Identification of essential loops and residues of glucosyltransferase V (GtrV) of Shigella flexneri

Figures & data

Table I.  Chimeras GtrV→GtrX and GtrX→GtrV constructed by a PCR mediated approach and homologous recombination. P, positive. This table is reproduced in colour in Molecular Membrane Biology online.

		Agglutination with antisera
	Construct model	V	X	3,4	Dual indicator coloration/Protein synthesis – assembly
pNV1066		+	−	+	Red/P
FL1460
-
pNV1067		+	−	+	Red/P
SFL1630
-
pNV1261		−	−	+	Blue/P
-
pNV1188		−	−	+	Blue/P
pNV1189		−	−	+	Blue/P
SFL1554
pNV1197		−	−	+	Blue/P
pNV1232		−	−	+	Blue/P
pNV1337		−	−	+	Blue/P
pNV1077		+	−	+	Blue/P
Agglutination example with serotype V antisera

Figure 1.  Functional analysis of mutants. Electron micrographs (X50 000 magnification) of SFL1444 containing pNV1066 and 1067 tested with type V antibody. Secondary antibody used is conjugated to 10nm gold particles. Presence of gold particles is indicative of O-antigen modification. Description of each strain is shown below the image.

Figure 2.  Western immunoblot of functional chimeras. Western immunoblot of chimeras pNV1066 and 1067 using mouse anti-E.coli PhoA antisera. pNV1105, positive control; pNV1060, negative control.

Figure 3.  Best fit analysis of Gtrs. (A) Best fit analysis of GtrV and GtrX. Consensus is shown below sequence and in boxes. Note consensus regions boxed (red), indicative of motifs containing negatively charged residues. In vertical boxes shaded grey are the residues that were targeted by site-directed mutagenesis. (B) Best fit analysis of first periplasmic loop No 2 of N-terminus present in all Gtrs. The conserved aspartic acid (D) is shown in italics (grey box). Also in bold are either aspartic or glutamic acids part of the shown motif. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 4.  Position of critical GtrV residues and western immunoblot. Targeted residues in respect to the recently solved topology of GtrV. (A) Highlighted residues (bold-underlined) were mutated to residues shown next or above them. Note that important residues ED in the first periplasmic loop No 2 (N-terminus) were mutated to QN respectively, while residue D in loop No 10 (C-terminus) was mutated to N as well as A. (B) Western immunoblot of site-directed mutants pNV1173 and 1168 using mouse anti-E. coli PhoA antisera. A 98kDa band which includes both GtrV and dual reporter is observed in both constructs. This Figure is reproduced in colour in Molecular Membrane Biology online.

Table II.  Site directed mutagenesis applied on selected GtrV residues. Mutations in bold are critical in function. P, Positive.

		Agglutination with Antisera		Dual Indicator Plate Coloration/Protein Synthesis	Agglutination slides
Strain/Wild type Residues	Mutated Residues	V	3.4		Mutant	Positive Control pNV1077
pNV1092/E42, pNV1094/ D43, pNV1173/E42D43	Q42, N43 Q42N43	−	+	Blue/P
pNV1085, pNV1187/D168	N168, A168	+	+	Blue/P	Positive	Positive
pNV1193/ C175	A175	+	+	Blue/P	Positive	Positive
pNV1087, pNV1168/ D380	N380, A380	+, −	+	Blue/P
pNV1180/E397	A397	+	+	Blue/P	Positive	Positive

Supplementary Table I. Bacterial strains and plasmids used in this study.

Strain or plasmid	Relevant characteristics^?	Source reference
E. coli K-12
DH5?	supE44 ΔlacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	(Hanahan [1983])
JM109	recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Δ(lac-proAB) F’ [ traD36 proAB+ lacI^q lacZ ΔM15]	(Yanisch-Perron et al. [1985])
XL1-Blue MRF’	supE44 hsdR17 recA1 endA1 gyrA46 thirelA1 lac- Δ(mcrA)183 Δ(mcr CB-hsd SMR-mrr)173	(Jerpseth et al. [1992])
S. flexneri
SFL124	Attenuated Shigella vaccine serotype Y (ΔaroD)	(Lindberg et al. [1988])
SFL1444	SFL124 carrying pNV1060	This study
SFL1616	SFL124 carrying pNV1241	A. Lehane
Plasmids
pBC SK	cloning vector	Stratagene
pMA632	pBluescript II SK (+) carrying ‘phoA-lacZ?’ cassette	(Alexeyev & Winkler [1999])
pNV323	SfV 3 gene cluster (gtrA, gtrB and gtrV in pUC19)	(Huan et al. [1997])
pNV574	SfX 3 gene cluster (gtrA, gtrB and gtrX in pBC KS II, Amp^r)	(Huan et al. [1997])
pNV1060	Amp^r, 1.64kb HindIII/BglII fragment containing gtrA and gtrB in low copy number pACYC177
pNV1066	chimeric gene consisting of gtrX 1-417bp and gtrV 418-1221, produced using pNV1063 by homologous recombination	’’
pNV1067	chimeric gene consisting of gtrX 1-378 and and gtrV 379-1224, produced using pNV1063 by homologous recombination	’’
pNV1077	Cm^r pBCSK containing SacI/XbaI gtrV (1.25kb) fragment	This study
pNV1085	Using pNV1077, gtrV mutated at amino acid 168 from D to N	’’
pNV1087	Using pNV1077, gtrV mutated at amino acid 380 from D to N	’’
pNV1092	Using pNV1077, gtrV mutated at amino acid 42 from E to Q	’’
pNV1094	Using pNV1077, gtrV mutated at amino acid 43 from D to N	’’
pNV1105	Cm^r derivative of pNV1090 carrying fusion of truncated gtrV and dual reporter phoA/lacZ at amino acid position 412 of GtrV	This study
pNV1168	pNV1105 carrying GtrV and dual reporter mutated at 380 amino acid GtrV from D to A	’’
pNV1173	pNV1105 GtrV mutated at amino acids 42 and 43 from E to Q and D to N respectively	’’
pNV1180	pNV1105 GtrV mutated at amino acids 397 from E to A	’’
pNV1187	pNV1105 GtrV mutated at amino acid 168 from D to A	’’
pNV1188	Chimeric gene consisting of gtrV 1-687bp and gtrX 688-1254, produced by PCR mediated method using pNV1082	’’
pNV1189	Chimeric gene consisting of gtrV 1-795bp and gtrX 796-1254, produced by PCR mediated method using pNV1082	’’
pNV1197	Chimeric gene constisting of gtrV 1-1134bp and gtrX1135-1245bp, produced by PCR mediated method using pNV1082	’’
pNV1193	pNV1105 GtrV mutated at amino acid 175 from C to A	’’
pNV1232	Double mutant consisting of gtrV 1-390, gtrX 391-696 reentrant loop, gtrV 697-816 gtrX 817-1275 produced using pNV1189	’’
pNV1241	Km^r, 1.64kb BamHI/PstI fragment containing gtrA and gtrB in low copy number pACYC177	A. Lehane
pNV1261	Chimeric gene consisting of gtrV 1-408bp and gtrX 409-1275, produced by PCR mediated method using pNV1082	This Study
pNV1337	Double mutant consisting of gtrV 1-390, gtrX 391-696 reentrant loop, gtrV 697-1272 produced using pNV1324	’’

^??The genetic nomenclature is that described by Bachman ([1990]). Allele numbers are indicated where known. Cm^r, chloramphenicol resistance; Amp^r, ampicillin resistance.

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