Topological Analysis of Glucosyltransferase GtrV of Shigella flexneri by a Dual Reporter System and Identification of a Unique Reentrant Loop
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m401316200
ISSN1083-351X
AutoresHaralambos Korres, Naresh K. Verma,
Tópico(s)RNA and protein synthesis mechanisms
ResumoLipopolysaccharide, particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-Antigen modification is mediated by glucosyltransferase genes (gtr) encoded by temperate serotype-converting bacteriophages. The gtrV gene encodes the GtrV glucosyltransferase, an integral membrane protein that catalyzes the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a. Analysis of the GtrV amino acid sequence using computer prediction programs indicated that GtrV had 9–11 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of GtrV to a dual reporter system composed of alkaline phosphatase and β-galactosidase. Sandwiched GtrV-PhoA/LacZ fusions were also constructed at predetermined positions. The enzyme activities of cells with the GtrV-PhoA/LacZ fusions and the particular location of the fusions in the gtrV indicated that GtrV has nine transmembrane segments and one large N-terminal periplasmic loop with the N and C termini located on the cytoplasmic and periplasmic sides of the membrane, respectively. The existence of a unique reentrant loop was discovered after transmembrane segment IV, a feature not documented in other bacterial glycosyltransferases. Its potential role in mediating serotype conversion in S. flexneri is discussed. Lipopolysaccharide, particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-Antigen modification is mediated by glucosyltransferase genes (gtr) encoded by temperate serotype-converting bacteriophages. The gtrV gene encodes the GtrV glucosyltransferase, an integral membrane protein that catalyzes the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a. Analysis of the GtrV amino acid sequence using computer prediction programs indicated that GtrV had 9–11 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of GtrV to a dual reporter system composed of alkaline phosphatase and β-galactosidase. Sandwiched GtrV-PhoA/LacZ fusions were also constructed at predetermined positions. The enzyme activities of cells with the GtrV-PhoA/LacZ fusions and the particular location of the fusions in the gtrV indicated that GtrV has nine transmembrane segments and one large N-terminal periplasmic loop with the N and C termini located on the cytoplasmic and periplasmic sides of the membrane, respectively. The existence of a unique reentrant loop was discovered after transmembrane segment IV, a feature not documented in other bacterial glycosyltransferases. Its potential role in mediating serotype conversion in S. flexneri is discussed. Bacillary dysentery or shigellosis is caused by the Shigella bacterium, which is of major concern in overcrowded areas of the developing world (1WHOWorld Health Forum. 1997; 18: 1-8PubMed Google Scholar, 2Kotloff K.L. Winickoff J.P. Ivanoff B. Clemens J.D. Swerdlow D.L. Sansonetti P.J. Adak G.K. Levine M.M. Bull. W. H. O. 1999; 77: 651-666PubMed Google Scholar). A major surface component of Shigella flexneri that contributes to pathogenesis is the lipopolysaccharide (3Okada N. Sasakawa C. Tobe T. Yamada M. Nagai S. Talukder K.A. Komatsu K. Kanegasaki Yoshikawa M. Mol. Microbiol. 1991; 5: 187-195Crossref PubMed Scopus (69) Google Scholar, 4Brahmbhatt H.N. Lindberg A.A. Timmis K.N. Sansonetti P.J. Pathogenesis of Shigellosis. Springer-Verlag, Berlin1992: 45-64Google Scholar, 5Sandlin R.C. Goldberg M.B. Maurelli A.T. Mol. Microbiol. 1996; 22: 63-73Crossref PubMed Scopus (73) Google Scholar, 6Sansonetti P.J. Jpn. J. Med. Sci. Biol. 1998; 51: 69-80Crossref Google Scholar, 7Sansonetti P.J. FEMS Microbiol. Lett. 2001; 25: 3-14Google Scholar). The lipopolysaccharide consists of three segments, lipid A, core polysaccharide, and the O-antigen (8Makela P.H. Stocker B.A.D. Rietschel E.T. Chemistry of Endotoxin. 1. Elsevier Science Publishers B. V., Amsterdam1984: 419Google Scholar). The O-antigen chain is composed of repeating sugar subunits that vary in their composition and so contribute to serotype diversity. For example, the different serotypes of S. flexneri arise from the addition of glucosyl or O-acetyl residues to the repeating sugar subunits. This specific attachment is thought to be mediated by glucosyl- and O-acetyltransferases (9Huan P.T. Bastin D.A. Whittle B.L. Lindberg A.A. Verma N.K. Gene. 1997; 195: 217-227Crossref PubMed Scopus (54) Google Scholar, 10Allison G.E. Verma N.K. Trends Microbiol. 2000; 8: 17-23Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 11Kenne L. Lindberg B. Petesson K. Katzenellenbogen E. Romanowska E. Eur. J. Biochem. 1978; 91: 279-284Crossref PubMed Scopus (91) Google Scholar, 12Verma N.K. Verma D.K. Huan P.T. Lindberg A.A. Gene (Amst.). 1993; 129: 99-101Crossref PubMed Scopus (28) Google Scholar). The glucosyltransferase genes gtrV, gtrI, gtrIV, and gtrX of S. flexneri serotypes 5a, 1a, 4a, and X, respectively, have been isolated and characterized (10Allison G.E. Verma N.K. Trends Microbiol. 2000; 8: 17-23Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 13Guan S. Bastin D.A. Verma N.K. Microbiology. 1999; 145: 1263-1273Crossref PubMed Scopus (104) Google Scholar, 14Mavris M. Manning P.A. Morona R. Mol. Microbiol. 1997; 26: 939-950Crossref PubMed Scopus (91) Google Scholar). The only glucosyltransferase genes that exhibit some degree of homology at the amino acid and nucleotide level are gtrV and gtrX (35.7% identity and 61.3% similarity). The exact model of GtrV operation is not known, although two other proteins, GtrA and GtrB, are thought to interact with GtrV mediating in serotype conversion (13Guan S. Bastin D.A. Verma N.K. Microbiology. 1999; 145: 1263-1273Crossref PubMed Scopus (104) Google Scholar, 14Mavris M. Manning P.A. Morona R. Mol. Microbiol. 1997; 26: 939-950Crossref PubMed Scopus (91) Google Scholar, 15Adhikari P. Allison G. Whittle B. Verma N.K. J. Bacteriol. 1999; 181: 4711-4718Crossref PubMed Google Scholar). This paper deals with a glucosyltransferase, GtrV, that catalyzes the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II of the O-antigen unit (10Allison G.E. Verma N.K. Trends Microbiol. 2000; 8: 17-23Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). According to hydropathy data, distribution of charged residues and possible turns, this protein consists of 9–11 hydrophobic segments, with the N terminus in the cytoplasm and the C terminus in the periplasm. The validity of the proposed model is examined by a genetic approach, which involves the construction of fusion proteins between reporter enzymes and GtrV. This study utilizes a newly developed dual reporter system consisting of alkaline phosphatase (phoA) which is in-frame with the β-galactosidase α fragment (lacZα) (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar). The signal sequence of PhoA 1The abbreviations used are: PhoA, alkaline phosphatase; LacZ, β-galactosidase; AP, alkaline phosphatase; BG, β-galactosidase; NAR, normalized activity ratio; Cm, chloramphenicol; GT, glycosyltransferase. 1The abbreviations used are: PhoA, alkaline phosphatase; LacZ, β-galactosidase; AP, alkaline phosphatase; BG, β-galactosidase; NAR, normalized activity ratio; Cm, chloramphenicol; GT, glycosyltransferase. can be replaced by export signals derived from other proteins such as GtrV, producing a chimeric protein that can be used to report cellular location according to the location of the protein domain under investigation (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar, 18van Geest M. Lolkema J.S. Microbiol. Mol. Biol. Rev. 2000; 64: 13-33Crossref PubMed Scopus (163) Google Scholar). Alkaline phosphatase (AP) is active only when located in the periplasm. This is based on the assumption that in the periplasm the mature part of PhoA is oxidized. The cysteine residues form disulfide bridges that enable the correct folding of PhoA. The enzyme becomes active after dimer formation is complete. The process of folding, and assembly of PhoA occurs only after export to the periplasm because various factors prevent the formation of disulfide bonds in the cytoplasm (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar, 18van Geest M. Lolkema J.S. Microbiol. Mol. Biol. Rev. 2000; 64: 13-33Crossref PubMed Scopus (163) Google Scholar, 19Manoil C. Methods Cell Biol. 1991; 34: 61-75Crossref PubMed Scopus (196) Google Scholar, 20Sugiyama J.E. Mahmoodian S. Jacobson G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9603-9607Crossref PubMed Scopus (78) Google Scholar, 21Boyd D. White S.H. Membrane Protein Structure: Experimental Approaches. Oxford University Press, Oxford1994: 144-163Crossref Google Scholar, 22Manoil C. J. Bacteriol. 1990; 172: 1035-1042Crossref PubMed Google Scholar, 23Manoil C. Beckwith J. Science. 1986; 233: 1403-1408Crossref PubMed Scopus (314) Google Scholar, 24Sarsero J.P. Pittard A.J. J. Bacteriol. 1995; 177: 297-306Crossref PubMed Google Scholar, 25Daniels C. Vindurampulle C. Morona R. Mol. Microbiol. 1998; 28: 1211-1222Crossref PubMed Scopus (157) Google Scholar). In contrast to AP, β-galactosidase (BG) is active in the cytoplasm. The enzyme is inactive in the periplasmic space since its proper folding is prevented by becoming trapped in the membrane crossing to the periplasm. Also, α-complementation should occur only when the enzyme (lacZα) is accessible to cytoplasmic located ω-fragment (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar, 18van Geest M. Lolkema J.S. Microbiol. Mol. Biol. Rev. 2000; 64: 13-33Crossref PubMed Scopus (163) Google Scholar, 19Manoil C. Methods Cell Biol. 1991; 34: 61-75Crossref PubMed Scopus (196) Google Scholar, 21Boyd D. White S.H. Membrane Protein Structure: Experimental Approaches. Oxford University Press, Oxford1994: 144-163Crossref Google Scholar, 24Sarsero J.P. Pittard A.J. J. Bacteriol. 1995; 177: 297-306Crossref PubMed Google Scholar, 25Daniels C. Vindurampulle C. Morona R. Mol. Microbiol. 1998; 28: 1211-1222Crossref PubMed Scopus (157) Google Scholar). Thus fusions to periplasmic sites will be inactive whereas fusions to cytoplasmic domains will be active. Alexeyev et al. (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar) report that by using this novel approach of PhoA/LacZ dual reporters, BG and AP activities can be measured at the same point simultaneously without resorting to genetic recombination to switch fusions (17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar). The normalized activity ratios (NAR) can correct for variable expression. Thus, determination of protein synthesis rates is not necessary. The system provides easily interpretable information about subcellular localization of the reporter (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar). Furthermore, α complementation is based upon the availability of ω-fragment, which is solely confined to the cytoplasm and not the periplasm, rendering the enzyme inactive if located in the periplasm. In this way the formation of toxic aggregates in the periplasm is eliminated. Finally, the use of dual indicator plates in conjunction with these reporters discriminates between non-informative fusions (white), cytoplasmic fusions (red), and periplasmic fusions (blue or purple) (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar). In this paper we report a topology model of GtrV based on analysis of data from computer prediction models and fusion constructs consisting of gtrV-phoA/lacZ and gtrV-phoA/lacZ-gtrV sandwich fusions. The model suggests that GtrV consists of nine transmembrane segments and a large N-terminal periplasmic hydrophilic loop. The N terminus is located in the cytoplasm whereas the C terminus is located in the periplasm. The model also reveals that GtrV contains a unique reentrant loop after transmembrane segment IV. To the best of our knowledge, this is the first report showing the presence of a reentrant loop in bacterial glycosyl transferases. Bacterial Strains and Growth Conditions—All bacterial strains used in this study are derivatives of Escherichia coli K-12. Their particular genotypes are described in Table I. The bacterial strains were grown aerobically at 37 °C in Luria broth (LB). LB agar plates were prepared as described previously (26Sambrook J. Russell W.D. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY2001: A2.2-A2.5Google Scholar). Ampicillin and chloramphenicol (Cm) were added to liquid and solid media at 100 and 30 μg/ml, respectively. The PhoA and LacZ colony phenotypes were identified on agar plates containing 1.5% Bacto-agar, 1% Bacto-tryptone, 0.5% yeast extract, 0.5% NaCl, 5-bromo-4-chloro-3-inolyl phosphate disodium salt (Sigma; X-phos 80 μg/ml), 6-chloro-3-indolyl-β-d-galactoside (Research Organics Red-Gal; 100 μg/ml), 1 mm isopropyl-1-thio-β-d-galactopyranoside, 80 mm K2HPO4 (pH 7.0), and 30 μg/ml Cm.Table IBacterial strains and plasmids used in this study Plasmids carrying gtrV-phoA/lacZ fusions and gtrV-phoA/lacZ-gtrV sandwich fusions are shown in Table III. kb, kilobase(s).Strain or plasmidRelevant characteristicsaThe genetic nomenclature is that described by Bachman (73). Allele numbers are indicated where known. Cmr, chloramphenicol resistance; Ampr, ampicillin resistanceSource referenceE.coli K-12DH5αsupE44 ΔlacU169 (Φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA169Hanahan D. J.Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8098) Google ScholarJM109recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Δ(lac-proAB) F′ [traD36 proAB + lacIq lacZ ΔM15]70Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11410) Google ScholarXL1-Blue MRF′supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac- Δ(mcrA)183 Δ(mcr CB-hsd SMR-mrr)17371Jerpseth B. Greener A. Short J.M. Viola J. Kretz P.L. Strategies Newsletter. 5. Stratagene, La Jolla, CA1992: 81-83Google ScholarS. flexneriSFL124Attenuated Shigella vaccine serotype Y (DaroD)72Lindberg A.A. Karnell A. Stocker B.A. Katakura S. Sweiha H. Reinholt F.P. Vaccine. 1988; 6: 146-150Crossref PubMed Scopus (61) Google ScholarSFL1444SFL124 carrying pNV1060This studyPlasmidspBC SKCloning vectorStratagenepMA632pBluescript II SK (+) carrying “phoA-lacZa” cassette16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google ScholarpNV323SfV 3 gene cluster (gtrA, gtrB, and gtrV in pUC19)9Huan P.T. Bastin D.A. Whittle B.L. Lindberg A.A. Verma N.K. Gene. 1997; 195: 217-227Crossref PubMed Scopus (54) Google ScholarpNV1060Ampr, 1.64-kb HindIII/BglII fragment containing gtrA and gtrB in low copy number pACYC177This studypNV1077Cmr pBCSK containing SacI/XbaI gtrV (1.25 kb) fragmentThis studypNV1081Cmr derivative of pNV1077 carrying gtrV (1.25 kb) mutated at PstI site to SphIThis studypNV1090Cmr derivative of pNV1081 containing gtrV (1.25 kb) fragment and phoA/lacZ dual reporter (1.47 kb)This studypNV1105Cmr derivative of pNV1090 carrying fusion of truncated gtrV (1.24 kb) and dual reporter phoA/lacZ at amino acid position 412 of GtrVThis studya The genetic nomenclature is that described by Bachman (73Bachmann B.J. Microbiol. Rev. 1990; 54: 130-197Crossref PubMed Google Scholar). Allele numbers are indicated where known. Cmr, chloramphenicol resistance; Ampr, ampicillin resistance Open table in a new tab Plasmids, Primers, PCR, and Sequencing—Plasmids used in this study are listed in Table I. Oligonucleotide primers used for PCR are listed in Table II. The primers were synthesized by Invitrogen. Double-stranded plasmid sequencing was performed at the Biomolecular Resources Facility, John Curtin School of Medical Research, Australian National University. Sequencing primer PHOSEQ specific for the 5′ end of the pho-lac fusion (5′-TCACCCGTTAAACGGCGAGCACC-3′) was used to determine the exact point of fusion (2Kotloff K.L. Winickoff J.P. Ivanoff B. Clemens J.D. Swerdlow D.L. Sansonetti P.J. Adak G.K. Levine M.M. Bull. W. H. O. 1999; 77: 651-666PubMed Google Scholar). PCR was carried out using Pfu polymerase (Stratagene) as specified by the manufacturer.Table IIOligonucleotide primers used in this study to create sandwich fusions and amplification of gtrV fragment from pNV323PrimerSequenceAnnealing siteRestriction SitesNruIFL21510GCAGGAGATGCTCGCGAATGGTATGCTATGGgtrVNruINruIRL21510CCATAGCATACCATTCGCGAGCATCTTCTGCgtrVNruIFL31786GTATCAGAAGTGCTCGCGAATGTTACTAATGgtrVNruINruIRL31786CATTAGTAACATTCGCGAGCACTTCTGATACgtrVNruIL41870CCTACAAATAATTATCGCGAAGCACACGATTATATAGCgtrVNruINruIL41870GCTATATAATCGTGTGCTTCGCGATAATTATTTGTAGGgtrVNruIL51970CTTATACAAAATCGCGAATCTACAAAAAAAATCgtrVNruINruIL51970GATTTTTTTTGTAGATTCGCGATTTTGTATAAGgtrVFgtrVNruI2056CTATTATCATGACATCTCGCGAGACCAGAGTTGgtrVNruIRgtrVNruI2056CAACTCTGGTCTCGCGAGATGTCATGATAATAGgtrVGtrVFSacIGATGAGCTCTGAGAAACAAAAATGAAAAAGCCgtrVGtrVRXbaIACGTCTAGAACCATTCAACATTAAGGCgtrVgtrV1230FsphIGTTCAACCCTGCATGCTGGACGATGACgtrVSphIgtV1230RsphIGTCATCGTCCAGCATGCAGGGTTGAACgtrV Open table in a new tab DNA Methodology—Restriction endonucleases and T4 DNA ligase were purchased from Amersham Biosciences and Promega, respectively, and used in accordance with the protocols supplied by manufacturers. Plasmids were maintained in strain JM109 and prepared using the Qiagen MiniPrep kit. Transformation of E. coli with plasmid DNA or ligation mixtures was performed using RbCl2 protocols (66Strahl-Bolsinger S. Scheinost A. Baulard A.R. Gurcha S.S. Engohang-Ndong J. Gouffi K. Locht C. Besra G.S. Girrbach V. Zeller T. Priesmeier M. Bartsevich V.V. Pakrasi H.B. J. Biol. Chem. 1999; 274: 9068-9075Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Sequence Analysis—Eight computer programs available on the Internet were used to examine the GtrV protein sequence for the presence of hydrophobic regions. Programs used were DAS (27Cserzo M. Wallin E. Simon I. von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar) (www.sbc.su.se/~miklos/DAS), HMMTOP (28Tusnady G.E. Simon I. J. Mol. Biol. 1998; 283: 489-506Crossref PubMed Scopus (942) Google Scholar) (enzim.hu/hmmtop), PHDhtm/PHDtopology (29Rost B. Casadio R. Fariselli P. Sander C. Protein Sci. 1995; 4: 521-533Crossref PubMed Scopus (641) Google Scholar, 30Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (532) Google Scholar) (www.emblheidelberg.de/predictprotein), PSORT (31Nakai K. Kanehisa M. Proteins. 1991; 11: 95-110Crossref PubMed Scopus (631) Google Scholar) (psort.nibb.ac.jp), SOSUI (32Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1540) Google Scholar) (sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM (33Sonnhammer E.L. von Heijne G. Krogh A. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar) (www.cbs.dtu.dk/services/TMHMM1.0), TMpred (34Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 374: 166-168Google Scholar) (www.isrec.isbsib.ch/software/TMPRED_form.html), TopPred2 (35Claros M.G. von Heijne G. Comput. Appl. Biosci. 1994; 10: 685-686PubMed Google Scholar, 36von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1395) Google Scholar) (www.sbc.su.se/~erikw/toppred2). Five of these programs (HMMTOP, PHDtopology, TMHMM, TMpred, and TopPred2) were used to predict the membrane topology of GtrV (37Nilsson J. Persson B. von Heijne G. FEBS Lett. 2000; 486: 267-269Crossref PubMed Scopus (93) Google Scholar). Also hydrophobicity profiles were generated using the method of Kyte and Doolittle, implementing a sliding window of 19 residues. Cloning of gtrV and Preparation for Reporter Gene Fusions—The wild type gtrV gene was amplified from plasmid pNV323 using the primers gtrVFSacI and gtrVRXbaI (Table II). Because these primers have the SacI and XbaI sites incorporated in them at the 5′ end, it was possible to ligate the amplified fragment into pBCSK using the same sites that gave rise to pNV1077. The nested deletion method requires the presence of two unique restriction enzymes such as PstI and BamHI, allowing protection of the phoA/lacZ dual reporter and initiation of gtrV deletion by ExoIII, respectively. To use PstI as one of these enzymes, the PstI site in gtrV (1230 bp) had to be mutated so that only one PstI would be left between gtrV and the dual reporter. This was done by site-directed mutagenesis (Stratagene) changing the PstI site to a SphI site using primers gtrV1230FSphI and gtV1230RSphI (Table II) according to the manufacturer's protocol. Functionality of the gtrV gene was checked by introducing the new plasmid pNV1081 into SFL1444. In brief, plasmid pNV1081 was introduced by electroporation into SFL1444, which is a derivative of SFL124 except that it carries pNV1060 (gtrA and gtrB). Because the whole three-gene cluster has to be present for complete serotype conversion, this allows functional examination of gtrV with gtrA and gtrB, which are carried on another plasmid. Transformants were plated onto dual LB agar plates containing Cm (30 μg/ml) and ampicillin (100 μg/ml). Serotype conversion was tested by slide agglutination. This assay detects the expression of various S. flexneri epitopes. A glass slide was divided into two sections, and one drop of the test bacteria resuspended in saline (0.9% NaCl) was placed at each end of the slide. On the right section of the slide, one drop of S. flexneri serotype V (SEIKEN) antiserum was added above the bacterial suspension. On the left side of the slide one drop of saline was added in place of the specific antiserum. This served as a negative control. Using a sterile loop both antigen and serum or saline drops were mixed. The glass slide was rocked, and the mixtures were observed for agglutination. Only agglutination that occurred within 1 min was taken as positive. The functional gtrV carried by pNV1081 was cut with EcoRV to permit ligation of the dual indicator phoA/lacZ, which was isolated from pMA632 using the EcoRV and SmaI sites. This gave rise to pNV1090 (Fig. 1). Construction of gtrV-phoA/lacZ—The nested deletion method described by Sugiyama et al. (20Sugiyama J.E. Mahmoodian S. Jacobson G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9603-9607Crossref PubMed Scopus (78) Google Scholar) was used to produce a series of fusion plasmids in which various lengths of the 5′ end of the gtrV gene were attached to the phoA/lacZ dual reporter (20Sugiyama J.E. Mahmoodian S. Jacobson G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9603-9607Crossref PubMed Scopus (78) Google Scholar). pNV1090 was linearized by cleavage of the unique PstI and BamHI sites located between the genes. This produced a 5′ overhang just downstream of the gtrV gene and a 3′ overhang positioned upstream of the phoA/lacZ gene. Exonuclease was used to progressively delete gtrV from its 3′ end via the 5′ overhang according to the method of Henikoff (38Henikoff S. Methods Enzymol. 1987; 155: 156-165Crossref PubMed Scopus (674) Google Scholar) as outlined in the instructions provided with the Promega kit. The reporter gene was protected from digestion since 3′ overhangs are resistant to ExoIII digestion. After treatment with Klenow fragment and the four deoxynucleotide triphosphates, the ends were ligated. Circularized DNA carrying truncated versions of gtrV plus the full dual reporter was transformed into JM109 and plated onto dual indicator plates. The plasmid DNA was isolated from blue, purple, and red colonies (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar), and the exact site of the fusion point was determined by restriction digests and confirmed by DNA sequencing carried out on the ABI 3730 capillary sequence analyzer using the Big Dye Version 3.1 sequencing protocol and the PHOSEQ primer (2Kotloff K.L. Winickoff J.P. Ivanoff B. Clemens J.D. Swerdlow D.L. Sansonetti P.J. Adak G.K. Levine M.M. Bull. W. H. O. 1999; 77: 651-666PubMed Google Scholar). Construction of gtrV-phoA/lacZ-gtrV Sandwich Gene Fusions— Unique NruI restriction endonuclease sites were introduced throughout the gtrV gene present in pNV1077 by oligonucleotide-mediated site-directed mutagenesis (Stratagene) as specified in the manufacturer's protocol. Primers with an NruI site incorporated are listed in Table II. The presence of the desired mutation was checked by NruI digests and sequenced using gtrVFSacI and gtrV1780FSeq primers. The phoA/lacZ dual reporter was excised separately from pMA632 (2Kotloff K.L. Winickoff J.P. Ivanoff B. Clemens J.D. Swerdlow D.L. Sansonetti P.J. Adak G.K. Levine M.M. Bull. W. H. O. 1999; 77: 651-666PubMed Google Scholar) using a combination of EcoRV-SmaI and StuI-NruI double digests. After ligation of the reporter insert to the desired construct carrying the NruI restriction site, the ligation mix was transformed in JM109 and then plated onto dual indicator plates. Colored colonies were picked and checked for inserts using digests and double-stranded sequencing to confirm the correct orientation, site of fusion, and maintenance of the correct reading frame. Assays of AP and BG Activities—Overnight cultures of E. coli JM109 bearing fusion constructs and unfused plasmid (background control) were diluted 1:20 in fresh LB containing 30 μg/ml Cm grown to A600 ∼ 0.5, at which point cultures were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 1 h, and activities were assayed as described previously (38Henikoff S. Methods Enzymol. 1987; 155: 156-165Crossref PubMed Scopus (674) Google Scholar, 44Traxler B. Boyd D. Beckwith J. J. Membr. Biol. 1993; 132: 1-11Crossref PubMed Scopus (90) Google Scholar). Background activities were subtracted from experimental data (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 19Manoil C. Methods Cell Biol. 1991; 34: 61-75Crossref PubMed Scopus (196) Google Scholar, 39Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 72-74Google Scholar). Topology of S. flexneri GtrV Based on Computational Analysis—Protein sequence analysis was performed initially by using computer programs as indicated under “Materials and Methods.” Two of the most commonly used programs are shown in Fig. 2. This includes Kyte-Doolittle and TMHMM, which when applied to GtrV predict a topology that consists of nine putative transmembrane segments with a large N-terminal periplasmic loop and the N and C termini located in the cytoplasm and periplasm, respectively. Topology of S. flexneri GtrV:gtrV-phoA/lacZ Fusions—The nested deletion system as described by Sugiyama et al. (20Sugiyama J.E. Mahmoodian S. Jacobson G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9603-9607Crossref PubMed Scopus (78) Google Scholar) was used to create different length hybrid protein fusions between gtrV and the dual reporter phoA/LacZ (20Sugiyama J.E. Mahmoodian S. Jacobson G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9603-9607Crossref PubMed Scopus (78) Google Scholar). In particular, exonuclease III was used to progressively delete gtrV from the 3′ end before ligating it back to the full dual reporter. Transformed cells were grown on media containing the chromogenic substrates Red-Gal (6-chloro-3-indolyl-β-d-galactoside) and X-phos (5-bromo-4-chloro-3-inolyl phosphate disodium salt) used by LacZ and PhoA, respectively. Coloration was produced due to in-frame fusion of the dual reporter to the truncated gtrV series of deletions. Red was produced due to LacZα being active in the cytoplasm, and blue was due to PhoA being active in the periplasm (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar). Purple colonies were also produced when fusions were adjacent to transmembrane segments or periplasmic domains (16Alexeyev M.F. Winkler H.H. J. Mol. Biol. 1999; 285: 1503-1513Crossref PubMed Scopus (71) Google Scholar, 17Alexeyev M.F. Winkler H.H. Biochemistry. 2002; 41: 406-414Crossref PubMed Scopus (17) Google Scholar). Fusions not carrying the dual reporter produced clear colonies on dual indicator plates, whereas no in-frame reporter fusions appeared clear, but prolonged incubation after 14–30 h produced red colonies, prob
Referência(s)