Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, The Australian National University, Canberra, ACT 0200, Australia1
Author for correspondence: Naresh K. Verma. Tel: +61 2 6125 2666. Fax: +61 2 6125 0313. e-mail: Naresh.Verma{at}anu.edu.au
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ABSTRACT |
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Keywords: bacteriophage, serotype conversion, lipopolysaccharide, glucosyltransferase
The GenBank accession number for the sequence reported in this paper is AF288197.
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INTRODUCTION |
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Infection with S. flexneri results in serotype-specific immunity (Phalipon et al., 1995 ). There are 13 different serotypes of S. flexneri, which differ in the nature of the O antigen component of the outer-membrane lipopolysaccharide. S. flexneri O antigens, except for serotype 6, have the same basic repeating tetrasaccharide unit, comprised of N-acetylglucosamine attached to three rhamnose units (Simmons & Romanowska, 1987
). The basic O antigen structure is referred to as serotype Y (Simmons & Romanowska, 1987
) (Fig. 1
). The addition of glucosyl and/or O-acetyl residues to the basic O antigen results in type- (i.e. I, II, III, IV, V) and group- (i.e. 3,4; 7,8; 6) specific antigenic determinants. The addition of an O-acetyl residue occurs only on rhamnose III of the basic tetrasaccharide unit, and confers group 6 O antigen modification. Acetylation is mediated by an O-acetyltransferase which is encoded by bacteriophage Sf6 (Clark et al., 1991
; Verma et al., 1991
). Glucosylation of the O antigen can occur on any of the residues in the tetrasaccharide unit and can vary in the nature of the linkage. The genes responsible for O antigen glucosylation of type I, II and V and group 7,8 serotypes have been identified (Adhikari et al., 1999
; Bastin et al., 1997
; Guan et al., 1999
; Guan & Verma, 1998
; Huan et al., 1997a
, b
; Mavris et al., 1997
; Verma et al., 1993
). In all cases, the factors required for serotype conversion or O antigen modification are encoded by temperate or cryptic bacteriophages. The organization of the genes encoding glucosylation is conserved (reviewed by Allison & Verma, 2000
): the first two genes, designated gtrAtype and gtrBtype, are highly homologous and interchangeable whereas the third gene, referred to as gtr(type), is unique and encodes the serotype-specific glucosyltransferase. In the phage genome, the glucosylation genes are typically located downstream of the integrase gene and attP site, which mediates integration of the phage into the prolac region of the host genome (Adhikari et al., 1999
; Guan & Verma, 1998
; Petrovskaya & Licheva, 1982
). The genes involved in O antigen modification are of interest because of their potential application in the development of S. flexneri vaccine strains expressing specific and, potentially, multiple serotypes (Guan & Verma, 1998
; Huan et al., 1995
; Verma et al., 1991
).
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METHODS |
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DNA techniques.
Plasmid DNA was routinely prepared by alkaline lysis (Sambrook et al., 1989 ). The BRESAClean DNA Purification Kit (Geneworks) was used to further purify plasmid DNA when required and was also used to gel-purify DNA fragments. Chromosomal DNA was prepared using the procedure outlined by Bastin et al. (1997)
. Restriction enzymes were used according to the manufacturers directions (Progen and Amersham Pharmacia).
Purified DNA was sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit in a GeneAmp 2400 thermal cycler (Perkin Elmer) according to the manufacturers protocol. Sequencing was carried out on an ABI Prism 377 Automated Sequencer at the Biomolecular Resource Facility in the John Curtin School of Medical Research, The Australian National University. Sequence analysis was performed using the GCG group of programs (University of Wisconsin) available through the Australian National Genomic Information System.
DNA for Southern hybridization was digested with the appropriate restriction enzymes and subjected to electrophoresis on a 0·6% agarose gel. The alkali blot method was used to transfer DNA onto Hybond-N+ membrane (Amersham Pharmacia) using the manufacturers protocol. The Gigaprime DNA Labelling Kit (Geneworks) was used to label probe DNA with [-32P]dCTP (Amersham Pharmacia). The DNA for the probes was prepared as follows: the 524 bp EcoRI fragment of pNV734 was used for the gtrAEc probe; the 957 bp EcoRV fragment of pNV677 [pBluescript containing the gtrAX and gtrBX genes of SfX (Guan et al., 1999
)] provided the gtrBX probe; and the 1527 bp EcoRI fragment of pNV734 provided the gtrIVEc probe. Southern hybridization was performed as outlined in Sambrook et al. (1989)
. The membrane was washed twice with 2xSSPE containing 0·1% SDS at 45 °C, and once with 1xSSPE containing 0·1% SDS at 65 °C.
For colony hybridization, overnight colonies were transferred and fixed to a sterile nitrocellulose filter (MSI) using the manufacturers protocols, and screened as described elsewhere (Sambrook et al., 1989 ).
PCR.
Colony PCR (Schuch & Maurelli, 1997 ) was used to amplify the putative serotype 4a O antigen modification genes (o120 o306 o443) from the E. coli K-12 genome using primers based on sequence accession number AE000323 (Blattner et al., 1997
): forward primer, TAATGGTACCACAGCAAGTATCGAT (nt 72537270); and reverse primer, TTAGGATCCCGCAATTCTATCAGGAG (complementary to nt 1001210029). KpnI and BamHI restriction sites introduced into the primers are underlined. The PCR reaction conditions were 94 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 65 °C for 4 min. PCR products were cloned directly into the pGEM-T Easy TA cloning vector (Promega).
Immunogold labelling and electron microscopy.
Immunogold labelling was conducted as described by Huan et al. (1997b) with the following modifications: the primary antibodies, MASF Y-5 (serotype-Y-specific) and MASF IV-2 (serotype-4a- and -4b-specific), both kindly supplied by Nils Carlin (Carlin & Lindberg, 1987
), were diluted 1:50 in PBS containing 0·4% BSA (PBS-BSA); and the secondary antibodies, goat anti-mouse IgM conjugated to 10 nm gold particles (MASF Y-5) and goat anti-mouse IgG conjugated to 20 nm gold particles (MASF IV-2) (British Biocell International), were diluted 1:9 in PBS-BSA. The samples were viewed under a Joel 2000X transmission electron microscope at 80 kV.
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RESULTS AND DISCUSSION |
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Based on these data, Allison & Verma (2000) renamed the E. coli o120 o306 o443 genes gtrAEc gtrBEc gtrIVEc, consistent with the newly proposed nomenclature system. To determine if these E. coli genes were capable of mediating serotype conversion in S. flexneri, the gtrAEc gtrBEc gtrIVEc genes were amplified from E. coli DH1 by colony PCR. The 2·78 kb PCR product was ligated directly into pGEM-T Easy and transformed into E. coli JM109. Plasmid DNA from three transformants, B831, B832 and B833, was subjected to restriction analysis and the corresponding plasmids, pNV734, pNV735 and pNV736, respectively, were found to contain the correct 2·78 kb fragment. The gtrAEc gtrBEc gtrIVEc genes were in the correct orientation to the lac promoter of the vector in pNV736, whereas the genes in pNV734 and pNV735 were in the opposite orientation.
Plasmids pNV734, pNV735 and pNV736 were transformed into serotype Y strain SFL124 to create recombinant strains SFL1258, SFL1259 and SFL1260, respectively. All three recombinant strains, SFL124 and NCTC 8296 (serotype 4a) were analysed by immunogold labelling with monoclonal antibodies MASF Y-5 (serotype-Y-specific) and MASF IV-2 (type-IV-specific) as the MASF antibodies have been shown to be serotype-specific when used in immunogold labelling and Western blots (Adhikari et al., 1999 ; Guan et al., 1999
; Huan et al., 1997a
, b
). The O antigen of the control strains SFL124 and NCTC 8296 was only recognized by MASF Y-5 and MASF IV-2, respectively (Fig. 2
). The O antigen of recombinant strains SFL1258, SFL1259 and SFL1260, however, was recognized by MASF IV-2 and MASF Y-5 antibodies, indicating the presence of both serotype Y and type IV antigens on the surface of the bacteria (the results for SFL1258 are shown in Fig. 2
). The degree of O antigen modification was confirmed by analysing bacteriophage sensitivity. Bacteriophage SfV is a serotype-converting bacteriophage (Huan et al., 1997a
, b
). While serotype Y strain SFL124, and strains partially converted to serotype 5a, are sensitive to SfV, recombinant strains of SFL124 which are completely converted to serotype 5a are resistant to SfV infection (Huan et al., 1997a
, b
). Similar results were obtained with complete and partial conversion to serotype 4a. While NCTC 8296 was resistant to infection by SfV, SFL1258 and SFL1260 remained sensitive (only 10-fold less sensitive than control strain SFL1257; data not shown). Taken collectively, the immunogold and phage sensitivity data confirm the partial serotype conversion of SFL1258, SFL1259 and SFL1260.
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To ensure that the partial serotype conversion observed for SFL1258, SFL1259 and SFL1260 was not the result of a PCR-induced mutation in either gtrA or gtrB, the gtrAEc gtrBEc genes from pNV734 were cloned in front of the gtrX gene, which encodes the glucosyltransferase responsible for group 7,8 glucosylation (Guan et al., 1999 ). The resulting recombinant plasmid, pNV781, was introduced into SFL124 to create recombinant strain SFL1350. Sensitivity to bacteriophage SfV and colony agglutination were used to determine if gtrAEc gtrBEc could completely complement gtrX. SFL1350 was resistant to infection by SfV (data not shown) whereas partially converted strain SFL1096 (SFL124 containing the gtrX gene only: Verma et al., 1993
; Huan et al., 1995
) was sensitive to SfV. These data were confirmed with colony agglutination where SFL1350 agglutinated strongly with group 7,8 sera compared to SFL1096, which reacted very weakly (data not shown). These data suggest that the GtrAEc and GtrBEc proteins are functional in SFL124. These results also suggest that the partial conversion observed for strains SFL1258, SFL1259 and SFL1260 is related to GtrIVEc activity.
The basic structure of the native E. coli K-12 O16 O antigen (Stevenson et al., 1994 ) differs from that of S. flexneri (Fig. 1
). While the rhamnose III-N-acetylglucosamine disaccharide is present in both, the bonds between these two sugars differ, with a 1,3-ß bond present in S. flexneri O antigen and a 1,3-
bond present in E. coli (Fig. 1
). The activity of GtrIVEc may be specific for the E. coli O antigen and it is possible that this activity and/or specificity is reduced when presented with the heterologous S. flexneri substrate. Differences in the GtrIVEc and GtrIVSf proteins (refer to results below) could be related to this substrate-specificity. Since partially converted recombinant vaccine strains do not confer complete protection (Huan et al., 1995
; Verma et al., 1991
), the E. coli genes are poor candidates for the development of recombinant vaccine strains. Consequently, we decided to isolate the type IV glucosylation factors from S. flexneri.
Induction of temperate bacteriophages from S. flexneri serotype 4a and 4b strains
The O antigen glucosylation genes encoding type II and V and group 7,8 (serotype X) modifications have been successfully isolated and characterized from bacteriophages SfII, SfV and SfX, respectively (Guan et al., 1999 ; Huan et al., 1997a
, b
; Mavris et al., 1997
; Verma et al., 1993
). A bacteriophage, designated
9725, was induced from serotype 4a strain NCTC 9725, but phages were not induced from serotype 4a strain NCTC 8296 and serotype 4b strain NCTC 8336. To determine if the phage genome contained the O antigen modification genes, attempts were initially made to create a
9725 lysogen in SFL124 that could be analysed for serotype conversion; however, a lysogen was not obtained. Subsequently, the phage DNA was subjected to Southern hybridization using the conserved gtrA and gtrB genes and gtrIVEc as probes. The
9725 genome failed to hybridize to any of the probes, with results from the gtrAEc hybridization shown in Fig. 3
. The absence of the conserved gtrA and gtrB genes associated with other serotype conversion loci suggests that the phage is not involved in serotype conversion. Phage
9725 is one of the few S. flexneri bacteriophages described in the literature that does not confer O antigen modification. Further studies will focus on characterizing the molecular biology of this phage.
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The fact that both gtrA and gtrB probes hybridized to the same fragment in all strains is consistent with the organization of other loci encoding O antigen modification, where the gtrA and gtrB genes are adjacent to one another. The hybridization data for gtrB are consistent with that of Mavris et al. (1997) , who reported that all serotypes of S. flexneri as well as E. coli K-12 hybridized to the gtrBII (bgt) probe. All the strains tested in this study also hybridized to the gtrAEc probe. Hybridization of SFL124 DNA to both gtrA and gtrB suggests that this strain may contain homologues of these genes, which may explain why partial serotype conversion is observed when only the serotype-specific gtr gene, or gtr in combination with only gtrA or gtrB, is introduced into SFL124 (Adhikari et al., 1999
; Guan et al., 1999
; Huan et al., 1995
, 1997a
, b
). The activity and/or specificity of the gtrA gtrB homologues in SFL124 may be reduced relative to that of the glucosylation genes and, therefore, results in partial serotype conversion. It is also interesting to note that the patterns of hybridization for all strains except NCTC 9725 (serotype 4a) and NCTC 8336 (serotype 4b) are different. These observations suggest that the nature and/or completeness of the serotype-converting phages, and/or overall chromosome organization, can vary significantly among different strains, including those of the same serotype.
The approximately 4 kb chromosomal fragment from NCTC 8296, which hybridized to the gtrA and gtrB probes, was theoretically large enough to contain all three serotype conversion genes and was a convenient size for cloning. Genomic DNA from NCTC 8296 was digested with BamHI and fragments from 3·6 to 4·2 kb were gel-purified, ligated into the BamHI site of pUC18, and transformed into E. coli JM109. White colonies (800) were restreaked and screened by colony hybridization using gtrBX as a probe. Seven transformants hybridized strongly to the probe and were analysed further. Plasmid DNA from B837 and B838 contained the approximately 4 kb BamHI fragment and the respective plasmids were designated pNV739 and pNV740.
To determine if pNV739 and pNV740 contained the serotype conversion genes, the plasmids were transformed into serotype Y strain SFL124 to create recombinant strains SFL1264 and SFL1265, respectively. Both recombinant strains, SFL124 (serotype Y) and NCTC 8296 (serotype 4a) were analysed by immunogold labelling using MASF Y-5 (serotype-Y-specific) and MASF IV-2 (type-IV-specific) monoclonal antibodies (Fig. 2). The O antigen of the control strains was only recognized by the respective serotype-specific antibody as described previously (Fig. 2
). The O antigen of recombinant strains SFL1264 and SFL1265 was no longer bound by MASF Y-5 but was recognized by MASF IV-2 in a pattern similar to that of NCTC 8296 (results for SFL1265 shown in Fig. 2
). These data indicate that the approximately 4 kb BamHI chromosomal fragment from NCTC 8296 contained the type IV O antigen modification genes, which conferred complete serotype conversion from Y to 4a in SFL124.
The complete BamHI fragment was sequenced and analysed. The sequence has been deposited in GenBank under accession number AF288197. The 3834 bp fragment was predicted to contain three complete open reading frames (orf1,2,3) and one incomplete ORF. All ORFs were transcribed in the same direction (Table 2). Putative promoter (-35 region, nt 830835; -10 region, nt 845850) and rho-independent terminator (nt 34783502) sequences were located upstream of orf1 and downstream of orf3, respectively, suggesting that these three ORFs are transcribed in an operon. When the sequencing data were compared to the restriction map of pNV739 and pNV740, it was determined that the putative operon was in both orientations, with the operon in pNV739 being in the correct orientation to the lac promoter of pUC18 and in the opposite orientation in pNV740.
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Organization of the serotype conversion genes in NCTC 8296
Upstream of gtrAIV, a 46 nt sequence showing 100% identity to the attachment sites of S. flexneri serotype-converting phages, and other related phages, was identified (Table 2). The incomplete ORF located immediately upstream of attP also showed significant homology to related integrase genes (Table 2
). In fact, the nucleotide sequence of int', the intervening non-coding attP region, gtrAIV, and the first approximately one-third of the gtrBIV gene in NCTC 8296 (nt 11580) is almost identical to the nucleotide sequence of the corresponding regions in phages SfII and SfV (98·9% identity). In the SfII and SfV genomes, the glucosylation genes are located downstream of the integration module in the order of int attP gtrA gtrB gtr. Upon integration of the phage into the host chromosome, the int and gtr genes are separated by the phage genome but are still transcribed in the same direction. In SfII and SfV lysogens, host DNA would be expected up- and downstream of the attL and attR sites, respectively. This organization was largely conserved in serotype 1a strain Y53 even though a large portion of the phage genome had been deleted (Adhikari et al., 1999
). The organization of the genes in this region of NCTC 8296, however, suggests that the int attP located upstream of the glucosylation genes belongs to a phage which has integrated adjacent to the type IV O antigen modification genes. These data suggest that this region of the NCTC 8296 genome has been derived from two bacteriophages, neither of which was induced using the UV induction.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 August 2000;
revised 20 November 2000;
accepted 14 December 2000.