170 , No. 9 o Identification of the Products and Nucleotide Sequences of Tw Regulatory Genes Involved in the Exogenous Induction of Phosphoglycerate Transport in Salmonella typhimurium YUN-LIU YANG , t DIANNE GOLDRICK , AND JEN-SHIANG HONG * Department of Cell Physiology , Boston Biomedical Research Institute , 20 Staniford Street , Boston , Massachusetts 02114 Received 25 January 1988/Accepted 8 June 1988 We describe the determination of the nucleotide sequence of two genes ( pgtB and pgtC ) contained within the 3.4-kilobase DNA segment sandwiched between the transporter gene , pgtP , and the regulatory gene , pgtA .
These two genes are involved in the regulation of expression of phosphoglycerate transport in Salmonella typhimurium .
The sequence indicates the presence of two large open reading frames , potentially coding for two polypeptides of 397 and 593 amino-acid-residues .
The two gene products were identified by using the bacteriophage T7 RNA polymerase-T7 promoter coupled system of Tabor and Richardson , and the observed apparent mass of 45 and 69 kilodaltons correlated well with the respective open reading frames .
The cellular location of these two polypeptides was directly determined , and the polypeptides were found to be associated with the membrane .
Although overall these polypeptides appear to be hydrophilic , there is one hydrophobic transmembrane segment in the smaller polypeptide and four such segments in the larger polypeptide which can account for their association with the membrane .
In the accompanying paper , we present genetic evidence that pgtB and pglC genes are involved in the induction of the pgtP expression by modulating derepressor activity .
Inc. and New England BioLabs , Inc. .
All chemicals were reagent grade and were obtained from commercial sources .
Plasmid DNA was prepared from cleared lysates by CsCI-ethidium bromide centrifugation as described by Davis et al. ( 1 ) .
The methods described by Maniatis et al. ( 8 ) were used for DNA manipulations .
Identification of gene products .
The phage T7 RNA poly-merase-T7 promoter coupled system of Tabor and Richardson ( 11 ) was used to identify gene products , except that labeling with [ 35S ] methionine was for 10 min instead of 5 min .
Sodium dodecyl sulfate ( SDS ) - polyacrylamide slab gels ( 12 % polyacryl-amide ) were used , and samples were boiled for 3 min prior to application .
A series of cross-linked cytochrome c 's were used as molecular mass standards .
Gels were run at a constant current of 30 mA for 4 h , stained with Coomassie brilliant blue , treated with enhancer ( Dupont , NEN Research Products ) , dried , and exposed to X-ray film for autoradiography .
Determination of cellular locations of pgt-encoded proteins .
Cells with [ 35S ] methionine-labeled , plasmid pJH583-en-coded proteins synthesized in the T7 RNA polymerase-T7 promoter coupled system ( 11 ) were fractionated as previously described ( 2 ) .
Membrane and supernatant fractions were examined by SDS-polyacrylamide gel electrophoresis .
We previously reported cloning , from Salmonella typhi-murium , a 14.4-kilobase ( kb ) DNA fragment carrying the genetic information for the exogenously induced phosphoglycerate transport system and its regulatory elements ( 5 ) .
We have identified and sequenced the transporter gene , pgtP , and a regulator gene , pgtA , contained in this fragment ( 2 , 13 ) .
These two genes are closely linked physically and are transcribed outwardly in opposite directions ( 2 , 13 ) .
In this paper , we describe the determination of the nucleotide sequence of the 3.4-kb DNA segment that separates the two genes and the identification of the products the contained genes encode and their cellular locations .
The pgtB and pgtC genes are involved in the induction of the pgtP expression ( 6 ) .
MATERIALS AND METHODS Bacterial strains and bacteriophages .
Strains used were Escherichia coli K-12 derivatives : BK9MDG ( F-thi hsdR hsdM endB metC ) ( 9 ) and JM103 ( thi pro leu endA ) .
Phages M13mpl8 and M13mpl9 were used for gene sequencing .
Plasmids used were derivatives of pBR322 , pT7-1 , or pT7-2 and were constructed by standard methodology .
Plasmid pGP1-2 was a gift of S. Tabor and C. C. Richardson of Harvard Medical School , Boston , Mass. .
Strains were grown in nutrient broth , YT , or medium E ( 12 ) containing 0.5 % succinate or 0.4 % 3-phos-phoglycerate .
When required , amino-acids were added to final concentrations of 30 to 50 , ug/ml .
Antibiotics were used at the following concentrations : ampicillin , 35 , ug/ml ; tetracycline , 15 , ug/ml ; and chloramphenicol , 30 jig/ml .
Restriction endonucleases and Bethesda Research Laboratories , DNA enzymes were from t Present address : Department of Microbiology , Shanghai Institute of Plant Physiology , Academia Sinica , Shanghai , People 's Republic of China .
RESULTS AND DISCUSSION Nucleotide sequence of pgtBC genes .
Figure 1A shows the restriction and genetic maps of plasmid pJH6 containing the genetic information necessary for inducible expression of the S. typhimurium phosphoglycerate transport system ( 2 ) .
The transporter gene , pgtP , is transcribed from right to left , and the activator gene , pgtA , is transcribed from left to right .
The nucleotide sequences of these two genes have previously been determined ( 2 , 13 ) .
We suspected that the region of 3.4 kb between the pgtA and pgtP genes contained genes involved in the regulation of pgtP gene expression , since removal of part of this segment altered the mode of expression of the pgtP gene ( 9 ) .
In order to facilitate studies on the regulatory role of genes encoded in this region , it was necessary to have structural information on the products of these genes .
Accordingly , the nucle-otide sequence of a series of deletion clones spanning this 3.4-kb ClaI-SalI segment was determined by the M13/di-deoxynucleotide-chain termination method ( 10 ) ( Fig. 1B and C ) and used to deduce an overall sequence ( Fig. 2 ) .
The sequence , when read in the ClaI-to-SalI direction ( Fig. 1 ) , contains two large open reading frames ( ORFs ) : one , from position 1 to position 1191 , could code for a polypeptide of 397 amino-acid-residues , and the other , from position 1417 to position 3195 , could encode a polypeptide of 593 amino-acid-residues .
The smaller of the two ORFs extends 82 base pairs ( bp ) beyond the HindIII site ( position 1104 ) and 50 bp beyond the BglII ( position 1136 ) site .
The larger ORF extends 61 bp beyond the SailI site ( position 3128 ) .
The sequence of this short segment had been previously determined and immediately precedes the pgtA gene ( 13 ) .
There are no ORFs of significant size in the opposite direction , i.e. , Sall to ClaI .
To determine if polypeptide products were encoded in the two ORFs identified in the sequence determined above , a series of restriction fragments were placed behind the phage T7 promoter of pT7-1 or pT7-2 at appropriate sites and the plasmid-encoded products were identified by the T7 RNA polymerase-T7 promoter coupled system of Tabor and Richardson ( 11 ) .
Plasmid pJH583 , carrying the 7.6-kb PstI sequence with the T7 promoter at the left , encoded , in addition to beta-lactamase , six peptides labeled as pB , B , A , pC , C , and E , with apparent masses of 69 , 65 , 46 , 45 , 41 , and 30 kilodaltons ( kDa ) , respectively ( Fig. 3A and B , lane 2 ) .
The 46-kDa polypeptide ( peptide A ) had been previously identified as the product of the pgtA gene ( 13 ) .
The 30-kDa polypeptide ( peptide E ) is the product of an adjacent gene downstream from pgtA and is nonessential for pgtP expression ( 13 ) .
The gene ( or genes ) encoding the 69-and 65-kDa products ( peptides pB and B , respectively ) was localized to the insert of the plasmid pJH591 ( Fig. 3A , lane 4 ) , since this plasmid expressed these two products and peptides A and E , but not pC or C .
It was further localized to the 3.0-kb HindIII-AsuII fragment carried on plasmid pJH598 , which was identical to the fragment on pJH591 , except for the deletion of the 1.2-kb AsuII-AsuII sequence , since pJH598 expressed peptides pB and but not A or C B , ( the band at 33 kDa was presumed to be a truncated pgtA gene product ) .
Since there was only one gene in the 3.0-kb HindIII-AsulI fragment large enough to encode a product of about 69 kDa , the presence of two products suggests that the smaller product , the 65-kDa polypeptide , might be derived from the larger one .
It is not known whether this reflects normal protein processing or nonspecific proteolytic degradation .
Whatever the reason , the results presented identified the larger of the two ORFs ( Fig .
as gene , 2 ) containing the pgtB which would extend from position 1417 near the HindIlI site to a site ( position 3195 ) slightly downstream from the Sall site ( Fig. 2 ) .
The gene encoding peptides pC and C was probably contained in the 3.0-kb PstI-HindIII fragment , since these two peptides were produced with pJH583 but not with pJH591 , which does not harbor this fragment .
This conclusion was further supported by the finding that plasmid pJH585 which harbored the 3.0-kb PstI-HindIII fragment encoded only one major peptide , a product of about 46 kDa ( Fig. 3 , lane 3 ) .
This peptide was slightly larger than the peptides pC and C observed with plasmid pJH583 ( lane 2 ) .
This suggests that peptides pC and C are encoded by the smaller of the two ORFs , which lies in the PstI-HindIII region but extends beyond the HindIlI site by 82 bp .
The 46-kDa peptide presumably was a fusion product resulting from expression of the pgtC gene ( up to the HindIII site ) plus some downstream sequence in the pT7-1 vector .
This interpretation is consistent with the finding that the ORF extends beyond the HindIII site by 82 bp .
Finally , the size of the ORF is compatible with a peptide product of 45 kDa .
As with peptides pB and B , peptide C might be derived from peptide pC via normal protein processing or nonspecific proteolytic degradation .
Plasmid pJH582 ( Fig. 3 , lane 6 ) carrying the same 7.6-kb PstI sequence as in plasmid pJH583 but with the insert in the opposite orientation , with the promoter at right , encoded only one product of 37 kDa , besides beta-lactamase .
The 37-kDa product had previously been identified as the product of the transporter gene pgtP ( 2 ) .
Figure 3C summarizes the locations of the pgtB and pgtC gene relative to the pgtPA genes and the apparent mass of the gene products observed .
pgtBC products are membrane bound .
To determine the cellular location of the pgtB and pgtC gene products , cells carrying plasmid pJH583 were labeled with [ 35S ] methionine in the T7 RNA polymerase-T7 promoter coupled system as above , sonicated , and centrifuged to separate the membrane fraction from the soluble fraction .
The products of the pgtB genes ( as well as the pgtA gene ) expressed by pJH583 were found mainly associated with the membrane fraction , and very little was in the soluble fractions ( Fig. 4 ) .
Repeated washing of the membranes with the buffer containing 0.4 M NaCl did not dissociate the proteins from the membranes .
429 A p , , , Sal Pst Ast 11 Mind III Cll Bg9111 p A B Pst 11 Cla c Sail Hind IIl FIG. 1 .
Restriction and genetic of plasmid pJH6 containing maps the pgt transport system , and the sequencing scheme for the 3.4-kb Clal-SalI region between pgtP and pgtA .
( A ) Locations of the transporter gene , pgtP ( P ) , and the activator gene , pgtA ( A ) , in the 7.6-kb PstI fragment of pJH6 , as determined previously ( 2 , 13 ) .
The locations of the two genes in the insert ( thick line ) within plasmid pJH6 are shown by the thin arrows , which also indicate the direction of their transcription .
( B and C ) Sequencing schemes for the 3.4-kb ClaI-SalI DNA .
To sequence the 1.3-kb Clal-HindlIl segment in the ClaI-to-HindIII direction , the 3.0-kb PstI-HindIII fragment was cloned into M13mpl9 at the PstI and HindIll sites ; to sequence the HindIII-SalI region in the HindIII-to-SalI direction , the 5.1-kb PstI-SalI fragment was cloned into M13mpl8 at the PstI and Sall sites ; to sequence the 3.4-kb SalI-ClaI in the SalI-to-Clal direction , the 5.1-kb SalI-PstI fragment was cloned into M13mpl9 at the SalI and PstI sites .
A series of unidirectional deletions from each clone was generated by controlled digestion with exonuclease III ( 4 ) .
Sequencing of overlapping deletion clones enabled a complete sequence to be determined .
All indicated regions were sequenced at least twice .
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] mMeethmibornianneelabeled , plasmid pJH583-encoded proteins synthesized in the T7 RNA polymerase-T7 promoter coupled system ( 11 ) were prepared sa1s l-described previously ( 2 ) and analyzed by SDS-polyacrylamide gel electrophoresis .
Membrane fractions containing approximately 12,000 cpm were used , except for the fraction in lane 6 , which had 20,000 cpm , and supernatant fractions , which cohtained at least 2,000 cpm .
Lane 1 , Whole cells lysed in the buffer ( 60 mM Tris hydrochloride [ pH 6.8 ] , l1o SDS , 1 % 2-mercaptoethanol , 10 % o glycerol ) ; lane 2 , supernatant after sonication ; lane 3 , membrane fractions after sonication ; lane 4 , supernatant of the first membrane washing ; lane 5 , supernatant of the second membrane washing ; lane 6 , membranes after two washings .
The location of the peptide bands are shown to the left of the gel .
LANE   l 2 pJi 3 3 565 591 s59 582 4 5 6 37 45 o 46 30 kD P C B A E FIG. 3 .
Identification ofpgt gene products and localization ofpgt genes .
( A ) Radioautogram of SDS-polyacrylamide gel electrophoresis of [ 35S ] methionine-labeled , plasmid-mediated proteins synthesized in the T7 RNA polymerase-T7 promoter coupled system .
Lane 1 , Plasmid pT7-1 ( control ) ; lanes 2 through 6 , plasmids carrying different pgt restriction fragments cloned into either pT7-1 or pT7-2 .
The location of the peptide bands are shown to the left of the gel .
Bla , Beta-lactamase ; KDa , kilodalton .
( B ) All plasmids were constructed by inserting the appropriate restriction fragments isolated from pJH6 ( arrowed bars ) into the vector pT7-1 or pT7-2 at appropriate sites in the polylinker behind the T7 promoter .
pJHS82 and pJH583 contained the 7.6-kb PstI fragment inserted at the PstI site of pT7-1 ; pJH585 contained 3-kb PstI-HindIII fragment at the PstI and HindIIl sites of pT7-1 ; pJH591 contained the 4.7-kb HindIII-PstI fragment at the HindtII and PstI sites of pT7-2 ; pJH598 was identical to pJH591 , except that the 1.2-kb AsuII fragment was deleted in the latter .
P , PstI ; S3 , Sau3A ; H , HindIII ; S , Sail ; A , AsuII .
( C ) Summary of the location , size , and direction of transcription of the genes identified .
The numbers above the bars indicate the apparent mass ( in kilodaltons [ kD ] ) of the gene products .
The letters below the bars specify the pgt genes .
We conclude that the pgtB and pgtC gene products are membrane-bound polypeptides .
Deduced amino-acid sequence and protein structure .
Examination of the deduced amino-acid sequences of PgtB and PgtC polypeptides indicates that these two polypeptides are very hydrophilic , as only 45 % of the total amino-acid-residues in each polypeptide contain nonpolar side chains .
This value is significantly lower than the figure ( 67 % ) for the hydrophobic membrane-bound transporter , PgtP ( 2 ) .
In spite of their overall hydrophilic nature , hydropathy analysis of these two polypeptides by the method of Kyte and Doolittle ( 7 ) , with a window of 19 , revealed the presence of potentially hydrophobic segments , each being at least 20 amino-acids residues long and therefore capable of spanning the membranes ( Fig. 5 ) .
For PgtB polypeptide , four such potential transmembrane segments are located at residues 19 to 44 , 263 to 282 , 487 to 510 , and 532 to 563 ; for the PgtC polypeptide , one such segment occUrs at residues 101 to 121 .
Since the cellular localizatioh experiment ( Fig. 4 ) indicated that the PgtB and PgtC polypeptides were associated with the membrane , we conclude that these two polypeptides most likely owe their association with the membrane to these transmembrane segmients .
The pgtBC genes preferentially use the degenerate codons found in the weakly expressed gene FIG. 3 .
Identification ofpgt gene products and localization ofpgt genes .
( A ) Radioautogram of SDS-polyacrylamide gel electrophoresis of [ 35S ] methionine-labeled , plasmid-mediated proteins synthesized in the T7 RNA polymerase-T7 promoter coupled system .
Lane 1 , Plasmid pT7-1 ( control ) ; lanes 2 through 6 , plasmids carrying different pgt restriction fragments cloned into either pT7-1 or pT7-2 .
The location of the peptide bands are shown to the left of the gel .
Bla , Beta-lactamase ; KDa , kilodalton .
( B ) All plasmids were constructed by inserting the appropriate restriction fragments isolated from pJH6 ( arrowed bars ) into the vector pT7-1 or pT7-2 at appropriate sites in the polylinker behind the T7 promoter .
pJHS82 and pJH583 contained the 7.6-kb PstI fragment inserted at the PstI site of pT7-1 ; pJH585 contained 3-kb PstI-HindIII fragment at the PstI and HindIIl sites of pT7-1 ; pJH591 contained the 4.7-kb HindIII-PstI fragment at the HindtII and PstI sites of pT7-2 ; pJH598 was identical to pJH591 , except that the 1.2-kb AsuII fragment was deleted in the latter .
P , PstI ; S3 , Sau3A ; H , HindIII ; S , Sail ; A , AsuII .
( C ) Summary of the location , size , and direction of transcription of the genes identified .
The numbers above the bars indicate the apparent mass ( in kilodaltons [ kD ] ) of the gene products .
The letters below the bars specify the pgt genes .
Hydropathy index of the PgtB and PgtC polypeptides .
This was determined by the method of Kyte and Doolittle ( 7 ) , with a window of 19 amino-acids .
The hydrophobic regions are above the horizontal zero line , whereas regions of relatively hydrophilic nature are below the zero line .
The amino-acid numbers are the same as in Fig. 2 .
according to the data compiled and analyzed by Grosjean and Fiers ( 3 ) , a feature characteristic of regulatory genes .
In particular , the so-called modulator codons , CGA/CGG/AGA for arginine codons and GGA/GGG for glycine codons , corresponding to minor tRNAs frequently used .
In the are accompanying genetic evidence is presented that paper , these two genes are in fact regulatory genes involved in the induction and signal transduction of inducer binding ( 6 ) .
We thank Brian Wallace for helpful discussion and comments on the manuscript .
This work 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 ,3 IONET National Computer Resource for Molecular Biology sponsored by the National Institutes of Health .
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