Portal de Periódicos da CAPES

Phosphotransferase-mediated Transport of the Osmolyte 2-O-α-Mannosyl-d-glycerate in Escherichia coli Occurs by the Product of the mngA (hrsA) Gene and Is Regulated by the mngR (farR) Gene Product Acting as Repressor

2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês

10.1074/jbc.m310980200

1083-351X

Maria Manuel Sampaio, Fabienne F. V. Chevance, Renate Dippel, Tanja Eppler, Anja Schlegel, Winfried Boos, Ying‐Jie Lu, Charles O. Rock,

Enzyme Structure and Function

2-O-α-Mannosyl-d-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100.UptakeismediatedbytheP-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 μm, and the Vmax was 0.65 nmol/min/109 cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with α-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system. 2-O-α-Mannosyl-d-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100.UptakeismediatedbytheP-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 μm, and the Vmax was 0.65 nmol/min/109 cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with α-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system. 2-O-α-Mannosyl-d-glycerate (MG) 1The abbreviations used are: MG2-O-α-mannosyl-d-glycerateIPTGisopropyl-β-d-thiogalactopyranosidePTSP-enolpyruvate-dependent phosphotransferase systemMMAminimal medium AX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside-Pphosphate. is a widespread compatible solute of thermophilic or hyperthermophilic bacteria and archaea (1Santos H. da Costa M.S. Methods Enzymol. 2001; 334: 302-315Crossref PubMed Scopus (59) Google Scholar) but not of mesophilic prokaryotes. MG has otherwise only been encountered in the red algae of the order Ceramiales (2Bouveng H. Lindberg B. Wickberg B. Acta Chem. Scand. 1955; 9: 807-809Crossref Google Scholar) where its function remains unclear. The highly preferential distribution of MG among prokaryotes requiring high temperatures for growth led to the hypothesis that it could play a role in the thermoprotection of cell components in vivo. At least in vitro,MG has been shown to be efficient in the protection of some enzymes against thermal inactivation (3Ramos A. Raven N.D.H. Sharp R.J. Bartolucci S. Rossi M. Cannio R. Lebbink J. Van der Oost J. de Vos W.M. Santos H. Appl. Environ. Microbiol. 1997; 63: 4020-4025Crossref PubMed Google Scholar, 4Borges N. Ramos A. Raven N.D.H. Sharp R.J. Santos H. Extremophiles. 2002; 6: 209-216Crossref PubMed Scopus (150) Google Scholar). Recently, we described a method to synthesize radiolabeled MG of high specific activity from radiolabeled mannose by the use of a genetically engineered strain of Escherichia coli (5Sampaio M.-M. Santos H. Boos W. J. Bacteriol. 2003; 69: 233-240Google Scholar). This prompted us to use this compound to study the potential uptake of MG in bacteria and archaea. Here, we report that carrier-mediated uptake of MG in E. coli occurs by the P-enolpyruvate-dependent phosphotransferase system (PTS) with the HrsA (now renamed MngA) protein as a specific EII complex substrate recognition site. So far, mngA has not been assigned to the uptake of a particular PTS sugar. However, the gene was recognized during a study of temperature regulation of the outer membrane porin OmpC (6Utsumi R. Horie T. Katoh A. Kaino Y. Tanabe H. Noda M. Biosci. Biotechnol. Biochem. 1996; 60: 309-315Crossref PubMed Scopus (10) Google Scholar). 2-O-α-mannosyl-d-glycerate isopropyl-β-d-thiogalactopyranoside P-enolpyruvate-dependent phosphotransferase system minimal medium A 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside phosphate. Another line of investigation that contributed to the present report is the regulation of fatty acid metabolism gene expression in E. coli. The FadR regulator has a dual function in repressing the enzymes of fatty acid β-oxidation and activating the transcription of the fabA and fabB genes of fatty acid biosynthesis (7Campbell J.W. Cronan Jr., J.E. J. Bacteriol. 2001; 183: 5982-5990Crossref PubMed Scopus (100) Google Scholar, 8Henry M.F. Cronan J.E. J. Mol. Biol. 1991; 222: 843-849Crossref PubMed Scopus (88) Google Scholar). FabR is a repressor that acts downstream of FadR in the transcriptional regulation of the fabA and fabB genes (9Zhang Y.M. Marrakchi H. Rock C.O. J. Biol. Chem. 2002; 277: 15558-15565Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The existence of a third fatty acid-responsive transcription regulator was proposed by Quail et al. (10Quail M.A. Dempsey C.E. Guest J.R. FEBS Lett. 1994; 356: 183-187Crossref PubMed Scopus (39) Google Scholar), who described a transcription factor adjacent to the citric acid gene cluster that was released from its DNA-binding site by either fatty acid or acyl-CoA. This gene was named farR (fatty acyl-responsive regulator). Here, we report that FarR (now renamed MngR) has no role in fatty acid metabolism but rather functions as a repressor for the adjacent and divergently oriented genes mngA and mngB. Transport of 14C-MG—Bacterial strains (Table I) were grown either in NZA medium (10 g of NZ amine (Sheffield Products Inc.) and 5 g of yeast extract/liter) or in minimal medium A (MMA) (11Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 354-432Google Scholar) with 0.4% glycerol as a carbon source in the presence or absence of 0.1% MG. After overnight growth the cultures were washed three times in MMA without carbon source and resuspended to an optical density at 578 nm of 1 corresponding to 7 × 108 cells/ml (11Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 354-432Google Scholar). [14C]2-O-α-Mannosyl-d-glycerate (296 mCi/mmol) (5Sampaio M.-M. Santos H. Boos W. J. Bacteriol. 2003; 69: 233-240Google Scholar), labeled uniformly in the mannose moiety, was added to a final concentration of 0.1 μm. 0.5-ml samples were withdrawn in a time span of 10 min and filtered, and their radioactivity was determined in a scintillation counter. The initial rate of uptake was extrapolated. The rate of transport in the standard assay (0.1 μm [14C]MG at an optical density of 1.0 of the bacterial culture resuspended in MMA) is given in pmol of MG/min/109 cells taken up at room temperature. To determine the Km and Vmax of uptake, the same concentration of 0.1 μm [14C]MG was mixed with different unlabeled MG concentrations prior to the assay, and the initial rate of uptake of 14C was determined.Table IBacterial strains (E. coli) and plasmids Strains other than E. coli were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany).Strain or plasmidKnown genotypeOrigin or referenceStrains BL21E. coli B F-, ompT hsdS (rB- mB-) gal dcmRef. 32Studier F.W. Moffat B.A. Mol. Biol. 1986; 189: 113-130Crossref Scopus (4842) Google Scholar DY330ΔlacU169 gal490(λ cl 857 Δ(cro-broA)Ref. 14Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed Scopus (1382) Google Scholar ET222MC4100 mngB::camThis study ET223MC4100 mngB::camThis study JA177MC4100 cdaR::Tn5Ref. 23Monterrubio R. Baldoma L. Obradors N. Aguilar J. Badia J. J. Bacteriol. 2000; 182: 2672-2674Crossref PubMed Scopus (33) Google Scholar KM522MC4100 malK-lacZ otsA::Tn10otsA::Tn10 allele obtained from A. Strøm (26Giæver H.M. Styrvold O.B. Kaasen I. Strøm A.R. J. Bacteriol. 1988; 170: 2841-2849Crossref PubMed Google Scholar) LYJ1PDJ1 ΔmngR::camThis study LYJ2PDJ1 Δ(mngR-mngB)::camThis study MC4100F-araD139 Δ(argF-lac)U169 deoC1 flbB5301 ptsF25 rbsR relA1 rpsL150Ref. 33Casadaban M.J. J. Mol. Biol. 1976; 104: 541-555Crossref PubMed Scopus (1305) Google Scholar MC4100 gudP-lacZMC4100 gudP-lacZ; needs tryptophan for growthRef. 23Monterrubio R. Baldoma L. Obradors N. Aguilar J. Badia J. J. Bacteriol. 2000; 182: 2672-2674Crossref PubMed Scopus (33) Google Scholar MC4100 garD-lacZMC4100 garD-lacZ; needs tryptophan for growthRef. 23Monterrubio R. Baldoma L. Obradors N. Aguilar J. Badia J. J. Bacteriol. 2000; 182: 2672-2674Crossref PubMed Scopus (33) Google Scholar MC4100 garP-lacZMC4100 garP-lacZ; needs tryptophan for growthRef. 23Monterrubio R. Baldoma L. Obradors N. Aguilar J. Badia J. J. Bacteriol. 2000; 182: 2672-2674Crossref PubMed Scopus (33) Google Scholar MG1655Sequenced E. coli wild type strainRef. 34Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V.M.R. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1462Crossref PubMed Scopus (6056) Google Scholar MS0112MC4100 ΔptsI::Tn5This study MS2112MC4100 Δcrr::Tn5This study PDJ1F-metB1 relA1 spoT1 gyrA216 recD::Tn10 λ- λRRef. 9Zhang Y.M. Marrakchi H. Rock C.O. J. Biol. Chem. 2002; 277: 15558-15565Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar RD25MC4100 ΔmngA::kanThis study RD26MC4100 ΔmngR::cam; allele from LYJ1This study XL1-BlueΔ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac(F′ proAB-lac1qZΔM15 Tn10(TetR) amy CamR)StratagenePlasmids and phage pACS31pTrc99b mngAThis study pACS32pTrc99b mngRThis study pTK18pUC18 mngR mngARef. 6Utsumi R. Horie T. Katoh A. Kaino Y. Tanabe H. Noda M. Biosci. Biotechnol. Biochem. 1996; 60: 309-315Crossref PubMed Scopus (10) Google Scholar pGS701pET16b harboring mngRRef. 10Quail M.A. Dempsey C.E. Guest J.R. FEBS Lett. 1994; 356: 183-187Crossref PubMed Scopus (39) Google Scholar pTrc99bAmprlacIqRef. 18Amann E. Ochs B. Abel K.-J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (881) Google Scholar pUC18AmprRef. 35Vieira J. Messing J. Gene (Amst.). 1982; 19: 259-268Crossref PubMed Scopus (3785) Google Scholar λNK1324Harboring mini cam casette and transposaseRef. 15Way J. Davis M. Morisato M. Roberts E. Kleckner N. Gene (Amst.). 1984; 32: 369-379Crossref PubMed Scopus (357) Google Scholar Open table in a new tab Construction of a mngR Deletion Strain—The strategy and outcome are outlined in Fig. 1A. The 934-bp fragment upstream of mngR was amplified by PCR with primers PS (5′-CGCGAGCTCCTCCTTGATGGCGACTTCAG, the SacI site is underlined) and PB (5′-CGCGGATCCTTAAGCTCGCCACGCGCAAT, the BamHI site is underlined). The SacI-BamHI-digested 934-bp fragment was cloned into the SacI-BamHI site of pUC19. The 809-bp fragment downstream of mngR was amplified similarly by PCR with primers PP (5′-CGCCTGCAGTTGAATATAGCCGCAACG, the PstI site is underlined) and PH (5′-CCCAAGCTTTCCATTCTGGAAGCCATC, the HindIII site is underlined). Then the PstI-HindIII-digested fragment was inserted into the PstI-HindIII site of the plasmid harboring the 934-bp upstream fragment. The BamHI and PstI double-digested chloramphenicol-resistant gene fragment was cloned into the BamHI-PstI site of the plasmid harboring both the upstream (934 bp) and downstream (809 bp) PCR fragments, yielding plasmid pUCMngRC. This plasmid was digested with AatII and Hin-dIII. The 3.0-kb fragment containing both PCR fragments, the chloramphenicol gene replacing mngR and a 350-bp fragment of pUC19, was purified from a 0.8% agarose gel. The 3.0-kb linear DNA (25 ng) was transformed into strain PDJ1 (recD::Tn10) (9Zhang Y.M. Marrakchi H. Rock C.O. J. Biol. Chem. 2002; 277: 15558-15565Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) by electroporation, and chloramphenicol-resistant transformants were isolated. The mngR::cam strain grew with the same doubling time as the wild type on both rich and minimal media. The location of the inserted element and the absence of the mngR gene were confirmed by PCR (Fig. 1B) and Southern blotting (Fig. 1C). PCR experiments were performed with the primer pair P1 (5′-CGGTTGCGTATCTTGCT GTA) and P2 (5′-CTCAGAACAGGCCATTGCAT) to amplify a fragment encompassing 1 kb both upstream and downstream of mngR. The PCR products from both wild type and ΔmngR strains were digested with enzymes ClaI, NcoI, and BamHI individually. Substitution of the chloramphenicol-resistant gene for mngR increased the size of the PCR product from P1 and P2 primers by 300 bp. Thus, the PCR product from the ΔmngR strain with P1 and P2 was 2.9 kb in length (Fig. 1B, lane 2) compared with the 2.6-kb product from wild type cells (Fig. 1B, lane 1). When these two PCR products were digested with three restriction enzymes (Fig. 1B, lanes 3 and 4, ClaI; lanes 5 and 6, NcoI; and lanes 7 and 8, BamHI), the products were of the predicted sizes (Fig. 1B). Southern blots were performed on genomic DNA isolated from both strain PDJ1 and strain LYJ1 (ΔmngR) by the phenol/chloroform/isoamyl alcohol extraction method (12Murray M.G. Thompson W.F. Nucleic Acids Res. 1980; 8: 4321-4325Crossref PubMed Scopus (9497) Google Scholar). Two different restriction enzymes, BglI and PvuI, were used to digest 5 μg of genomic DNA. The digested genomic DNA was separated using a 0.8% agarose gel. The probe was the 320-bp NruI-PvuII fragment downstream of mngR (Fig. 1A) and was labeled with [α-32P]dCTP. Southern transfer, hybridization, and washing were carried out by standard procedures. The bands were detected with a Molecular Dynamics Storm 860 imager, and their intensity was determined using the ImageQuant 5.1 program. The results from Southern blot analysis with the 320-bp NruI-PvuI fragment corroborated the PCR results, confirming that the chloramphenicol gene replaced the mngR gene in strain LYJ1 in the correct location without compromising the neighboring genes. The probe hybridized with a 2.9-kb fragment of wild type genomic DNA digested with BglI (Fig. 1C, lane 1), whereas the mutant had a 3.2-kb fragment (Fig. 1C, lane 2). The probe also recognized bands of the correct sizes in the PvuI digests (Fig. 1C). In the case of the ΔmngR strain, the fragments were larger by 650 bp because of the loss of the PvuI digestion site upon insertion of the chloramphenicol gene. The mngR deletion in LYJ1 was transduced into MC4100 by P1vir-mediated transduction selecting for chloramphenicol resistance yielding strain RD26. Construction of Strain LYJ2 Δ(mngR-mngB)—The construct was made using the same approach as used for the mngR knockout construct (Fig. 1A). The 846-bp fragment downstream of mngB was amplified by PCR with primers PS2 (5′-CGCGAGCTCATCGCATGAAGACTCCGAGA, the SacI site is underlined) and PB2 (5′-CGCGGATCCTTACCGGCTTGCCTGAATAG, the BamHI site is underlined). The PCR product was ligated to PCR 2.1 vector to generate PCRmngB and sequenced to be right in the Hartwell Center with ABI Prism 3700 DNA analyzer. This plasmid PCRmngB and plasmid pUCMngRC (mngR deletion construct) were double-digested by SacI and BamHI, and the released 846-bp fragment from PCRmngB was cloned into the SacI-BamHI site of pUCMngRC to yield a triple knockout construct (pUC-triC) that replaced mngR, mngA, and mngB with the chloramphenicol resistance gene. This plasmid was digested with AatII and HindIII. The 3.0-kb fragment containing both PCR fragments, the chloramphenicol gene replacing the three genes and a 350-bp fragment of pUC19, was purified from a 0.8% agarose gel. The linear DNA (25 ng) was transformed into strain PDJ1 (recD::Tn10) by electroporation, and chloramphenicol-resistant transformants were isolated. Two pairs of primers, pair P3 and PL and pair P4 and PR, were used to amplify the genomic region of the isolated strain and sent to sequence to confirm that mngR, mngA, and mngB were successfully deleted. Construction of Strain RD25 Δ(mngA)—We used the technique of Datsenko and Wanner (13Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). Plasmid pKD13 harboring a kanamycin-resistant cassette was used as template for creating a PCR product encompassing the kanamycin cassette equipped with the DNA sequence 3′ and 5′ adjacent to the mngA gene. Strain DY330 (14Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed Scopus (1382) Google Scholar) was transformed with this linear and purified DNA fragment selecting recombinants that were resistant against kanamycin. The successful deletion of mngA was verified by PCR using one primer initiating within the kanamycin cassette and the other from the neighboring mngB gene (Fig. 1A). The deletion was transduced by P1vir transduction (selection for kanamycin resistance) into MC4100, yielding strain RD25. Mini-transposon Mutagenesis—MC4100 (gudP-lacZ) cells were grown at 37 °C overnight in 10 ml of trypton broth (11Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 354-432Google Scholar) containing 0.2% maltose and 10 mm MgSO4. They were washed and resuspended in 1 ml of trypton broth. 100 μl of cells were incubated with phage λNK1324 (harboring a choramphenicol insertion together with the gene for the transposase) for 15 min at room temperature and for a further 15 min at 37 °C (15Way J. Davis M. Morisato M. Roberts E. Kleckner N. Gene (Amst.). 1984; 32: 369-379Crossref PubMed Scopus (357) Google Scholar). λNK1324 can only be multiplied in an amber suppressor strain (XL1 from Stratagene was used). The multiplicity of infection was kept between 0.1 and 1 phage/cell. The cells were washed twice in 5 ml of LB containing 50 mm sodium citrate and were allowed to grow for 1 h at 37 °C. 100-μl samples were plated on LB containing sodium acetate (20 mm), chloramphenicol (15 μg/ml), XGal (40 μg/ml), and MG (0.5 mm) to grow overnight at 39 °C. Approximately 10,000 colonies were screened for light blue color. After one time purification of 40 such colonies, they were tested for the ability to take up [14C]MG. Two were found to exhibit 3-4-fold reduced MG transport in comparison with the parent strain. The mutation was transduced into MC4100 selecting for chloramphenicol resistance yielding ET222 and ET223. Sequencing by Inverse PCR—Chromosomal DNA (1-2 μg) of the mutants was digested by Sau3a (2 units) (New England BioLabs), and DNA fragments were separated on a 1% agarose gel (SeaKem). DNA fragments between 1000 and 2000 base pairs were cut out of the agarose gel and extracted (Qiagen). The DNA concentration was determined by UV spectrophotometry. The fragments (diluted to a concentration of 0.3 μg/ml in ligation buffer) were circularized overnight by T4 ligase (1 unit) (New England BioLabs) at 16 °C. DNA was then treated by an equal volume of phenol/chloroform. The upper phase was removed, and the DNA was precipitated with 1/10 volume of 3 m sodium acetate and 2-fold volume of 100% ethanol and collected by centrifugation. After washing the DNA with 70% ethanol, PCR was performed with 0.2 μg of circularized DNA in the presence of 0.2 mm dNTPs and 5 pmol of each primer, ah1 (5′-GATTTTTACCAAAATCATTAGGGGATTCATC-3′) and ah2 (5′-CATTAAGTTAAGGTGGATACACATCTTG-3′) (16Ochman H. Gerber A.S. Hartl D.L. Genetics. 1988; 120: 621-623Crossref PubMed Google Scholar, 17Triglia T. Peterson M.G. Kemp D.J. Nucleic Acids Res. 1988; 16: 8186Crossref PubMed Scopus (725) Google Scholar). These primers are complementary to the 3′ and 5′ ends of the insertion and permit direct elongation into the adjacent chromosomal DNA. We used an initial denaturation step at 95 °C for 4 min followed by 35 cycles comprising denaturation at 95 °C for 30 s, primer annealing at 56 °C for 1.5 min, and extension by Taq polymerase (Eppendorf) at 72 °C for 2 min. The resulting PCR product was electrophoresed through a 1% agarose gel, cut out, purified (gel purification kit; Qiagen), and sequenced using the primers ah1 and ah2 (GATC Biotech AG) (Fig. 1A). Construction of pACS31 Harboring mngA under lac Promoter Control—Plasmid pACS31 harboring mngA under an IPTG-inducible promoter was cloned using pTK18 (6Utsumi R. Horie T. Katoh A. Kaino Y. Tanabe H. Noda M. Biosci. Biotechnol. Biochem. 1996; 60: 309-315Crossref PubMed Scopus (10) Google Scholar) as template for PCR. mngA was amplified with two flanking primers containing a BamHI cleavage site at the 5′ end (underlined) and a HindIII cleavage site (underlined) at the 3′ end. The first primer was 5′-AAAGGATCCATGGTATTGTTTTATCGGGC-3′, and the second primer was 5′-AAAAAGCTTTTATGGCATTACGCCATCA-3′. The PCR product was digested with BamHI and HindIII and ligated into pTrc99B (18Amann E. Ochs B. Abel K.-J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (881) Google Scholar) that had been opened with the same two restriction endonucleases. Construction of pACS32 Harboring mngR under lac Promoter Control—mngR was amplified by PCR using chromosomal DNA of MC4100 as template and flanking primers containing a BamHI cleavage site (underlined) at the 5′ end and a HindIII cleavage site (underlined) at the 3′ end. The first primer was 5′-AAAGGATCCATGGGACACAAGCCCTT-3′ and the second primer was 5′-AAAAAGCTTTTATCGCGATGATTTTCG-3′. The PCR product was digested with BamHI and Hin-dIII and ligated into pTrc99B that had been opened with the same restriction endonucleases. The resulting plasmid carrying mngR under an IPTG-inducible promotor was named pACS32. Microarray Analysis—An overnight culture of E. coli MC4100 grown in MMA plus 0.4% glycerol was diluted 1:100 into the same medium for the control and in MMA, 0.4% glycerol plus 0.1% MG for the induced culture condition. The cells were harvested at an optical density of 0.49 for the control and 0.53 for the MG medium (6.6 h after inoculation). Extraction of the RNA was conducted following the Gross lab method (www.microarrays.org/protocols.html). Further purification was performed using Qiagen columns (RNeasy Mini protocol for RNA clean up). The quality of the RNAs was assessed by visualization on an agarose gel and the purity by ratios of the absorbance at 260 and 280 nm in 10 mm Tris-HCl, pH 7; these values were found to be 2.1. Reverse transcription of the RNA preparation was conducted using random hexamers (Amersham Biosciences) as primers and Fluoroscript reverse transcriptase (Invitrogen). Reverse transcription of RNA (26.5 μg) from the control culture was labeled with cyanine-3 fluorescent dNTPs, whereas that from the MG culture was labeled with cyanine-5 fluorescent dNTPs. The reaction was performed at 50 °C for 90 min. The obtained cDNAs were purified using the fluoroscript kit purifying columns (Invitrogen). The quality of the cDNAs was assessed on agarose gels and quantified by absorbance at 260 nm. Equal quantities of labeled cDNA were mixed, and the solution was concentrated to 1-4 μl by freeze drying. Hybridization was performed on full E. coli K12 genome microarrays (Affymetrix). The concentrated solution of cDNAs was dissolved in 120 μl of hybridization buffer and heated for 3 min at 90 °C. The hybridization solution was placed on the chip using the MWG gene frame and incubated for 16 h at 42 °C under mild agitation and protected from light. The chip was washed by successive incubations at 30 °C, under mild agitation, in the three following washing solutions: washing buffer 1 (2× SSC buffer (SSC buffer: 0.15 m sodium chloride, 0.015 m sodium citrate, pH 7.0) plus 0.1% SDS); washing buffer 2 (1× SSC); and washing buffer 3 (0.1× SSC). Excess water was eliminated by centrifuging the microarray at 2000 rpm for 2 min. Detection of cyanine-3 and cyanine-5 fluorescence of the hybridized microarray was performed using the Affymetrix 428 Array scanner and processing of the data using the image analysis software (Imagene and Genesight software from Biodiscovery). Essentially the same protocol was used for the identifying the gene expression of the ΔmngR strain and to test the response of the cloned mngA gene on pACS31 in comparison with the vector plasmid (control culture). Strain MC4100 gudP-lacZ harboring plasmid pACS31 or the vector pTrc99B was grown on MMA 0.4% glycerol, 0.1 mm tryptophan, and 100 μg/ml ampicillin. β-Galactosidase Activity—The β-galactosidase activity in reporter fusions was tested according to Miller (11Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 354-432Google Scholar) in cells grown overnight in MMA and 0.2% glycerol as a carbon source in the presence or absence of 0.1% glycerate or 0.5 mm MG. Specific β-galactosidase activity is given in μmol of o-nitrophenyl-β-d-galactopyranoside/min/mg of protein hydrolyzed. The protein content was estimated by the optical density of the culture at 578 nm. According to Miller (11Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 354-432Google Scholar) an optical density of 1.4 corresponds to 150 μg/ml. α-Mannosidase Activity—The α-mannosidase activity assay was performed as described previously (19Li Y.T. J. Biol. Chem. 1967; 242: 5474-5480Abstract Full Text PDF PubMed Google Scholar). E. coli strains (PDJ1, LYJ1, and LYJ2) were grown overnight in M9 minimal medium (11Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 354-432Google Scholar) supplemented with 0.4% succinate, 0.01% methionine, and 0.0005% thiamin. Then 5 mmp-nitrophenyl-α-d-mannoside or 5 mmp-nitrophenyl-β-d-mannoside was added into each cell suspension, and incubation was continued for 3 h at 37 °C. The supernatant was collected by centrifugation at 10,000 × g for 10 min, and the adsorption at 405 nm was measured. In Vitro MG Phosphorylation—Strain MC4100 harboring plasmid pACS31 or no plasmid was grown overnight in LB supplemented with ampicillin (100 μg/ml). No IPTG was added for induction when growing MC4100/pACS31, allowing just basal expression of mngA. The cells were washed three times with 10 mm Tris-HCl, pH 7.6, plus 100 mm NaCl and broken in a French pressure cell at 16,000 p.s.i. The extract was shortly centrifuged to remove unbroken cells. The resulting supernatant was ultracentrifugated (90,000 × g for 60 min at 4 °C) to separate membranes from the crude extract. The membranes and crude extract were dialyzed separately overnight at 4 °C against 10 mm Tris-HCl, pH 7.6. The phosphorylation assay contained 12 μl of an equal mixture of buffer A (10 mm Tris-HCl, pH 7.6, 25 mm dithiothreitol, 125 mm NaF) and buffer B (10 mm Tris-HCl, pH 7.6, 10-100 mm P-enolpyruvate, 50 mm MgCl2), 15 μl of crude extract (30 μg protein), and 3 μl of membrane suspension (3-4 μg of protein). The reaction was started by the addition of 5 μl of [14C]MG (0.025 μCi; 296 mCi/mmol) (5Sampaio M.-M. Santos H. Boos W. J. Bacteriol. 2003; 69: 233-240Google Scholar). 10-μl samples were removed at different time intervals and spotted on a TLC (silica) plate. Chromatograms were developed with a solvent system composed of n-propanol/25% ammonia (1:1, v/v). TLC plates were autoradiographed for 2-3 days. The control assays were done with membranes from uninduced MC4100 not harboring mngA. When the dependence of P-enolpyruvate was measured, unlabeled MG (100 μm) was added to the complete assay mixture 10 min before the addition of [14C]MG. To ensure that the product formed was MG-P, in one assay before spotting the samples onto the TLC plate, the proteins were precipitated by adding 5 μl of 12% trichloroacetic acid to the 10-μl samples. The mixture was kept on ice for 15 min and centrifuged for 2 min at 20,000 × g. The acidic supernatant was neutralized by adding concentrated KOH. After reaching neutrality, 1 unit of alkaline phosphatase was added, and the samples were incubated for 30 min at 37 °C. After the treatment, the samples were spotted on the TLC plate, and the chromatograms were developed using the same solvent system. Growth Curves—Strain KM522 is defective in the synthesis of internal trehalose and is therefore highly sensitive to salt. It was grown in MMA and 0.4% glycerol as carbon source. It was diluted 1:100 into fresh medium, one with and one without 0.5 mm MG. After 6 h the cultures were split, and 500 mm salt was added to one portion. The optical density at 600 nm was followed. Transport of MG in E. coli K12 Strain MC4100 —We teste