Department of Microbiology, Ruhr-Universität Bochum, NDEF-06, D-44780 Bochum, Germany1
Author for correspondence: Wolfgang Hengstenberg. Tel: +49 234 7004247. Fax: +49 234 709 4620. e-mail: wolfgang.hengstenberg{at}ruhr-uni-bochum.de
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ABSTRACT |
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Keywords: glucose-specific phosphotransferase system, Staphylococcus carnosus, membrane protein, regulation, kinetics
Abbreviations: CRE, catabolite-responsive element; NTA, nitrilotriacetic acid; PEP, phosphoenolpyruvate; PTS, phosphotransferase system
a Present address: Department of Microbiology, Biozentrum, University of Basel, Basel, Switzerland.
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INTRODUCTION |
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Recently, we reported the cloning and sequencing of two EII-coding genes, glcA and glcB, from Staphylococcus carnosus and their expression in E. coli (Christiansen & Hengstenberg, 1996 ). Both genes showed high similarities to members of the glucose-specific subfamily of PTS permeases. Despite their close proximity, sequence analysis indicated an independent individual regulation of gene expression for glcA and glcB.
The three EII domains are fused in both permeases (EIICBA). Each protein is physiologically active in E. coli WA2127ptsG::Cmr and was shown to restore glucose fermentation in the E. coli mutant. Since EII permeases show overlapping specificity, we addressed the question whether these two glucose transporters have different substrates. To study the regulation of glucose uptake and metabolism in the Gram-positive bacterium S. carnosus, an important organism in food production, a further characterization of the two highly similar permeases is necessary. Here, we describe the purification of the two proteins EIICBAGlc1 and EIICBAGlc2 to apparent homogeneity, and the characterization of their substrate specificities in an in vitro fluorimetric assay. Additionally, the potential to initiate transcription of the two promoters preceding glcA was analysed by deletion studies.
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METHODS |
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Bacterial strains, plasmids and growth conditions.
E. coli TG1 (Sambrook et al., 1989 ) was used for cloning experiments. Expression of the EIICBAGlc proteins was performed in E. coli WA2127
ptsG::Cmr (
ptsG ptsLPM Cmr his leu met lac supE±
) (Buhr et al., 1994
), obtained from B. Erni, Bern. S. carnosus TM300 (Schleifer & Fischer, 1982
) was obtained from F. Götz, Tübingen. pUC18/19 (Vieira & Messing, 1985
) were used for cloning experiments. LB medium and growth conditions were as described previously (Christiansen & Hengstenberg, 1996
). Fusion of histidine hexapeptide to the gene products was achieved by using the pQE vectors from Qiagen.
Preparation of cell membranes.
Cells of E. coli WA2127ptsG::Cmr were grown overnight in 2 l LB medium and harvested by centrifugation. The cell paste (approx. 10 g) was resuspended in 2 vols standard buffer Sb (0·05 M Tris/HCl, pH 7·5, 10-4 M DTT, 10-4 M PMSF, 10-4 M NaN3, 10-4 M EDTA). The cells were disrupted by sonication with a Branson sonifier B12. The crude extract was centrifuged for 15 min at 10000 g and 4 °C. Membranes were collected at 170000 g for 4 h (4 °C), resuspended in 20 vols Sb and sedimented under the same conditions to remove remaining cytoplasmic contaminants. The washed membrane fractions were suspended in the required volume of Sb.
In vitro EIIGlc activity test and inhibitor kinetics.
The PTS-dependent phosphorylation of glucose was measured by a coupled reaction of glucose-6-phosphate dehydrogenase in 250 µl samples at 37 °C, containing 6 mM MgCl2, 0·5 mM NADP+, 1 mM PEP, 5 µg EI from S. carnosus (80 pmol), 5 µg HPr from S. carnosus (530 pmol), glucose-6-phosphate dehydrogenase (0·25 units), 10 µl membrane resuspension (1 mg membrane fraction is resuspended in 10 µl Sb), and varying amounts of glucose (3·125100 nmol). The reaction was started by adding HPr. Increments of NADPH concentration were detected in an Eppendorf fluorimeter 1030 (primary filter 313366 nm, secondary filter 4003000 nm).
The specificity of the EIIGlc was tested by inhibition of the reaction by potential substrate analogues. A negative control was performed by adding the inhibitors as substrates.
The reaction was performed using a glucose concentration five times higher than the calculated Km value, and a 10-, 100- or 1000-fold excess of the potential inhibitor was added. In the case of an inhibition, glucose-specific MichaelisMenten kinetics were repeated in the presence of different concentrations of inhibitor (1-, 2-, 5- and 100-fold the Ki value) and the inhibition constants were calculated by nonlinear curve-fitting. In the case of a quenching effect by the inhibitor on the detection of fluorescence (o- and p-nitrophenyl glucosides), the concentration-dependent factor was determined and taken into account.
Construction of plasmids.
For expression of His-tagged EIIGlc1 and EIIGlc2, the corresponding genes were cloned into the BamHI restriction site of pQE, upstream of the histidine-hexapeptide coding region. Therefore, PCR was performed using the pUC universal primer and the oligonucleotides 3'-ATCAATTTAACCTAGGGGTA-5' (glcA) or 3'-CCACTTATTACCTAGGACAA-5' (glcB) (annealing temp. 42 °C) to introduce a BamHI restriction site (underlined bases) with concomitant removal of the termination codons of the genes. A 192 bp EcoRVBamHI 3' fragment (glcA) and a 157 bp HincIIBamHI 3' fragment (glcB) of the corresponding PCR products were isolated, sequenced and used to substitute the corresponding sequence in glcA and glcB, respectively. The modified genes were ligated into pQE plasmids, so the gene products were extended by the amino acids LDRS(H)6 (EIIGlc1) or GS(H)6 (EIIGlc2), respectively.
Two potential promoters P0 and P1 are located upstream of glcA. P1 is flanked by the two restriction sites HinfI and MnlI, which facilitated its removal (glcA-P1). MnlI was used to delete both promoters (glcA-
P01), whereas P0 was deleted by unidirectional deletion of DNA by exonuclease III and mung bean nuclease (glcA-
P0).
Purification of EIICBAGlc1 and EIICBAGlc2.
Both EIIGlc permeases were purified by using the Ni2+-NTA metal chelate system according to the procedure of the manufacturer. To improve the expression of the fusion proteins, the coding sequences were recloned into pUC18.
Alkaline wash of membranes.
Membrane fractions (1 g) of E. coli WA2127ptsG::Cmr, expressing the desired gene product of glcA or glcB, were resuspended in 10 vols alkaline buffer [0·05 M Tris/HCl, pH 8·0, 10-4 M PMSF, 10% (v/v) glycerol, 10 mM glucose, 5 mM 2-mercaptoethanol]. The pH was adjusted to 12 (EIIGlc1-His) and 11·5 (EIIGlc2-His), respectively. After incubation for 15 min at room temperature, the pellet was harvested by centrifugation at 170000 g, 4 °C, for 3·5 h.
Solubilization of membrane proteins.
The membrane fraction after the alkaline wash (approx. 300 µg) was resuspended in 10 vols solubilization buffer (0·05 M Tris/HCl, pH 7·5, 10-4 M PMSF, 10-4 M NaN3, 5 mM 2-mercaptoethanol). Triton X-100 (final concn 1%, w/v) and NaCl (final concn 100 mM) were added and the proteins solubilized for 1 h with continuous stirring at 4 °C. After centrifugation at 170000 g for 1 h, the supernatant was suitable for Ni2+-NTA affinity chromatography.
Ni2+-NTA affinity chromatography.
The supernatant containing the solubilized proteins was applied to a column containing 0·5 ml Ni2+-NTA suspension, equilibrated with 20 vols solubilization buffer containing 1% Triton X-100. After washing with 20 vols solubilization buffer +0·8% Triton X-100 and 6 vols solubilization buffer +0·8% Triton X-100 and 25 mM imidazole, EIIGlc proteins were eluted with 4 vols elution buffer (solubilization buffer +0·8% Triton X-100, 100 mM imidazole).
Ion-exchange chromatography.
In case of residual contaminating proteins, an ion-exchange chromatography step was included. The eluted fractions of the Ni2+-NTA column were supplied to a column containing 1 ml Fractogel EMD DMAE 650 (S), equilibrated with 10 vols solubilization buffer +0·8% Triton X-100. After washing with 10 vols of the same buffer, proteins were eluted with a linear gradient of NaCl (00·4 M, 5 ml).
PAGE.
SDS-polyacrylamide gels were prepared according to Laemmli (1970) . Protein concentration was determined according to the method of Peterson (1977)
.
Blotting to PVDF membranes.
Protein transfer from SDS-PAGE gels to PVDF membranes was carried out in a Biometra Semi-dry Fast Blot. The transfer buffer contained 0·09 M Tris/borate, pH 8·0, 1 mM EDTA, 20% methanol, 0·05% SDS. Gels were stained in 50% methanol, 0·1% Coomassie R250, and destained in 50% methanol.
N-terminal sequencing of proteins.
Purified proteins EIICBA1Glc-His and EIICBA2Glc-His (approx. 20 µg) were blotted to PVDF membranes and, after staining, protein-containing membrane slices were cut out and sequenced on a gas-phase sequencer according to Hewick et al. (1981) .
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RESULTS AND DISCUSSION |
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To enable a fusion of the histidine hexapeptide coding region of the pQE plasmids to the 3' end of glcA or glcB, the stop codon was removed and the genes were cloned into pQE as described in Methods. After expression using the pQE system, both proteins (EIIGlc1-His and EIIGlc2-His) complemented the ptsG deletion mutant E. coli WA2127ptsG::Cmr. Membrane fractions were collected and shown to phosphorylate glucose in the PEP-dependent reaction of the in vitro assay, although 15 times more slowly than after expression via the pUC system (see Fig. 2
; data shown for pQE17-glcA.)
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Alkaline washing of the isolated membranes at pH 12·0/pH 11·5 retained 15·5%/17·4% of the proteins and 74%/78% of the EIIGlc activity in the case of EIIGlc1-His/EIIGlc2-His. Approximately 55% of the membrane-bound EIIGlc activity could then be solubilized with Triton X-100. Varying amounts of Triton X-100 and NaCl did not improve the yield of solubilized activity, whereas other detergents were even less efficient. After adsorption to Ni2+-NTA and washing with buffer containing 25 mM imidazole, 5060% of the total activity could be eluted with 100 mM imidazole. Residual contamination with proteins of a lower molecular mass (Fig. 1b, lane 5) was eliminated by ion-exchange chromatography on Fractogel EMD DEAE 650 (S). The yield of this additional purification step was 95%. The data are summarized in Table 1
; Fig. 1
shows the purity reached in each of the chromatography steps. The identity of the purified proteins was verified by 10 cycles of Edman degradation.
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That similar proteins show either an increased or decreased electrophoretic mobility after heating prior to SDS-PAGE has been shown for modified variants of the OmpA protein. Whereas the wild-type protein has a lower mobility, the R236V variant lacks modifiability and the membrane-spanning N-terminus (OmpA171) has an increased mobility after heat treatment (Ried et al., 1994 ). In contrast, a circular permutation (2341) of the OmpA N-terminus has a decreased mobility under the same conditions (Koebnik & Krämer, 1995
).
With the purification method described, we were able to isolate 200 µg (EIIGlc1) or 160 µg (EIIGlc2) purified protein from 10 g wet cell paste. A large-scale purification would provide enough purified proteins for production of antibodies and for crystallization of the proteins to study expression, structure and function. Crystallization seems to be more likely for EIICBA fusion proteins compared to EIICB molecules because of the higher proportion of hydrophilic amino acids. Structure analysis would give insight into function of the widely distributed bacterial PTS transporters which are also involved in bacterial signal transduction.
In vitro test of EIICBAGlc activity and kinetics of inhibitors
After expression of pUC18-glcA and pUC18-glcB in E. coli WA2127ptsG::Cmr, isolated membranes had, respectively, a 45-fold and 15-fold increased rate of phosphorylation of glucose compared to equal amounts of cell membranes from S. carnosus (Fig. 2
). The Km values of the glucose phosphorylation were 12 µM and 19 µM for cell membranes after expression of pUC18-glcA and pUC18-glcB, respectively, similar to the Km value obtained for the same reaction performed by use of membrane fractions isolated from S. carnosus (approx. 5 µM; data not shown). The addition of the His-tag did not alter the kinetic properties. However, incubation with Triton X-100 led to slightly increased Km values of PEP-dependent glucose phosphorylation by the products of glcA and glcB, whether membrane-bound or purified. Closer analysis of the Triton X-100-dependent kinetics of the EII proteins showed that glucose phosphorylation at lower substrate concentration is identical, but in the presence of Triton X-100, the activity at higher glucose concentrations, and thus the Vmax value, increases up to 120% (Fig. 2
), resulting in an increased Km value. One possible reason for this may be the existence until solubilization of closed membrane vesicles, which may be rate-limiting in cellular membrane suspensions (Lengsfeld et al., 1973
), thus slightly changing the apparent kinetic properties of the EII proteins.
For determination of inhibition constants, the influence of different carbohydrates on the glucose-phosphorylation reaction by purified EII proteins was investigated. In the case of an inhibition, the kinetics of the glucose phosphorylation were measured in the presence of different inhibitor concentrations, and the Ki values were calculated by nonlinear curve-fitting. Table 2 summarizes these experiments. The monosaccharides galactose, fructose and mannose, as well as the tested disaccharides cellobiose, sucrose, maltose, lactose, melibiose and trehalose, and also N-acetylglucosamine did not show any inhibition of glucose phosphorylation by either EIIGlc, and thus seem not to be substrates for either glucose transporter.
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pH-stat measurements showed that the carbohydrate analogues tested do not result in an acidification of the environment when supplied to S. carnosus cells in an unbuffered medium (data not shown). Thus, they are not likely to be metabolizable by S. carnosus or even to be natural substrates.
Sequence analysis of EIICBAGlc1 and EIICBAGlc2
By mutagenesis of glucose-specific IIC domains, several amino acids have been suggested to play a role in binding or translocation of the substrate (Buhr et al., 1992 ; Ruijter et al., 1992
; Begley et al., 1996
). Alignment of the two EIIGlc from S. carnosus with all known glucose permeases EIICB(A) shows that M17, G149, R203, I296 and D343 are conserved in all the sequences, adding evidence to the above suggestion (the numbers are taken from the E. coli sequence; Fig. 3). In position 206 the amino acids V, I, L and G occur, indicating importance of hydrophobicity.
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Further investigation using site-directed mutagenesis or construction of genes encoding chimeric transporters could lead to the identification of regions containing the binding sites. Identification of stacking aromates or changing substrate specifity by site-directed mutagenesis have been shown for other sugar-binding proteins (Iobst & Drickamer, 1994 ; Strokopytov et al., 1994
).
Sequence analysis of the glc region and deletion of the promoters P0 and P1
While glcB is preceded by a single putative promoter, two putative promoters, P0 and P1, are located upstream of glcA. Together with the intergenic putative termination loop, this indicates independent expression of glcA and glcB.
To prove the ability to initiate transcription of glcA, the two upstream promoters P0 and P1 were deleted as described above. After expression of the constructs in E. coli WA2127ptsG::Cmr, cell membranes showed impaired in vitro glucose phosphorylation compared to glcA (glcA-
P0, 25%; glcA-
P1, 63%; glcA-
P01, 5%, Fig. 2
). The residual activity of glcA-
P01 can be explained by transcription products initiated at the upstream bla promoter of the pUC18 vector combined with its high copy number. A control plasmid lacking both promoters, the ribosome-binding site and the codons of the first 38 amino acids of EIIGlc1 caused no detectable glucose phosphorylation (data not shown).
Thus, both promoters, P0 and P1, are able to initiate the transcription of glcA in the heterologous host E. coli. P0 shows a higher activity (63%) than P1 (25%). We expect both putative promoter regions to be involved in regulation of EIIGlc1 expression in S. carnosus, in which during growth on glucose the EIIGlc activity of membrane fractions increases about five- to sevenfold (data not shown). Although catabolite repression via CRE elements and RAT specific antitermination is not probable in the heterologous host E. coli, it cannot be ruled out that these elements influence the residual promoter activities. CRE1 is missing in all three constructs P0,
P1 and
P01; the putative RAT sequence is only absent in
P01. To confirm the role of P0 and P1, as well as the role of the existence of two EIIGlc coding genes in regulation of glucose uptake in S. carnosus, further work should focus on genetical analysis in the homologous organism, e.g. disruption of genes or regulative elements, following the expression pattern using reporter genes.
Catabolite repression in Gram-positive bacteria is mediated by HPr after ATP-dependent phosphorylation of HPr on a seryl residue. Recently, the genes of the HPr kinase hprK and the phosphatase hprP have been identified in E. coli (Galinier et al., 1998 ; Reizer et al., 1998
). A complex of CcpA, HPr-Ser~P and, at least in some cases, fructose 1,6-bisphosphate binds to the CRE which is found near the transcription start of many catabolic genes, resulting in repression of their expression (Hueck et al., 1994
).
Sequence analysis revealed the existance of three CRE-like elements in the region of glcA and glcB (Fig. 4), suggesting that binding of a CcpA/HPr-Ser~P to these regions leads to an additional control of the PTS. One possibility is a negative regulation to protect cells from a toxic effect of accumulated sugar phosphates.
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Recently, a regulation of the ptsG gene in B. subtilis via antitermination was described following the presented mechanism (Stülke et al., 1997 ). In S. carnosus a stemloop (TAACTAATTCGATTAGGCATGAGTGA) with 85% identity to the glucose-specific RAT sequence of B. subtilis is located between the putative glcT gene and glcA, followed by a putative weak termination loop (AAGTTTGGAGCAATCCAACTTTTTT).
In conclusion, we have cloned a genomic region of S. carnosus encoding two very similar PTS permeases with the common substrate glucose but a clearly distinguishable substrate specifity towards glucosides. The identified and putative regulatory elements suggest a complex regulatory mechanism of glucose metabolism involving differential gene expression, transcriptional regulation, catabolite repression and antitermination.
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ACKNOWLEDGEMENTS |
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Received 22 January 1999;
revised 24 May 1999;
accepted 7 June 1999.