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1 Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada
2 Department of Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada
Correspondence
Miguel A. Valvano
mvalvano{at}uwo.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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uppP-ybjG-yeiU-waaL has a growth defect and abnormal cell morphology, suggesting that accumulation of unprocessed Und-PP-linked O antigen polysaccharides is toxic for these cells. Together, the results support the notion that YbjG, and to a lesser extent YeiU, exert their enzymic activity on the periplasmic side of the plasma membrane and are implicated in the recycling of periplasmic Und-PP molecules.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Burda & Aebi, 1999; Helenius & Aebi, 2002; Helenius et al., 2002; Valvano, 2003). A 55-carbon isoprenoid, undecaprenyl phosphate (Und-P), is the lipid carrier of biosynthetic intermediates for cell wall peptidoglycan, O antigen, enterobacterial common antigen, teichoic acids and other bacterial carbohydrate polymers. Und-P arises from the dephosphorylation of its precursor undecaprenyl pyrophosphate (Und-PP), which in turn results from the addition of isoprene units onto farnesyl pyrophosphate, a reaction catalysed by the UppS synthase (Apfel et al., 1999; Shimizu et al., 1998). Since UppS is a soluble protein these biosynthetic steps must occur in the cytosol or in close proximity to the cytosolic face of the plasma membrane. The isoprene molecules isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the five-carbon universal basic building blocks of all isoprenoids (White, 1996). Two distinct biosynthetic pathways exist for the de novo synthesis of IPP and DMAPP: the mevalonate pathway in eukaryotes and archaea and the 2-C-methyl-erythrito1 4-phosphate (MEP) pathway in eubacteria, green algae and plant chloroplasts (Kuzuyama, 2002; Rohmer et al., 1993; Rohmer, 1999). The MEP pathway (Fig. 1) starts with the synthesis of 1-deoxy-xylulose 5-phosphate from pyruvate and glyceraldehyde 3-phosphate, a reaction catalysed by the 1-deoxy-xylulose 5-phosphate synthase encoded by the dxs gene (Lois et al., 1998; Sprenger et al., 1997). In the second step, MEP is synthesized by the 1-deoxy-xylulose 5-phosphate reductoisomerase encoded by the dxr gene (Takahashi et al., 1998). This reaction is specifically inhibited by the antibiotic fosmidomycin (Kuzuyama et al., 1998a; Steinbacher et al., 2003). Subsequent enzymic steps result in the formation of IPP and DMAPP, the precursors of Und-PP (Fig. 1).
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; Goldman & Strominger, 1972). The availability of Und-P is a limiting factor in the biosynthesis of O polysaccharides, since this lipid carrier is made in very small amounts, and it is also required for the biosynthesis of multiple carbohydrate polymers. Therefore, the recycling pathway for Und-P synthesis from preformed Und-PP released at the periplasmic side of the plasma membrane may also contribute to the total Und-P pool available for the initiation of lipid-linked glycan biosynthesis (Fig. 1
). The de novo synthesis and regeneration of Und-PP at opposite sides of the membrane suggests the requirement for pyrophosphatases whose active sites are in cytosolic and periplasmic environments, respectively. Recently, El Ghachi et al. (2004, 2005) have identified four genes in Escherichia coli K-12 encoding membrane proteins with Und-PP pyrophosphatase activities. Among these, UppP (formerly BacA) accounts for 75 % of the cellular Und-PP pyrophosphatase activity (El Ghachi et al., 2004), while the proteins YbjG, YeiU and PgpB account for the remaining activity (El Ghachi et al., 2005). However, it is not clear which of these proteins function in Und-PP recycling.
The recycling of pyrophosphoryl polyisoprenols is not only confined to bacteria. In eukaryotes and also in archaea, dolichol phosphate serves as a lipid anchor for the assembly of glycan moieties that are further added to proteins (Bugg & Brandish, 1994; Burda & Aebi, 1999). After the transfer of the glycan to the glycosylation site of the protein, released dolichol-PP is recycled to dolichol-P by dephosphorylation (Abeijon & Hirschberg, 1992; Fernandez et al., 2001). Fernandez et al. (2001) have shown that Cwh8, a membrane protein in the endoplasmic reticulum of Saccharomyces cerevisiae, has a dolichol-PP pyrophosphate phosphatase activity and possesses a luminally oriented active site. Homologues of this protein are also found in mammalian cells (Rush et al., 2002). The topology of Cwh8 agrees with its proposed function in the formation of dolichol-P from dolichol-PP intermediates arising from the oligosaccharyl transfer reaction during N-linked protein glycosylation, which occurs on the luminal side of the endoplasmic reticulum membrane. Cwh8 has a segment of approximately 122 amino acids that corresponds to an acid phosphatase domain (Fig. 2). This domain, shared among lipid phosphatases and non-specific acid phosphatases, comprises three motifs (Neuwald, 1997; Stukey & Carman, 1997). The conserved His from motif 3 is proposed to attack the phosphate group of the substrate, leading to the formation of a covalent phosphoenzyme intermediate that is stabilized by the neighbouring Arg in motif 3, while Lys and Arg in motif 1 together with Ser, Gly and His in motif 2 have an important role in holding the phosphate group of the substrate close to the His from motif 3 (Ishikawa et al., 2000; Neuwald, 1997).
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) (El Ghachi et al., 2005). However, their function in the recycling of Und-PP remains to be determined. In this work, we show that the acid phosphatase motifs in YbjG and YeiU face the periplasmic space, and provide experimental evidence suggesting that these proteins are implicated in the recycling of periplasmic Und-PP molecules. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Plasmids were introduced into recipient cells by calcium chloride transformation (Cohen et al., 1972) or electroporation (Dower et al., 1988). Bacteria were grown at 30, 37 or 42 °C in Luria–Bertani (LB) medium supplemented with 100 µg ampicillin ml–1, 40 µg kanamycin ml–1, 80 µg spectinomycin ml–1 and 0.2 % or 0.02 % (w/v) arabinose, as appropriate. For growth curves, overnight cultures of mutants and parental E. coli strains were diluted to an OD600 of 0.03 (corresponding to a bacterial density of approximately 104 c.f.u. ml–1), and the growth was monitored every half hour in a 100-well microtitre plate using a Bioscreen C automated microbiology growth curve analysis system (MTX Lab Systems). This system is a computerized bacterial incubator that measures growth continuously by optical density (Löwdin et al., 1993). The results were analysed statistically by two-way ANOVA and the Bonferroni post-test using the Prism 4 software package (GraphPad Software).
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. PCR fragments for mutagenesis were obtained using primers containing 40–45 nucleotides corresponding to regions adjacent to the gene targeted for deletion, plus 20 more nucleotides that annealed to the template DNA from plasmid pKD4, which carries a kanamycin-resistance gene flanked by FLP recognition target sites. PCR products were transformed into E. coli strains carrying pKD46 that were grown in LB plus 0.5 % (w/v) arabinose. Kanamycin-resistant colonies were screened for the insertion of the kanamycin-resistance cassette into the targeted gene by PCR with primers annealing to regions outside of the mutated gene. This resulted in the complete replacement of the parental gene by the kanamycin phosphotransferase gene (aph). If required, the antibiotic gene cassette was excised using pCP20, which encodes the FLP recombinase. Plasmids pKD46 and pCP20 are thermosensitive for replication and they were cured at 42 °C.
Cloning strategies.
PCR products were cloned into pBADHis (Table 1) using specific restriction sites included in the cloning primers. The primers used in this work are listed in Table 2. We used the Stratagene Quick Change kit and appropriate mutagenesis primers to replace His145 by Ala in YbjG. The correct replacement was confirmed by DNA sequencing.
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). Overnight cultures grown at 30 °C on LB plates were used to inoculate Mueller–Hinton (MH) broth followed by incubation at 30 °C until an OD600 of approximately 1. As indicated above for the growth curves, aliquots of these cultures were adjusted to an OD600 of 0.03 in 100-well microtitre plate containing 300 µl MH broth with various Fm concentrations ranging from 0 to 480 ng ml–1. The Bioscreen C was set at low constant shaking at 37 °C and OD600 measurements were automatically recorded every 30 min during 18 h. The percentage survival for each strain was calculated at each time point using the optical density at 0 ng ml–1 Fm as 100 % growth. For complementation experiments with strain LDT30, cells were grown as indicated before in MH broth also containing 100 µg ampicillin ml–1 and 0.02 % (w/v) arabinose to induce recombinant protein expression. In all cases survival data were analysed at the 6 h time point from multiple repeats.
LPS analysis.
LPS was prepared as described earlier (Marolda et al., 1990) and samples were separated by 14 % Tricine SDS-PAGE. The gels were stained with silver nitrate (Marolda et al., 1990). For some experiments, bands were transferred to nitrocellulose membranes for immunoblot analysis. The membranes were treated with either anti-O16 (The Gastroenteric Disease Center, Wiley Laboratory, University Park, PA, USA) or anti-LPS core monoclonal antibodies (HyCult Biotechnologies). Fluorescent secondary antibodies (IRDye800CW affinity-purified anti-rabbit IgG antibodies) were used to detect the positive bands by monitoring the fluorescence with an Odyssey infrared imaging system (LI-COR Biosciences).
Preparation of crude membrane extracts.
Bacterial cultures grown overnight in 5 ml LB with the appropriate antibiotic were diluted in 15 ml fresh medium to an OD600 of 0.05 and grown to an OD600 between 0.5 and 0.7. At this point recombinant protein expression was induced by adding arabinose to a final concentration of 0.2 % or 0.02 % (w/v) and cultures were incubated for another 3 h. Bacteria were harvested by centrifugation at 7741 g for 10 min at 4 °C and pellets stored at –20 °C. Bacterial pellets were resuspended in 4 ml 20 mM phosphate buffer pH 7.2 containing an EDTA-free cocktail of protease inhibitors (Roche), and lysed by sonic disruption with a Branson Digital Sonifier model 450 on ice using 10–15 s pulses at an amplitude of 40 %. Cell lysates were cleared of cell debris by centrifugation in a tabletop centrifuge at full speed (13 000 r.p.m.) for 15 min at room temperature. The membranes were collected from the clear lysate by another centrifugation at 39 191 g for 30 min at 4 °C using a Beckman high-speed refrigerated centrifuge, and total protein concentration was determined by the Bradford assay (Bio-Rad), using BSA as a protein standard.
Protein expression analysis.
Membrane proteins were separated by electrophoresis in 14 % SDS-polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes by standard procedures. Membranes were blocked overnight with 5 % skim milk dissolved in TBS pH 7.6 (50 mM Tris and 150 mM sodium chloride), incubated for 2 h with monoclonal anti-alkaline phosphatase antibodies (Sigma-Aldrich) at a dilution of 1 : 12 000. Specific bands were detected by fluorescence using Alexa Fluor 680 anti-mouse IgG antibodies (Molecular Probes) in the Odyssey infrared imaging system. The GFP fusions were detected with rabbit anti-GFP polyclonal antibodies (Chemicon International; 1 : 1000). The protein with a C-terminal His6 epitope was detected with rabbit polyclonal (Sigma) anti-His6 antibodies. Alexa Fluor IRDye800 CW affinity-purified anti-rabbit IgG antibodies (Rockland) were used as the secondary antibody for the detection of specific bands by fluorescence.
Alkaline phosphatase assay.
The expression of hybrid proteins with alkaline phosphatase (PhoA) activity was qualitatively assessed by examining the blue-colony phenotypes on LB plates containing 40 µg ml–1 of the chromogenic substrate 5-bromo-3-chloro-indolyl phosphate (XP, Sigma). To quantify the alkaline phosphatase activity, 1 : 100 culture dilutions were grown for 3 h to an OD600 of 0.5–0.6 and then induced with 0.02 % arabinose for 3 h, after which the cells were harvested and assayed as described by Manoil (1991) with the addition of 1 mM iodoacetamide in all the buffers to avoid folding of cytoplasmic PhoA (Derman & Beckwith, 1995).
Microscopy.
Overnight cells expressing GFP fusions were diluted in LB to OD600 0.1, grown to OD600 0.5 and than induced with 0.2 % arabinose for 3 h. At this point the cells were kept on ice for 1–2 h to facilitate GFP folding, and then visualized using an Axioscope 2 (Carl Zeiss) microscope with an X100/1.3 numerical aperture Plan-Neofluor objective and a 50 W mercury arc lamp with a GFP band pass emission filter set (Chroma Technology) with a 470±20 nm excitation range and a 525±25 nm emission range. Images were digitally processed using the Northern Eclipse version 6.0 imaging analysis software (Empix Imaging).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) predicted six, four and five transmembrane domains, respectively, with the C-terminal end of PgpB facing the cytosol and the C-terminal ends of YbjG and YeiU located towards the periplasmic space (data not shown). The computer-generated model of PgpB placed the acid phosphatase motif facing the periplasmic space. However, the models generated for YbjG and YeiU placed approximately half of the acid phosphatase motif shown in Fig. 2 within a cytosolic loop and the other half within a periplasmic loop (data not shown). Such an arrangement would render the enzyme inactive, suggesting the model was not accurate. We constructed C-terminally fused derivatives of YbjG and YeiU proteins with GFP to experimentally confirm the orientation of their C-termini. E. coli W3110 cells expressing the recombinant proteins YbjGGFP or YeiUGFP exhibited fluorescence with varying degrees of intensity, which was distributed around the perimeter of each individual cell (Fig. 3a, b). In contrast, the fluorescence of cells transformed with the control plasmid expressing soluble GFP was homogeneously distributed within the cytoplasm (Fig. 3c). The distribution of fluorescence around the cell perimeter agrees with a membrane location of the chimeric proteins. Since GFP cannot fold into a chromophore within the periplasm (Drew et al., 2002; Feilmeier et al., 2000) we concluded that the C-terminus in both proteins faces the cytosolic compartment. This conclusion was supported by a fusion experiment with PhoA, which folds into an enzymically functional form only in the periplasmic space. Bacteria expressing YbjGPhoA or YeiUPhoA had a white phenotype on agar plates containing the colour indicator XP, and cell lysates did not show any detectable alkaline phosphatase activity. Together, absence of PhoA activity and GFP fluorescence demonstrated unequivocally the cytoplasmic location of the C-terminal segment of YbjG and YeiU.
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). We confirmed that the fusion proteins were properly expressed by Western blotting with an anti-PhoA monoclonal antibody (data not shown). Recombinant plasmids expressing the PhoA fusion proteins were transformed into the PhoA-deficient strain E. coli CC118 (Table 1) and transformants examined for a blue-colony phenotype, which results from the degradation of the indicator dye XP by those bacteria with periplasmic exposed fusions. The PhoA enzymic activity was quantified as described in Methods and normalized to the amount of PhoA protein detected densitometrically from Western blots. PhoA fusions to amino acids Val85 and His105 in YbjG (YbjGV85-PhoA and YbjGH105-PhoA), and Thr123 and Asp144 in YeiU (YeiUT123-PhoA and YeiUD144-PhoA) resulted in proteins with detectable normalized PhoA activities ranging from 284 to 1495 units (Table 3), indicating that these residues are exposed to the periplasmic space. However, a PhoA fusion to the predicted catalytic His145 residue in YbjG did not show detectable enzymic activity, while a fusion to the His190 in YeiU (corresponding to the catalytic His residue in this protein) gave 198 units. The expression of both protein fusions as detected by Western blotting was low, suggesting the possibility of folding problems leading to protein degradation. We therefore constructed a PhoA ‘sandwich’ fusion in YbjG such that the PhoA protein was inserted between Arg139 and Val140 (YbjGR139-PhoA-V140; Table 3). This recombinant protein gave 245 units of activity, demonstrating a periplasmic location for these two residues, and suggesting that the entire loop containing the catalytic His145 is exposed to the periplasm. From these results, together with the analysis of the hydrophobicity of the intervening regions and the distribution of positively charged amino acids that are usually located in cytosolic loops (Heijne, 1986), we constructed a revised topological model for YbjG and YeiU (Fig. 4). The location of the essential residues of the conserved acid phosphatase domain in both proteins indicates that the catalytic site is oriented towards the periplasmic side of the plasma membrane, supporting the notion that the enzymic activity of YbjG and YeiU resides in the periplasmic compartment.
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; Zeidler et al., 1998), the second step in the Und-PP de novo synthesis pathway (Kuzuyama et al., 1998b; Rodriguez-Concepcion & Boronat, 2002; Takahashi et al., 1998). We reasoned that in comparison to the parental strain, E. coli uppP mutants with additional defects in the Und-PP recycling pathway would exhibit higher susceptibility to Fm since de novo synthesis of Und-P and recycling of Und-PP would both be compromised. Therefore, we constructed in W3110 a series of mutants containing single, double and triple deletions of pyrophosphatase genes implicated in the metabolism of Und-P (Table 1) and grew them in increasing concentrations of Fm as described in Methods. The antibiotic concentrations used in these experiments were subinhibitory for the parental strain W3110, which has a MIC50 of 900 ng Fm ml–1. Table 4 shows the percentage survival of these strains at 6 h in the presence of Fm concentrations of 160, 320 and 480 ng ml–1. This time point was chosen as it allowed for detection of maximal differences among the mutant strains. The results show that single deletion mutants in uppP and ybjG genes, and double deletion mutants in either uppP and yeiU or uppP and ybjG, exhibited less than 50 % survival, especially at 480 ng Fm ml–1 (Table 4). Furthermore, a triple uppP-yeiU-ybjG deletion mutant reproducibly exhibited 35 % survival, indicating that lack of these genes creates an additive effect on the mutants’ viability in the presence of Fm. In contrast, the survival of mutants with single deletions in either yeiU or pgpB was much higher than 50 % and even closer to that of the parental strain (Table 4). The observed differences in percentage survival among parental and mutant strains were not due to growth rate differences or general changes in bacterial cell permeability, as the parental E. coli W3110 and all mutant derivatives grew similarly in the absence of antibiotic and did not differ with respect to their sensitivity to other antibiotics (data not shown). From these data we concluded that ybjG, and yeiU to a lesser extent, are required for Und-P synthesis when the de novo pathway is partially blocked by Fm. In contrast, the function of pgpB appears not to be required under similar experimental conditions.
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uppP-yeiU-ybjG triple mutant (LDT30) was transformed with pLDT68, which expresses the YbjGHis6 protein under the control of the arabinose-inducible promoter. Fig. 5(a) shows that the percentage survival at 6 h of LDT30(pLDT68) exposed to various Fm concentrations was similar to that of the parental strain W3110. In contrast, LDT30 transformed with pLDT67 expressing YbjGH145A, which has an amino acid substitution of the phosphatase catalytic site (Ala replacing His), exhibited poor survival under the same conditions, suggesting that functional complementation required an enzymically active protein. The lower survival found with LDT30(pLDT67) relative to LDT30 with no plasmid is probably due to a general growth defect caused by protein expression under arabinose induction. Indeed, LDT30 cells carrying pLDT67 or pLDT68 displayed a growth rate delay in the presence of arabinose in the medium, compared to the growth rate of the same strains in arabinose-free medium (Fig. 5b). The growth rate delay was maximal at 6 h, which is the same as the time point chosen for the Fm survival experiments.
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shows that the parental and mutant proteins are expressed and migrate as a 24 kDa polypeptide, in agreement with the predicted molecular mass of YbjG. Polypeptides of higher molecular mass were detected; these are likely the result of protein aggregates due to the mild denaturing conditions used for electrophoretic separation of membrane proteins (see Methods), which are required to prevent complete protein aggregation that would preclude detection by Western blotting. From these results we concluded that the lack of complementation by the plasmid expressing the YbjGH145A polypeptide is not due to lack of protein expression or membrane localization, suggesting that YbjGH145A does indeed have a defect in enzymic activity. Collectively, the results of the experiments described in this section suggest that a functional YbjG protein is required to restore survival of strain LDT30 exposed to subinhibitory Fm concentrations to similar levels to those of the parental strain, supporting the notion that YbjG might be required for Und-PP metabolism.
O16 LPS expression in a waaL-uppP-yeiU-ybjG mutant causes a growth defect
E. coli K-12 W3110 does not normally produce O antigen due to an insertion element inactivating the rhamnosyltransferase gene wbbL (Stevenson et al., 1994; Yao & Valvano, 1994). Complementation of the wbbL defect in trans with plasmid pPR1474 (wbbL+) reconstitutes the synthesis of O16 LPS in E. coli W3110 (Feldman et al., 1999; Liu & Reeves, 1994), as detected by silver staining and Western blots with antibodies specific for O16 and the E. coli lipid A-core oligosaccharide (Fig. 6). In contrast, a deletion of the O antigen ligase waaL gene in strain CLM24 (W3110 waaL) results in the synthesis of O16 antigen polymers that are poorly detectable by silver staining but clearly detected with anti O16 antibodies (Fig. 6, top and central panels). However, these polymers do not react with the lipid A-core oligosaccharide-specific antibody (Fig. 6, bottom panel). This indicates that the O16-specific material produced by the ligase-deficient control strain CLM24 (waaL) containing the high-copy number plasmid pPR1474 (wbbL+) corresponds to polymerized O units not linked to lipid A-core oligosaccharide, which most likely remain linked to Und-PP. Therefore, we reasoned that the introduction of the waaL gene deletion into the triple deletion uppP-yeiU-ybjG strain would increase the synthesis of Und-PP-linked O units that cannot be processed any further, resulting in reduction of the available pool of Und-PP for recycling into Und-P. This prediction is supported by the results in Fig. 6 that show increased amounts of O16 polymeric bands only detectable by silver staining and anti-O16 antibodies.
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uppP-yeiU-ybjG-waaL (strain LDT53), containing plasmid pPR1474 (wbbL+), grew significantly slower than the parental strain, the triple uppP-yeiU-ybjG mutant (strain LDT30) with or without pPR1474, and the LDT53 quadruple mutant without pPR1474 (Fig. 7 and data not shown). No growth defects were observed in the pyrophosphate phosphatase single, double and triple mutants in strains not expressing O16 antigen, or in the waaL mutant containing pPR1474 (data not shown). Phase-contrast microscopy also revealed changes in the morphology of bacterial cells, especially cells transformed with pPR1474. Unlike W3110(pPR1474), LDT30 and LDT53 cells containing pPR1474 were very long with a filamentous appearance, and abundant empty sacculi plus cellular debris were noticed, particularly in LDT53 (data not shown). Together, these results suggest that in cells expressing O16 antigen but lacking the O16 antigen ligase WaaL and the UppP, YgjG and YeiU pyrophosphatases, sequestration of Und-PP-linked O16 that cannot be ligated to the lipid A-core oligosaccharide delays growth rate and compromises cell viability. This phenotype would be consistent with an expected reduction in the Und-PP pool available to the recycling pathway, which cannot be compensated by de novo synthesis due to the deletion of the uppP gene.
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PgpB is also a membrane pyrophosphate phosphatase of the same family as YbjG and YeiU (Fig. 2). However, this protein may have another function, since deletions in the pgpB gene did not cause any observable phenotype under the same conditions in which deletions of ybjG and yeiU did. In contrast to the other three phosphatases, UppP lacks any features of a typical acid phosphatase. However, this protein accounts for approximately 75 % of the cellular Und-PP phosphatase activity, and it has been suggested to catalyse the conversion of de novo-synthesized Und-PP into Und-P (El Ghachi et al., 2004). This conclusion also agrees with the observation that the enzymes for the earlier steps of Und-PP synthesis are all present in the cytosolic compartment, including UppS, which catalyses the condensation of isopentenyl-P units into Und-PP. A predicted topological model for UppP reveals a large cytosolic loop containing the motif IGxxQ(E)xxA(S,G)xxxPGxSRS(A,G)G that is conserved in more than 500 bacterial homologues of this protein (M. A. Valvano, unpublished observations). No other proteins are detected from PSI-BLAST and linear hidden Markov models analyses (Hughey & Krogh, 1996), suggesting that UppP belongs to a unique family of Und-PP pyrophosphatases. Therefore, the biochemical evidence demonstrating the phosphatase activity on Und-PP (El Ghachi et al., 2004) together with the predicted membrane topology of UppP suggests that this protein carries out its function in the cytosolic compartment. Based on the available genetic and biochemical evidence we suggest a model as depicted in Fig. 8, which implicates UppP in the de novo synthesis pathway of Und-P while YbjG and YeiU are implicated in the recycling pathway of periplasmic Und-PP. How dephosphorylated periplasmic Und-P molecules return to the biosynthesis pathway on the cytosolic side of the plasma membrane also remains a mystery and awaits further investigations.
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ACKNOWLEDGEMENTS |
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Edited by: M. S. Ullrich
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Received 23 January 2007;
revised 15 April 2007;
accepted 18 April 2007.
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