Department of Bacteriology, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726-4087, USA
Correspondence
Jorge C. Escalante-Semerena
escalante{at}bact.wisc.edu
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ABSTRACT |
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Present address: Plant Pathology Department, UW-Madison, 1630 Linden Dr., Madison, WI 53706, USA.
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INTRODUCTION |
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METHODS |
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Plasmids pCOBS4 and pNLA1.
The construction of these plasmids was described by Maggio-Hall & Escalante-Semerena (1999).
Plasmids pCOBS1 and pCOBS6.
Plasmid pCOBS1 was derived from pJO21, a plasmid previously described (O'Toole et al., 1993). The HincIIHindIII fragment of pJO21 was cloned into cloning vector pGEM3Zf() (Promega) cut with the same restriction enzymes. To construct pCOBS6, the EcoRIHindIII fragment of pCOBS1 was cloned into pT7-5 cut with the same restriction enzymes.
Plasmids pCOBS9 and pCOBS10.
Plasmids pCOBS9 and pCOBS10 encode N-terminally truncated products of the cobS gene, directing translation to begin at the second (pCOBS9) or third (pCOBS10) codons for methionyl residues of the gene sequence (Roth et al., 1993). These plasmids were constructed by site-directed mutagenesis of plasmid pCOBS2 (Maggio-Hall & Escalante-Semerena, 1999
) to create NdeI restriction sites adjacent to each of the new start codons using the reverse primer (5'-AGCTATGACCATG-3') and 5'-CGTTTTGGCATATGCTCGCT-3' (for pCOBS9) or 5'-GTGGGATCCATATGTTTCCC-3' (for pCOBS10). The new cobS alleles were cloned into plasmid pT7-7, which provides a strong ribosome-binding site (Tabor, 1990
). Bases shown in bold-type face identify the engineered NdeI site in both primers.
Plasmid pCOBS16.
The M. thermoautotrophicum strain H ORF1112, predicted by sequence homology to encode a cobalamin synthase orthologue, was amplified from chromosomal DNA using PCR protocols. The 5' primer used created an NdeI restriction site immediately upstream of the predicted start codon of ORF1112 to facilitate cloning into pT7-7 (Tabor, 1990
). The primers used were: 5'-GAGGGGGGTGCCATATGAACGTCACC-3' and 5'-CGAAGGCCCCTGCAGTGAG-3'. Bases shown in bold-type face identify the engineered NdeI site.
Plasmids encoding CobSLacZ and CobSPhoA fusion proteins.
PstI restriction sites were engineered into the cobS gene of pCOBS6 for insertion of phoA or lacZ genes at desired positions using Stratagene's QuikChange XL Site-Directed Mutagenesis kit according to the manufacturer's instructions. All plasmids were sequenced to verify the location of the restriction site. Due to the extensive list of plasmids generated, they are not detailed here; specific information on fusion plasmids is available from the authors upon request.
The phoA-containing PstI fragment from plasmid pCH40 (Hoffman & Wright, 1985) or the lacZ-containing PstI fragment of plasmid pSKS107 (Casadaban et al., 1983
) was cloned into the engineered PstI sites of cobS in plasmid pCOBS6. The ligation reactions were then transformed into a phoA lacZ null strain (CC118). Since the phoA or lacZ fragment could insert in either orientation, plasmids were screened by restriction analysis to determine the presence and orientation of the phoA or lacZ gene. Properly oriented genes were confirmed by DNA sequencing. The resulting plasmids encoded CobSPhoA or CobSLacZ fusion proteins in which the phoA or lacZ gene was preceded by varying lengths of cobS in-frame. A list of the plasmids used in these studies is presented in Table 3
.
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Protein purification.
A cell-free extract was prepared as previously described from cells from a culture of E. coli BL21(DE3) carrying plasmid pCOBS4 harvested 4 h after induction (O'Toole & Escalante-Semerena, 1995). Purification of the overproduced protein was monitored by SDS-PAGE (Laemmli, 1970
). Unless otherwise noted, all chromatography resins used were equilibrated with 0·1 M Tris/HCl buffer pH 8 at 4 °C. SDS-PAGE/Coomassie blue staining was used to identify fractions containing overproduced protein; these fractions were pooled and further purified.
Step 1.
Anion-exchange chromatography was performed on Toyopearl DEAE-650M resin (TosoHaas; 25x82 mm, 40 ml bed volume). A total of 11·5 mg protein was loaded per ml resin. The column was developed with a 00·3 M KCl linear gradient in buffer at a rate of 2 column volumes h1.
Step 2.
Hydroxyapatite chromatography (Bio-Gel HTP Gel, Bio-Rad) was performed on a 25x60 mm (30 ml bed volume) column equilibrated with 10 mM potassium phosphate, pH 7. Pooled fractions from step 1 (14·5 mg protein) were applied after dialysis in 4 litres 0·1 M Tris/HCl buffer pH 8 at 4 °C. Proteins adsorbed to the column were eluted with a 10400 mM linear gradient of potassium phosphate buffer, pH 7, at a flow rate of 2 column volumes h1.
Step 3.
Gel filtration chromatography was performed on a 15x283 mm (50 ml bed volume) Sephacryl S300 column (Amersham Pharmacia). The void volume of the column was calculated using Blue Dextran (Sigma). Fractions containing overproduced protein from step 2 (4 mg protein) were concentrated and applied to the column at a flow rate of 10 ml h1.
Step 4.
A B12 agarose affinity chromatography column (Sigma; 10x30 mm, 2·5 ml bed volume) was used as the last step in the purification protocol since the protein of interest was initially believed to be cobalamin synthase. Fractions containing the overproduced protein from step 3 were pooled and loaded onto the column (1 mg protein). After loading, the column was washed with 3 bed volumes of equilibrating buffer followed by buffer containing 1 mM cyanocobalamin. Proteins that remained bound to the column were desorbed with buffer containing 0·5 M NaCl.
N-terminal sequencing.
Fractions from step 3 that contained the overexpressed protein were subjected to SDS-PAGE. The PAGE gel was blotted onto an Immobilon P membrane using a ProBlot apparatus (Bio-Rad) according to the manufacturer's instructions. The blot was stained with Coomassie brilliant blue R-250 (Sigma) (Sasse, 1991). The protein band corresponding to the 26 kDa protein was excised, dried under a stream of N2, and the N-terminal sequence determined at the Protein Research Laboratory at the University of Illinois-Chicago.
In vitro enzyme activity assays.
Succinate dehydrogenase (Sdh) activity assays were performed as described by Markwell & Lascelles (1978). One unit (U) of Sdh activity was defined as the amount of enzyme that synthesizes 1 µmol product min1. Cobalamin synthase (CobS) assays were performed as previously described (Maggio-Hall & Escalante-Semerena, 1999
). Cobalamin synthase activity of the Methanobacterium thermoautotrophicum enzyme was routinely assayed at 50 °C. One unit (U) of cobalamin synthase activity was defined as the amount of enzyme required to generate 1 µmol of product min1.
-Galactosidase activity assay.
Whole-cell -galactosidase assays were performed in 96-well microtitre dishes using the fluorogenic substrate 3-carboxyumbelliferyl
-D-galactopyranoside (CUG; Molecular Probes) according to the manufacturer's instructions. Cultures were grown overnight with aeration at 37 °C in LB medium containing ampicillin. A sample (0·02 %, v/v) from the overnight culture was used to inoculate 5 ml of the same medium, grown for 2 h (approximate OD650 0·3) and concentrated threefold into sterile 0·145 M NaCl. Cells were permeabilized by the addition of 50 µl CHCl3 to 300 µl of cells followed by vortexing; 50 µl of permeabilized cells was used per assay. E. coli
-galactosidase (Sigma) was used to generate a standard curve ranging from 1 ng to 10 pg. Reactions were started by the addition of 110 nmol CUG substrate (100 µl 0·1 M sodium phosphate buffer pH 7·3, at 25 °C, containing 1·1 mM CUG, 1 mM MgCl2 and 45 mM
-mercaptoethanol). Reaction mixtures were incubated at 30 °C for 30 min, and reactions were stopped by the addition of 10 µmol Na2CO3 (50 µl 0·2 M Na2CO3). Commercially available 7-hydroxycoumarin-3-carboxylic acid (Molecular Probes), the fluorescent product of the hydrolysis of CUG, was used as standard. Fluorescence was measured using a high-throughput SpectraMAX GeminiEM spectrofluorometer (Molecular Devices) with the emission wavelength set at 460 nm and the excitation wavelength set at 390 nm. The amount of cells used per assay was determined by viable cell counts. The number of active molecules of
-galactosidase was calculated taking into consideration that the active form of the enzyme is a tetramer.
Alkaline phosphatase assay.
Whole-cell alkaline phosphatase assays were performed in 96-well microtitre dishes using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUP; Molecular Probes). The above procedure for the -galactosidase assay was modified for the use of MUP and the buffers used were as described by Brickman & Beckwith (1975)
. Cells were grown under the same conditions as described above and 50 µl of the prepared cells was used per assay. It was not necessary to permeabilize the cells prior to assaying. Shrimp alkaline phosphatase (Promega) was used to generate a standard curve ranging from 1 ng to 10 pg. Reactions were started with the addition of 100 nmol MUP substrate (100 µl 1 mM MUP in 1 M Tris/HCl buffer pH 8·0, at 25 °C). Reaction mixtures were incubated at 30 °C for 30 min and stopped by the addition of 50 µmol K2HPO4 (50 µl 1 M K2HPO4). Commercially available 7-hydroxy-4-methylcoumarin (Molecular Probes), the fluorescent product of the hydrolysis of MUP, was used as standard. Fluorescence was then measured using the high-throughput SpectraMAX GeminiEM spectrofluoremeter (Molecular Devices) with the excitation wavelength set at 360 nm and the emission wavelength set at 449 nm. The number of cells used per assay was determined by viable cell counts. The amount of active alkaline phosphatase per cell was determined taking into consideration that the active form of the enzyme is a dimer.
Cell fractionation of S. enterica
Procedure 1. Preparation of crude ribosomes.
Crude ribosomes were prepared as described by Spedding (1990) from cells of strain TR6583. Briefly, an S30 extract was obtained from cells disrupted using a French pressure cell (Aminco) and centrifuged at 30 000 g. The extract was then centrifuged at 100 000 g using a 70.1 Ti rotor in a Beckman L8-70 refrigerated ultracentrifuge (Beckman Coulter). The resulting pellet containing the ribosomes was brown and translucent. Both pellet and supernatant were assayed for cobalamin synthase activity.
Procedure 2. Preparation of salt-washed ribosomes.
A more stringent purification of ribosomes was accomplished by centrifuging the S30 extract described above through a cushion containing 1·1 M sucrose and 0·5 M NH4Cl, as described by Spedding (1990). The resulting pellet was clear. Both pellet and supernatant were assayed for Sdh and CobS activities. This procedure was also repeated without addition of NH4Cl to the sucrose cushion. The pellet obtained was similar to but smaller than that found in the crude preparation, and both supernatant and pellet were assayed for Sdh and CobS activities.
Procedure 3. Spheroplast formation and membrane fractionation by isopycnic density ultracentrifugation.
The procedure used to convert S. enterica cells to spheroplasts has been described (Osborn & Munson, 1974). Attempts to generate spheroplasts from cells grown on minimal NCE medium were unsuccessful. Although spheroplasts of strain TR6583 grown on LB medium were obtained, these cells did not contain quantifiable levels of CobS activity because of the low expression of the cob operon in rich medium (Escalante-Semerena & Roth, 1987
). Strain JE5896, which carried cobS on a multi-copy number plasmid (pNLA1), was used instead. It was found that low but detectable levels [0·01 U (mg protein)1] of CobS were expressed from plasmid pNLA1 in strains that did not carry a T7 polymerase despite the fact that transcription from plasmid pNLA1 was driven by a T7 promoter. This level of CobS activity was approximately sevenfold higher than that obtained by inducing the chromosomal cobUST genes. Spheroplasts of this strain were lysed by osmotic shock and membranes were isolated by centrifugation (144 000 g). Inner and outer membranes were separated by isopycnic ultracentrifugation as described by Osborn & Munson (1974)
. Fractions (250 µl each) were collected after piercing the bottom of the polyallomer centrifuge tube (Beckman) with a needle. The A280 was measured for every fraction using a quartz microtitre plate and SpectraMAX Plus spectrophotometer (Molecular Devices). Fractions were assayed for Sdh and CobS activities. Western blot analysis employing anti-CobS antibodies (see below) was also used to identify the cellular location of CobS. The fractionation procedure was performed twice.
Cell fractionation of M. thermoautotrophicum strain H.
Frozen cell pellets (3 g) were thawed and resuspended in 10 ml 50 mM MOPS/NaOH buffer pH 7 containing 10 mM MgCl2. Cells were disrupted using a French pressure cell, and cell debris was removed by centrifugation (10 000 g, 30 min). Cleared cell-free extract was subjected to ultracentrifugation at 125 000 g for 3 h. Pellets were resuspended in MOPS buffer and centrifuged again under the same conditions for an additional 2 h.
Generation of rabbit anti-CobS antibodies and Western blot analysis.
Polyclonal antibodies were raised against a synthetic 25 amino acid peptide of CobS (DTCDGIFSARRRERMLEIMRDSRLG) generated at the Peptide Synthesis Facility at the Biotechnology Center of the University of Wisconsin-Madison. Four peptides were conjugated to a branching lysine core molecule to form a multiple antigenic peptide (Posnett et al., 1988). Antibodies were elicited in a New Zealand White rabbit at the Animal Care Unit at the Medical School of the University of Wisconsin-Madison. Serum from the sixth bleed was pre-cleared (Suh, 1994
) using strain JE877 (Table 1
). Western blot analysis was performed as described previously (Rondon & Escalante-Semerena, 1997
), using a 1 : 10 000 dilution of CobS antibody.
Other methods.
Protein concentrations were determined as described by Kunitz (1952). Non-radioactive DNA sequencing of plasmid constructs was performed using the ABI PRISM BigDye terminator cycle sequencing kit v3.1 (PerkinElmer Life Sciences) according to the manufacturer's instructions. DNA sequences were determined at the Biotechnology Center at the University of Wisconsin-Madison.
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RESULTS |
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Residues exposed to the periplasmic space.
The model predicted that three loops of the CobS protein would be exposed to the periplasm. Amino acid residues 131 and 199 were chosen as fusion sites. Cells containing CobS199LacZ or CobS199PhoA fusion proteins had low levels of -galactosidase activity per cell but the number of active molecules of alkaline phosphatase indicated that these residues were exposed to the periplasm (Table 3
). Surprisingly, cells making CobS131LacZ fusions had 16-fold higher-than-background activities of
-galactosidase. This unexpected result was difficult to explain, especially in light of the strong activity level measured in cells making the corresponding CobS131PhoA fusion protein. The PhoA data for this location were consistent with the predicted periplasmic exposure of residue L131 to the periplasm.
Residues of CobS embedded in the membrane.
The low level of PhoA activity measured in cells making CobS55PhoA protein suggested that residue L55 was likely to be embedded in the membrane, but the PhoA protein fused to it could still reach the periplasmic space and become active. Alkaline phosphatase and -galactosidase activities in cells making CobS185 and CobS243LacZ/PhoA fusion proteins were consistent with these residues being embedded in the membrane. Residue L196 was also placed within the membrane. The high level of PhoA activity of the CobS196PhoA fusion protein was explained by the closeness of L196 to the periplasm. A similar argument was made to interpret the data obtained with CobS179LacZ protein. In the model shown in Fig. 5(b)
, residue R179 is located very close to the cytosol, but still within the membrane. Activity measurements of fusions to residues L74 and L157 were ambiguous, but their location within the membrane would be consistent with the model. Activity measurements with the CobS115 protein fusions suggested that residue L115 was embedded in the membrane but sufficiently close to the cytosol for LacZ to reach it and become active.
The CobS orthologue from the archaeon M. thermoautotrophicum strain H is also a membrane protein
As mentioned above, the cobS gene of the methanogenic archaeon M. thermoautotrophicum strain H compensated for the lack of CobS in S. enterica cobS strains during B12-dependent growth (Fig. 2
). This result suggested that the archaeal protein was localized to the membrane. To investigate this possibility, spheroplasts of strain JE6002 (S. enterica cobS/M. thermoautotrophicum pCOBS16 cobS+) were obtained. Total cell membranes of strain JE6002 were isolated following lysis by osmotic shock. This membrane preparation contained 3·5x103 U cobalamin synthase activity per mg protein when assayed at 50 °C. An S30 extract of strain JE6002 had a 10-fold lower specific activity (3·5x104 U per mg protein), indicating that the protein accumulated in the membrane. The activity of the M. thermoautotrophicum cobS orthologue was twofold lower when assayed at 37 °C, the temperature at which it complemented a S. enterica cobS mutant.
We used ultracentrifugation to fractionate M. thermoautotrophicum strain H cell-free extracts (Gärtner et al., 1993
; Heiden et al., 1994
). Ninety percent of the total cobalamin synthase activity was detected in the washed pellet, distributed as 64 % in the tight pellet and 26 % in the gelatinous pellet. These pellets are both considered to be part of the membrane fraction (Fischer et al., 1992
; Heiden et al., 1994
). The specific activities for these pellets were 2·5x104 and 3·8x104 U per mg protein, respectively, and were more than 10-fold higher than that found for the supernatant (2·2x105 U per mg protein).
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DISCUSSION |
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The topological analysis of CobS performed using protein fusions to alkaline phosphatase or -galactosidase as reporters was not unambiguous for all the fusions analysed; hence detailed knowledge of the association of CobS to the membrane is still incomplete. However, as a whole, the data provide good evidence to conclude that CobS is an integral membrane protein. The most intriguing question raised by these results is: why should coenzyme B12 synthesis be associated with the membrane? The CobS protein is not the only membrane-associated enzyme of the pathway. Interestingly, the cobinamide-phosphate synthase (CbiB) enzyme is also an integral membrane protein (C. L. Zayas & J. C. Escalante-Semerena, unpublished results). The CbiB enzyme catalyses a step of the pathway that is key to salvaging of cobyric acid from the environment. Whatever the reason for the association of these two enzymes with the membrane, it is not unique to Salmonella enterica. To date, all of the genome sequences of B12-producing prokaryotes that have CobS or CbiB are predicted to be very hydrophobic proteins, and hence likely to be associated with the cell membrane. Here we provided support for this idea by looking at the location of one CobS orthologue in the methanogenic archaeon M. thermoautotrophicum strain
H. Answers to the question raised above will provide insights into the strategy prokaryotes need to use to successfully synthesize this important coenzyme.
Is there a connection between membrane stress and high levels of CobS protein?
In many instances protein overproduction reveals stress responses that are consistent with physiological strategies aimed at maintaining some proteins at low levels in the wild-type strain. It is too early to tell why CobS cannot be overproduced. So far, it appears that CobS overproduction stresses the cell membrane, affecting its structure and/or function. This idea is indirectly supported by the overproduction of the membrane-stress indicator protein PspA that results from cobS overexpression. Physiological responses associated with membrane stress may be a mechanism used by S. enterica for maintaining cobamide biosynthesis at low levels.
Conflict with previously reported work
The localization of the CobS enzyme to the cell membrane of S. enterica conflicts with results from previous work in E. coli, in which cobalamin synthase activity was reported to be associated with the ribosomal L18 protein (Pezacka & Walerych, 1981). In considering the physiological relevance of the putative cobalamin synthase activity of the L18 protein one must bear in mind that: (i) the L18 protein failed to use
-ribazole and
-ribazole phosphate, the documented substrates of cobalamin synthase (Walerych et al., 1968
); (ii) the work on the L18 protein predated the identification of the cob genes in any prokaryote, and since the discovery of the cob genes, cobalamin synthase activity has been shown to be encoded by the cobS gene of S. enterica and the cobV gene of P. denitrificans (Cameron et al., 1991
; Maggio-Hall & Escalante-Semerena, 1999
); (iii) cobS mutant strains of S. enterica lack cobalamin synthase activity and are unable to convert AdoCbi to AdoCbl (Jeter et al., 1984
; O'Toole et al., 1993
). If the L18 protein had a physiologically relevant cobalamin synthase activity, no phenotype would be observed; (iv) in spite of the use of an extremely sensitive bioassay, cobalamin synthase activity was not detectable in cell-free extracts of strains carrying a deletion of the cobS gene (Maggio-Hall & Escalante-Semerena, 1999
); and (v) wild-type chromosomal levels of cobalamin synthase activity in S. enterica were three orders of magnitude higher than the activity reported for purified E. coli ribosomes (Pezacka & Walerych, 1981
).
Although the work reported here did not investigate the putative cobalamin synthase activity of the L18 protein in S. enterica, on the basis of previously reported genetic and biochemical evidence (Maggio-Hall & Escalante-Semerena, 1999; O'Toole et al., 1993
) and this work, it is concluded that the cobalamin synthase activity of the CobS protein is essential for AdoCbl synthesis, and is associated with the inner membrane in this bacterium, and probably in all corrinoid-producing prokaryotes.
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ACKNOWLEDGEMENTS |
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Received 1 December 2003;
revised 22 January 2004;
accepted 26 January 2004.
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