Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri
© Nair et al; licensee BioMed Central Ltd. 2011
Received: 12 July 2011
Accepted: 22 December 2011
Published: 22 December 2011
The three bacteriophage genes gtrA, gtrB and gtr (type) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr (type) was found to be specific to each serotype, leading to the hypothesis that the Gtr(type) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Based on the confirmed topologies of GtrI, GtrII and GtrV, such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm.
In this study, the topology of GtrIV was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. Though this topology differs from that of GtrI, GtrII, GtrV and GtrX, it is very similar to that of GtrIc. Furthermore, both the N-terminal periplasmic and the C-terminal periplasmic loops are important for GtrIV function as shown via a series of loop deletion experiments and the creation of chimeric proteins between GtrIV and its closest structural homologue, GtrIc.
The current study provides the basis for elucidating the structure and mechanism of action of this important O-antigen modifying glucosyltransferase.
Shigellosis, or bacillary dysentery, is caused by members of the genus Shigella. This genus belongs to the Gram-negative bacterial family Enterobacteriaceae, and is divided into 4 species: S. flexneri, S. dysenteriae, S. boydii, and S. sonnei, all of which cause shigellosis. Shigella flexneri, the species responsible for the highest mortality rate, is endemic in most developing countries. There are 15 known S. flexneri serotypes, which differ in their virulence, prevalence, distribution and O-antigens [1, 2]. The O-antigen is the distal capping moiety of the bacterial
lipopolysaccharide (LPS), a molecule that extends from the bacterial surface. The O-antigen backbone consists of repeating units of the tetrasaccharide N-acetylglucosamine-rhamnose I-rhamnose II-rhamnose III . This basic backbone, serotype Y, is present in all serotypes except serotype 6 and 6a. The addition of glucosyl and/or O-acetyl groups to different sugars in the tetrasaccharide unit by one of several linkages gives rise to different serotypes. Glucosylation can occur in any one of the four residues that are present in the tetrasaccharide unit. O-acetylation was thought to occur only on the rhamnose III residue, resulting in the group 6 epitope . However, recent studies have revealed that a degree of O-acetylation may be occurring on the other sugars as well [5, 6].
The three genes involved in O-antigen glucosylation are gtrA, gtrB, and gtr (type) . They are encoded by temperate bacteriophages and located downstream of the attP site in the bacteriophage genome . While gtrA and gtrB, which encode for GtrA and GtrB, are highly conserved and interchangeable among serotypes [7–10], gtr (type) , which encodes for Gtr(type) protein, is serotype-specific and is unique to each bacteriophage. It is hypothesised that GtrB catalyses the transfer of glucose from UDP-glucose to bactoprenol phosphate to form UndP-β-glucose in the cytoplasm . This molecule is then flipped by GtrA into the periplasm before the glucosyl residue is attached by the Gtr(type) to the growing O-antigen unit . This attachment is thought to take place in the periplasm. GtrIV adds a glucosyl residue to N-acetylglucosamine of the O-antigen repeat unit via an a1,6 linkage, thus converting serotype Y to serotype 4a.
In this study, a dual reporter system consisting of alkaline phosphatase (phoA) which is in-frame with the β-galactosidase a-fragment (lacZα) developed by Alexeyev and Winkler  was used to determine the membrane topology of GtrIV. Alkaline phosphatase (AP) is an E. coli enzyme that is only active when it is localised in the periplasm as the mature part of PhoA is oxidised such that the cysteine residues are able to form disulfide bridges. This enables PhoA to fold correctly . In contrast to AP, β-galactosidase (BG) is only active in the cytoplasm as the a-fragment must interact with the cytoplasmically expressed w-fragment of the enzyme to cause α-complementation .
Here we report that GtrIV clearly differs from the topologies of the other Gtrs (GtrI, GtrII, GtrV, GtrX) but is strikingly similar to the confirmed topology of GtrIc, a newly discovered glucosyltransferase, which has 11 transmembrane regions, a short cytoplsmic C-terminal tail and two large periplasmic loops . Based on the fact that glucosylation is thought to occur in the periplasm , we also show that the two large periplasmic loops of GtrIV are important for its stable assembly in the membrane and function by creating GtrIV proteins that have either the N-terminal periplasmic loop or various segments of the C-terminal periplasmic loop deleted, resulting in the abolishment of O-antigen modification from serotype Y to serotype 4a.
Growth conditions of bacterial strains
All bacterial cultures were grown aerobically at 37°C on either liquid Luria-Bertani (LB) agar plates or in liquid LB medium. Antibiotics such as chloramphenicol (Sigma), kanamycin (Sigma) and ampicillin (Sigma) were added to solid and liquid media when required. The final concentrations of these antibiotics were 25 mg/mL, 50 mg/mL, and 100 mg/mL, respectively. Bacterial plates were stored for up to 3 weeks at 4°C in the cold room. The E. coli strains used in this study are derivatives of E. coli K-12. XL1-Blue MRF' E. coli was used for routine cloning procedures while JM109 E. coli was used for cloning of topology constructs to be screened on Dual indicator (DI) plates that contain the chromogenic substrates X-phos (5-bromo-4-chloroindol-3-inolyl phosphate disodium salt, Sigma) for alkaline phosphatase and Red-Gal (6-chloroindol-3-inolyl-β-D-galactoside, Research Organics) for β-galactosidase as outlined by Alexeyev and Winkler .
DNA cloning techniques
All plasmids constructed in this study were derived from the cloning vector pBC SK+ (Stratagene). They were maintained in JM109 cells and isolated by means of using either the QIAGEN MiniPrep Kit or the alkaline lysis method adapted by Sambrook and Russell  from Birnboim and Doly . Restriction enzymes were purchased from Fermentas or New England Biolabs (NEB). Amplification of specific genes was performed via PCR by using Pfu ultraII poymerase (Stratagene) and specific oligonucleotide primers purchased from Sigma-Aldrich (Additional file 1, Table S1). T4 DNA ligase (Promega) was used for all ligation reactions as specified by Promega. Electrocompetent cells for bacterial transformations were prepared according to Dower et al.  and transformations were carried out as described by Sambrook and Russell  and Dower et al. . DNA sequencing was performed at the Biomolecular Resources Facility, John Curtin School of Medical Research, The Australian National University. Point mutations within gtrIV were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The resulting amplicon was then treated with Dpn I before being introduced into XL1-Blue MRF' E. coli that repairs nicks. The mutant constructs were sequenced to confirm the desired mutation.
Computer analysis of protein topology
Six topology prediction programs that were available online were used to examine the GtrIV protein sequence for the presence of hydrophobic regions. They were HMMTOP [http://www.enzim.hu/hmmtop/] , SOSUI [http://bp.nuap.nagoya-u.ac.jp/sosui/] , TMpred [http://www.ch.embnet.org/software/TMPRED_form.html] , DAS [http://www.sbc.su.se/~miklos/DAS/] , TopPredII [http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html]  and finally, TMHMM [http://www.cbs.dtu.dk/services/TMHMM/] .
Creating a topology template and generation of constructs for topology studies
Before exonuclease III (Exo III) deletion and PCR-based fusions can be carried out to create gtrIV-phoA/lacZ truncations, an appropriate template had to be created such that gtrIV is in tandem with the phoA/lacZ dual reporter. pNV1090, constructed by Korres and Verma , contains gtrV in tandem with phoA/lacZ. The majority of pNV1090, with the exception of gtrV, was amplified with forward primer 1090Xba IFnew containing an Xba I site that anneals directly upstream of phoA/lacZ in pNV1090 and reverse primer 1090Nhe IRnew containing anNhe I site that anneals upstream of gtrV. Similarly, gtrIV was amplified from pNV739 (containing gtrIV) using a forward primer gtrIVNheIFnew (5'-TCAGCTAGC CTCGGTGGTGTGCAGCTC-3') and a reverse primer gtrIVXbaIRnew (5'-TCATCTAGA CCCCCCAGGATAACTGTGGG-3'). Ligation of both amplicons created pNV1473 (Additional file 2, Figure S1) which contains a Pst I site closest to phoA/lacZ that leaves an Exo III-resistant 3' overhang and a Bam HI site closest to gtrIV that provides an Exo III-susceptible 5' overhang. Upon creation of pNV1473, the plasmid was linearised by a Pst I/Bam HI double digestion and progressively deleted from the end of gtrIV using the Erase-a-base kit from Promega. PCR was also used to create gtrVI-phoA/lacZ fusions. In this approach, the majority of pNV1473 was amplified using the forward primer phoF (5'- GTTCTGGAAAACCGGGCTGCTCAG-3'), which anneals at the beginning of the phoA/lacZ sequence, and a reverse primer that anneals at the point of interest in gtrIV.
In order to make sandwich fusions, Nru I sites were first incorporated into the gtrIV sequence in pNV1473 via site-directed mutagenesis. Nru I digest screening was used to identify successfully mutated constructs and they were tested for functionality by slide agglutination (as described below). phoA/lacZ was excised from pMA632 by either Sma I/Eco RV, Ehe I/Ecl 136II or Stu I/Nru I double digests, such that phoA/lacZ fragment would be in-frame when ligated with the Nru I digested fragments. JM109 was transformed with the ligation mixtures and plated onto DI plates. Colonies that displayed colouration were investigated further using restriction digests and then verified by sequencing using M13R or the PHOSEQNewR primers.
Quantifying alkaline phosphatase and β-galactosidase activities
Alkaline phosphatase (AP) and b-galactosidase (BG) assays were carried out to quantify the AP and BG activities, respectively. Both assays were performed in parallel with duplicates for each experiment. After obtaining the AP and BG activities for each fusion in the data set, the normalised activity ratios (NAR) were calculated as follows [24, 25]:
NAR = (Alkaline phosphatase activity/highest Alkaline phosphatase activity)/(b-galactosidase activity/highest b-galactosidase activity)
Functional analysis of GtrIV
The function of Gtr proteins were tested by transformation into SFL1616, a serotype Y Shigella strain containing chromosomally-encoded gtrA and gtrB. Serotype conversion (from serotype Y to serotype 4a) indicated a functional GtrIV protein. Slide agglutination assays were carried out by bacteria grown to log phase at 37°C (A600 0.8-1.0), mixing the culture gently on a glass slide with Type IV antisera (Denka Seiken) and observing for agglutination .
Bacterial lipopolysaccharide (LPS) preparation
This method was adapted from Hitchcock and Brown  with several modifications. Overnight cultures were diluted 1/100 in LB containing the appropriate antibiotics and incubated for 2 - 3 h at 37°C until OD600 of 0.6 was reached. 1.5 ml of culture was then spun down and the pellet was resuspended in 80 μl sample loading buffer (4% SDS, 160 mM Tris-HCl, 20% glycerol, 10% β-mercaptoethanol). Proteinase K was then added to each sample at a concentration of 50 mg/ml and the samples were incubated overnight at 56°C and stored at -20°C. Before samples are run on 12% SDS-PAGE gel, 0.5 μl β-mercaptoethanol was added to the samples and boiled for 10 min.
Membrane protein preparation
The membrane protein isolation method is a modified version of that described by Morona et al. . Cells were grown in LB to mid-log (OD600 of 0.6) and pelleted using a Sorvall SLA1500 rotor (7,000 rpm, 10 min, 4°C) and resuspended in 1 ml of 20% (w/v) sucrose, 30 mM Tris-HCl pH 8.1, transferred to SS-34 tubes and chilled on ice. 0.1 ml of 1 mg/ml lysozyme in 0.1 M EDTA pH 7.3 was added to the cells for 30 min on ice. The cells were collected again as described above using a Sorvall SS-34 rotor and the pellet frozen for 30 min at -80°C. The pellet was thawed and resuspended vigorously in 6 ml 3 mM EDTA, pH 7.3. The cells were completely lysed by passing them twice through a French Press at 15,000 psi. Unlysed cells and inclusion bodies were removed by slow centrifugation as described above (7,000 rpm, 10 min, 4°C). Membrane proteins were sedimented by high speed centrifugation using a 50Ti or 80Ti rotor spun at 35, 000 rpm for 90 min at 4°C. The pellet was then resuspended in 200 μl sterile Milli Q H2O.
Western blotting of proteins
Western blotting was carried out as described by Thanweer et al . For membrane preparations 10 μl of each 2 mg/ml sample was loaded and for LPS samples, 10 μl of each LPS extraction was loaded. Primary antibodies used were either Mouse anti-alkaline phosphatase (Chemicon) diluted 1:1000 for membrane protein preparations, Type IV antisera (Denka Seiken) diluted 1:100 or serotype Ic-specific MASFIc monoclonal antibody (Reagensia) diluted 1:500 for bacterial LPS extractions. The secondary antibodies used were Goat anti-mouse IgG horse radish peroxidase (HRP)-conjugated (Sigma) diluted 1:8000 for membrane protein preparations while Goat anti-rabbit HRP-conjugated Ig (Sigma) diluted 1:1000 and anti-mouse IgM peroxidise conjugate (Sigma) diluted 1:5000 were used for profiling bacterial LPS extraction. The reactions were detected either by X-ray film (GE Healthcare) or viewed under the Fusion Chemiluminescence Camera (Fisher Biotech).
Results and discussion
Determination of GtrIV topology
A predicted topology model for GtrIV was created based on the results given by the various web-based prediction programs (Additional file 1, Table S2). There was no common prediction amongst the programs. HMMTOP and TMPred both predicted that GtrIV has 8 transmembrane helices. TopPred and TMHMM predicted 9 transmembrane helices while SOSUI and DAS predicted 10 transmembrane helices and 11 transmembrane helices, respectively. Of all the programs, HMMTOP and TMHMM both predicted that the N-terminus was cytoplasmic. TopPred predicted that the N-terminus was in the periplasm while the rest of the programs did not provide any information about the localisation of the N-terminus. From the information that has been gathered from the confirmed topologies of GtrI, GtrII and GtrV, a large periplasmic C-terminal tail was expected. By collating the data from all the programs, a consensus model was created. In this model, GtrIV is shown to have 10 transmembrane helices, a cytoplasmic N-terminus, a small cytoplasmic C-terminal region, and two large periplasmic loops between transmembrane helices I and II, and transmembrane helices VII and VIII (Additional file 3, Figure S2).
Analysis of gtrIV-phoA/lacZ fusions and gtrIV-phoA/lacZ-gtrIV sandwich fusions for GtrIV topology determination
Location on Model8
Random C-terminal Fusions
311 ± 9
25 ± 2
356 ± 47
357 ± 44
296 ± 47
195 ± 28
3 ± 1
133 ± 29
4 ± 1
383 ± 25
9 ± 91
15 ± 2
22 ± 4
132 ± 6
292 ± 51
394 ± 20
660 ± 39
962* ± 59
553 ± 30
385 ± 55
426 ± 21
4 ± 1
589 ± 15
1 ± 1
269 ± 34
9 ± 1
12 ± 3
8 ± 2
121 ± 17
1: > 100
953 ± 35
7 ± 1
6 ± 2
71 ± 9
79 ± 17
1: > 100
177 ± 40
5 ± 1
155** ± 8
1: > 100
10 ± 2
1: > 100
14 ± 2
1: > 100
135+ ± 25
1 ± 1
47++ ± 7
1: > 100
The topology of GtrIV was further elucidated by using two more strategies. These include a PCR-based approach and the use of sandwich fusions. Using the PCR-based fusion technique, it allowed us to exactly fuse the dual reporter to predetermined points in the protein. The second method involved the construction of sandwich fusions. A sandwich fusion can indicate a more accurate representation of topology since the whole protein is present with the dual reporter sandwiched in the middle. These two methods generated a host of constructs. Fusions D146, D406 and K438 were obtained via PCR based fusions. R93, K117, A181 and R375 were generated using gtrIV/phoA-lacZ/gtrIV sandwich fusions. A NAR value of 1: > 100 for the PCR-constructed red fusion K438 confirms that the C-terminal end is cytoplasmic. Fusion D406 with a NAR value of 20:1 indicated that loop No. 10 is in the periplasm. While both these fusions satisfied the consensus model, D146 was observed to display blue colouration with a NAR value of 21:1. This fusion was in contradiction to the hypothetical model, which predicts D146 to be localised in the cytoplasm between transmebrane helices IV and V. Sandwich fusion K117 displayed a NAR value of 1: > 100 that corresponded with its red colouration and confirms its localisation to the cytoplasm.
Identifying regions critical for GtrIV function
Investigating periplasmic loop function of GtrIV by using chimeric proteins
Predicted structures of the four chimeric proteins created in this study between GtrIc and GtrIV
In parallel to investigating the role of GtrIV loop No. 2, we also investigated the role of the C-terminal periplasmic loop No. 6 of GtrIV. Gtrs I, II, V and × have long periplasmic C-terminal ends that have been hypothesised to be responsible for conferring serotype specificity. In contrast, as both GtrIV and GtrIc have short cytoplasmic C-terminal ends, their specific function is thought to be performed by the large periplasmic loops No. 6 and No. 10 for GtrIV and GtrIc, respectively. By using the same PCR-based approach for the creation of the loop No. 2 chimeras, loop No. 6 of GtrIV was replaced by loop No. 10 of GtrIc and vice versa (Table 2). It was postulated that the hybrid GtrIV containing loop No. 10 of GtrIc would be able to convert serotype 1a to serotype 1c while the GtrIc-GtrIV loop No. 6-GtrIc chimera would be able to convert serotype Y to serotype 4a. Slide agglutination and LPS Western blots revealed absence of modification to respective serotypes. As both large periplasmic loops contain over 100 amino acids each (123 amino acids in loop No. 6 of GtrIV and 120 amino acids in loop No. 10 of GtrIc), each loop can possibly accommodate its own unique tertiary structure that would contribute to the serotype specificity of O-antigen modification. Therefore, in absence of this, GtrIV and GtrIc would not be able to carry out their specific functions. Alternatively, the structural integrity of each chimeric protein may have been compromised with the addition of the foreign loop. To confirm for assembly in the membrane, Western blots (using anti-alkaline phosphatase antibodies) were carried out on the membrane protein extracts of each chimeric protein (Table 2). Of the four chimeric proteins created, only GtrIc-GtrIV loop No. 2-GtrIc chimera was not detected by the blot. Its non-functional hybrid GtrIV counterpart however, was shown to have been assembled in the membrane strongly implying that specific interactions between residues present in its native loop No. 2 are required to form a catalytic site that facilitates O-antigen modification. The absence of assembly in the bacterial membrane of the GtrIc-GtrIV loop No. 2-GtrIc chimera suggests that GtrIV loop No. 2 has compromised the structural integrity of the GtrIc protein. Similarly, loss of function for GtrIc-GtrIV loop No. 6-GtrIc and GtrIV-GtrIc loop No. 10-GtrIV chimeras can be attributed to the importance of the two C-terminal periplasmic loops in conferring serotype specificity through the addition of glucosyl residues to the O-antigen in a site and linkage specific manner. As both loops span more than 100 amino acids each, this would facilitate intra-loop and inter-loop interactions that may contribute to serotype specificity and O-antigen modification.
The present study provides experimental evidence that GtrIV shares structural similarities with GtrIc, thus differing from the rest of the Gtrs. Further studies using site directed mutagenesis of amino acids between D260 to W269 and use of structure definition techniques should allow us to identify critical residues and further define the overall structure which will provide us with critical information to better understand its mechanism of action and catalytic site.
The structural similarity between GtrIV and GtrIc has been shown in this study by confirming the presence of a periplasmic loop No. 2, a large periplasmic loop No. 6 and a short cytoplasmic C-terminal end in GtrIV. The existence of a re-entrant loop, similar to that seen in GtrIc has also been observed. Two periplasmic regions were identified that could be involved in the attachment of the glucosyl group to the O-antigen. The non-critical nature of the conserved acidic residues coupled by the structural difference of GtrIV compared to the other Gtrs, except GtrIc, could indicate that its mechanism of action may be different from the rest of the Gtrs and may be similar to that of GtrIc. By sequentially deleting loop segments in loop No. 6, the presence of a potential catalytic site located between residues D260 to W269 was hypothesised. To further investigate the roles of conserved or specific functions of the two periplasmic loops of GtrIV, loop swap experiments between the N-terminal periplasmic loops and the C-terminal periplasmic loops were undertaken. The resulting hybrids lost their native function and were unable to substitute function to the other protein. This signifies the importance of both loops in GtrIV function. Furthermore, the identification of critical residues in these regions and further structural studies will provide the basis for the localization of the active site and the elucidation into the mechanism of action of GtrIV.
Normalised activity ratio
- Exo III:
We thank Herbert Winkler for providing pho-lac constructs. This work was funded by the National Health and Medical Research Council of Australia.
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