Regulation of type 1 fimbriae by unlinked FimB- and FimE-like recombinases in uropathogenic Escherichia coli strain CFT073

Abstract

Genomic DNA sequence analysis of the uropathogenic Escherichia coli strain CFT073 revealed that besides the fimB and fimE recombinase genes that control the type 1 pilus fim phase switch, there are three additional fimB- and fimE-like genes: ipuA, ipuB, and ipbA. Alignment of the predicted amino acid sequences showed that the five recombinases range in sequence similarity from 63 to 70%. An epidemiological survey indicates that ipuA and ipuB are present and linked next to the dsdCXA locus in 24 of 67 uropathogenic E. coli strains but are found in only 1 of 15 normal human fecal isolates. The ipbA sequence located next to the betABIT locus was found in 42 of 67 uropathogenic isolates and 8 of 15 of the commensal strains. We show that two of these recombinases, those encoded by ipuA and ipbA, can function at the type 1 pilus fim switch. In a CFT073 deletion mutant lacking all five recombinase genes, recombinant ipuA or ipbA provided in trans inverted the fim element from the off state to the on state. When a fim OFF CFT073 DeltafimBE mutant was used to infect the urinary tracts of mice, a switch to the fim on state was detected within 24 h in bacteria recovered from urine, the bladder, and the kidneys. A fim OFF CFT073 DeltafimBE ipuB ipbA mutant also demonstrated the ability to switch from the fim off state to the on state during mouse infection. CFT073 recombinase mutants derived from isolates in either the fim on or off state showed a reciprocal relationship for motility. Switches from a nonmotile to a motile phenotype and from a fim on to off genotype were observed in fim ON CFT073 DeltafimBE ipuAB ipbA mutants when ipuA or fimB was provided in trans. Together these results indicate that ipuA has fimB-like on-to-off and off-to-on fim switching activity and that ipbA has the ability to switch fim from the off to the on orientation.

FIG. 1.

The CFT073 genetic maps (top) and predicted protein alignments (bottom) of fimBE and the three fimBE-like recombinases, ipuA, ipuB, and ipbA. Boldface residues in the alignment represent the four catalytic amino acids for tyrosine recombinases. Asterisks represent identical amino acids, while periods represent similar amino acids. Dashes within a sequence indicate gaps used to maximize alignment.

FIG. 2.

The ipuA and ipbA genes function in trans at the fim switch. Ethidium bromide-stained agarose electrophoretic gels containing various SnaBI-digested fim switch PCR products are shown. Lanes 1 to 4 contain the DNA size standard and PCR products from CFT073 fim locked on (WAM2727) (labeled ON), CFT073 fim locked off (WAM2728) (labeled OFF), and CFT073 (WAM2266) (labeled WT) strains, respectively. Lanes 5 to 10 contain PCR products from the CFT073 ΔfimBE ipuAB ipbA fim ON (top panel) and CFT073 ΔfimBE ipuAB ipbA fim OFF (bottom panel) backgrounds transformed with recombinant plasmids carrying the respective recombinase genes listed along the bottom panel. The ipuA(−) and ipuA (+) designations refer to absence or presence of arabinose induction for the ipuA recombinant plasmid pWAM2770 based on the pBAD30 vector. Not shown, but where no switching was observed, are fim OFF WAM2920 and fim ON WAM2921, controls in which all five recombinases are deleted.

FIG. 3.

Inverse PCR detection of the on and off orientations of the CFT073 fim switch. A picture of an ethidium bromide-stained agarose electrophoretic gel containing PCR products is shown. Lanes: 1, the DNA size standard; 2, a nontemplate PCR control; 3, CFT073 (WAM2266); 4, fim ON CFT073 ΔfimBE ipuAB ipbA (WAM2921); 5, fim ON CFT073 ΔfimBE ipuB ipbA (WAM3038); 6, fim ON CFT073 ΔfimBE ipuA ipbA (WAM2925); 7, fim ON CFT073 ΔfimBE ipuAB (WAM2901); 8, fim OFF CFT073 ΔfimBE ipuAB ipbA (WAM2920); 9, fim OFF CFT073 ΔfimBE ipuB ipbA (WAM2986); 10, fim OFF CFT073 ΔfimBE ipuA ipbA (WAM2924); 11, fim OFF CFT073 ΔfimBE ipuAB (WAM2959).

FIG. 4.

In vivo off-to-on switching of fim OFF CFT073 ΔfimBE. Shown in the three panels are ethidium bromide-stained agarose electrophoretic gels containing various SnaBI-digested fim switch PCR products derived from DNA harvested from urine, bladders, and kidneys of infected mice. The first three lanes from left to right contain the DNA size marker and the SnaBI digest of the fim switch PCR product derived from CFT073 fim locked on (WAM2727) and CFT073 fim locked off (WAM2728). The remaining lanes contain the SnaBI digests from the three strains listed to the right: fim ON CFT073 ΔfimBE ipuAB ipbA (WAM2921), fim OFF CFT073 ΔfimBE ipuAB ipbA (recombinase-negative controls), and fim OFF CFT073 ΔfimBE (WAM2958). DNA was extracted from urine, bladders, or kidney samples that were pooled from four to six infected mice. Along the bottom are the postinfection times at which urine was harvested, including the input inocula labeled as INOC. At the far right are SnaBI-digested fim switch PCR products taken from DNA extracted from bladders (B) and kidneys (K) at 72 h postinfection.

FIG. 5.

Quantification of bacteria from urine of infected mice. Urine samples were pooled from four to six mice infected with CFT073 (WAM2266), fim ON and OFF controls of recombinase-negative CFT073 ΔfimBE ipuAB ipbA (respectively WAM2921 and WAM2920), and fim ON and OFF CFT073 ΔfimBE ipuB ipbA strains with ipuA alone (WAM3038 and WAM2986). Urine was collected in preweighed tubes with 500 μl of PBS and enumerated on LB plates. Histogram bars represent numbers of colony-forming units per gram of urine. This graph is from a single experiment but is representative of two to four experiments for each of these strains.

FIG. 6.

ipuA alone can mediate the fim off-to-on switch in vivo. Shown in the three panels are ethidium bromide-stained agarose electrophoretic gels containing various SnaBI-digested fim switch PCR products derived from DNA extracted from infected mouse urine. The first three lanes from left to right contain the DNA size marker and the SnaBI digest of the fim switch PCR product derived from digest controls of CFT073 fim locked on (WAM2727) and CFT073 fim locked off (WAM2728). The orientation of the fim switch in bacteria in pooled urine from mice infected with CFT073 (WAM2266) (top panel), CFT073 recombinase negative fim ON and OFF controls (WAM2921 and WAM2920) (middle panel), and CFT073 strains where only ipuA remains in backgrounds of fim on and off orientations (WAM3038 and WAM2986) (bottom panel). The figure is representative of the results from at least two separate experiments. In one experiment, WAM2986 was observed to be a mixed ON/OFF population at 24 h postinoculation. The experiment with WAM2920 was conducted four times. For this strain alone, a PCR product was not obtained directly from DNA in the urine at 48 h postinoculation, although bacteria were detected by growth in the urine on LB plates. Therefore, the WAM2920 PCR product at 48 h was obtained from DNA taken from colonies on the LB agar.

FIG. 7.

Reciprocal status of motility and type 1 pilus expression in CFT073. Shown in the top two photographs (A and B) are zones of swimming motility in 0.3% agar tryptone plates. Plate A contains fim OFF CFT073 ΔfimBE (WAM2958) (1); CFT073 fim locked OFF (WAM2728) (2); fim OFF CFT073 ΔfimBE ipuAB ipbA(WAM2920) (3); fim ON CFT073 ΔfimBE (WAM2849) (4); CFT073 fim locked ON (WAM2727) (5); fim ON CFT073 ΔfimBE ipuAB ipbA (WAM2921) (6); CFT073 (WAM2266) (7). Plate B contains fim ON CFT073 ΔfimBE ipuAB ipbA (WAM2921) (1); fim OFF CFT073 ΔfimBE ipuAB ipbA (WAM2920) (2); fim ON CFT073 ΔfimBE ipuAB ipbA/pWAM3579, which encodes ipuA (WAM3610) (3); fim ON CFT073 ΔfimBE ipuAB ipbA/pWAM3580, which encodes ipuB (WAM3611) (4); CFT073 (WAM2266) (5). The bottom photograph (C) shows an ethidium bromide-stained agarose electrophoretic gel containing SnaBI-digested fim switch PCR products from bacterial DNA extracted from cells on motility plates shown in panels A and B. Lanes: 1, CFT073 (WAM2266); 2, fim ON CFT073 ΔfimBE ipuAB ipbA (WAM2921); 3, fim ON CFT073 ΔfimBE ipuAB ipbA/pWAM3579, which encodes ipuA (WAM3610); 4, fim ON CFT073 ΔfimBE ipuAB ipbA/pWAM3580, which encodes ipuB (WAM3611); 5, fim OFF CFT073 ΔfimBE ipuAB ipbA (WAM2920); 6, fim OFF CFT073 ΔfimBE ipuAB ipbA/pWAM3579; 6, fim OFF CFT073 ΔfimBE ipuAB ipbA/pWAM3580.