolism of propionate in S. typhimurium (20). The second transcriptional unit
contains the prpBCDE gene cluster, which is organized as an
operon that encodes propionate-degrading enzymes (13, 14).
Work with E. coli has identified PrpC as the 2-methylcitrate
synthase (9, 32). The 2-methylcitric acid cycle was first
discovered in Yallowia lipolytica (2, 29, 30),
and in this cycle propionyl-coenzyme A (CoA) is 
-oxidized to
pyruvate (Fig. 1).
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Our laboratory discovered that in addition to the prpRBCDE genes, catabolism of propionate in S. typhimurium requires the activity encoded by the cobB gene (34). The CobB gene product was first identified as an activity required for the expression of a phosphoribosyltransferase enzyme that restored synthesis of adenosylcobalamin in strains defective in the late steps of the biosynthesis of this coenzyme (33).
We have also reported that the inability of cobB mutants to
catabolize propionate was corrected by the induction of the
pdu genes required for the catabolism of 1,2-propanediol
(1,2-PDL) (34). This observation was of interest to us
because propionate, or an activated form of it (not clear at this
point), is the end product of the pathway responsible for the
catabolism of 1,2-PDL (4, 16, 37). We noted that induction
of the pdu operon, without catabolism of 1,2-PDL, was
sufficient to correct the Prp
phenotype of
cobB mutants. This finding was interpreted to mean that
1,2-PDL-dependent correction of this phenotype was due to the synthesis
of a Pdu protein that compensated for the lack of CobB when propionate
was the sole source of carbon and energy (34).
To better understand the implications of these results, it was essential to learn more about the roles of the Pdu and CobB proteins in propionate catabolism. These proteins may be enzymes that catalyze the same reaction, they may be regulatory proteins, or they may be both. Of particular interest to us was the possibility that CobB plays a regulatory role in the catabolism of propionate, since compensating for its absence through the induction of the pdu genes would provide evidence for pathway networking.
The regulation of expression of the prpBCDE operon is complex. PrpR is required, and at this point we assume that it has a coregulator, although it has not yet been identified. Propionate per se is not the coregulator, since it fails to induce transcription of the operon. This finding was shown by using transcriptional fusions of MudI1734 (lacZ+) elements (5) under the control of the prpBCDE promoter (PprpBCDE) (11). Expression of the fusion was observed only in merodiploid strains that carry a wild-type copy of prpBCDE and a second copy of the operon, into which the MudI1734 element was inserted. These results were interpreted to mean that a catabolite of propionate was the signal for the presence of propionate in the environment.
In this paper, we show that CobB, PrpC, and PrpE are needed for transcription of the prpBCDE operon. We also show that the pdu function that allows growth of cobB mutants on propionate does so by fully restoring transcription of the prpBCDE operon. We suggest that 2-methylcitrate, the proposed product of the reaction catalyzed by PrpC (9, 32) or a derivative of it, is the coregulator needed for PrpR to activate transcription of the operon. Consistent with the requirement for PrpC activity, reduction of the intracellular levels of propionyl-CoA in a prpC+ strain mimicked the effect of a prpC mutation on the expression of the prpBCDE operon. The roles of CobB and of the uncharacterized Pdu protein are discussed within the framework of metabolic pathway integration and physiological strategies used by the cell to ensure expression of target genes in response to multiple environmental stimuli.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Bacterial strains, medium, and growth conditions. All strains used in this work were derivatives of S. typhimurium LT2, and their genotypes and all plasmids used are listed in Table 1. Cells were grown as detailed in Table 1 or the figure legends. The no-carbon E (NCE) medium was used as minimal medium. Nutrients and their concentrations in the medium were as follows: propionate, 30 mM; 1,2-PDL, 12 mM; glycerol, 22 mM; methionine, 0.5 mM; D-(+)-arabinose, 500 µM; and MgSO4, 1 mM. Antibiotics were used at concentrations previously reported (8).
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Genetic techniques. (i) Transductions. All genetic crosses were performed with the high-level-transduction mutant phage P22 HT105 int-201 (25, 26) as described elsewhere (7). Transductants were freed of phage by streaking on indicator plates (6).
(ii) Complementation analysis of prp point mutants. The prpB, prpC, prpD, and prpE mutants used in experiments aimed at determining the involvement of prp functions in the expression of the prpBCDE operon were isolated after localized chemical mutagenesis with hydroxylamine (7, 12) as previously described (13). It was assumed that a single mutation was responsible for the inactivation of a gene product; however, it was not determined how many mutations were present in each affected gene. It should be noted that each one of these mutants was complemented by plasmids carrying the wild-type allele of the appropriate gene under the control of the arabinose-inducible promoter ParaBAD (13). The construction of these plasmids has been reported previously (13, 14). Plasmids were introduced into recipient strains by transformation (31). Inheritance of the plasmid was ensured by selecting for the antibiotic resistance carried by the cloning vector. Transformants were replica printed to NCE minimal medium supplemented with magnesium, propionate, and methionine.
(iii) Construction of a cobB mutant carrying a
duplication of the prpRBCDE genes.
A
Tn10-held duplication of the S. typhimurium LT2
spanning the 8- to 26-centisome region of the chromosome
(DUP1033[proA-pyrC]) was moved by transduction
into a strain carrying a deletion of cobB by selecting for
tetracycline resistance. The insertion
prpC114::MudJ was placed by transduction into one
of the copies of the prpBCDE operon present within the
duplicated region (Fig. 2). As reported elsewhere, the resulting cobB mutant was unable to grow on
propionate (34). cobB mutant strains containing
this duplication displayed a Prp phenotype and were
routinely grown in the presence of tetracycline to avoid segregation of
the duplicated material (1).
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Recombinant DNA techniques. (i) DNA sequencing.
Plasmid DNA
was isolated with a QIAprep Spin Plasmid Miniprep kit of Qiagen Inc.
(Chatsworth, Calif.) by following the manufacturer's instructions
without modifications. PCR sequencing reaction mixtures were prepared
with an ABI PRISM Dye Terminator Cycle Sequencing kit (Perkin-Elmer,
Norwalk, Conn.) according to the manufacturer's instructions. Reaction
mixtures were purified in AutoSeq G-50 columns (Pharmacia Biotech,
Piscataway, N.J.), dried in a SpeedVac concentrator (Savant
Instruments, Farmingdale, N.Y.), and sequenced at the Biotechnology
Center of the University of WisconsinMadison.
(ii) Plasmid constructions. A ca.-790-bp EcoRI fragment from plasmid pPRP8 (13) containing PprpBCDE was cloned into the EcoRI site of plasmid pRS551 (28) to place the promoterless lacZ+ gene in pRS551 under the control of PprpBCDE. The resulting plasmid, pPRP25 (Fig. 3), was used as a reporter of promoter activity. The orientation of this fragment was confirmed by DNA sequencing. Plasmid pPRP35 carries a wild-type allele of prpC under the arabinose-inducible promoter ParaBAD. The construction of this plasmid has been reported previously (13).
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(iii) Electroporation. Plasmids were introduced into recipient cells by electroporation under conditions described elsewhere (19). Resistance to the antibiotic encoded by the plasmid was used to assess inheritance. Plasmid DNA was isolated as described above.
-Galactosidase enzyme activity assay.
-Galactosidase
activity assays were performed as described elsewhere (8). A
unit of activity was defined as the amount of enzyme required to
catalyze the hydrolysis of 1 nmol of
o-nitrophenyl--D-galactopyranoside (ONPG) per
min. Specific activity was reported as units per unit of absorbance at
650 nm (A650).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In a previous report, we showed that expression of a prp-lacZ transcriptional fusion occurs only in merodiploid strains that carry a wild-type copy of the prpBCDE operon. This result suggested that the activity of one or more of the Prp proteins was needed to generate the catabolite of propionate required for activation of transcription of the operon (11).
Identification of prp functions encoded by the prpBCDE operon needed for expression of prpBCDE. To determine which functions encoded by the prpBCDE operon were required for its transcription, we investigated the effect that missense mutations in prpB, prpC, or prpD had on the expression of a plasmid-encoded lacZ reporter gene placed under the control of PprpBCDE (plasmid pPRP25). Allele prpE213, a kanamycin resistance cassette, was inserted into prpE by recombinant means (14). This insertion element ensured inactivation of prpE function, and since prpE is the most downstream gene in the operon, polarity of this insertion was not a concern.
Strains carrying mutations in prpB, prpC, or prpD displayed a severe Prp phenotype that, in
all cases, was corrected by introducing into the strains a plasmid
carrying a wild-type allele of the gene affected by the mutation.
prpE mutants with an otherwise wild-type genetic background
did not display a Prp phenotype due to redundant
functions in the background (13, 14). When these functions
were inactivated, however, the Prp phenotype of
prpE mutants was complemented by a wild-type allele of
prpE.
Complementation of the Prp phenotype of prpB,
prpC, prpD, and prpE mutant strains by
a single-gene plasmid showed that the mutations tested did not affect
the synthesis of other gene products in the operon. Except for
prpE, two independently isolated alleles of each gene were
used in these studies to help validate the conclusions drawn. All
strains tested carried a wild-type allele of prpR, the
putative activator of prpBCDE transcription (13).
Loss of prpC function resulted in a severe reduction (ca.
95%) in the level of expression of the operon relative to the level of
expression in the wild-type strain (Table
2). Lack of any of the remaining
prp functions did not have as severe an effect. Noteworthy
was the effect observed in strains lacking PrpD. In these mutants the
level of prpBCDE expression was reduced ca. 66% (Table 2).
Lack of PrpE reduced the level of expression by 30% (Table 2), and
lack of PrpB resulted in a ca. 10% reduction in the level of
expression of the operon relative to that in the wild type (Table 2).
The roles of PrpD and PrpE are discussed further below.
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PrpC provided in trans restores activity of the prpBCDE promoter in a haploid strain. Data presented in Table 3 demonstrate that the lack of PrpC activity was the reason why the prpC114::MudJ transcriptional fusion present in the chromosome was not expressed if the strain did not carry an additional, wild-type copy of the prpBCDE operon (11). When prpC+ was provided in trans, a 28-fold increase in expression of the prpC114::MudJ fusion was measured.
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phenotype of a prpC missense mutant;
that is, complementation of function was not observed in the absence of
arabinose (data not shown). These results demonstrated the ability of
arabinose to substantially increase the level of PrpC in the cell. As
expected, the growth phenotype of strain JE4199
(prpC114::MudJ/pPRP35 ParaBAD prpC+) was not corrected in the presence or absence of
arabinose (data not shown but see reference 13).
These results were consistent with the need for prpD and
prpE gene products to catabolize propionate. Neither PrpD
nor PrpE is synthesized in strain JE4199 due to the polar effect of the
MudJ element on prpD and prpE.
The dramatic increase in expression of the fusion in the absence of
prpD and prpE gene products strongly suggested
that the propionate catabolite needed for expression of the
prpBCDE operon was either 2-methylcitrate (Fig. 1) or a
derivative of it.
The pathway shown in Fig. 1 predicted that expression of the
prpBCDE operon in a strain deficient in the synthesis of
propionyl-CoA would not be restored by providing
prpC+ in trans. This prediction was
tested and proven to be correct. The results of the experiments
addressing this point are discussed below.
Propionyl-CoA is a precursor of the catabolite that signals the presence of propionate in the environment. Figure 4 shows the effect of the lack of propionyl-CoA on the expression of the prpBCDE operon. Work from our laboratory recently demonstrated that the prpE gene encodes a specific propionyl-CoA synthetase (14). It was also demonstrated that prpE mutants compensate for the lack of this enzyme through the activity of the acetyl-CoA synthetase encoded by the acs gene.
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-galactosidase activity (mean ± standard deviation, 1,661 ± 407 U/A650 unit) was
reduced 92% relative to that in the wild-type strain JE4044
(20,696 ± 850 U/A650 unit). In strain
JE4397 the level of activity was reduced 89% relative to the level in
the control strain JE4044 (2,286 ± 206 versus 20,696 ± 850 U/A650 unit). Thus, the acetyl-CoA-synthesizing
enzymes in S. typhimurium can activate propionate into
propionyl-CoA. In addition, these results mimic those obtained with
strains carrying mutations in prpC, indicating that PrpE
(propionyl-CoA synthetase) activity is also required for expression of
the prpBCDE operon.
It should be pointed out that a strain deficient in the synthesis of
PrpE and Acs, but wild type for ack and pta, did
not grow on propionate (14). In such a strain, expression of
the prpBCDE operon was reduced only by a third relative to
the level expressed by the wild-type strain (Fig. 4), and yet this
strain failed to grow on propionate. To help explain this result, we argue that in the context of growth, Ack and Pta are poor substitutes for Acs or PrpE and that these enzymes cannot keep up with the demand
of the pathway for propionyl-CoA to sustain cell growth. On the other
hand, Ack and Pta can clearly generate enough propionyl-CoA to activate
transcription of the prpBCDE operon. These results warn us
about the risks of concluding that a high level of transcription of any
gene necessarily means that the function of the corresponding gene
product is sufficient for the cell to grow.
Although transcription of the prpBCDE operon in a prpE
ack double mutant (strain JE4521) was only 39% of the level
measured in the wild-type strain JE4044 (8,148 ± 171 versus
20,696 ± 850 U/A650 unit), strain JE4521
grew well on propionate, indicating that, unlike Ack and Pta, Acs can
satisfy the demand of the pathway for propionyl-CoA by itself and that
the cell can grow on propionate. In the acs ack double
mutant (strain JE4524), transcription of prpBCDE was high
(11,196 ± 785 or 50% of wild-type expression) and the strain
grew well on propionate. These results were not surprising since strain
JE4524 was wild type for prpE.
cobB function is required for transcription of the prpBCDE operon. As alluded to in the introduction, one possible explanation for the inability of cobB mutants to grow on propionate was that CobB activity is required for the expression of the prpBCDE operon. To test this idea we measured the expression of the prpC114::MudJ transcriptional fusion in a merodiploid strain as a function of CobB. Data in Table 4 clearly show that cobB mutants fail to express the prpBCDE operon (Table 4). In the absence of CobB, expression of the operon was reduced 24-fold. This drastic reduction was due exclusively to the lack of CobB, since when a wild-type allele of cobB was provided in trans, transcription of the operon was restored to the levels observed in the cobB+ strain (Table 4). Plasmid pCOBB5 carries only a wild-type allele of cobB (35).
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Induction of the propanediol utilization (pdu) operon restores expression of prpBCDE and growth on propionate in cobB mutants. As mentioned above, we previously reported that induction of the propanediol utilization (pdu) genes by 1,2-PDL was sufficient to restore the ability of cobB mutants to grow on propionate as the carbon and energy source (34). Data in Table 4 show that induction of the pdu operon in a cobB mutant resulted in expression of the prpBCDE operon to levels observed in strains carrying a wild-type allele of cobB (Table 4). Consistent with previous phenotypic observations (34), introduction of the insertion element pdu-8::MudA into cobB mutant strain JE4265 completely eliminated the ability of the resulting strain (JE4359) to express the prpBCDE operon; hence, strain JE4359 failed to grow on propionate as the sole carbon and energy source in spite of the presence of 1,2-PDL in the medium (Table 4). These results further define our understanding of the role of CobB in propionate catabolism and provide a good example of pathway networking in the cell.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The work reported herein makes two important contributions to our understanding of the physiology of S. typhimurium: (i) it provides insights into the complex regulation of transcription of the prpBCDE operon, which encodes functions required for the catabolism of propionate in this bacterium, and (ii) it provides strong evidence for metabolic pathway integration.
Propionyl-CoA synthetase (PrpE), 2-methylcitrate synthase (PrpC), and CobB activities are needed to activate prpBCDE operon transcription. Data reported in this paper show that three proteins are required for expression of this operon, namely, PrpE, PrpC, and CobB or its alternative, an uncharacterized Pdu protein. All these proteins are needed in addition to PrpR, the sigma-54 activator encoded by prpR (13, 20).
Two of these proteins have documented biochemical activities associated with them: PrpE has propionyl-CoA synthetase activity (14) and PrpC has 2-methylcitrate synthase activity (9, 15, 32). The role of PrpE and PrpC in prpBCDE transcription can be rationalized on the basis of the proposed pathway for propionate catabolism in this bacterium (Fig. 1). On the basis of our data, we hypothesize that the catabolite of propionate that signals the presence of this fatty acid in the environment is either 2-methylcitrate or a derivative of it. This metabolite may be the coregulator needed by PrpR to activate transcription of prpBCDE. Since PrpC catalyzes the synthesis of 2-methylcitrate from propionyl-CoA and oxaloacetate, it is clear that PrpE activity is central to the generation of one of the substrates for PrpC. That is why the effect of blocking propionyl-CoA synthesis in prpC+ strains mimics the effect that mutations in prpC have on the expression of the prpBCDE operon.Role of the PrpD and PrpB proteins in prpBCDE expression. Our data on the involvement of PrpB in the regulation of the prpBCDE operon suggest that this putative 2-methylisocitrate lyase enzyme activity most likely does not play a role in the generation of the catabolites that signals the presence of propionate in the environment.
Although the data for PrpD are less clear, we also conclude that PrpD activity is not needed for the generation of the signal. The data show a significant reduction in prpBCDE expression in prpD mutants that suggests that PrpD activity may be required to generate the signal needed to activate prpBCDE transcription. However, this reduction should be interpreted with caution. The same effect would be observed if the absence of PrpD had the net effect of reducing the amount of 2-methylcitrate made by PrpC, in which case, the observed reduction of prpBCDE operon expression in prpD mutants would be the result of suboptimal concentrations of the signal needed by PrpR to activate transcription of the operon. An additional argument supporting the idea that PrpD activity may not be needed to generate the signal comes from the experiments that analyzed the expression of the prpC114::MudJ fusion in a haploid strain with or without prpC+ in trans (Table 3). In these experiments, PrpC was sufficient to restore full expression of the fusion in a strain where expression of prpD was presumably eliminated by the MudJ element upstream of this gene. At this point, we cannot rule out the possibility that a small but sufficient amount of PrpD was synthesized in this strain. Such a small amount of PrpD would have to be sufficient to allow regulation at the wild-type level to occur. One important difference between these experiments and the ones performed with strains carrying duplications of the prpBCDE operon should be kept in mind. In the experiments with the haploid strain, prpC+ was carried by a high-copy-number plasmid; thus, the concentration of PrpC protein was greater than the concentration afforded by a single chromosomal copy of prpC in the strain with the duplication. This high concentration of PrpC, we argue, is likely to generate sufficient 2-methylcitrate to make PrpR fully functional in the absence of PrpD.Roles of the CobB and Pdu proteins in prpBCDE operon expression. The roles that the CobB protein and its alternative, Pdu protein, play in the transcription of prpBCDE are not obvious. The CobB and Pdu proteins may have one or more enzymatic activities needed for the generation of a metabolite required for prpBCDE transcription activation. Alternatively, they may be proteins without any enzymatic activity that play some role in either transcription activation or attenuation, or in a more complex scenario these proteins may have one or more enzymatic activities in addition to playing a more direct role in transcription activation and/or posttranscriptional regulation of the prpBCDE operon expression. How these proteins affect expression of the prpBCDE operon remains under investigation.
Integration of the 1,2-PDL and propionate catabolism pathways. Regardless of how the CobB and Pdu proteins affect transcription of the prpBCDE operon, we believe that the existence of these redundant functions reflects physiological strategies to ensure that synthesis of the propionate degrading enzymes occurs under different environmental conditions. Since 1,2-PDL catabolism is likely to proceed via propionyl-CoA, it is clear that expression of the prpBCDE operon is needed for the cell to be able to use 1,2-PDL as a carbon and energy source.
It appears that the Pdu alternative for CobB function is a mechanism that the cell has evolved to ensure the timely synthesis of the PrpB, -C, -D, and -E enzymes to further degrade propionyl-CoA into central metabolites. Figure 5 illustrates how we think the expression of the prpBCDE operon is ensured when either 1,2-PDL or propionate is present in the environment. When 1,2-PDL is the sole carbon and energy source, it binds to the PocR protein (21, 22) and the PocR-1,2-PDL complex activates transcription of the cob or pdu regulon (23, 24). Since this Pdu function is available only upon induction of the operon by 1,2-PDL, S. typhimurium would be unable to use propionate as a carbon and energy source in an environment devoid of 1,2-PDL. One way to solve this problem would be to have a redundant function encoded outside of the pdu operon, with the expression of such a gene being independent of the presence of 1,2-PDL. In this case, the redundant function is encoded by cobB. Learning more about how the CobB and Pdu proteins affect prpBCDE operon expression will shed light on one mechanism used by this bacterium to integrate its metabolism.
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ACKNOWLEDGMENTS |
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This work was supported by NSF grant MCB9724924 and USDA Hatch grant WIS3765 to J.C.E.-S.
We acknowledge the participation of Michael Smits in the initial stages
of this work. Michael Smits was a participant of the 1997 REU Summer
Research Program of the Department of Bacteriology at the University of
WisconsinMadison, which is sponsored by the NSF, the Graduate School,
and the College of Agricultural and Life Sciences of the University of
WisconsinMadison. We thank R. LaRossa for strains.
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FOOTNOTES |
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*
Corresponding author. Mailing address: Department of
Bacteriology, University of WisconsinMadison, 1550 Linden Dr.,
Madison, WI 53706-1567. Phone: (608) 262-7379. Fax: (608) 262-9865. E-mail: jcescala{at}facstaff.wisc.edu.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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