Structural and functional divergence of the newly identified GtrIc from its Gtr family of conserved Shigella flexneri serotype-converting glucosyltransferases

Figures & data

Table I.  Dual reporter activity for fusions within GtrIc.

E. coli strain	Tagged residue	Domain	DI colony colour	Relative % Activity		NAR(PhoA:LacZ)
				PhoA	LacZ
C-terminal truncation fusions
B2169	F20	Helix I	Blue	2.6	0.3	9:1
B2232	S50	Loop 1	Blue	66.4	2.3	29:1
B2170	N53	Loop 2	Blue	20.7	0.8	26:1
B2171	S89	Loop 2	Blue	100.0	33.7	3:1
B2172	F98	Loop 2	Blue	29.6	32.3	1:1
B2181	A216	Loop 5	Red	0.0	35.6	1:> 100
B2173	R258	Loop 6	Blue	38.8	22.7	5:3
B2174	A291	Helix VI	Purple	1.1	1.7	1:1
B2233	N301	Loop 7	Red	3.7	15.3	1:4
B2175	C318	Helix VII	Blue	14.7	0.4	37:1
B2234	L333	Loop 8	Blue	13.7	0.3	46:1
B2235	K361	Loop 9	Red	1.3	32.2	1:25
B2176	L374	Helix IX	Blue	6.6	2.3	3:1
B2177	Y402	Loop 10	Blue	4.0	0.6	7:1
B2178	L417	Loop 10	Blue	1.5	1.4	1:1
B2179	T440	Loop 10	Blue	22.7	6.6	7:2
B2180	I483	Loop 10	Blue	10.2	0.4	26:1
B2182	C-terminus	Loop 11	Red	0.0	100.0	1:> 100
Sandwich fusions
B2236	L6	Loop 1	Red	1.5	100.0	1:67
B2183	V132	Loop 3	Red	0.5	37.7	1:75
B2237	P155	Loop 4	Blue	100.0	0.4	> 100:1
B2184	V179	Loop 5	Red	0.0	20.0	1:> 100
B2238	A196	Loop 5	Red	0.1	1.2	1:12

JM109 hosts were used to express pho-lac activity. Strain codes are provided respective to the fusion product they harbour. The location of each fusion point is provided according to the tagged GtrIc residue substituted with the dual reporter and its representative protein domain in reference to the finalized topology model (Figure 2b). Assessment of DI plate colony colour under ambient light of JM109 strains containing fusion constructs are also provided. PhoA and LacZ activities are shown as percentage values relative to the maximum activity for each enzyme. The PhoA:LacZ NARs for each fusion is shown rounded to the nearest integer. Each construct was examined in duplicates per enzyme assay. Values are representative of two independent experiments.

Figure 1.  DAS predictions for potential presence of membrane spanning segments within GtrIc. Twelve transmembrane α-helices were detected at the loose cut-off (dotted red line). Peak signals are annotated according to these measurements. At the strict cut-off (solid blue line), 13 transmembrane α-helices were identified. DAS is used to represent the consensus output, with consistent plots also observed in HMMTOP, PHDhtm, TMHMM, and TMpred. This Figure is reproduced in colour in the online version of Molecular Membrane Biology.

Figure 2.  Determination of the GtrIc membrane topology. (a) Various strategies were used to construct pho-lac fusions at different codons of gtrIc. Replacement of the C-terminus of full length gtrIc by PCR (top); random truncation fusions obtained by nested deletion (middle); pho-lac inserted in-frame into gtrIc sequence as a sandwich fusion (bottom). (b) Experimentally verified topology model of GtrIc resolved through computerised modelling and corrected by scanning with pho-lac fusions. Truncation gtrIc/pho-lac fusions (spots) and sandwich gtrIc/pho-lac/gtrIc fusions (encapsulated spots) are coloured according to DI plate observations with their respective residue position. The location of D/ExD/E and KK motifs copies are also provided (green boxes). A larger periplasmic loop 2 was detected relative to the consensus model, along with a double membrane dipping loop centred between the N- and C- termini and ten transmembrane α-helices. This Figure is reproduced in colour in the online version of Molecular Membrane Biology.

Figure 3.  Alignment of Gtr_[type] periplasmic sequences. The topologies of GtrII and GtrV were experimentally elucidated in previous studies [14,15]. Identification of loop 2 and large C-terminal loop sequences in GtrI, GtrIV and GtrX was based on preliminary analyses by Lehane et al. [15] by using predictive software and sequence homologies between GtrI and GtrX with GtrII and GtrV, respectively. (a) The large N-terminal loop 2 of all known Gtr_[type] members shows a conserved D/ExD/E motif. (b) Conserved KK (dilysine) motifs are seen in the large C-terminal loops of all Gtr_[type] proteins except for GtrIV. Two KK motif copies are detected in the same loop of GtrI, GtrIc and GtrV. This Figure is reproduced in colour in the online version of Molecular Membrane Biology.

Figure 4.  Functional analysis of GtrIc mutants. Transformed SFL1416 serotype 1a harbouring mutated GtrIc constructs were assayed for host 1a to 1c serotype conversion. GtrIc point mutations are annotated in parentheses with their SFL1416-derived host. Comparisons of mutant strains can be made with the SFL1961 containing empty pBCSK+ vector and with SFL1905 containing wild-type gtrIc in pNV1650. (a) Colony immunoblots showing detection of S. flexneri serotype 1c LPS in all mutant strains. (b) Slide agglutinations complemented colony immunoblot readings, showing GtrIc activity in all mutants. Binary scoring was used for positive and negative agglutination tests.

Korres H, Verma NK. 2004. Topological analysis of glucosyltransferase GtrV of Shigella flexneri by a dual reporter system and identification of a unique reentrant loop. J Biol Chem 279(21):22469–22476.

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Lehane AM, Korres H, Verma NK. 2005. Bacteriophage-encoded glucosyltransferase GtrII of Shigella flexneri: Membrane topology and identification of critical residues. Biochem J 389(Pt 1):137–143.

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