418
Views
11
CrossRef citations to date
0
Altmetric
Original

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

&
Pages 407-419 | Received 13 Mar 2006, Published online: 09 Jul 2009

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.

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 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 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 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.

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.

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

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.