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The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.
Shiga-toxigenic Escherichia coli (STEC) serotype O157:H7 carries O157 and H7 antigens, so these traits are extensively used in identification (1). Strain MA6, isolated from beef in Malaysia (8), carries the O157:H7 virulence factor genes, including the Shiga toxin 2 gene (stx2), the γ intimin allele (γ-eae), the enterohemolysin gene (ehxA), and the +93 uidA single nucleotide polymorphism (SNP) found only in O157:H7 strains (1). Multilocus sequence typing also showed MA6 to have the most common sequence type (ST-66) for O157:H7 strains. However, and in spite the fact that MA6 had per gene sequences essential for O157 antigen synthesis (2), no O157 antigen is expressed (O rough), and therefore, it is undetectable with serological assays used in O157:H7 analysis.
The biosynthesis and assembly of E. coli O antigen are highly complex (9). The rfb operon (12 genes) (16), along with 3 ancillary genes outside of the rfb, is required for the biosynthesis of the 4 sugar nucleotide precursors and the assembly of the O unit (11). This is then linked to the core antigen, comprising an inner and an outer component, which require 3 other operons for biosynthesis and assembly (9). As defects in any of these genes could produce the O-null phenotype (13), we systematically examined these genes (Table (Table1)1) to elucidate the cause of the absence of O157 expression in MA6.
PCR and sequencing primers for the individual genes were designed from sequences for the O157:H7 strain EDL933 (GenBank accession no. AE005174). The 50-μl PCR mix contained 5 U of HotStar Taq (Qiagen, Valencia, CA), 1× polymerase buffer, 2.5 to 3.5 mM MgCl2, 400 μM each dNTP, 300 nM of each primer, and ~100 ng of template DNA from either MA6 or the EDL931 reference strain. The “touchdown” PCR (10) consisted of 95°C for 15 min and 10 cycles of 95°C for 30 s, 69 to 60°C (−1°C/cycle) for 20 s, and 72°C for 1.5 min, followed by 35 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 1.5 min, with a single step of 72°C for 1 min for final extension. Products were examined on a 1% agarose gel in Tris-borate-EDTA (TBE) buffer. Comparison of amplicons from respective genes from MA6 and EDL931 showed that no gross differences in size were observed for any of the rfb or related genes, suggesting the absence of major insertions or deletions. Consistently, contigs assembled from the MA6 amplicon were identical in sequence to those of EDL933 in GenBank, indicating the absence of base mutations in either the promoter or any of the open reading frames (ORF). One exception was the gne gene, encoding UDP-acetylgalactosamine (GalNAc)-4-epimerase, which is essential for the synthesis of one of the oligosaccharide subunits in the O antigen (14). When PCR primers that bound upstream of the putative promoter and downstream of the gne gene were used, an expected ~1,400-bp product was obtained from EDL931 (Fig. (Fig.1,1, lane 3), but the MA6 amplicon was ~2,700 bp (Fig. (Fig.1,1, lane 4). PCR of other O157:H7 strains all yielded the ~1,400-bp product, while MA6 consistently produced the larger amplicon. Comparison of sequences to that of EDL933 showed the presence of a 1,310-bp insertion within the MA6 gne ORF at +385 that shared 96% homology to the insertion sequence 629 (IS629) (accession no. X51586) element. Furthermore, the deduced protein sequences for the putative orfA and orfB genes on the insert were 100% and 99% identical to those of the IS629 transposase in O157:H7 strains Sakai (accession no. NC_002695), and EDL933 and EC4115 (accession no. NC_011353), respectively.
To determine whether gne::IS629 (accession no. GU183138) caused the absence of O157 expression in MA6, the wild-type EDL931 gne ORF was amplified using primers that added BamHI and SacI restriction sites at the 5′ and 3′ termini, respectively. The purified amplicon was digested accordingly, ligated into pTrc99A vector (Stratagene, La Jolla, CA), and electroporated into E. coli DH5α (10). Transformants were selected on LB plates with 100 μg/ml ampicillin (Amp). Colonies that were Amp resistant (Ampr) were PCR amplified with vector-specific primers, and those carrying the insert were sequenced to confirm the presence of the wild-type gne insert in the construct (pGNE). For trans-complementation studies, pGNE was electroporated into MA6. Ampr transformants were PCR amplified with vector-specific primers as well as primers that annealed to sequences outside the gne gene and also not present on the vector, to confirm that they carried both pGNE and the gne::IS629 locus. Serological testing with the RIM O157:H7 latex kit (Remel, Lenexa, KS) confirmed that the Ampr MA6 transformants expressed O157 antigen.
These results confirmed that gne::IS629 caused the O rough phenotype of MA6. Originally isolated from Shigella sonnei (7), IS629 has since been found, often in multiple copies, to cause gene disruptions in other enteric bacteria (6). fliC::IS629 caused nonmotility of an E. coli O111 strain (17), and wbaM::IS629 resulted in an O rough Shigella boydii strain (15). The IS629 recognition site remains unknown (5), so it is uncertain that there is an IS629 hot spot within the O157:H7 gne ORF. Other bacteria, like O157:H7, also have the gne gene positioned upstream of the rfb operon (12), but no gne::IS629 rough strains of these have been reported. This suggests that the IS629 insertion site within the gne of MA6 may have occurred as a result of a random mutation and that MA6 appears to be the only naturally occurring O rough O157:H7 strain that resulted from the gne::IS629 insertion.
The O antigen is not required for growth but does confer protection (9), so the loss of the O antigen has been reported to make pathogens serum sensitive or less virulent (4). If that is so, we would expect MA6 to be less pathogenic than O157:H7; consistent with that speculation, MA6 has not been implicated in illness. Even so, while no O rough O157:H7 strains have caused disease, other O rough STEC strains have caused illnesses (3); hence, the virulence potential of MA6 remains undetermined.
In conclusion, the absence of O157 antigen expression by MA6 is caused by gne::IS629. Occurrence of O rough:H7 strains like MA6 in food or clinical samples is of concern, as they are undetectable by the serological assays used to identify O157:H7. However, the IS629 insertion site within the O157:H7 gne ORF appears to have been due to a random mutational event, and therefore, MA6-like O rough mutants of O157:H7 are thus far uncommon.
This project was supported by an appointment to the Research Fellowship Program for the Center for Food Safety and Applied Nutrition administered by the Oak Ridge Associated Universities through a contract with the FDA.
Published ahead of print on 30 November 2009.