Nucleotide Sequence and Transcription Start Point of the Phosphoglycerate Transporter Gene of Salmonella typhimurium DIANNE GOLDRICK , GUAN-QIAO YU , t SHU-QIN JIANG , AND JEN-SHIANG HONG * Department of Cell Physiology , Boston Biomedical Research Institute , 20 Staniford Street , Boston , Massachusetts 02114 Received 7 December 1987/Accepted 27 April 1988 We identified the phosphoglycerate transporter gene of Salmonella typhimurium and its polypeptide product and determined the nucleotide sequence of the gene .
The predicted translation product was a protein of 406 amino-acid-residues and was extremely hydrophobic , a feature that is consistent with its role in membrane transport .
Hydropathy analysis suggested that there are eight transmembrane segments of at least 20 amino-acid-residues for the protein .
The transcription start point was mapped to lie at position -44 relative to the putative translational initiation start point .
Comparison of PgtP with UhpT and GlpT , the membrane-bound proteins involved in the transport of hexose-6-phosphate and glycerol-3-phosphate , respectively , revealed a very high degree of amino-acid sequence similarity among them , reflecting not only similar structures and functions among these polypeptides but also a common evolutionary origin for them .
Phosphoenolpyruvate , 2-phosphoglycerate , and 3-phos-phoglycerate ( 3-PG ) are transported into Salmonella typhi-murium via the inducible transport system pgt ( 14 ) .
Induction of transport occurs only when inducer is present extracellularly ; no induction occurs in the absence of inducer , even though phosphoglycerates are present intracellularly at millimolar concentrations ( 14 ) .
The pgt system has been cloned previously ( 11 ) .
In this report we describe the identification of the transporter gene pgtP , its polypeptide product and cellular location , the nucleotide sequence of the gene , and the transcription start point .
Expression of the pgtP gene requires a functional pgtA gene .
The nucleotide sequence of this gene , which encodes an activator protein , has been determined previously ( 21 ) .
MATERIALS AND METHODS Bacterial strains and phages .
The bacterial strains used in this study were all Escherichia coli K-12 derivatives : BK9MDG ( F-thi hsdR hsdM endB metC ) ( 13 ) and JM103 ( thi pro leu endA ) .
Phages M13mpl8 and M13mpl9 were used for gene sequencing .
The plasmids used in this study were derivatives of pBR322 , pACYC184 ( 2 ) , and pT7-1 or pT7-2 ( 17 ) and were constructed by standard methodologies .
Plasmid pGP1-2 was a gift from S. Tabor and C. C. Richardson ( Harvard Medical School , Boston , Mass. ) .
The bacterial strains were grown in nutrient broth , YT , or medium E ( 18 ) containing 0.5 % succinate or 0.4 % 3-PG .
When required , amino-acids were added to final concentrations of 30 to 50 , ug/ml .
The following antibiotics were used at the indicated concentrations : ampicillin , 35 , ug/ml ; tetracycline , 15 pxg/ml ; chloramphenicol , 30 pug/ml .
Restriction endonucleases and DNA enzymes were obtained from Bethesda Research Laboratories ( Gaithersburg , Md. ) and New England BioLabs , Inc. ( Beverly , Mass. ) .
All chemicals were reagent grade and were obtained from commercial sources .
t Present address : Department of Molecular Genetics , Shanghai Institute of Plant Physiology , Academia Sinica , Shanghai , People 's Republic of China .
Plasmid DNA was prepared from cleared lysates by CsCl-ethidium bromide centrifugation , as described by Davis et al. ( 4 ) .
The methods described by Maniatis et al. ( 12 ) were used for DNA manipulations .
Identification of gene products .
The phage T7 RNA poly-merase-T7 promoter coupled system of Tabor and Richardson ( 17 ) was used to identify gene products encoded by pgtP , with the exception that labeling with [ 35S ] methionine was done for 10 min instead of 5 min .
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis .
For sodium dodecyl sulfate-polyacrylamide gel electropho-resis , 12 % slab gels were used , and samples were boiled for 3 min prior to application .
A series of cross-linked cyto-chrome c 's were used as molecular weight standards .
Gels were run at 30 mA of constant current for 4 h , stained with Coomassie brilliant blue , treated with En3Hance ( New En-gland Nuclear Corp. , Boston , Mass. ) , dried , and exposed to X-ray film for autoradiography .
Strain CSR603 harboring particular plasmids was grown at 37 °C in minimal-medium ( medium E ) that contained 0.5 % succinate as a carbon source and that was supplemented-with thiamine , threonine , leucine , proline , arginine , and the appropriate antibiotics .
When growth reached the exponential phase , cells were collected by centrifugation and washed twice with and suspended in medium E to an optical density at 660 nm of 3.0 .
When induction of the pgt transport system was required , 0.2 % 3-PG was added to exponentially growing cells , and the cells were harvested 2 h later .
3-PG transport was measured as follows .
A portion ( 25 RIl ) of the cell suspension prepared as described above was incubated at 37 °C for 2 min , when 1 pAl of 250 mM glucose was added .
Fifteen seconds later , 1 ptl of 3-phos-pho [ 14C ] glycerate ( 2.3 mM ; specific activity , 55 mCi/mM ) was added , and incubation was continued for the desired time intervals .
To terminate transport , the mixture was diluted with 2 ml of medium E. Cells were collected on cellulose acetate membranes ( pore size , 0.45 , um ; Schleicher & Schuell , Inc. , Keene , N.H. ) and washed once with 2 ml of medium E. Membranes were dried and counted in toluene-based Omnifluor ( New England Nuclear ) in a liquid scintillation counter .
Determination of cellular locations of pgt proteins .
Cells ( 10 ml ) with [ 35S ] methionine-labeled , plasmid pJH587-encoded proteins synthesized in the T7 RNA polymerase-T7 promoter coupled system ( 17 ) were pelleted by centrifugation , suspended in 0.6 ml of 0.1 M Tris hydrochloride ( pH-7.6 ) , and subjected to sonication 4 times for 15 s each time , with a 1-min cooling interval between sonications .
After low-speed centrifugation ( 4,000 rpm in a rotor [ RC-5B ; Ivan Sorvall , Inc. , Norwalk , Conn. ] ) for 10 min to remove unbroken cells , the supernatant was layered on top of a 0.2-ml-thick cushion in a tube containing 0.25 M sucrose , 10 mM Tris hydrochloride , ( pH-7.6 ) , 50 mM KCl , 10 mM MgCl2 , and 0.01 % L-methionine and subjected to high-speed centrif-ugation ( 37,000 in SW50 .1 rotor ) for 2 h .
The rpm an membrane fraction was suspended in 0.2 ml of 0.1 M Tris hydrochloride ( pH-7.6 ) containing 0.4 M NaCl and then subjected to centrifugation as described above .
Washing of the membrane with the buffer containing 0.4 M NaCl was repeated once .
Transcription start point determination .
The site of transcription initiation was determined by the primer-extension method of Hu and Davidson ( 10 ) .
RNA was isolated from plasmid pJH6-harboring strain BK9MDG grown on minimal-medium containing 3-PG as the sole carbon and energy source by the method of Chen et al. ( 3 ) , with the modification that phenol replaced m-cresol .
The RNA was hybridized to single-stranded M13mpl8 carrying the 3.0-kilobase-pair ( kbp ) HindIII-PstI fragment , and the RNA-DNA complex was used as a template for the extension by T4 DNA polymerase of the 32P end-labeled hexadecameric primer 5 ' - TTCACCACACCCTTCA-3 ' ( corresponding to positions -157 to -142 in Fig. 2 ) that were annealed to it .
The reaction mixture was subjected to polyacrylamide gel electrophoresis with a control from which RNA was omitted and in parallel with corresponding sequence ladders , as described previously ( 10 ) .
342 RESULTS AND DISCUSSION Subcloning .
We previously described ( 11 ) the cloning of the phosphoglycerate transport system of S. typhimurium LT-2 into pBR322 .
The plasmid , pBR322-pgt2 , which contained a 14.4-kbp insert in pBR322 at the BamHI site , was found to be capable of conferring on E. coli K-12 the ability to transport 3-PG and to utilize 3-PG as the sole carbon and energy source , suggesting that the genes for the entire pgt system are contained in the 14.4-kbp insert ( 11 ) .
This plasmid was renamed pJH5 .
Subcloning of the 14.4-kbp insert was undertaken to localize genes of the pgt system .
A series of subclones was constructed from plasmid pJH5 , and their ability to confer H E p I s p H5 3 - PG Transport I I EH , B E B P HP HPS3 H S P '' S. .
I pJH5 PJH12 pJH13 ¬ pJ H14 pJH6 I pJH71 pJH72 pJH7 ¬ C ~ ~ pJH8 C pJH501 pJH502 pgtP pgtA FIG. 1 .
Construction ofpgt subclones from plasmid pJH5 .
Plasmids pJH12 , pJH13 , and pJH14 were derived from pJH5 by deleting EcoRI , HindIll , and Sall fragments , respectively .
Plasmid pJH6 was constructed by inserting the 7.6-kbp PstI fragment into pBR322 at the PstI site ; plasmid pJH71 was constructed by inserting the 5.1-kbp BamHI-HindIII fragment at the BamHI and HindIll sites of pBR322 ; and pJH72 was constructed by inserting the 7.6-kbp EcoRI-SalI fragment at the EcoRI and Sall sites of pBR322 .
Plasmid pJH501 was constructed by cloning the 4.5-kbp HindIII-SalI-PstI fragment from pJH6 into pACYC184 .
A 2.6-kbp Sall fragment was then removed from pJH501 , to yield pJH502 , which then contained only a 2.7-kbp SalI-PstI fragment of the original 7.6-kbp insert of pJH6 .
Construction of pJH7 and pJH8 are described in the text .
Restriction sites are abbreviated as follows : B , BamHI ; E , EcoRI ; H , HindIII ; P , PstI ; Hp , HpaI ; S , Sall ; S3 , Sau3A .
The broken bars indicate the locations of the pgtP and pgtA genes .
Transport phenotypes are indicated as inducible ( I ) , constitutive ( C ) , or nonexpression ( - ) FIG. 2 .
The nucleotide sequences of the pgtP gene and its 5 ' - flanking region and the deduced amino-acid sequence of PgtP polypeptide .
The antisense ( mRNA-like ) strand is shown .
The 3.0-kbp PstI-HindIII and the 3.0-kbp PstI-BglII fragments from pJH6 were cloned into M13mpl9 at the PstI and HindIII sites and the PstI and BamHI sites , respectively .
A series of deletions from each clone was generated by the method of Henikoff ( 9 ) .
Nucleotide sequences were determined by the M13-dideoxynucleotide chain-termination method ( 15 ) .
A complementary universal 15-bp oligodeoxynucleotide was used as primer , and [ 35S ] dATP was used to label the products .
Fractionation of the single-stranded DNA products of the primer elongation reaction was performed on 8 % polyacrylamide gels .
The numbers in parentheses indicate amino-acid-residues beginning from the N terminus .
TSP indicates the transcription start point ( see Fig. 3 ) .
The potential catabolite activator protein binding site is underlined with lines , with arrows , and the potential -10 and -35 promoter regions are indicated with thin lines .
3-PG transport was examined ( Fig. 1 ) .
Deletion of the 3.3-kbp EcoRI fragment from the left arm of pJH5 yielding pJH12 had no effect on the inducible expression of 3-PG transport , indicating that this fragment contains none of the information needed for 3-PG transport or its regulation .
However , deletion of the 9.0-kbp HindIII fragment from the left arm of pJH5 , yielding pJH13 , abolished 3-PG transport , indicating that the right half of the deleted fragment , namely , the 4.9-kbp EcoRI-IlindIII fragment , contains information that is required for 3-PG transport , regulation , or both .
Deletion of the 3.9-kbp Sall fragment from the right arm of pJH5 , yielding pJH14 , also abolished 3-PG transport .
When the 7.6-kbp Pstl fragment was subcloned into pBR322 at the PstI site , the resulting clone conferred inducible 3-PG transport .
However , subclones with inserts containing less than the full complement in pJH6 , such as pJH71 , pJH72 , pJH501 , or pJH502 , conferred no 3-PG transport .
Thus , it is evident that the genetic information necessary for inducible expression of 3-PG transport is contained in a 7.6-kbp PstI fragment .
We have previously identified ( 21 ) a regulator gene that is needed for the expression of 3-PG transport in this region ; this gene , pgtA , encodes an activator protein and is located on the right arm of the 7.6-kbp PstI fragment .
Constitutive transport conferred by pJH7 and pJH8 ( see below ) suggests that a regulatory sequence is localized in the region of Sau3A-HindIII-SalI-PstI .
Localization of the transporter gene pgtP .
To localize the structural gene ( s ) for the 3-PG transporter pgtP , subclones that were capable of conferring constitutive expression of 3-PG transport were sought .
For this purpose , plasmid pJH5 was partially digested with Sau3AI .
After electrophoresis fragments of 2 to 3 kbp in length were purified and ligated to pBR322 at the BamHI site .
The ligation mixture was then used to transform strain BK9MDG , and transformants that were able to utilize 3-PG as a source of carbon and energy were selected .
Several of the 3-PG + clones were grown on minimal succinate medium in the absence of inducer ( 3-PG ) and assayed for their ability to transport 3-PG .
The two smallest plasmids , pJH7 and pJH8 , which contained 2.2-and 3.3-kbp inserts , respectively , conferred a constitutive 3-PG transport ability ( Fig. 1 ) .
Restriction analysis indicated that the inserts in these two plasmids correspond to the 1.6-kbp left arm of the 7.6-kbp insert in pJH6 , as shown in Fig. 1 , plus a short segment contiguous to its left end .
For pJH7 this segment was 0.6 kbp , and for pJH8 it was 1.7 kbp .
Thus , the structural gene ( s ) for the 3-PG transporter pgtP is contained within the 1.6-kbp PstI-HpaI-HpaI-Sau3AI sequence .
The sequences to the left of the 1.6-kbp arm are not present in pJH6 and are therefore not required for pgt expression .
The location of the pgtA gene reported previously ( 21 ) is also indicated in Fig. 1 .
Located between the pgtP and pgtA genes are two pgt genes that are involved in the induction process of the pgtP gene expression ( unpublished data ) .
Sequencing of these genes is in progress .
Identification of the gene product .
To identify the pgtP gene product that is encoded in the insert of plasmid pJH7 and the transcriptional direction of the gene , the 1.8-kbp PstI-HindIII fragment of pJH7 was placed behind the phage T7 promoter of plasmids pT7-1 and pT7-2 at the PstI and HindIII sites , generating pJH586 and pJH587 , respectively , and the plasmid-encoded products were identified by the T7 RNA polymerase-T7 promoter coupled system of Tabor and Richardson ( 17 ) .
Plasmid pJH587 , which carried the insert with the T7 promoter proximal to the Hindlll site , encoded a rather diffused , 37-kilodalton product , but no product was observed with pJH586 , which carried the insert in the opposite orientation ( data not shown ) .
Thus , it is evident that the 37-kilodalton product is the 3-PG transporter encoded by pgtP and that the direction of transcription of the pgtP gene is from right to left in Fig. 1 .
The product of the pgtP gene is membrane bound .
To determine the cellular location of the pgtP gene product , cells carrying plasmid pJH587 were labeled with [ 355 ] methi-onine in the T7 RNA polymerase-T7 promoter coupled system as described above , sonicated , and centrifuged to separate the membrane fraction from the soluble fraction .
The transporter expressed by pJH587 was found to be associated with the membrane fraction , whereas the mature periplasmic P-lactamase was found in the soluble fraction , as expected ( data not shown ) .
Repeated washing of the membranes with buffer containing 0.4 M NaCl did not dissociate the proteins from the membranes ( data not shown ) .
Nucleotide sequence of pgtP gene .
The entire nucleotide sequence of the 3.0-kbp PstI-HindIII fragment containing the pgtP gene was determined by using the M13-dideoxynu-cleotide chain-termination method ( 15 ) .
Both strands were sequenced .
The sequences of the pgtP gene and its flanks are presented in Fig. 2 .
The pgtP gene is encoded in the sequence from positions 1 to 1218 with 406 amino-acid-residues .
A putative Shine-Dalgarno sequence AGGTG at -10 to -6 precedes the coding frame .
Sequences at positions -56 to -51 ( 5 ' - CACTCT ) and -78 to -72 ( 5 ' - TT GAATT ) are potential -10 and -35 promoter regions .
At 10 bp upstream from these sequences ( positions -106 to -89 ) is the 18-bp sequence 5 ' - TGAGTCAATTTTGACACA-3 ' , which is a potential catabolite activator protein-binding site .
The transcription start point of the pgtP gene was determined by the examination of RNA transcripts by hybridization mapping of mRNA isolated from the pJH6-carrying strain grown on 3-PG .
We used T4 DNA polymerase to extend a radiolabeled primer annealed to a single-stranded DNA template in the presence of mRNA ( 10 ) .
Because of the inability of T4 DNA polymerase to displace a RNA hybridized to DNA , primer-extension should stop at the 5 ' terminus of the hybridized mRNA , with the 3 ' end of the growing DNA chain thereby marking its position .
The site of the first termination of primer-extension in the presence of mRNA was at position -44 , whereas no termination was observed in that region in the absence of added mRNA ( Fig. 3 ) .
Thus , the transcription start point of pgtP probably lies at position -44 relative to the translational initiation start point .
Significant primer-extension occurred beyond position -44 , probably because of the existence of partially degraded mRNA molecules and possibly because of limited mRNA displacement by T4 DNA polymerase .
Amino acid sequence and protein structure .
Examination of the deduced amino-acid sequence of the PgtP polypeptide indicates that the polypeptide is extremely hydrophobic , because 67 % of its total amino-acid-residues contain nonpolar side chains .
The PgtP protein appears to be highly positively charged , with a total of 35 arginine plus lysine residues but only 17 aspartic plus glutamic-acid residues .
The calculated Mr of 44,800 for PgtP is larger than the apparent Mr of 37,000 observed on sodium dodecyl ` sulfate-polyacrylamide gel electrophoresis .
Such anomalous behavior can be attributed to the high degree of hydrophobicity of the protein and is apparently characteristic of hydrophobic membrane transport proteins , such as the lactose permease ( 1 ) and glucose-6-phosphate carrier of E. coli ( 7 , 19 ) .
Examination of the PgtP protein for potential transmembrane method of Engelman et al. ( 6 ) indicated the regions by the presence of eight such regions ( Fig. 4 ) , suggesting that the polypeptide traverses the membrane 8 times .
The amino terminus , which is relatively hydrophilic in composition ( Fig. 4 ) , is assumed to lie in the cytoplasm .
Of the 61 codons , 2 ( AGG and GAC ) were not used in the pgtP gene .
From the analysis of Grosjean and Fiers ( 8 ) , the pgtP gene appears to preferentially use the degenerate codons found in the weakly expressed genes and has a codon preference statistic of 0.38 , which was calculated as described by Sharp and Li ( 16 ) .
This suggests that the pgtP gene belongs to a group of genes with a low codon bias .
Amino acid sequence homology with components of hexose-6-phosphate and glycerol-3-phosphate transport systems .
The PgtP polypeptide ( 406 amino-acid-residues ) has a high degree of amino-acid sequence similarity with the UhpT polypeptide amino ( 463 acid residues ) , the membrane-bound transporter for the hexose-6-phosphate transport system , which , like the pgt system , is expressed only in the presence of exogenous inducers ( 7 , 19 ) ; with the UhpC polypeptide ( 219 amino-acid-residues ) , a membrane-bound regulatory protein that is presumably involved in inducer recognition and binding in the regulation of uhpT expression and which has a high degree of sequence homology with UhpT ( 7 ) ; and with the GlpT polypeptide ( 452 amino-acid-residues ) , a membrane-bound glycerol-3-phosphate transporter ( 5 ) .
Allowing for a few small gaps and a misalignment by one amino-acid-residue at the N terminus , the sequences of PgtP and UhpT aligned well , with identical amino-acid-residues occupying 31 % of the positions ( Fig. 5 ) .
Approximately the same degree of sequence similarity was observed between PgtP and UhpC polypeptides ( comparison not shown ) .
The sequences of PgtP and GlpT also aligned well , with identical amino-acid-residues occupying 37 % of the positions .
As expected from the pairwise similarity observed between GlpT here and that and UhpT reported previously ( 5 , 7 ) , a high degree of similarity was observed among GlpT , PgtP , and UhpT polypeptides ; 17 % of the 406 positions were occupied by identical amino-acids ( Fig. 5 ) .
In addition to the sequence similarities among GlpT , PgtP , and UhpT , the hydropathy profiles of these polypeptides were also extremely similar ( data not shown ) .
Eiglmeier et al. ( 5 ) have noted previously that the hydropathy profiles between GlpT and UhpT are similar .
These observations reflect not only similar structures and functions among these polypeptides but also a common evolutionary origin for them .
3423 pgtP GENE OF S. TYPHIMURIUM COCTCAiA * CtC1 ACTCAT CCGATC OCTTWTA TMTTMMAA TC = AGT4A 2 !
o-45 ZO CW = -30 Q -250 AAAACTACOrTATC 77ThIW `` CSCI.UACACMATTl `` TA SJM U TAAW -20,0 -1 SQ - t -100 TGACTcATr VCMcTOrrrAT ATTTTACCTT-SC5 CAMCT C ACtcATCA TC AMT 1TA ACA ATA TL MAA ACA GCC CM TOG CCG CAT AM GTC CCA CCOCI AGM CTC CAC CCC ACA TAT OCT CIA TAT COT Net Lou Tbr Ile Lou Lys Thr Gly Gln Sr Ala His Lys Val Pro Pro Glu Lys Vol 01G Ala Tbr Tyr Cly Ag Tyr ArL f ' * ( 20 ) ATA CAG CCA TSC TTA ACTGSTA TTT CYC CA TAT CTC GCT TAC TAT ATA CTC MA MC MC TIC ACA OT7 TaLOGA CT TAT Gln Ile Ala Lou Lou Sor Va1 Pb .
Lou Gly Tyr Lou Ala Tyr 17rIle Vol Ar Loa Ang Pn.o br Lou Sor Tb : Pto Tyr ( 40 ) 2090 TTA MA GAG CM TIC CAT CTC AGC GCr ACC CAA ATC21C CTC CIC AMT AMC ?
CT AOT COT ATT CCT TAC OCA ATC MT AM Lou Lye C1u GIn Pb .
Aep Lou Ser Ale Thr Clo Ile Cly Lou Lou Sor Sor Cyc Net Lou Ile Ale Tyr Gly Ile Sor Ly ( 60 ) ( 60 ) 2SQ 300 OGC MTA AGC AMT CTC CCC CAT MA CCC AMC CCA AM OTC TIC ATC CCI TCC OCT CSC CTA CTC OCG CCI ALT OTT MC Gly Vol Net Sor Sor Lou Ale Asp Lye Ala Sor fto Lye Vol he Net Ale Cye Cly Lou Val Lou Cye Ale Ile TVo Le ( 100 ) CTT COC C2C OCA TTC MCT A G C CC ATA TV ?
CCC GCT CSC GTCGC TIC M CCC CO TO CM GGCC ATO OS Vol Gly Lou Gly bo Sor : Sor Ala te Trp II1Pb .
Ala Ala Lou Vol Vol Pbo Lse Cly Lou Pb CIs Gly Net Arg Lvg ( 120 ) CCCCSC CT ?
TAT TAC TATTCC MA CMG CCT CCT CIT COG GAG COC CI CIC CTA CCC CCC TIC TO MAT ATC TOO CAT AC * 59 Pro Lou Vol Tyr Tyr Tyr Cys Lye Lou Vol Pro Avg Avg Glu Avg Gly Avg Vol Cly Al. .
Pb Tip Le Ile So : IUs L ( 140 ) ( 160 ) 509 35 GCT CCC CCC OCG AC CC ATC GCC CCG GCI CC TT CC ATA TIC CC CMC CLA CAC TO CAA LC ACC MOT TAT Val Gly Cly Gly Ile Val Ale Pro Ile Vol Cly Ale Al. .
Ile Lou Cly Sor CGI lie rp G1s Mm Ala S Tr ( 190 ) 60Q ATC COT CCC CCC CTC CTC CCC CTC AT ?
TT GCc TA AT ?
GCT CSC CCC TIC OCA AM GOT TOG MGC CC AMLGOT GIl Ile Vol fro Ala Cye Val Ale Val Ile Pb .
Ale Lou Ile Va1 Lou Val The Cly Lye Sly So : fro Arg Lye Lop Gly 1s (200)-650-709 CCC TCC CT CM CAC A2C AM CCC CMGCA GMMCTC TA CSC AM ACIG C T M CAC AM cCI cCI CA M ATO LOG fro Sor Lou Glu Gln Net Net Pro Glu Glu Lye Vol Val Lou Lye Tbr Lye ALo Tbr Ale Lye Ale tro Glu Lo Net Sor ( 220 ) ( 240 ) CCA MCC CMA ATC TIC SCT ACN TAT CTC CTG CCC MT AM AAT CCC TCC TAT LT TOG CM TCG CAT COCT6C CCC TAT ATO Ala Trp GlC Ile Ph-Cye Tbr Tyr Val Vol Arg ALe Lye ALe Ala Trp Tyr Ile go : Lou Val ALp Vol Pb Val Tyr Net ( 260 ) 850 OTC CIG mT GGC A2C ATT AGC 2CC TIC CCT ATC TAT CTC STC ACA CA AMA CAC m TCA AM GAL CMU ATO MC OTC0C0 Vol Avg Pb .
Glu Net Ile Ser Trp Lou Pro Ile Tyr Lou Lou Thr V.l Lye Hli Pb .
Sor Lye Clu Glo net BSr Val Ala (280)-909-959 mT CT OT TIC CM TOG GCG CCC LTI CCC TCC ACG CTL CCC CA CCC 2CC CCT TCA CAT MAA CM IT AMC GCC COC mAA Pb .
Clu Trp Ale AlJ Ile Pro Ser Thr Lou Lou Ale Gly Trp Lou Sor ALp Lys Lou Pb .
Lye Gly Arg Avg ( 300 ) (320)-1009-1059 A2C COc TTA CCC OTG ATT mCC TCS CC CSC ATT TT ?
COTGT M LT CCA TAC TOG AM AMT CM TCV TIC C ATO GTA Net Pro Lou Ale Het Ile Cye Not Al Lou Ile Pb .
Val Cye Lou Ile Gly Tyt Trp Ly S : r Glu So : Lou Lou Net Val ( 340 ) ACG AT ?
TC CCC CCC LTT CTA CGT ?
CT CM ATT TAC CCC CCC CA IIC CT COCG CC CCA CACGM A ATO CM GTA CCC Thr Ile Pbe Ala Al. .
Io Vol Cly Cye Lou Ioe Tyr Vol Pro Gl Pb .
Leu Ale Ser Vol GI Thbr Net Clu Io Vol fto ( 360 ) 11S 120 AGC TV ?
CC CGA CCL * CC GCC CTC CCT 7TI CIT CGG mTC ATG MC TAT AT ?
COC MCC MC C Tc m Ser Pb .
Vol GCy Ser Ala Voi Cly Lou Arg Giy Phb Net Ser Tyr II .
Seo Lou Gly Tb : Sor Lou Pb .
( 380 ) (400)-1250-130 QCCC 2CT TOGTcG7ATAMAAcrCCCTCGTAcGGwTT CTTICTCATGGC .
T C i CACC V ?
CCGCCTrCC2TCAICT Cy DEn 1372 LCTCGCCACCCACGACMTCCGTACATMTCAGACTCACTCCA FIG. 2 .
The nucleotide sequences of the pgtP gene and its 5 ' - flanking region and the deduced amino-acid sequence of PgtP polypeptide .
The antisense ( mRNA-like ) strand is shown .
The 3.0-kbp PstI-HindIII and the 3.0-kbp PstI-BglII fragments from pJH6 were cloned into M13mpl9 at the PstI and HindIII sites and the PstI and BamHI sites , respectively .
A series of deletions from each clone was generated by the method of Henikoff ( 9 ) .
Nucleotide sequences were determined by the M13-dideoxynucleotide chain-termination method ( 15 ) .
A complementary universal 15-bp oligodeoxynucleotide was used as primer , and [ 35S ] dATP was used to label the products .
Fractionation of the single-stranded DNA products of the primer elongation reaction was performed on 8 % polyacrylamide gels .
The numbers in parentheses indicate amino-acid-residues beginning from the N terminus .
TSP indicates the transcription start point ( see Fig. 3 ) .
The potential catabolite activator protein binding site is underlined with lines , with arrows , and the potential -10 and -35 promoter regions are indicated with thin lines .
Mapping of the transcription start point of the pgtP gene .
The transcription start point was identified by primer-extension analysis with T4 DNA polymerase , as described in the text .
Lane 1 , Primer extension in the absence of mRNA ; lane 2 , primer-extension in the presence of mRNA ; lanes 3 to 6 , sequence ladders made by the dideoxynucleotide sequencing method with the same primer ( unlabeled ) .
Part of the nucleotide sequence deduced from the sequencing lanes is shown on the right , and the shortest extended primer segment is indicated with an asterisk 3425 VOL .
170 , 1988 pgtP GENE OF S. TYPHIMURIUM ' - I - I Pgt P D 20-cbto 0 I 0 c 0 co1 C : CI -- 20 - ii p I a - ) 24I 0-Iu Il II 300 360 180 240 Residue 4 60 120 FIG. 4 .
Analysis of the 3-PG transporrter amino-acid sequence for potential transmembrane regions .
This was determined by the method of Engelman et al. ( 6 ) by using a window of 20 amino-acids .
The potential transmembrane hydroph ( ) bic regions are above the horizontal zero line , whereas regions writh a relatively hydrophilic nature are below the zero line .
The amin ( oacid numbers are the same as those in Fig. 2 .
65 MLSIIrlKPAPNKlAULF PIU ATIORLU I IS a1 A fFiALAMPTLV3QCISRDL 181 tl * lI II t o1 I I 1 8 NLTSLITOQSANKIP I Q l glt-s ^ es , t AN1S1L LSTITLK3QIDL5ATQ NLAILNQYUKPnL & iYLtUtKxnPf?d3tJYtiJW T3dL pIAQDMI STTGLSNTQ GJpT 65 66 Igt ?
UbpT 129 125 - AL3 Ies $ f03 , * xISIS , .
LS3CX ZIIIPPVCMSL * Ahlh1 cIVNtt CLCGS3AIV ----- IFAALVV L SOLOTq FG LsT Y3t TtK Pr S C3ILG3ASNC3G3OYIATTLPLAI 131 c ; eNO1WP ' ?
CRTNYUVWSQK I5TS T SPPLLILLO3AVIWDVWAALY3 192 I SYAISVPLVG-ACK T F C C LpAlSWlAAFILCSQNQQSAS Ise L Tj3?GO3C8TR3 -1 * S3TIWT ; la LCG 1 z fL3ACAACTALIGANTLIDCNV -- 191 q-i ANNRDT?Q3 -- CULIIS3TKWD1tDDTN -- Q -- ZLTAI TXI 1 inLvYIOG3IXCDOLtILIQNN?IEKVVLKTK?TFI34I -- .
D TUSTDNTK 1iVI PAICAILVALW T * TY?CTAV ; - ; GMFIYFSI ; 2 248-251-254 i AIAW KCIOIV T L KPIALDXS WATYL AG ?? L 313 MVAtl SYLDiII * 34IS 4l LTVKU SR3QNIIL AhAhhItt I ~ l1V ~ ` LCCANJUDTWIQZLKLSKAVAIQCOTL GALV 316 319 lI24iJ MNPAGNPTVDNICNIV Ih I GLHALILA K SZLLNVT -- IAASAI1 TY SVQ * TJ IS YS HASN!TL -- ASLLWA GVGA GVAAVCGY VtT ATCVIIN VTSATSI LISjS ŜLANSCHA CLSI 37S 379 LA LVACLAL IATLG 381 443 1 ; t P. II SLCTSLIC 406 II 7 pSFAKLCLGNI ADGSTVYGtTGVAGTIAALD I AAIGCI CLtlAIVAVM 446 IAul 452 QLLQZRNGG h SRKSRR3t R GGLTVA 463 FIG. 5 .
Alignment of amino-acid sequences of PgtP , UhpT , and GlpT polypeptides .
Identical positions between a pair of polypeptides are indicated by double dots , and those among the three polypeptides are boxed .
Gaps were introduced to optimize the alignment , which was generated as described by Wilbur and Lipman ( 20 ) We thank Brian Wallace for helpful discussions and comments on the manuscript .
This study was supported by Public Health Service grant GM31836 from the National Institute of General Medical Sciences .
Computer resources used to carry out our studies were provided by the National Institutes of Health-sponsored BIONET National Computer Resource for Molecular Biology .
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