Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland1
Author for correspondence: Peter Owen. Tel: +353 1 6081188. Fax: +353 1 6799294. e-mail: powen{at}tcd.ie
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ABSTRACT |
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Keywords: antigen 43, Escherichia coli, phase variation, multiple alleles
Abbreviations: Ag43, antigen 43; Dam, deoxyadenosine methylase; EPEC, enteropathogenic E. coli; DIG, digoxigenin
The GenBank accession numbers for the sequences reported in this paper are AF233271AF233273.
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INTRODUCTION |
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The gene (agn43 or flu) encoding Ag43 has been sequenced from both E. coli K-12 and ML308-225 strains (Blattner et al., 1997 ; Henderson & Owen, 1999
) and is located on the K12 chromosome between min 44·6 and min 44·8. The primary translation product of agn43 is processed via an N-terminal signal peptidase and by internal cleavage to generate the mature
43 [predicted Mr 49789; apparent Mr (by SDS-PAGE) 60000] and ß43 [predicted Mr 51642; apparent Mr (by SDS-PAGE) 53000] subunits. Sequence comparisons and secondary structure analysis have provided compelling evidence that Ag43 is a member of the autotransporter family (Henderson et al., 1998
) with
43 representing the passenger domain involved in autoaggregation and ß43 the translocation domain organized as an 18-stranded ß-barrel pore (Henderson & Owen, 1999
). Reversible phase switching of agn43 is regulated by a novel mechanism involving competition between deoxyadenosine methylase (Dam) and OxyR (a LysR-type transcriptional activator/repressor) for three unmethylated 5'-GATC-3' sites in the regulatory region of the gene. Thus, in contrast to the phase-ON/OFF state observed for parental strains, dam mutants are locked-OFF for Ag43 expression whereas oxyR mutants are locked-ON (Henderson et al., 1997b
; Henderson & Owen, 1999
; Haagmans & van der Woude, 2000
).
Although agn43 is known to be present in single copy on the chromosome of E. coli K-12 (Blattner et al., 1997 ), the situation in wild-type E. coli strains is less clear. In this context, previous analysis of a panel of enteropathogenic E. coli (EPEC) strains by immunofluorescence microscopy and Western immunoblotting revealed the presence of phase-variable outer-membrane proteins that cross-reacted with anti-
43 antiserum (Owen et al., 1996
). Significantly, strains were identified that expressed multiple
43-like subunits in the Mr 5400060000 and Mr 94000 ranges. Cross-reactive subunits in the Mr 5400060000 range displayed properties reminiscent of native
43 and could be released from outer membranes when heated at 60 °C. In contrast, those of Mr 94000 displayed features anticipated of a hypothetical uncleaved (covalently bonded)
43-ß43 monomer. Based on these data, it has been suggested that Ag43 may be a member of a family of related phase-variable outer-membrane proteins (Owen et al., 1996
).
In this study, we demonstrated that E. coli ML308-225 possesses duplicate copies of agn43, both of which are subject to reversible phase variation. Sequencing and Western blotting revealed the genes to be 98% identical at both nucleotide and amino acid levels and to express size-variable subunits. Furthermore, we showed by Southern blot analysis of EPEC strains known to express multiple anti-43 cross-reactive proteins that ML308-225 is not unique in possessing multiple copies of agn43.
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METHODS |
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Genetic nomenclature.
agn43 alleles were labelled alphabetically in order of decreasing size of their respective EcoRV/SpeI restriction fragments. Ag43 proteins were assigned the same letter as the encoding allele.
Construction of agn43 fragments and DNA probes.
Selected fragments of the agn43 coding region (F1F5; see Fig. 1) were used in the construction of knockout mutants and of probes for Southern hybridization experiments. The following forward and reverse oligonucleotide primers (Sigma-Genosys) complementary to selected agn43 sequences and containing, where appropriate, engineered 5'-SacI, 5'-EcoRI, 3'-KpnI and 3'-SalI restriction enzyme cleavage sites (underlined) were used to amplify agn43 fragments F1 and F3F5: F1 (551 bp), 5'-CGCGAGCTCTGCTGGCTGCTGACATTGTTGTGC-3' (forward) and 5'-GGGGGTACCAGTGGTATTTGCCGTTCCTTCAGC-3' (reverse); F3 (400 bp), 5'-GTAAGGGTATTCAGGTGGTTG-3' (forward) and 5'-CCGGCAACCTCTGTTCTCATC-3' (reverse); F4 (569 bp), 5'-CCGAATTCACGGTGAACAACGATACCCTGCAA-3' (forward) and 5'-CCGTCGACCGCCAGCCCCGACGCACTGTTGCC-3' (reverse); F5 (1065 bp), 5'-ACGGTAAATGGCGGACTGTT-3' (forward) and 5'-CCGGCAACCTCTGTTCTCATC-3' (reverse).
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Construction of plasmids and knockout mutants.
Amplified fragment F1 (see Fig. 1) was digested overnight with KpnI and SacI and subsequently ligated with KpnI/SacI-digested
pir suicide vector pJP5603 or pJP5608 to give plasmid constructs designated pAJR003 (3·7 kb) or pAJR005 (7·2 kb), respectively. In a similar manner, EcoRI/SalI-digested fragment F4 was ligated with appropriately digested
pir suicide vector pGP704 to generate pAJR004 (4·2 kb).
Single and double knockout mutants in agn43 were constructed in both wild-type (oxyR+) and oxyR backgrounds by mating appropriate E. coli ML308-225 derivatives with E. coli S17.1 pir containing one of the three suicide vectors pAJR003pAJR005 (see Table 1
). Vector integration via single crossover events resulted in formation of interrupted alleles. The relevant genotypes of these mutants were confirmed by Southern blot analysis of randomly chosen transconjugants, using EcoRV/SpeI-restricted total genomic DNA with F1 and plasmid probes (see Fig. 2
). Thus, disruption of agn43A in both wild-type and oxyR backgrounds using pAJR004 (4·2 kb; Apr) resulted in disappearance of the larger (7·5 kb) of the two F1-hybridizing bands and the appearance of a band of the anticipated size (12 kb) hybridizing with both F1 and plasmid probes (see Fig. 2
, lanes 1, 3, 5, 7, 10, 12, 14 and 16). Similarly, disruption of agn43B in a wild-type background using pAJR003 (3·7 kb; Kmr) resulted in selective increase in size of the smaller EcoRV/SpeI fragment to generate an approximately 8 kb doublet, the upper component of which (disrupted agn43B) hybridized with both F1 and plasmid probes. The lower component of this doublet corresponds to the original agn43A restriction fragment (Fig. 2
, lanes 1, 2, 10 and 11). Inactivation of agn43B in an oxyR (Kmr) background required the use of an alternative suicide vector (pAJR005; 7·2 kb; Tcr) carrying a different antibiotic selection marker. This vector possesses an EcoRV site which results in mutated agn43B alleles giving rise to two restriction fragments (approx. 3·6 and 7 kb) capable of hybridizing with both F1 and plasmid-specific probes. The upper band is similar in size to the native agn43A restriction fragment (Fig. 2
, lanes 5, 6, 14 and 15). Southern blot analysis of double (agn43AB) mutants constructed in wild-type and oxyR backgrounds using pAJR003/4 and pAJR004/5, respectively, again yielded the predicted pattern of hybridizing bands (see Fig. 2
, lanes 1, 4, 5, 8, 10, 13, 14 and 17). The slight increase in anticipated size of the mutated agn43A fragment (Fig. 2
, lanes 4 and 13) is most likely the result of a duplication event involving the integrated vector.
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The upstream regions of each gene could not be amplified using the above strategy, presumably due to the presence of an EcoRV or SpeI cleavage site at the 5' end of the gene. Accordingly, a different strategy was devised to amplify the region encoding the signal sequence of both genes. This involved use of purified total genomic DNA from single knockouts AJR1 (ML308-225 agn43B::pAJR003) and AJR2 (ML308-225 agn43A::pAJR004) as templates in PCR reactions utilizing forward and reverse primers complementary to sequences upstream of the signal codons and downstream of the mutated agn43 allele, respectively, and extension periods selectively favouring amplification of the (smaller) wild-type allele. The clear size difference between products derived from mutated and wild-type alleles additionally ensured the unambiguous identification of agn43A-specific PCR fragments (produced from AJR1 template DNA) and agn43B-specific PCR fragments (produced from AJR2 template DNA) following their analysis by agarose gel electrophoresis.
DNA sequencing, performed with custom-made primers, was carried out on PCR-generated gel-purified DNA fragments according to the manufacturers instructions using the Thermosequenase kit (Amersham). Sequencing reactions were resolved using an ABI 373A sequencer and resultant sequences were analysed by Seqed (Applied Biosystems). Analysis of completed sequences was carried out using the GCG programs (University of Wisconsin Genetics Computer Group).
Protein analysis.
SDS-PAGE was performed using 12·5% (w/v) polyacrylamide separating gels and a 4·5% polyacrylamide stacking gel (Laemmli, 1970 ). Samples were routinely heated for 3 min at 100 °C in Laemmli sample buffer (Laemmli, 1970
) prior to electrophoresis. Proteins were detected by staining with Coomassie brilliant blue. Molecular masses were determined from the relative mobilities of 15 standard molecular mass marker proteins (BenchMark protein ladder; Gibco-BRL). Immunofluorescence microscopy, colony and Western immunoblotting, and determination of switch frequencies were carried out using anti-
43 antiserum in accordance with established procedures (Eisenstein, 1981
; Caffrey et al., 1988
; Henderson et al., 1997a
). The Antigenicity Index program devised by Jameson & Wolf (1988)
was used to identify potential epitopes. This method is based on an algorithm that integrates the predicted influence of hydropathy and surface probability with flexibility factors.
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RESULTS |
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Analysis of the various knockout mutants by Western immunoblotting (Fig. 4), immunofluorescence microscopy (Fig. 5
) and colony immunoblotting (not shown) indicated that disruption of agn43A or agn43B alone was not sufficient to suppress expression of immunoreactive antigen. Thus, phase-ON populations of single mutants AJR1 (agn43B) and AJR2 (agn43A), as well as cells of AJR4 (agn43B oxyR) and AJR5 (agn43A oxyR) clearly expressed a polypeptide (apparent Mr 60000) capable of reacting with anti-
43 serum, whereas phase-OFF populations of the single mutants, together with double mutants AJR3 (agn43AB) and AJR6 (agn43AB oxyR) did not (Fig. 4
). These results were confirmed and extended in immunofluorescence and colony-immunoblotting experiments which clearly revealed that AJR1 (agn43B) and AJR2 (agn43A) expressed Ag43 in a phase-variable manner similar to that observed for control strains, whereas expression of the antigen in AJR4 (agn43B oxyR) and AJR5 (agn43A oxyR) was locked-ON in a manner similar to that evidenced by the parental (oxyR) derivative, with all cells in the population expressing antigen (see Fig. 5
). These data clearly demonstrate that ML308-225 possesses duplicate copies of agn43 viz. agn43A and agn43B, both of which are capable of expressing Ag43 homologues in a phase-variable manner. They also indicate that the agn43A::pAJR004 constructs (see Table 1
) containing insertions within the ß43A-encoding region are incapable of producing stable
43A, as anticipated from consideration of the roles and properties attributed to the translocation (ß) domain of autotransporters (Henderson et al., 1998
; Maurer et al., 1999
).
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Sequence analysis of agn43AML308-225 and agn43BML308-225
A sequence for agn43ML308-225, based on analysis of cloned PCR products amplified from total genomic DNA, has previously been published by workers in this laboratory (Henderson & Owen, 1999 ). However, in view of the demonstration of two agn43 homologues in this strain and the obvious potential for cross-amplification, it became necessary to refine this work and independently sequence each gene using more stringent procedures. The strategy involved is detailed in Methods. Comparative analysis of the resulting agn43A and agn43B sequences (GenBank accession nos AF233271 and AF233272, respectively) revealed 98% identity at both nucleotide and predicted protein levels. Both sequences showed minor differences to that previously published for agn43 (U24429; Henderson & Owen, 1999
). The 3120 bp coding region of each allele (agn43A and agn43B) differed at 40 nucleotide positions, nine occurring in the
43-encoding region, four in the ß43-encoding region and the remainder in the region encoding the signal sequence. This resulted in similarly sized (1039 residue) primary translation products of predicted Mrs 106979 (Ag43A) and 106940 (Ag43B) containing, respectively, 4, 3 and 12 amino acid differences between the two
43 domains, the two ß43 domains and the two signal sequences. The predicted Mrs for the
43A,
43B, ß43A and ß43B subunits were 49904, 49795, 51554 and 51633, respectively. Of interest are differences observed in the two surface-expressed
43 subunits. Notably, similar changes from alanine residues (
43B) to threonine residues (
43A) at two neighbouring positions (226 and 230) of the primary translation product significantly increase the surface probabilities [from 0·7 (
43B) to 1·5 (
43A)] of one of the predicted (Jameson & Wolf, 1988
) cell-surface epitopes. The small sequence variations may also account for the minor differences in the electrophoretic mobilities of the two
43 subunits detected following SDS-PAGE (see Fig. 4
). The differences predicted in the amino-terminal sections of the two precursor proteins do not appear to radically alter the properties of the principal domains/motifs characteristic of the extended signal sequence observed for autotransporters (Henderson et al., 1998
).
Both genes contained previously identified motifs, viz. those for an aspartyl protease active site, O-glycosylation attachment, a P-loop and RGD motifs (Henderson & Owen, 1999 ). Additional motifs identified as a result of this study include (a) a leucine zipper motif (L500L521) implicated in protein dimerization (Phizicky & Fields, 1995
) and located at the C terminus of each
43 subunit and (b) the presence, within 53 residues of the P-loop motif of both ß43 subunits, of a sequence (R692DSDESWY) characteristic of a tyrosine phosphorylation site. These observations suggest possible mechanisms for subunitsubunit associations/autoaggregation and autotransport, respectively. Numerous (17) potential N-glycosylation sites were dispersed throughout both
43 and ß43subunits.
Southern blot analysis of EPEC strains
Previous studies from this laboratory have documented the presence within certain EPEC strains of multiple anti-43 cross-reactive proteins (Owen et al., 1996
). In view of the current demonstration of duplicate agn43 genes in ML308-225, this panel of EPEC strains was analysed by Southern blotting using agn43-specific gene probes (F1, F3 and F5). Resultant blots revealed several strains to possess multiple (up to four) hybridizing EcoRV/SpeI fragments. Some strains [e.g. NCTC 10089 (Fig. 6
), NCTC 8603 and NCTC 9114 (data not shown)] possessed only one hybridizing fragment and NCTC 8621 none at all (see Fig. 6
). Identical hybridization patterns were obtained for all three probes except in the case of NCTC 8007, where the smallest hybridizing band had an approximate size of 6 kb and 3 kb when probed with F3/5 and F1, respectively, indicating the presence of an EcoRV or SpeI site between regions F1 and F5 of the agn43D allele of this strain. These data strongly suggest the presence of varying numbers of agn43 alleles within clinical EPEC isolates. The number of predicted gene copies was always equal to or greater than the number of anti-
43 cross-reactive proteins detected by Western immunoblotting (see Fig. 6
and Table 1
). Taken together, these results suggest the presence of a family of Ag43 proteins encoded by multiple chromosomal alleles.
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DISCUSSION |
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The differences between the two agn43 alleles of E. coli ML308-225 are subtle rather than striking. Both genes are clearly capable of expressing product. Both are subject to phase variation, and both appear to be regulated by a similar mechanism in which OxyR competes with Dam for unmethylated 5'-GATC-3' sites in the regulatory region of agn43 (Henderson et al., 1997a ; Henderson & Owen, 1999
; Haagmans & van der Woude, 2000
). Certainly, the locked-ON phenotype observed for both oxyR agn43A and oxyR agn43B derivatives (Fig. 5
) and the total absence of any Ag43-expressing cells in dam agn43A+B+ derivatives (Henderson & Owen, 1999
) are fully compatible with this proposition. In the absence of allele/product-specific probes it is difficult to determine whether agn43A and agn43B are capable of simultaneous expression in any one cell. However, comparison of fluorescent intensities in parental and single knockout mutants suggests that this may be possible (A. J. Roche, M. Meehan & P. Owen, unpublished data). The observed differences in OFF
ON and ON
OFF switch frequencies for the two alleles may reflect a mechanism that allows for selection of populations expressing either Ag43A or Ag43B in vivo, and strongly suggests that Ag43B is the more dominant of the two antigens during growth under standard laboratory conditions.
Whether or not the Ag43A and Ag43B of E. coli ML308-225 are antigenically/functionally variant has not been definitively established. Autoaggregation and colony morphology changes induced by Ag43 (Diderichsen, 1980 ; Henderson et al., 1997a
) occur only in certain genetic backgrounds (e.g. E. coli K-12) and are not pronounced properties of E. coli ML308-225 derivatives, a phenomenon probably related to the presence of different types of surface structures, for example fimbriae and lipopolysaccharide (Hasman et al., 1999
, 2000
). However, the relatively small number of amino acid differences in the surface-exposed
43 subunits does result in changes in molecular size and apparent epitope expression. It is also conceivable that the principal differences observed, A(
43B)226T(
43A) and A(
43B)230T(
43A), result in changes in O-glycosylation patterns. Certainly, there is growing evidence that bacterial surface structures can be glycosylated (Stimson et al., 1995
; Brimer & Montie, 1998
; Forest et al., 1999
; Lindenthal & Elsinghorst, 1999
; Sleytr & Beveridge, 1999
). Additional experimentation, involving refined chemical and immunological analysis, is required to fully resolve these issues.
Size-variable 43 subunits suggestive of antigenic variation have been identified in this study of E. coli strain ML308-225 and also in previous studies of EPEC isolates (Owen et al., 1996
). Our present studies confirm and extend these latter observations and demonstrate the probable presence of multiple agn43 alleles in certain EPEC strains. It is also clear from sequence analysis that, whereas the agn43 genes of strains ML308-225 and K-12 show very high homology (indeed the primary sequence of
43B from ML308-225 and
43 from K-12 are identical), those of other strains (e.g. agn43NCTC9114) can encode
43 subunits which differ more substantially, especially towards the N terminus, and can lack putative functional motifs. Ongoing sequence analysis of the genome of the uropathogenic E. coli isolate CFT073 confirms the thrust of our data and indicates the presence in this strain of two ORFs showing strong homology with agn43 and predicted to encode proteins lacking RGD motifs (Blattner et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 15 June 2000;
revised 10 August 2000;
accepted 23 August 2000.