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 ⌬fimBE 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 ⌬fimBE 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 ⌬fimBE 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.

SummaryAlthough once thought to be unique to bacteria, Damino acids are also produced by mammals. For example, D -serine is excreted in human urine at concentrations ranging from 3.0 to 40 m m m m g ml ----1. An epidemiological survey demonstrated that urine isolates of E. coli are more likely to catabolise D -serine via expression of D -serine deaminase, DsdA than enteric disease isolates. The urosepsis strain, CFT073, and an isogenic dsdA mutant have similar growth kinetics in minimal or complex media. However, relative to the wild type, the dsdA mutant has a pleiomorphic cell shape and a prolonged, 4-6 h lag phase when grown in human urine. This suggests that D -serine catabolism provides a growth advantage in the urinary tract. Unexpectedly, in a direct competition model of urinary tract infection, the dsdA mutant was recovered 300-times more frequently than the wild type in the bladders of mice 48 h after infection. A new model of E. coli uropathogenesis is proposed where growth and gene expression are modulated in response to environmental D -serine levels. In support of this, the CFT073 dsdA mutant is hyperflagellated and more motile than the wild type indicating that intracellular levels of D -serine may directly or indirectly influence the expression of regulons associated with E. coli uropathogenesis.

The genome sequences of Escherichia coli pathotypes reveal extensive genetic variability in the argW-dsdCXA island. Interestingly, the archetype E. coli K1 neonatal meningitis strain, strain RS218, has two copies of the dsdCXA genes for D-serine utilization at the argW and leuX islands. Because the human brain contains D-serine, an epidemiological study emphasizing K1 isolates surveyed the dsdCXA copy number and function. Forty of 41 (97.5%) independent E. coli K1 isolates could utilize D-serine. Southern blot hybridization revealed physical variability within the argW-dsdC region, even among 22 E. coli O18:K1:H7 isolates. In addition, 30 of 41 K1 strains, including 21 of 22 O18:K1:H7 isolates, had two dsdCXA loci. Mutational analysis indicated that each of the dsdA genes is functional in a rifampin-resistant mutant of RS218, mutant E44. The high percentage of K1 strains that can use D-serine is in striking contrast to our previous observation that only 4 of 74 (5%) isolates in the diarrheagenic E. coli (DEC) collection have this activity. The genome sequence of diarrheagenic E. coli isolates indicates that the csrRAKB genes for sucrose utilization are often substituted for dsdC and a portion of dsdX present at the argW-dsdCXA island of extraintestinal isolates. Among DEC isolates there is a reciprocal pattern of sucrose fermentation versus D-serine utilization. The ability to use D-serine is a trait strongly selected for among E. coli K1 strains, which have the ability to infect a wide range of extraintestinal sites. Conversely, diarrheagenic E. coli pathotypes appear to have substituted sucrose for D-serine as a potential nutrient.It is remarkable that aside from the normal intestinal commensal strains, there are genotypes of Escherichia coli that can cause different diseases in the intestine and others that infect and cause disease at extraintestinal sites. This diversity suggests that different genotypes of E. coli have the ability to respond successfully to different environments by expression of relevant colonization factors, immune avoidance mechanisms, and advantageous nutrient acquisition strategies. Comparison of the sequenced genomes of uropathogenic E. coli (UPEC) strain CFT073, laboratory K-12 strain MG1655, and enterohemorrhagic E. coli (EHEC) strain EDL933 showed that only 39.2% of the predicted proteins were common among all three strains (40). Aside from virulence-and colonization-related genes that are specific to the niches for each strain, these strains vary in some specific catabolic capabilities. Our laboratory has recently focused on the argW-dsdCXA chromosomal region that corresponds to the E. coli K-12 MG1655 53Ј genetic map position. In this region, EDL933 possesses phagelike genes and cscRAKB, which permit non-phosphotransferase (PTS)-mediated sucrose utilization; and MG1655 and CFT073 have genes (dsdCXA) that encode the ability to use D-serine as a sole carbon and nitrogen source. Until recently, it was thought that naturally occurring D-amino acids were found only in bacterial cell w...

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