Apr 5, 2018 - A L-rhamnose transport-negative strain of Esche- richia coli was generated by Mu d(ApR,lac)I mutagen- esis. This strain was used to isolate a ...
Vol. 267, No. 10, Issue of April 5, pp. 69234932,1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping, Cloning, Expression, and Sequencing of the rhaT Gene, Which Encodes a Novel L-Rhamnose-H+ TransportProtein in Salmonella typhimurium and Escherichia coli* (Received for publication, November 18, 1991)
Christopher G. Tate, Jennifer A. R. MuiryS, andPeter J. F. Henderson From the Department of Bwchemistv, Universityof Cambridge, Tennis Court Road, Cambridge CB2 lQW, United Kingdom
A L-rhamnose transport-negative strain ofEschetranscription of four defined promoters in the rha operon richia coli was generated byMu d(ApR,lac)I mutagen- (Tobin and Schleif, 1990a); under the same conditions RhaR esis. This strain was used to isolate a clone of Salmo- bound to an inverted repeat upstream from the rhaS gene nella typhimurium DNA that encoded L-rhamnose-H+ (Tobin and Schleif, 199Ob). However, RhaS stimulates trantransport activity, the gene for which, rhaT, was se- scription of rha promoters only weakly and its physiological quenced. The rhaT gene was mapped on the E. coli role is uncertain. sodA at 87.9 min, inichromosome between rhaR and The gene encoding L-rhamnose-H+ symport activity, rhuT, tially by Southern blotanalysis and then by the isola- was located by deletion mapping of the rha locus in S. typhition, expression, and sequencing of rhaTgene. the Both murium. It was found to be situated adjacent to therhaC gene 344 (Al-Zarban et al., 1984) as follows. rhaT genesencodedahydrophobicproteinof amino acids(91%identical) that contained 10 putative transmembrane regions. The RhaT protein represents rhaD rhaA rhaB rhaC rhaT a novel class of sugar transport protein. The RhaT transportsystem can transport L-mannose and Llyxose in addition to L-rhamnose, but atreduced rates (Muiry, 1989; Badia et al., 1991). It seems that RhaT catalyzes the L-Rhamnose (6-deoxy-~-mannose) canbe utilized as sole uptake of L-rhamnose with the influx of protons i.e. it is a Lcarbon and energy source by many groups of microorganisms, rhamnose-H+ symporter (Muiry, 1989). Steady state kinetic which is a reflection on the relative abundance of L-rhamnose analysis of 14C-labeledL-rhamnose transport gave apparent in the environment (Cheshire, 1979). The metabolism of L- K,,, values between 16 and 43 p M and apparent V,,, values rhamnose hasbeen extensively studied in Escherichia coli and between 11 and 17 nmol/min/mg dry mass (Muiry, 1989). Salmonella typhimurium. L-Rhamnose enters the cell by a The kinetic data yielded only one set of kinetic constants, transport system (RhaT) andis then converted to rhamnulose which suggested that there is only a single L-rhamnose transby L-rhamnose isomerase (RhaA; Wilson and Ajl, 1957a; port system in E. coli. Apart from L-mannose and L-lyxose, Takagi and Sawada, 1964a). Rhamnulose is metabolized via no other L-sugar, nor any D-sugar, tested were inhibitors of rhamnulose-l-phosphate to dihydroxyacetone phosphate and L-rhamnose uptake. Thus, for a sugar to be transported by L-lactaldehyde by the sequential action of rhamnulokinase RhaT, the configuration at C2, C3, C4, and C5 of the sugar (RhaB; Wilson and Ajl, 1957b; Chiu and Feingold, 1964; must be identical to L-rhamnose. Unlike most sugar/cation Takagi and Sawada, 1964b) and rhamnulose-l-phosphate al- transport proteins, RhaT is not inhibited by N-ethylmaleimdolase (RhaD; Takagi andSawada, 1964c; Chiu and Feingold, ide, neither is it inhibitedby cytochalasin B nor by forskolin,’ 1965). The genes encoding these proteins aregrouped at 87.7 both of which are inhibitors of other sugar-H+ symporters min on the E. coli chromosome in thefollowing order (Power, (reviewed by Henderson, 1990). 1967; Bachmann, 1983; Badia et al., 1989). This paper describes the isolation, characterization, and sequencing of rhaT clones from S. typhimurium and E . coli. rhaD rhnA rhaB rhaC An E. coli strain was constructed that was defective in LThe rhaC locus of E. coli was sequenced (Tobin andSchleif, rhamnose uptake by Mu d(ApR,lac)Imutagenesis; this strain 1987) and found to encode two proteins RhaS and RhaR. was used to screen a S. typhimurium C5 cosmid library by Genetical experiments originally identified the rhuC gene as complementation. A single plasmid that complemented the a positive regulator of transcription of the rha genes (Power, lesion in the rhaT gene was obtained. A fragment of DNA 1967). In the presence of L-rhamnose RhaR stimulated the that expressed a protein with L-rhamnose transport activity was subsequently isolated and sequenced. This clone was used * This study was supported by Grant GRF/17759 from the Science to map the position of the rhaT gene between rhuR and s o d and Engineering Research Council and by a grantfrom the Wellcome at 87.9 min on the E. coli chromosome by the isolation, Trust for equipment and technical assistance for oligonucleotide expression, and sequencing of the E. coli rhaT gene.
synthesis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBLData Bankwith accession number($ M85157 and M85158. $ Recipient of a studentship from the Science and Engineering Council. Present address: Stratagene Ltd., 140 Science Park, Cambridge CB4 4GF, United Kingdom.
EXPERIMENTAL PROCEDURES
Genetical Techniques-The E. coli strains used are listed in Table I. Mu d(ApR,lac)Imutagenesis was performed as described by Casadaban and Cohen (1979). Samples of the infection mixture were spread onto minimal media plates (Miller, 1972) containing 10 mM S. A. Bradley, J. A. R. Muiry, G. E. Martin, and P. J. F. Henderson, unpublished observations.
6923
6924
T h e rhaT genes of S. typhimurium and E. coli
L-rhamnose, 5 mM lactose, 80 pg/ml histidine, 100 pg/ml ampicillin t o select for insertion of the Mu phage into the rhu operon. PI transductions were carried out as described by Miller (1972). The E. coli strain used for the preparation of P1 phage to produce an fdp+ strain was CSH25, and strain PB13 was used to generate a recAstrain. @-GalactosidaseAssays-Quantitative &galactosidase assays were performed as described by Miller (1972); the method of Davis et al. (1984) was followed when performing plate assays for @-galactosidase. Screening a Cosmid Library-The cosmid library was made by ligating a Sau3A partial digest of S. typhimurium C5 DNA into a BamHI-digested plasmid pHC79 (Hohn andCollins, 1980) and was a kind gift from Dr. C. Hormaeche (Department of Pathology, University of Cambridge). The cosmid library was amplified using standard protocols (Maniatis et al., 1982). DNA Sequencing-The S. typhimurium rhuT gene was sequenced by generating partial restriction digests of the 3-kb2 EcoRV/PuuII fragment from plasmid pJAR6 with either AluI, HaeIII, or Sau3A. In addition, anumber of specific fragments were generated by digestion with combinations of HincII, PuuII, EcoRV, and BglII. All the DNA fragments were ligated into bacteriophage M13mp18 and sequenced by the dideoxynucleotide chain termination method (Sanger et al., 1980). In addition, two oligonucleotides were synthesized to the rhuT gene sequence and were used as primers to obtain complete coverage of the DNA sequence. Single-stranded DNA was produced (Messing, 1983)and sequenced from universal primers using Sequenase (United States Biochemicals Ltd.). The E. coli rhaT gene was sequenced by cloning the 1.6-kb SmaI/ XmnI fragment from plasmid pCGT6 into EcoRV-digestedBluescript plasmid (Stratagene), to make plasmid pCGT12. The sequencing strategy was to make unidirectional deletions in the fragment by using exonuclease 111 (Nested Deletion Kit, Pharmacia) followed by double-stranded sequencing of the product. To make deletions in the XrnnIISrnaI fragment, to be sequenced with the M13 universal primer, plasmid pCGT12 was digested with BarnHI and Sac1 prior to treatment with exonuclease 111. Deletions were made in the opposite direction, to be sequenced using the M13 reverse primer, by digesting plasmid pCGT12 with Hind111 and KpnI followed by digestion with exonuclease 111. Preparations of double strandedplasmid DNA (Johnson, 1990) weresequenced using Sequenase (United States Biochemicals Ltd.). The double-stranded DNA sequencing protocol was exactly as described in the Sequenase protocol manual except that NaOH was removed after the denaturation step using a spun column (0.3 ml of Sepharose CL-GB 200in a 0.5-ml microcentrifuge tube with a small hole in the bottom) rather than by precipitation. Additional DNA sequence was obtained from two oligonucleotide primers synthesized to the E. coli rhuT DNA sequence; oligonucleotides were synthesized on a Milligen “Cyclone” machine operated by M. Wheldon, Biochemistry Department, Cambridge University. Electrophoresis of sequencing gels was performed on a Pharmacia 2010 Macrophor sequencing apparatus at a constant voltage of 1200 V. DNA sequence data was analyzed using the Staden DNA sequencing programs (Bishop and Rawlings, 1987) processed by a DEC Micro Vax 3100 operated by the University of Cambridge Departments of Biological sciences. DNA Manipulations-Cosmid and plasmid DNA was prepared by the alkaline lysis method (Maniatis et al., 1982). Restriction digests were carried out according to the Manufacturers’ recommendations (Amersham, Pharmacia, or New England Biolabs), and ligations were performed according to Maniatis et al. (1982). The CaC12method for preparing competent cells was routinely used (Maniatis et al., 1982). Restriction fragments were isolated from agarose gels either by electrophoresis onto WhatmanDE81 ion exchange paper (Dretzen etal., 1981) or by excising the desired band and isolating the DNA by the “glass milk” method (Vogelstein and Gillespie, 1979). Transport Assays-Transport of “C-labeled sugar and pH measurements of sugar-H+symport activities were carried out asdescribed elsewhere (Henderson et al., 1977;Henderson and Macpherson, 1986). Initial rates of sugar uptake were determined from the 15-s time point. Southern Blots-Restriction enzyme-digested E. coli DNAwas transferred by capillary blotting (Southern, 1975) onto Hybond N membranes (Amersham). A 400-bp HincIIIBalI restriction fragment (STRHAl) from the S. typhimurium rhaT gene was labeled with 32P using the random priming method (Feinberg and Vogelstein, 1983, 1984). Southern blots were probed with restriction fragment STRHAl The abbreviations used are: kb, kilobase pair(s); bp, base pair(s).
(Maniatis et al., 1982) and washed at high stringency in 0.3 X SSC (20 X ssc: 3 M NaCl, 0.3 M tri-sodium citrate, pH 7) at 51 “C. Restriction digests of h clone 4B6 (Kohara et al., 1987)were probed with a gel-purified 32P-labeledoligonucleotide (Maniatis et al., 1982) and with the 400-bp HincIIIBalI restriction fragment STRHAl. The oligonucleotide (ORHA1) corresponded to nucleotides 2135-2152 of the DNA sequence downstream from the rhuR gene sequence (Tobin and Schleif, 1987). The restriction-digested h DNA was immobilized in the agarose gel and then it was probed with the 32P-labeledDNA. The preparation and drying of the agarose gel were as described by Thein and Wallace (1986). The dried agarose gel probed with oligonucleotide ORHAl was washed at it’s T, (52 “C) in 5 X SSPE (20 X SSPE: 3 M NaCl, 180 mM NaH2P04.2H20,20 mM EDTA, pH 7.4). The dried agarose gel probed with restriction fragment STRHAl was washed at high stringency in 0.3 X SSC at 50 “C. RESULTS AND DISCUSSION
Construction ofa L-Rhamnose Transport-negative Strainof E. coli-The strategy employed to produce a L-rhamnose transport-negative strain of E. coli was to use Mu d(ApR,lac)I mutagenesis in a fdp host strain. The Mu d(ApR,lac)Iphage inserts randomly into the host chromosome, inactivating the gene itinserts into. In addition, the lac operon in Mu d(Ap’,lac)I, which lacks its own promotor, can be expressed from a suitablypositioned promoter in the host chromosome. An E. coli host strain with the f d p lesion was used, because the presence of gluconeogenic carbon sources such as Lrhamnose or L-fucose results in the accumulation of toxic intermediates due to theabsence of fructose 1,6-bisphosphate1-phosphatase. Therefore a Mu d(ApR,lac)Iinsertion into a gene required for L-rhamnose utilization will make the fdp strain resistant to L-rhamnose. E. coli strain JM2463 (Table I) wasused as the parent strain for mutagenesis; the strainwas fdp and could not grow on L-rhamnose, L-fucose, or glycerol. The ability to grow on L-rhamnose and L-fucose wasrestored by P1 phage-mediated transduction to fdp+ (strain JAR3, Table I), which showed that strain JM2463 contained functional rha and f u c operons. A Mu d(Ap’,lac)I phage lysate was prepared from the Mu lysogen E. coli strain MAL103 (Table I) and was used immediately to infect strain JM2463. Ampicillin-resistant colonies were screened for L-rhamnose inducible growth on lactose. A single isolate, strain JAR1(Table I) had the phenotypes ApR, Lac-, Rha-, and (Rha + Lac)+. Plate assays for @-galactosidase activity indicated that lac2 was induced by 1 mM Lrhamnose in strain JARl but not in strain JM2463 or strain JAR3. Therefore, strain JARl appeared to have a Mu d(Ap’,lac)I insertion in a rha gene. To facilitate further investigation of the Mu insertion in strain JAR1, the strain was cured of the lesion in f d p by P1mediated transduction, using phage P1 propagated on strain CSH25 (fdp+).Transductants were selected on minimal medium containing glycerol as sole carbon source; all the Gly’ colonies also grew on L-fucose but were negative for growth on 10 mM L-rhamnose (strain JARZ, Table I).The Mu d(ApR,lac)I phage could have inserted into any of the rha genes to give the phenotype of strain JAR1; therefore, Lrhamnosetransport assays were performed to determine whether strain JAR2 was indeed impaired in L-rhamnose transport. Characterization of the L-Rhamnose Transport-negative E. coli Strain JAR2-The transport of 14C-labeledL-rhamnose or L-fucose into strain JAR2 was compared with transport intostrainJAR3(no Mu d(ApR,lac)I insertion)and with strain JM2513 (Table 11). E. coli strain JM2513 contained a rha gene encoding a metabolic Xplac Mu Iinsertionina enzyme? Cultures were grown on minimal medium with glycM. C. Jones-Mortimer, unpublished data.
rhuTgenes The
of
S. typhimurium and E. coli
6925
TABLEI E. coli strains Strain
Genotwe
Derivation
"
AD5827 F-, ilu,
his, supo, strA, proC:TnlO, gal OPZSl, Ab& (XBamN+ cIffi7H1 [cro-RAJ-bis] uurB) AR120 XN99, cI+, A-gal, nad::TnlO, T n l l A(cI1uurB) CSH25 supF, tyrT, thi, pro ara-, leu, pro, lacY1,gln, galK, recA, rpsL, xyl, mtl, thi, HU835 F-, hsdS, XcIffi7, b2,redp3, S7 Ahis-gnd, Alac, araDC, rpsL, ptsF, ptsM JM2418 Ahis-gnd, Alac, araD, rpsL, ptsF, ptsM, fdp, (P1 clr, 100 Cm) JM2463 As JM2463 but rha9XplacMuI JM2513 MAL103 F-, Muc'" dI(ApR,h),Muc'", @roAB, lacZPOZYA)XIII, str cysE, recA, s r k T n l 0 PB13 A(lac-pro),supE, thi, hsdD5 [F'traD36, proA+B+,ladq, TG1 lacZAM151 A(hc-pro), TG2 hsdD5 supE, thi, A(srl-recA)306::Tn10(tetR) [F'traD36, proA+B+,&Iq,lacZAM151 JAR1 Ahis-gnd, Alac, araD, rpsL, ptsF, ptsM, fdp, (P1 clr, 100 Cm) rha @Mud(ApR,hc)I JAR2 As JAR1, fdp+ but JAR3 As JM2463, fdp+ but JAR62 As JAR2, but Arha9Mu d(ApR,lac),Aps JAR66 As JAR62, but srl-recA::TnlO, TcR Genetics Department, Cambridge University. Biochemistry Department, Cambridge University. Laboratory of Molecular Biology, Cambridge University.
TABLE I1 Sugar transport assays in a control strain (JAR3) and two Mu lysogen strains (JM2513 and JAR2) Uninduced cultures were grown on minimal medium with glycerol as carbon source. Cultures induced with either L-rhamnose or Lfucose weregrown in minimal medium with glycerol and 10 mM inducer. Cultures were grown overnight at 30 "C; preparation of the cell suspension for the uptake experimentsand @-galactosidaseassays are as described in Henderson et al. (1977) and Miller (1972). The results are averages of duplicate measurements from three different experiments.
A. Das4 A. Das4 Miller (1972) P. Oliver" M. C. Jones-Mortimer3 M. C. Jones-Mortimer3 M. C. Jones-Mortimer3 Casadaban and Cohen (1979) P. Brittonb T. Gibson'
T. Gibson' This work This work This work This work This work
nose induction had taken place despite the low transport activities observed (Table 11). Neither L-rhamnose nor Lfucose could betransported by any of the uninduced strains. Additional evidence that theMu d(ApR,Zuc)I phage in strain JAR2 was inserted in the L-rhamnose-H+ symporter gene was obtained by measuring the sugar-H+symport activity of strain JAR2 after induction with L-rhamnose (Fig. 1).On addition of L-rhamnose to de-energized JAR3 cells (positive control) a rapid alkalinization of the external mediumwas observed followed by a rapid acidification (Fig. 1).This is characteristic of a sugar being imported into the cell via a sugar-H+ sym''gar uptake @-Galactosidase Strain Inducer porter followedby metabolism of the sugar to acidic end L-Rhamnose L-Fucose activity products that are excreted into the external medium (Hennmol/min/mg dry m a s s Miller units derson and Macpherson, 1986). In contrast, L-rhamnose-in2.4 0.0 JAR3 None 0.5 duced JAR2 cells did not elicit a rapid alkalinization on 25.3 14.6 0.1 L-Rhamnose 0.4 0.1 L-Fucose 33.5 addition of L-rhamnose, but only a slow acidification. This JM2513 None 0.5 2.0 0.3 apparent metabolism was at a reduced rate compared with L-Rhamnose 680.6 0.1 6.4 that observed for strain JAR3 and would have been unlikely 6.8 0.8 L-Fucose 4.7 to obscure sugar-H+ symport activity if it had been present. 1.o 0.4 JAR2 None 1.6 L-Rhamnose induction of the rha operon in strain JAR2 could 2.0 0.4 L-Rhamnose 672.7 L-Fucose 0.2 13.0 10.8 be observed by measuring the activity of Lacy, encoded by the Mu phage and expressed from a rha promoter. On addition for erol as carbon source and were induced with either L-rham- of isopropyl-1-thio-B-D-galactopyranoside(asubstrate nose or L-fucose, or not induced. The positive control strain Lacy) to JAR2 cells induced with L-rhamnose, a rapid alkaJAR3 exhibited L-rhamnose-inducible uptake of both 14C- linization of the medium was observed (Fig. 1).This is not labeled L-rhamnose and 14C-labeledL-fucose (Table 11); a followed by acidification because isopropyl-1-thio-(3-D-galaccommon product of L-rhamnose and L-fucose metabolism is topyranoside cannot be metabolized (Fig. 1).This experiment the true inducer of the fuc operon (Chen et aZ., 1987). Strain showed that the absence of L-rhamnose-H+ symport activity JAR2 showed no L-rhamnose-inducible transport of either L- from strain JAR2 was not due to leakiness of the membrane. Additional manipulations were required before strain JAR2 rhamnose or L-fucose, despite having a functional fuc operon as evidenced by the presence of fucose-inducible L-fucose could be used to screen a genomic library to isolate the rhuT transport. A single lesion in the L-rhamnose transport gene gene. It was first made recA- by phage P1-mediated transducwould be expected to give this phenotype. In contrast, strain tion; the P1 lysate was obtained from P1-infected strain PB13 JM2513 had a L-rhamnose-inducible L-rhamnose transport (Table I). The derived recA- strain JAR62 subsequently had activity, but L-rhamnose did not induce the fucose trans- the Mu phage excised from the L-rhamnose transport gene to porter; this is consistentwith the Mu insertion being in a rha produce strain JAR66 with a stable genetic background for gene encoding a metabolic enzyme rather than a transporter. the screening of a genomic library using ampicillin for plasmid @-Galactosidaseassays carried out in parallel to the sugar selection. During excision of the Mu phage there is often uptake assays indicated that both strains JAR2 and JM2513 deletion of genomic DNA which was adjacent to the phage, contained Mu insertions in the rha operon and that L-rham- thus producing a stable deletion in the E. coli chromosome.
6926
The rhaTgenes of S. typhimurium and E. coli
IPTG
L-Rhamnose
I a.
FIG.1. Sugar-H+ symport activities in E. coli strains JAR2, JARS, JAR66(pJAR2), and JAR66. E. coli strains were grown in minimal medium containing 20 mM glycerol, 10 mM Lrhamnose, and BO pg/ml histidine and grown overnight at 30 "C.Cells were harvested and prepared for sugar-H+ symport assays as described elsewhere (Henderson et al., 1977;Henderson and Macpherson, 1986).After calibration with 3 pl of 0.01M NaOH, 20 pl of a 0.5 M sugar solution was added to thecell suspension a t the points indicated by the arrows. An upward deflection of the trace represents an increase in the alkalinity of the external medium, indicative of Hf influx into the cells (Henderson et al., 1977; Henderson and Macpherson, 1986). The E. coli strains used in each experiment are as follows: a and b, JAR3 (Rha+); c and d, JAR2 (rhaaMu d(ApR,lac)I;e and f, JARM(pJAR2); g and h, JAR66 (Rha-).
b.
-"-
L-Rhamnose
\
d.
Y
L-Rhamnose
lmin
H
L-Mannose
T
9. Strain JAR66 was AD', Rha-, Lac- and no longer had Lrhamnose-inducible &galactosidase activity. Interestingly, strain JAR66 would not grow a t even high concentrations (100 mM) of L-rhamnose, whereas strain JAR2 could grow on L-rhamnose as sole carbon source if the concentration was greater than 40 mM. This suggested that additional genes had been deleted during the excision of Mu d(ApR,lac)I. Screening a S. typhimurium Genomic Library to Isolate the rhuT Gene-A genomic library prepared from S. typhimurium C5 DNA (see "Experimental Procedures") was amplified in strain HU835 and then used to infect the L-rhamnose transport-negative strain JAR66. 3000 ApRcolonies were screened by replica plating for growth on 10 mM L-rhamnose as sole carbon source. Three colonies grew on L-rhamnose. Restriction analysis of the plasmids from the three Rha' colonies indicated that they were identical (pJAR1). Plasmid pJARl (Fig. 2) fully compensated for the deletion at the rhu locus in strain JAR66. Strain JAR66(pJARl) grew on minimal medium with L-rhamnose as sole carbon source and effected the uptake of I4C-labeledL-rhamnose following induction by L-rhamnose. Plasmid pJAR2 (SalI-digested pBR322 ligated to the 9.8-kb SalI fragment from plasmid pJAR1) fully complemented the deletion in pJAR66; L-rhamnose-H+ symport activity was also observed (Fig. 1).Plasmid
I
& pJAR2 was used as a source of DNA for further subcloning of the rhaTgene. Expression of the rhaT Gene from a X Promoter and the Localization of the rhaT Gene-Plasmid pAD2M4 contains the X P L promoter on a 2.4-kb HindII/BamHI X DNA fragment ligated into plasmid pBR322. Plasmid constructs derived from pAD284 can be maintained in either of two X lysogenic E. coli strains which produce cI repressor to prevent potentially lethallevels of expression from h P L during routine growth of strains. E. coli strain AR120 contained wild type X as lysogen; the X PLpromoter could be induced by nalidixic acid (Mott etal., 1985). E. coli strain AD5827 was a X lysogen that expressed the cIs57 gene product; increasing the cell culture temperature from 33 to 42 "C resulted in the inactivation of the thermolabile cIS7 protein, thus allowing expression from the X PL promoter. The 2.4-kb HindIII/BamHI fragment from plasmid pAD284 containing the X PI, promoter was ligated to HindIII/BamHI-digested plasmid pJAR2, producing plasmid pJAR3. Only one orientation of the 9.8-kb SalI insert in relation to the X PLpromoter was obtained with this method. To obtain the opposite orientation(pJAR4), plasmid pJAR3 was digested with SalI to remove the 9.8-kb
'A. Das, unpublished data.
The rhaTgenes of S. typhimurium and E. coli
6927
RHAMNOSE TRANSPORT a.
pJARl
positive
b.
pJAR2
positive positive
pJAR3 c. d.positive pJAR5
positive pJAR6 e. negative pJAR7 f.
egative
g.
+
pJAR9 A Pv
h. pJARl0
positive
P H
-
BI
VY
BSEBI Hc
A
I
1.
FIG.2. Identification of a DNA fragment encoding the rhaT gene. Plasmid constructs that contained different restrictionfragments of the cloned S. typhirnurium genomic DNA were tested for their ability to complement the lesion in JAR66 as measured by uptake of [14C]~-rhamnose and sugar-H+ symport experiments. Plasmids pJARl and pJAR2 ( a and b ) both complemented the rha- lesion in JAR66, as indicated by growth of strains JAR66(pJARl) andJAR66(pJAR2) onminimal medium that contained L-rhamnose as sole carbon source. Uptake activity and L-rhamnose-H+ symport assays were performed on strains JAR66(pJARl) and JAR66(pJAR2) after growth in minimal media that contained L-rhamnose as sole carbon source. Plasmids pJAR3-10 (c-h) contained partial deletions of the cloned S. typhirnuriurn genomic DNA downstream from the X promoter PLin plasmid pAD284. Induction of the X PLpromoter upstream from the rhaTgene was achieved using both nalidixic acid induction for the plasmids in E. coli strain AR120 and by heat shock for the plasmids in E. coli strain AD5827. The ability of induced strains to exhibit uptake activity and L-rhamnose-H+ symport activity is indicated by “positive” or “negative” in the column labeled “rhamnose transport.” The rate of [14C]~-rhamnose uptake in positive strains varied between 1.7 and 14.7 nmol/min/mg dry mass, whereas negative strains typically showed uptake rates between 0.0 and 1.1 nmol/min/mg dry mass. a and b, linear diagrams of plasmid constructs pJARl and pJAR2. The hatched box in the diagram of plasmid pJARl represents part of plasmid pHC79, and thestippled box in plasmid pJAR2 represents part of plasmid pBR322. c-h, linear diagrams of plasmid constructs pJAR3 to pJAR10. Filled boxes represent part of plasmid pAD284 (Fig. 5). The arrow beneath the filled boxes represents the position and direction of transcription of the X PLpromoter. Partial restriction maps are given: A, AuaI; B, BarnHI; El, BglII; E, EcoRV; H, HindIII;Hc, HincII; N, NcoI; P, PstI; Pu, PuuII; S, SalI. The two EcoRV sites are labeled x and y for clarity. i, a restriction map of the 9.8-kb SalI fragment in plasmid pJAR3 with the position and direction of transcription of the rhaT gene (filled box) as deduced from c-h shown below. Transcription of the rhaT gene should start between the EcoRV and BglII sites (open box) and end between the HincII and AvaI sites (hatched box). insert followed by re-ligation of the insert with phosphatasetreated vector. Nalidixic acid induction of strains AR120(pJAR3) and AR120(pJAR4) resulted in expression of L-rhamnose transport activity (4.2 f 0.3 nmol/mg/min) only in strain AR120 (pJAR3). The L-rhamnose transport activity in nalidixic acid induced strain AR120(pJAR4) was 0.5 f 0.4 nmol/mg/min. This defined the direction of the transcription of the rhaT gene as BglII to AuaI (Fig. 2). To locate the rhaTgene within the 9.8-kb SalI fragment, a series of plasmids were constructed from plasmid pJAR3 with deletions in the insert DNA. Each construct was transformed into E. coli strains AR120 and AD5827 and tested for induction of L-rhamnose transport activity. The results (Fig. 2) suggested the presence of the
rhaT gene between the EcoRV site (proximal to a BglII site) and the AuaI site, with transcriptionstarting somewhere between the EcoRV site and the BgZII site. Thus, we have located the rhaT gene to a 2.4-kb portion of DNA within the original 9.8-kb fragment (Fig. 2i). Sequencing of the S. typhimurium rhaT Gene-A 3-kb EcoRV-PuuII fragment from plasmids pJAR6 and pJAR10, which contained the complete rhaT gene, was sequenced (see “Experimental Procedures”). A contiguous sequence of 3052 bp was assembled; this comprised 2377 bp of S. typhimurium DNA (Fig. 3) between the EcoRV and AuaI sites in plasmid pJARlO (Fig. 2i) and 675 bp of plasmid pBR322 DNA sequence (data notshown) between the AuaI and PuuII restriction sites. The S. typhimurium DNA sequence was determined
The rhuT genes of S. typhimurium and E. coli
6928
a. ATCAGMGAAACGGCGTACATATTCAAAACGATAGCGATAGCGCGCGCCMCCCACCAGCTTTCATTT~TGTCCACGTCGCGCGGMGTMGGCTTATAGGTAGAGTMCCGTTGCCTGTTTCCA 120 GCACGAAACCCGGTTGCCAGGCGACATCACCGGTTTTAAATTGGTAGCTTATCGTATATTCGTTGCCCTGTGTTTCTAGGTCATTAAACGCTTTATTGGTGTCATCCCCGCCTGATTTTG 240 AGATAGCTTCTATCGCAAAACGATAGCCMCACCATTCGCAAACCGATGAGAAATATACGCACGGTCATAGTTAGCTTTACTGTCATCAAGATATTCATGACGMCATCGACATATACTGCATGCG 360 CATTTGTTACMCMGAGGTAATAGTMG~GAGATTTTCTCAT~CAGMTCCATTT~TTTAAAACGATAGTT~CGTTGTTTCATGTCAGACCGTTACMCCATMTACGCMTGTGAA 480 TGGCGACMTATAGACAAAGATCTTTMTTTAAAACGATAGTTGGGATMTGTTC~GCAAAACGATAGTAAAACGATAGTAAATGAAATTCATATTCGTCATATAAAACGATAGT~TCAAAACGATAGCACCATCMCMCG600 CCMTATAGGGATTTCCTTATATTTATATTTMTTGATTCCTMCGTTMTTTCMTATTTTAAATTGTAAATACAAATAAAACGATAGCMCCAAATMTTACTGTATMCTACCCTTTTATCM 720 ACGGTAAGTCAGACTATTCCCATTATGTGACCTCACGCAGCGTTTTCTTTGCATTGTTGCCAGCTMGTCGTTTCGTGTCGTTATGATGMGGTATTTTAGAAAACGATAGCAAATATTACCCACT 840
-
___)
GATTTTAAATMCATCCAGACGGTCACGATGCGCTCCGACTTTTTT~CGTGMGTTMTCACTTC~TTMCGTGCTGATTGCCAGMTACACCC~CGGCGGCMTCGTTATMGGT
rhaT
-
*****
I)
960
****** M S N A I T M G I F W H L I G T A C C A G G C T G C G T M T T T T T A A A C T ~ T G C A A A C G G M ~ T A T C C C G T C C A T ~ T M T G A G G M C A T G T C A T G A G T M C G C G A T T A C G A T G G G T A T T T T C T G G C A T T T G A T A1080 GG A A S A A C F Y A P F K Q V K Q W S W E T M W S V G G I V S W L I L P W T I S A GGCGGCCAGTGCAGCCTGCTTCTATGCCCCGTTCAAGCMGTGAAACAGTGGTCATGGGAAACCATGTGGTCAGT~CGGCATCGTCTCATGGCTTATTCTGCCGTGGACMTTAGC~ 1200 L L L P D F W A Y Y G Q F N L S T L L P V F L F G A M W G I G N I N Y G L T M R TCTGTTACTGCCTGATTTCTGGGCCTATTATGGGCAGTTTMCCTCTCCACCCTTTTACCGGTTTTTCTGTTCGGCGCCATGTG~ATCGGCMTATTMCTACGGCCTGACCATGCG 1320 Y L G M S M G I G I A I G I T L I V G T L M T P I I N G N F D V L I H T E G G R TTATCTCGGCATGTCGATGTATCGGCATCGCTATCGGCATTACGCTT~CGTCGGCACGCTGATGACGCCTATCATCMCGGTMCTTCGATGTGTT~TTCATACCGMG~GACG 1440 M T L L G V F V A L I G V G I V T R A G Q L K E R K M G I K A E E F N L K K G L CATGACGCTACTTGGCGTTTTTGTCGCGCTGATCGGCGTC~ATTGTGACGCGCGCCGGACAGTTAAAACGATAGGAACGCAAAACGATAGTGGGCATTAAAGCGGAGGAGTTCMTCTGMGAAA~CT 1560 L L A V M C G I F S A G M S F A M N A A K P M H E A A A A L G V D P L Y V A L P TCTGCTGGCAGTGATGTGCGGTATTTTCTCGGCGGGMTGTCTTTTGCCATGMCGCCGCGAAACCGATGCATGATGCATGMGCTGCTGCCGCGCTTGGCGTTGACCCGCTCTATGTCGCGCTGCC 1680 S Y V V I M G G G A L V N L G F C F I R L A K V Q N L S I K A D F S L A R P L I GAGTTACGTGGTGATTATGGGCGGCGCGCTGGTGMCCTCGGTTTCTGTTTTATCCGCCTGGC~GTACAAAACGATAGTCTGTCGAT~GCCGACTTCTCGCTGGCMGACCGTTGAT 1800 I S N I L L S A L G G L M W Y L Q F F F Y A W G H A R I P A Q Y D Y M S W M L H TATCAGCMTATTCTGCTGTCCGCGCTTGGCGGTCTGATGTGGTATTTGCAGTTCTTTTTCTATGCCTGGGGTCACGCGCGCATTCCAGCGC~TATGACTACATGAGCTGGATGCT~A1920 M S F Y V L C G G L V G L V L K E W K N A G R R P V A V L S L G C V V I I I A A CATGAGCTTCTATGTGCTGTGCGGGGGGCTTGTCGGTCTGGTGCTAAAACGATAGGAGTGG~TGCT~CGCCGTCCCGTTGCTGTATTMGCCTCGGCTGCGTGGTMTTATTATCGCGGC 2040 N I V G L G M A S * GAATATTGTCGGTTTAGGCATGGCCAGTTMTCGCTTTGATTACTGAGATGACGCCATTGGCTCGGCGT~TCCCGGTTTCCCGCGTGMCACCACTG~TMTTACTATCTTCAAA 2160 ACCGCACTGCATCGAAATCTCGCTMTCATCAGCGTATGCTGMGCAGATACTGCGCGTGGCAAATACGGACCTGGCGTAAATATTGATT~TCGTCATCCCGGTTTGGGCGCG~2280 CTGTGCCGCAGCACCCGTTCGCTGCATTGTTCCTGCTGGCAAAACGATAGCGCATCCAGCGCAAAGGACACTCM~TATTAGCC~GCGGTMTTMTTTATCCMCA~ 2388
-
b. 3x
* -*
2‘4 2%)
7x
b
-----
5 x ”
+
______)
I
Bgll I
4 -
Due I
e 4
-
“
x
2
)
4 x +
rhaT
-+
2
x
I
BstE I I
Hfncl I
“
2X)
*
4
4x 2X 2x
*
h
Ball Duel
-4
4
3x
”
x 2
4
2x
-
+
___)
I
I
4-
4
-*
i *
2 x
+ 2x + 2X)
”
”
+*
4x4x
c-
4
4
3x 4x 3x 3x
2x
200bp
44
FIG. 3. The DNA sequence and sequencing strategy of the rhaT gene from S. typhimurium. a, the RhaT protein sequence is given in the single-letter amino acid code above the DNA sequence. A putative ShineDelgarno sequence is shown by a line above the DNA sequence. Possible -10 and -35 regions are indicated by asterisks, and a potential CRP binding site is shown by a box. DNA sequences predicted to form hairpin loop structures, or palindromic repeats, are shown by arrows. b, the DNA sequence was determined on both strands of the DNA except for nucleotides 1-54. The filled box represents the coding region of the r h T gene. A partial restriction map is shown below. Arrows represent a sequence of DNA derived from one sequencing reaction. If a particular sequence was obtained more than once, this is indicated by a number at the startof the arrow.
on both strands of the DNA, except for nucleotides 1-54 which were upstream from the rhaT gene (Fig. 3). A single open reading frame was identified by measuring the codon usage in all of the possible reading frames. The single open reading frame was in the 5‘ to 3’ orientation, as predicted from the L-rhamnose-transport data derived from the expression of the rhaT gene in pJAR3 andpJAR4 (Fig. 2). However, the reading frame started much further downstream from the BgDI site than expected. The reading frame is 1032 nucleotides long, whichcorresponded to a proteinof 344 amino acids (Fig. 3). Upstream from the putative initiation codon of the rhaT gene there was a good match to the consensus ShineDelgarno sequence (nucleotides 1025-1029), potential -10 and -35 regions (nucleotides 980-985 and 960-966, respec-
tively) and apossible CRP binding site (nucleotides 888-908). The poor match of the potential -10 and -35 regions to the consensus is typical of bacterial genes whose expression is controlled by a positive transcriptional activator, such as RhaR controlling expression of the rhaS/R genes (Tobin and Schleif, 1987, 1990b). In addition, there was a short palindromic repeat (nucleotides 920-926 and 944-950) adjacent to and between the potential ribosome and CRP binding sites. This palindromic repeat could be the binding site for a positive acting transcriptional element (RhaR or RhaS?), but is it not homologous to the RhaR binding site in E. coli (Tobin and Schleif, 1990b). At the 3‘ end of the rhaT gene, 16 bp after the termination codon, there was an inverted repeat (nucleotides 2088-2096 and 2105-2113) which could forma stemloop
genes of S. typhimurium and E. coli
rhaT The
structure comprising a 9-bp stem and eight-nucleotide loop. It is unlikely that thisstem loop represents a rho-independent terminator, because there areno contiguous thymine residues in theDNA sequence downstream from the loop, but it could be a rho-dependent terminator (reviewed by Platt, 1986). Identification of the rhaT Gene in E. coli-With the publication of a complete restrictionmap for the E. coli K12 chromosome (Kohara etal., 1987), it is comparatively easy to map the location of a new gene of known DNA sequence by Southern blot analysis. This technique was used to map the position of the rhuT gene. E. coli K10 genomic DNA was digested singly and in combination with various restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis and blotted onto a membrane. The Southern blots were probed with a 32P-labeled400-bp HincIIIBalI restriction fragment (STRHA1) corresponding to the C-terminal portion of the RhaT protein. A single band in each lane was seen on autoradiographyof the Southernblot (results not shown). The sizes of the restriction fragments produced by BamHI, EcoRI, PstI, HindIII, PuuII, and KpnI digestions were consistent with only one region of the E. coli DNA restriction map (Fig. 4), adjacent to the rhu locus. However, fragments derived by EcoRV digestion did not concur with the published data. The X clone 4B6 which encompassed this region was obtained from Dr. Y. Kohara, Nagoya University,
b.
a. 23
C.
I
kb
-
9.46.6-
44-
2.2 2.0
--
1.4-
-
1.1
1 2 3 4 5 6 78 1 2 3 4 5 6 71 2 3 4 5 6 7
d.
E I
B1
-
B
E
P
B
I
I
I 1
I
I
1
BI
81,Pv
BlPv
B1
PV
B1
H
I OOObp
rmi m a w s
FIG. 4. Mapping of the E. coli rhaT gene. DNA from the bacteriophage clone 4B6 (Kohara et al., 1987) was digested with restriction enzymes and thefragments were separated by gel electrophoresis. The gel was probed with a “P-labeled HincIIIBalI restriction fragment of the S. typhimurium rhaT gene and a ”P-labeled oligonucleotide complementary to the sequence downstream of the rhaR gene (nucleotides 2135-2152 (Tobin and Schleif, 1987)). A single agarose gel that contained two identical sets of restriction digests was electrophoresed and stained with ethidium bromide (a, lanes 1-8). Half of the gel was probed with the S. typhimurium rhaT gene fragment (b, lanes 1-7), the other half was probed with the oligonucleotide (c, lanes 1-7). Lanes containing bacteriophage 4B6 DNA were digested with the following restriction enzymes: lane 1, BamHI; lane 2, HindIII; lane 3, EcoRI; lane 4, B g k lane 5, PstI; lane 6, PuuII. Lane 7 contained 2.5 ng of BalIlBstEII-digested S. typhimurium rhaT gene. Lane 8 contained HindIII-digested X DNA and HaeIII-digested @X174 DNA, as markers. d, restriction map supplied with X clone 4B6, with the position of the rhaT gene (filled box) and the rhaS/R genes (open box) below; the direction of transcription is indicated by an arrow. Abbreviations for restriction enzymes are as follows:B, BamHI; Bl, BglII; E, EcoRI; H, HindIII; P, PstI;Pu, PuuII.
6929
Japan. Todefine the position of the rhaTgene in relation to the sequenced rhaSIR genes (Tobin and Schleif, 1987), an oligonucleotide (ORHA2) was synthesized to a region downstream of the rhaR gene. A dried agarose gel containing restriction digests of the bacteriophage clone was probed with 32P-labeledoligonucleotide ORHA2 and the S. typhimurium rhuT gene fragment,STRHA1, also labeled with 32P(see “Experimental Procedures”). Autoradiography of the probed gel indicated that the rhaTgene was on the same 3-kb PuuII fragment asthe region downstream from the rhaR gene. Computerized analyses of the DNA sequences for S. typhimurium rhaT and E. coli rhaR genes identified the region downstream from the E. coli rhaR gene as the C terminus of RhaT. However, there was not a single open reading frame in the region downstream from the rhuRgene (Tobin and Schleif, 1987) that might encode RhaT; this was ascribed to a sequencing error in this region of DNA (see below). Expression of the E. coli rhaT Gene under Control of a X Promoter-To confirm the presence of the rhuT gene on the 3-kb PuuII fragment, a restriction fragment that contained the r h T gene was ligated into plasmid pAD284 downstream of the X PLpromoter (Fig. 5). The 3-kb E. coli genomic DNA PuuII fragment from X clone 4B6 (Kohara et al., 1987) was first subcloned into plasmid pBR322 (pCGT6); aderived 1.6kb XmnIISmaI restriction fragment predicted to contain the rhaT gene was subsequently cloned from plasmid pCGT6 into the HpaI siteof plasmid pAD284 (Fig. 5). The SmaIsite was predicted to be at the end of the rhuR gene (Tobin and Schleif, 1987), whereas the XmnI site was in the pBR322 DNA, 35 nucleotides from the PuuII site(Fig. 5). Two orientations of the insert were obtained; plasmid pCGTlO was predicted to contain the insert in theright orientation for its expression by the X PLpromoter, whereas in plasmid pCGTll the rhaT gene was predicted to be in thewrong orientation for expression (Fig. 5). Plasmids pCGTlO and pCGTllwere transferred into E. coli strain T G ~ ( ~ C which I~~~ expressed ), the temperature-sensitive CI gene product, cIeS7(Remaut et al., 1983). Thus, at33 “C theX PLpromoter was inactive due to thecIs57 protein binding at the operator sites, whereas at 42 “C CIs57 protein was nonfunctional and the X P L promoter expressed the DNA inserted downstream from it. After induction at 42 “C, only strain TG2(pCGT10/pc1~,~~) produced a protein which could effect the uptakeof radiolabeled L-rhamnose into cells (Fig. 5). This confirmed that therewas a functionalopen reading frame in the XmnIISmaI fragment, despite the lack of an open reading frame in the region downstream from the rhaR gene (Tobin and Schleif, 1987). The XmnIISmaI fragment was also cloned into EcoRV-digested Bluescript plasmid (plasmid pCGT12) so that the E. coli rhuT gene could be sequenced. DNA Sequence of the E. coli rhuT Gene-The 1.6-kb XmnIl SmaI fragmentcontaining the E. coli rhaT genewas sequenced directly from plasmid pCGT12 (see “Experimental Procedures”). The DNA was sequenced a t least once on both strands; Fig. 6 depictsthe 1560 nucleotides of DNA sequenced between the PuuII and SmaI restriction sites. Three open reading frames were identified in the sequence. A single complete open reading frame that encoded a 344-amino acid protein was found; the orientation of the open reading frame corresponded to the orientation of the rhaT gene deduced from the induction of the X P L promoter inplasmid pCGT10. The protein was 91% identical to the RhaT protein predicted from the S. typhimurium gene sequence. Upstream from the putative initiator Met codon of the E. coli rhaT gene was a good match to the consensus Shine-Delgarno sequence (nucleotides 462-466), possible -35 and -10 regions (nucleotides
The rhaTgenes of S. typhimurium and E. coli
6930 a.
a.
CTGGTCCAGTTTGGTGATCAGCTCTTCMCCGGCAGGTTGGCAAATTCTGGCAGGCTTTC
Y PVUl
60
Q D L K T I L E E V P L N A F E P L S E CAGCGCCGCGTTGGCGTTGTTTACGTRGGTCTGATGGTGTTTGGTGTGGT~TTTCCAT 120 L A A N A N N V Y T Q H H K T H H I E M GGTCTGCTTATCGAAGTGCGGTTCCAGGGCATCGTAAGCATACGGCAGGGATGGCAGGGT 180 T Q K D F H P E L A D Y A Y P L S P L T A T A G C T C A T A T T C A T C T C C A G T A T T G T C ~ G ~ C G A T T G T T A A T ~ C ~ G T A A G C A240 GT
PStl
S
M
+ soli4
ex Pvul I
I
TGGTTCATTATAGTTAATTATGATA*GAAAATGATT~~CAATGCC~ACTTTTCGTA300 360
AGGGTATGGTTTTGCAGGATGCCCGAGATGTGAAGCAAATCACCCACTTAATGCCGT
J f
*..*et
Smal
GATTGCCAGTAAATCGACAACGGCGGCAACAGGCGAAAGGTTAATCGACAGCACGATTTT
rhaT
Hlndl I I
d.
8gll I
Aval
Aval
BamH I
BamH
~
e.
I50 (
200
rnln )
420
T***.I.* A C A C T C A T C T C G T C G G A G A T G T G A C G C G A C G ~ T G A T ~ A T A A G A A GMA T G SA G T A 480 N A I T M G I F W H L I G A A S A A C F ACGCGATTACGATGGGGATATTTTGGCATTTGATCGGCGCGGCCAGTGCAGCCTGTTTTT 5 4 0 Y A P F K K V K K W S W E T M W S V G G ACGCTCCGTTCAlVUlULGTAAAAAAATGGTCATGGGAAACCATGTGGTCAGTCGGTGGGA 600 I V S W I I L P W A I S A L L L P N F W TTGTTTCGTGWLTTATTCTGCGTGGGCCRTCllGCGCCTGTT~TACCGAATTTCTGGG 660 A Y Y S S F S L S T R L P V F L F G A M CGTATTACAGCTCGTTTAGTCTCTCTACGCGACTGCCTGTTTTTCTGTTCGGCGCTATGT 720 W G I G N I N Y G L T M R Y L G M S M G G&SGGATCGGTAATATCATACGGCCTGACCATGCGTTATCTCGGCATGTCGATGGGAA 780 I ~ I A I G I T L I V G T L M T P I I N TTG~ATCGCCATTGGCATTACGTTGATTGTCGGTACGCTGATGACGCCMTTATCMCG 840 G N ' > , F D V L I S T E G G R M T L L G V L GCAATT~CGATGTGTTGATTAGCACCGAAGGCGGACGCATGACGTTGCTCGGCGTTCTGG 900 V A c s , , I G V G I V T R A G Q L K E R K M TGGCGCT TTGGCGTAGGGATTGTAACTCGCGCCGGGCAGTTGAAAGAGCGCAAGATGG 960 G I K T E E F N L K K G L V L A V M C G GCATTM~GAAGAGTTCMTCTGAAAAAAGGGCTGGTGCTGGCGGTGATGTGCGGCA 1020 I F S A I G M S F A M N A A K P M H E A A TTTTCTCTGCC&GATGTCCTTTGCGATGAACGCCGCAAAACCGATGCATGAAGCCGCTG 1180 A A L G V D P L Y V A L P S Y V V I M G CCGCACTTGGCGTCGATCCACTGTATGTCGCTCTGCCMGCTATGTTGTCATCATGGGCG 1140 G G A I I N L G F C F I R L A K V K D L GCGGCGCGATCATTAACCTCGGTTTCTGTTTTATTCGTCTGGCAGTGAAGGATTTGT 1200 S L K A D F S L A K S L I I H N V L L S CGCTAGCCGACTTCTCGCTGGCAAAATCGCTGATCATTCACAATGTGTTACTCTCGA 1260 T L G G L M W Y L Q F F F Y A W G H A R CACTGGGCGGGTTGATGTGGTATCTGCAATTCTTTTTCTATGCCTGGGGCCACGCCCGCA 1320 I P A Q Y D Y I S W M L H M S F Y V L C TTCCGGCGCAGTATGACTACATCAGTTGGATGCTGCaGCG 1380 G G I V G L V L K E W N N A G R R P V T GCGGTATCGTCGGGCTGGTGCTGAAAGAGTGGAACMTGCAGGACGCCGTCCGGTAACGG 1440 V L S L G C V V I I V A A N I V G I G M TGTTGAGCCTCGGTTGTGTGGTGATTATTGTCGCCGCTAACATCGTCGGCATCGGCATGG 1500 A N ' ____) CGAATTMTCTTTCTGCGAATTG~ATGACGCCACTGGCTGGGCGTCATCCCGGTTTCCC 1560 ~
50 100 TIME AFTER INDUCTION
*
~
~
~~
~~
-
FIG. 5. Plasmid constructs containing the E. coli rhaT gene and its L-rhamnose uptake activity expressed from a X PL promoter. a, plasmid pAD284 (6.41kb)was constructed by ligating * D K I S N L H R W Q S P T M G T E + ~ ~ ~ R a 2.4-kb HindIII/BamHI X DNA fragment containing OLPL,into 200bp HindIIIIBamHI-digested plasmid ~ B R 3 2 2b, . ~plasmid pCGT6 (7.36 d b. the kb) was constructed by ligating 3-kb PuuII the fragment from X > clone 4B6 into PuuII-digested plasmid pBR322. One PuuII site was csodA rha T-+ crhaR not regenerated after the ligation; was this due to a 2-bpdeletion at e x X the PuuIIsite in the vector sequence (data not shown). c and d, ( (ex-smal) e 4 plasmids pCGTlO (8.01 kb) pCGTll and (8.01 kb) were constructed 4*+3-" by ligating the SmaI/XmnI 1.6-kb restriction fragment pCGT6 from into HpaI-digested pAD284.The AuaI site was used to determine the FIG. 6. The DNA sequence and sequencing strategy of the orientation of the insert. e, rate of 14C-labeled L-rhamnose uptake in from E. coli. a, the RhaT protein sequence rhaT gene is given in heat-induced strains TG2(pCGT10/pcIBs7) and TGB(pCGTll/pcI%7). the single-letter amino acid code above DNAthe sequence.A putative Plasmid pcIBs7 encoded the temperature sensitive cIg57protein. Shine-Delgarno sequence is shownby a line above DNA thesequence. Possible -10 and -35 regions are indicated by asterisks,a and 401-407 and 421-426, respectively), andapotential CRP potential CRP binding site is shown by a box. DNA sequences structures, repeats, are binding site (nucleotides 329-349). A palindromic repeat predicted to form hairpin loopor palindromic are downstream from the rhaT gene (nucleotides 1508-1533 and shown by arrows. The SodA and RhaR protein sequences shown DNA sequence.b, the DNA sequence was determined on 1542-1557) could represent a stem loop structure that may below the rhaT gene both strands of D Nthe A . The filled box represents the act asa rho-dependent termination site, similar to that in the coding region, the boxhatched represents part of the sodA gene coding S. typhimurium gene. of the coding region of the region, and open thebox represents part Another open reading frame (nucleotides 1-189) at the start rhuR gene. The arrow next to thename gene depicts the direction of arrows represent a single gel reading from a of the DNA sequence encoded the N terminus of a predicted transcription. Unlabeled of the arrow corresponds to the strand protein identical to the published sequences of the man- sequencing gel, the direction DNA read. A partial restrictionmap of theDNA sequence is ganese-containing superoxide dismutase SodA (Steinman, of shown;bracketed restriction enzyme siteswere present in the original 1978). The DNA sequence of the sodA gene has also been X clone but were destroyed during cloning.
; -+ +*
published (Takeda and Avila,1986); nucleotides 1-329 are identical to the published sequence except for a base change at nucleotide 16. Nucleotides 1508-1560 were part of an open reading frame that encode the C terminus of RhaR identical to that published by Tobin and Schleif (1987). The DNA sequence of their rhaR gene included 325 nucleotides of sequence downstream from the rhaR gene, which should have encoded the rhaT gene. However, the presence of a compression resulted
-
v *-*
in the omission of a single base (nucleotide 1306,Fig. 6), thereby disruptingthe coding sequence of the rhaTgene. A Comparison of the RhuT Protein and Gene Sequences from S. typhimurium and E. coli-An alignment of the E. coli and S. typhimurium RhaT protein sequences showed that the proteins are 91% identical (Fig. 7); out of 344 amino acids, thereare15 conservative changes, 10 semi-conservative
6931
The rhaT genes of S. typhimurium and E. coli ~ Z M ; I F W H L I G W i ? W E Y A P ~ I V S W I I 50 LP
IIIl111111111111111l11l1111.11.111111111111111:111 % N " D G I E W H L I ~ ~ I V S W 50 L I L E ' 50
"
S
S
F
s
l.IIIIIII:IIIII:
~
~
1.111
I
m
G
L
100
~
b.
IIIIIIIIIIIIIIIIIIIIIIIIIII
WTI~Dm"1mGL-
100
150
200
250
300
CYTOPLASM 7
100
M X G I A I G I T L I V W L K C P I I I S ~ V A L I ~150 I
IIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIII:IIIIIIII M
;
I
G
I
A
I
G
I
T
L
~
I
I
~ 150I
~
I
"vLAvMxIF~"HFl200
IIIIIIIIIIIIIIIIIIIIIIIII:IIIIIIIIIIIIIIIIIIlIIIII EH MW "x M v"
200
AAAAU;VDPLYW&PSIVVIM;GGAIINU;FCFIRWNKDLSLKADFSL250
IIIIIIIIIIIIIIIIIIIIIIIII::IIIIIIIIIIII.:II:IIIIII A A A A L G V D P L Y W & P S I ~ I ~ F 250 S L
PERIPLASM 7
FIG. 9. Hydropathy plot of the RhaT protein from S. typhi-
A K s L I I H M I L L S ~ P I Q Y D Y 300 I ~ murium and a putative model of its orientation in the mem-
l:.lll
I:III.IIlIIIIIIIIIIIIIIIIIIIIIII:IIIIIIIIII
A R P L I I S N I ~ A Q Y D s n T F Y V 300 L
C
G
G
T
"
1
r
344
n
IIII:IIIIIIII.IIIIIIl.II/IIIIIII:IIlIII:IlI. LCGGLV[;LVLKEWKMU;RRPVAVLSLGCWII~ 344 FIG. 7. Alignment of the RhaT protein sequences. The E. coli RhaT protein sequence is on the top line, and the S. typhimurium RhaT protein sequence is on the bottomline. The alignment was obtained using the program BESTFIT (Devereux et al., 1984). a. F TTT F TTC L TTA L TTG
7. 11.
2. 9.
S TCT S TCC S TCA S TCG
2. 1. 2. 7.
Y TAT Y TAC
8.
TAA TAG
1. 0.
C TGT C TGC TGA W T&
4.
~
L CTT L CTC L CTA L CTG
1. 6. 2.
21.
I ATT 20. I ATC 13. 1. I ATA M ATG 19. -~ v GTT 4 . v GTC 8 . 5. V GTA V GTG 10.
R CGT R CGC R CGA R CGG
7. 7. 13.
S AGT
1.
T T T T
ACT ACC ACA ACG
1. 3. 1.
N AAT N AAC
7.
K AAG
2.
A A A A
GCT
5.
n
GCC
15.
GAT n GAC
3. 2.
E GAA E GAG
4.
GCA
6.
GCG
9.
S TCT S TCC
1. 2.
S TCA S TCG
3.
H CAT H CAC
Q CAA Q CAG
K AAA ~~
~~
3.
0. 12.
~~~~~~~
3. 2. 1. 2.
P CCT P CCC P CCA P CCG
0. 3. 6.
~
3. 2.
3. 5. 1. 0.
5. S AGC 5. R ACA -~ 0... R AGG 0.
~
G GGT G GGC G GGA G GGG
6. 21. 3. 9.
b. F F L L
TTT TTC TTA TTG
L CTT L CTC L CTA L CTG I ATT I ATC I ATA M ATG V GTT v GTC V GTA V GTG
6. 13.
6. 3. 8. 5.
2. 21. 11. 12. 2.
20. 4. 7.
3. 11.
4.
TAT Y TAC *TAA * TAG Y
9.
3. 1. 0.
P CCT P CCC P CCA P CCG
2. 1. 1.
H CAT H CAC CAA
3. 2. 3.
1.
Q CAG
4.
T T T T
ACT ACC AWL ACG
0.
N AAT N AAC
6.
A A A A
GCT
6. 12.
GCC Gw\
GCG
4.
1.
6.
4. 14.
Q
KASA K AAG
D GAT n GAC E GAR E GAG
7. 10. 2.
2. 3. 4. 3.
C TGT C TGC * TGA W TGG ~~
R CGT R CGC R CGA R CGG
1. ~
4.
0.
12. 2. 6. 0.
0.
S AGT S AGC
4.
RAGA R AGG
1.
5. 0.
G GGT
9.
G GGC
21. 3.
G GGA G GGG
__7 .
FIG. 8. Codon usage in the rhaT genes from S. typhimurium and E. coli. a, codon usage for the E. coli rhaT gene. b, codon usage for the S. typhimurium rhuT gene.
changes, and 4 nonconservative changes. The majority of the differences seem to be in the hydrophilic regions and not in the putative transmembrane domains. The codon usage tables (Fig. 8) show that many non-preferred codons are used in the rhaT genes. The frequency of optimal codon usage (Ikemura, 1981) for RhaT is 0.59, compared with 0.61 for Lacy, 0.57 for MelB, 0.65 for XylE, and 0.74 for AraE. Highly expressed proteins tend tohave a higher frequency of optimal codon usage; for example, OmpA has anoptimal codon usage
brane. a, the hydropathy plot was derived using the algorithm of Kyte and Doolittle (1982) with a window size of 11.b, the orientation of the putative model of RhaT was determined by the overall charge on each hydrophilic loop. Asp and Glu residues are indicated by a negative sign,whereas Argand Lys residues are indicated by apositiue sign.
frequency of0.92 (Ikemura, 1981). TheRhaT protein is extremely hydrophobic; the E. coli RhaT protein contains 73.3% hydrophobic amino acids with a hydropathic index of 0.82 (Kyte andDoolittle, 1982). Alignment of the DNA sequences from the two organisms revealed a region of 1236 nucleotides with 81% identity (nucleotides 329-1560 of the E. coli sequence aligned with nucleotides 888-2123 of the S. typhimurium DNA sequence). The sequence upstream from the rhaT gene in S. typhimurium did not contain a sequence which corresponded to thes o d gene and was not homologous to any DNA sequence in computer databases. The region downstream from the rhaT gene in S. typhimurium was homologous to the rhaR gene from E. coli. However, two nucleotides in different locations were absent from the S. typhimurium rhaR gene, which resulted in the disruption of the reading frame for RhaR. This region was sequenced adequately on both strandsof the DNA (Fig. 3), so it is unlikely that the differences are due to a sequencing error. Therefore these changes may be a cloning artifact or may really represent the truerhaR gene sequence in S. typhimurium C5. This strainof S. typhimurium cannot grow on Lrhamnose as sole carbon source and does not possess a Lrhamnose-inducible RhaT activity, despite having a fully functional rhaT gene (see Figs. 1 and 2); anonfunctional rhaR gene in S. typhimurium C5 could explain these observations. CONCLUSIONS
We have isolated and sequenced the rhaT genes from S. typhimurium and E. coli. Expression of the rhaT genes from a X P L promoter resulted in an E. coli strain that could effect the uptake of 14C-labeledL-rhamnose and showed sugar-H+ symport activity. The rhaT gene maps between the sodA and rhaR genes at 87.9 min (3605 kb) on the E. coli chromosome restriction map (Koharaet al., 1987). It encodes an extremely hydrophobic protein of 344 amino acids; the S. typhimurium and E. coli RhaT proteins are 91% identical. TheRhaT protein sequence has been compared with protein sequences in computer databases, but no significant homology was detected. The model for RhaT in Fig. 9b was deduced from a
6932
The rhaTgenes of S. t:vphimurium and E. coli
hydropathy plot (Fig. 9a) which identified 10 clearly defined Dretzen, G., Bellard, M., Sassone-Corsi, P., and Chambon, P. (1981) Anal. Biochem. 1 1 2 , 295-298 hydrophobicregionsin the protein that could span a lipid bilayer. The orientation of RhaT in the membrane was de- Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochern. 132,6-13 Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 1 3 7 , 266ducedfrom the “positive inside” rule (von Heijne, 1986), 267 which predicts that hydrophilic loops in the cytoplasm gen- Henderson, P. J. F. (1990) J. Bioenerg. Biomembr. 2 2 , 525-569 erally have a net positive charge. A model of RhaT (Fig. 9) Henderson, P. J. F., and Macpherson, A. J. S. (1986) Methods Enzymol. 125,387-429 with the N and C terminus on the periplasmic face of the membrane conforms to the positive inside rule; all the hydro- Henderson, P. J. F., Giddens, R. A., and Jones-Mortimer, M.C. (1977) Biochem. J. 162, 309-320 philicloops in the cytoplasmhavea net positive charge, Hohn, B., and Collins, J. (1980) Gene (Amst.) 11,291-298 whereas all the loops in the periplasm have a net negative Ikemura, T. (1981) J. Mol. Biol. 157,389-409 charge or are uncharged.The proposed topology ofthe RhaT Johnson, K. R. (1990) Anal. Biochem. 190,170-174 protein is therefore completely differentto the 12 transmem- Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50,495-508 Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 5 7 , 105-132 brane domainmodels proposed for other sugar-H+ symporters (reviewed by Henderson, 1990). In addition, the amino acid Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, sequence of the RhaT proteinis not homologous to any other Cold Spring Harbor, NY protein. Thus the L-rhamnose-H+symporterrepresents a Messing, J. (1983) Methods Enzymol. 101, 20-78 novel type of sugar transport protein. Current is work focusing Miller, J. H. (1972) in Experiments inMolecular Genetics, Cold Spring on the overexpression of rhaT and the use of P-lactamase as Harbor Laboratory, Cold Spring Harbor, NY atopologicalreporter to define the structureof the RhaT Mott, J. E., Grant, R. A., Ho, Y.-S., and Platt, T. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,88-92 protein. Acknowledgment-We are grateful for the invaluable help of Dr. J. A. Ellis in the preparation of this manuscript.
REFERENCES AI-Zarban, S., Heffernan, L., Nishitani, J., Ransone, L., and Wilcox, G. (1984) J. Bacteriol. 158,603-608 Bachmann, B. J. (1983) Microbiol. Reu. 4 7 , 180-230 Badia, J., Baldoma, L., Aguilar, L., and Boronat, A. (1989) FEMS Microbiol. Lett. 6 5 , 253-257 Badia, J., Gimenez, R., Baldoma, L., Barnes, E., Fessner, W. D., and Aguilar, J. (1991) J. Bacteriol. 1 7 3 , 5144-5150 Bishop, M., and Rawlings, C. (1987) Nucleic Acid and Protein Sequence Analysis, IRL Press Ltd., Oxford, United Kingdom Casadaban, M. J., and Cohen, S. N. (1979) Proc. Natl. Acad. Sci. U. S. A . 76,4530-4533 Chen, Y.-M., Tobin, J. F., Zhu, Y., Schleif, R. F., and Lin, E. C. C. (1987) J. Bacteriol. 1 6 9 , 3712-3719 Cheshire, M. V. (1979) Nature and Origin of Carbohydrates in Soils, Academic Press, London Chiu, T. H., and Feingold, D. S. (1964) Biochim. Biophys. Acta 9 2 , 489-497 Chiu, T. H., and Feingold, D. S. (1965) Biochem. Biophys.Res. Commun. 19,511-516 Davis, E. O., Jones-Mortimer, M. C., and Henderson, P. J. F. (1984) J. Biol. Chem. 2 5 9 , 1520-1525 Devereux, J., Haeberli, P., and Smithies, 0.(1984) Nucleic Acids Res. 12,387-395
Muiry, J. A. R. (1989) The Bacterial Transport Systems for L-Rhamnose and L-Fucose. Ph.D. thesis, University of Cambridge, United Kingdom Power, J. (1967) Genetics 55,557-568 Platt, T. (1986) Annu. Reu. Biochem. 55,339-372 Remaut, E., Tsao, H., and Fiers, W. (1983) Gene (Amst.) 2 2 , 103113 Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980) J. Mol. Biol. 1 4 3 , 161-178 Sawada, H., and Takagi, Y. (1964~)Biochim. Biophys. Acta 9 2 , 2632 Steinman, H. M. (1978) J. Biol. Chem. 253,8708-8720 Southern, E. M. (1975) J. Mol. B i d . 98,503-517 Takagi, Y., and Sawada, H. (1964a) Biochim. Biophys. Acta 9 2 , 1017 Takagi, Y., and Sawada, H. (1964b) Biochim. Biophys. Acta 9 2 , 1825 Takagi, Y., and Avila, H. (1986) Nucleic Acids Res. 14,4577-4589 Thein, S. L., and Wallace, R. B. (1986) in Human Genetic Diseases, A Practical Approach (Davies, K.E., ed) pp. 33-50, IRL Press, Oxford, United Kingdom Tobin, J. F., and Schleif, R. F. (1987) J. Mol. Biol. 1 9 6 , 789-799 Tobin, J. F., and Schleif, R. F. (1990a) J. Mol. Biol. 2 11, 1-4 Tobin, J. F. and Schleif, R.F. (1990b) J. Mol. Biol. 2 1 1 , 75-89 Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci.U. S. A . 76,615-619 von Heijne, G. (1986) EMBO J. 5,3021-3027 Wilson, D. M., and Ajl, S. (1957a) J. Bacteriol. 73, 410-414 Wilson, D. M., and Ajl, S. (1957b) J. Bacteriol. 73,415-420
