Contact-dependent growth inhibition causes reversible metabolic downregulation in Escherichia coli.

Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI, encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Deltap), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Deltap is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Deltap, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.

The essential role of the promoter-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcription by CAP from -215.

Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP-CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP-dependent activation of papBA requires binding of the leucine-responsive regulatory protein (Lrp) at binding sites that extend from -180 to -149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP-dependent promoters. Also, like class I promoters, the C-terminal domain of the alpha-subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an 'oriented heterodimer' approach with wild-type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter-distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H-NS. The results presented here show that AR1 of the promoter-proximal CAP subunit was required for papBA transcription even in the absence of the histone-like protein H-NS. These results show that the promoter-proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C-terminal domain of the alpha-subunit of RNA polymerase.

Regulation of uropathogenic Escherichia coli adhesin expression by DNA methylation.

Pap pili play an important role in the pathogenesis of upper urinary tract infections by enabling uropathogenic Escherichia coli to adhere to host epithelial cells. Pap pili are coded for by the pyelonephritis-associated pili (pap) operon, which consists of 11 genes required for the expression and assembly of Pap pili. Expression of Pap pili is regulated by a phase variation mechanism in which the pili expression state of the bacterial population is skewed between phase-on (expression positive) and phase-off (expression negative) states. Pap phase variation is controlled by the cooperative binding of leucine-responsive regulatory protein (Lrp) to two sets of Lrp binding sites in the upstream regulatory region of the pap operon. A single GATC sequence, which is the target site for deoxyadenosine methylase (Dam), is centrally located within each Lrp binding region. Dam plays a critical role in the expression of Pap pili via the formation of DNA methylation patterns. Methylation of GATC-I reduced the affinity of Lrp for pap DNA by about twofold. Conversely, Lrp specifically blocked methylation of pap GATC-I in vitro. These data support the hypothesis that Lrp and Dam compete for binding to GATC-I, and are consistent with previous results indicating that methylation of GATC-I is important for stability of the phase-off state.

Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor.

The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26 degrees C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23 degrees C. The histone-like nucleoid structuring protein H-NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H-NS or a factor regulated by H-NS bound within the pap regulatory region at 23 degrees C but not at 37 degrees C, as evidenced by H-NS-dependent inhibition of methylation of the pap GATC sites designated GATC-I and GATC-II. These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H-NS bound specifically to a pap DNA fragment containing the GATC-I and GATC-II sites. Moreover, H-NS blocked methylation of these pap GATC sites in vitro: H-NS blocked pap GATC methylation at 1.4 microM but was unable to do so at higher concentrations at which non-specific binding occurred. Thus, non-specific binding of H-NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H-NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H-NS with pap regulatory DNA. We hypothesize that a function of H-NS such as oligomerization was altered at 23 degrees C, which enabled H-NS to repress pap gene expression through the formation of a specific nucleoprotein complex.

Use of a two-color genetic screen to identify a domain of the global regulator Lrp that is specifically required for pap phase variation.

The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as ilvIH, activation of papBA transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp's were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity for pap Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-locked pap derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp's are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.

Epigenetic phase variation of the pap operon in Escherichia coli.

Expression of the pyelonephritis-associated pilus (pap) operon in Escherichia coli is regulated by a complex epigenetic phase-variation mechanism involving the formation of differential DNA-methylation patterns. This review discusses how DNA-methylation patterns are formed by protein-DNA interactions and how methylation patterns, in turn, control pap gene expression.

Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli.

Pyelonephritis-associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC-I and GATC II, is essential for the transition between active and inactive pap transcriptional states. Here, we identify six Lrp binding sites within the pap regulatory DNA, each separated by about three helical turns. Lrp binds with highest affinity to three sites (1, 2 and 3) proximal to the papBAp promoter. A mutational analysis indicates that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the --35 and --10 RNA polymerase binding region of papBAp. The addition of PapI decreases the affinity of Lrp for sites 1, 2 and 3 and increases its affinity for the distal Lrp binding sites 4 and 5. Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activates pap transcription. The pap GATC-I and GATC-II methylation sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation.

Specific binding of PapI to Lrp-pap DNA complexes.

Expression of pyelonephritis-associated pili (Pap) varies between transcriptionally active (ON) and inactive (OFF) phase states. Pap phase variation is controlled by the binding of leucine-responsive regulatory protein (Lrp) to two pap regulatory DNA regions, each containing a deoxyadenosine methylase site and designated GATC-I and GATC-II. Methylation of these GATC sites modulates binding of Lrp and plays an essential role in phase variation. PapI, an 8.8-kDa pap-encoded regulatory protein, plays a key role in the switch between OFF and ON transcription states. In the absence of PapI, Lrp binds to sites overlapping the papBA promoter and inhibits transcription. Addition of PapI results in a translocation of Lrp binding to sites over 100 bp upstream, resulting in the ON transcription state. Gel shift analysis using radiolabeled PapI shows that PapI binds with high specificity to Lrp-pap DNA complexes but binds only weakly to free Lrp. Protein cross-linking studies indicate that Lrp and PapI directly interact with each other. On the basis of these data, we present a hypothesis in which PapI facilitates the transition between OFF and ON transcription states by binding to Lrp and altering Lrp's affinity for the pap GATC-I and GATC-II regions.

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli.

We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC1028 and GATC1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC1028 blocks formation of the ON state by inhibiting the binding of Lrp and PapI regulatory proteins to this site. Conversely, methylation of GATC1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Regulation of pyelonephritis-associated pili phase-variation in Escherichia coli: binding of the PapI and the Lrp regulatory proteins is controlled by DNA methylation.

Expression of pyelonephritis-associated pili (Pap) in Escherichia coli is under a phase-variation control mechanism in which individual cells alternate between pili+ (ON) and pili- (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC1028 and GATC1130) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC1028 site of phase ON cells is non-methylated and the GATC1130 site is fully methylated. Conversely, in phase OFF cells the GATC1028 site is fully methylated whereas the GATC1130 site is non-methylated. Two transcriptional activators, PapI and Lrp (leucine-responsive regulatory protein), are required for this specific methylation inhibition. DNA footprint analysis using non-methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC1130 site, whereas PapI does not appear to bind to pap regulatory DNA. However, addition of Lrp and PapI together results in an additional DNaseI footprint around the GATC1028 site. Moreover, Dam methylation inhibits binding of Lrp/PapI near the GATC1028 site and alters binding of Lrp at the GATC1130 site. Our results support a model in which Dam and Lrp/PapI compete for binding near the GATC1028 site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.

Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap.

Pyelonephritis-associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp and Papl bind to a specific non-methylated pap regulatory DNA region containing the sequence 'GATC' and facilitate the formation of an active transcriptional complex. Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam. However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited. These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states. The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved 'GATC-box' domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.

Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.

DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation.

Pyelonephritis-associated pili (Pap) are important in the pathogenesis of ascending, unobstructive Escherichia coli-caused renal infections because these surface bacterial organelles mediate digalactoside-specific binding to host uroepithelial cells. Pap are composed of many different polypeptides, of which only the tip proteins mediate specific binding. The PapA moiety polymerizes to form the bulk of the pilus structure and has been employed in vaccines despite its lack of Gal alpha(1-4)Gal receptor specificity. Animal recipients of PapA pilus-based vaccines are protected against experimental pyelonephritis caused by homologous and heterologous Gal-Gal-binding uropathogenic E. coli strains. Specific PapA immunoglobulin G antibodies in urine are correlated with protection in these infection models. The nucleotide sequences of the gene encoding PapA were determined for three E. coli clones expressing F7(1), F7(2), and F9 pili and were compared with corresponding sequences for other F serotypes. Specific rabbit antisera were employed in enzyme-linked immunosorbent assays to study the cross-reactivity between Gal-Gal pili purified from recombinant strains expressing F7(1), F7(2), F9, or F13 pili and among 60 Gal-Gal-binding wild-type strains. We present data which corroborate the concept that papA genes are highly homologous and encode proteins which exhibit greater than 70% homology among pili of different serotypes. The differences primarily occur in the cysteine-cysteine loop and variable regions and constitute the basis for serological diversity of these pili. Although there are differences in primary structures among these pili, antisera raised against pili of one serotype cross-reacted frequently with many other Gal-Gal pili of different serotypes. Furthermore, antisera raised against pili of the F13 serotype cross-reacted strongly or moderately with 52 (86%) of 60 wild-type Gal-Gal-binding E. coli strains. These data suggest that there are common immunogenic domains among these proteins. These additional data further support the hypothesis that broadly cross-protective PapA pilus vaccines for the immunoprophylaxis of pyelonephritis might be developed.

Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli.

Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states. Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter. DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site. papI and papB are two regulatory genes in the pap operon. Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site. We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites. The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E. coli chromosome. The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter. E. coli containing mbf::mTn10 and phase-off mbf+ E. coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E. coli cells. These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses. Moreover, transposon insertion within mbf greatly reduced Pap pilus expression. The mbf locus was isolated on a low-copy-number cosmid, pMBF1. Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon. The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.

Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells.

Legionella pneumophila is a gram-negative facultative intracellular parasite that causes Legionnaires disease. To explore the interactions between L. pneumophila and host cells, we have developed a continuous cell line model of infection. We show that about 80% of Chinese hamster ovary (CHO) cells were associated with L. pneumophila after incubation for 3 h at a multiplicity of infection of 20 bacteria per cell. Within 3 to 4 h of incubation with L. pneumophila, protein synthesis of CHO cells was markedly inhibited, as shown by the reduction of incorporation of radiolabeled amino acids into proteins. L. pneumophila did not inhibit transport of amino acids or cause degradation of newly synthesized proteins in CHO cells. Cytochalasin D blocked internalization of L. pneumophila by CHO cells, yet CHO cell protein synthesis was inhibited. These results indicated that L. pneumophila could inhibit host protein synthesis from the cell exterior. L. pneumophila that had been killed with antibiotics prior to incubation with CHO cells still inhibited protein synthesis, indicating that the inhibition of CHO cell protein synthesis occurred in the absence of de novo protein synthesis by L. pneumophila.

Regulation of pap pilin phase variation by a mechanism involving differential dam methylation states.

Transcription of the pap pilin (papA) gene in Escherichia coli is subject to control by a heritable phase variation mechanism in which alternation between transcriptionally active (phase on) and inactive (phase off) states occurs. Our results suggest that phase switching occurs without DNA rearrangement of pap DNA sequences, distinguishing this system from those described for E. coli type 1 pili and Salmonella flagellar phase variation. Analysis of the regulatory region upstream of papA in DNAs isolated from phase off and phase on cell populations showed that two deoxyadenosine methylase (Dam) sites, GATC1028 and GATC1130, were present. Southern blot analysis of MboI and DpnI restriction digests of DNAs showed that the GATC1028 site was unmethylated only in DNA isolated from phase on populations. Conversely, GATC1130 sites were unmethylated in DNA isolated from phase off populations. The presence of unmethylated GATC sites in E. coli is unusual and to our knowledge has not been previously reported. These results suggest that the methylation states of GATC1028 and GATC1130 may regulate pap transcription. Consistent with this hypothesis, Dam methylase levels affected the regulation of pap transcription; papA transcription was absent in dam- E. coli. Moreover, transition from the phase off to phase on state was not observed in E. coli expressing aberrantly high levels of Dam. A basic model is presented which outlines a possible mechanism by which alternation between phase off and phase on methylation states could occur.

Identification of an Escherichia coli genetic locus involved in thermoregulation of the pap operon.

We previously showed, using a single-copy papBAp-lac fusion (previously designated papBA-lac), that pyelonephritis-associated pili (pap) pilin gene transcription is subject to both phase variation and thermoregulatory control mechanisms (L. B. Blyn, B. A. Braaten, C. A. White-Ziegler, D. H. Rolfson, and D. A. Low, EMBO J. 8:613-620, 1989). At 37 degrees C, Escherichia coli strains carrying the papBAp-lac fusion displayed both Lac+ and Lac- colony phenotypes. In contrast, at 23 degrees C, colonies displayed a uniform Lac- phenotype, suggesting that pilin was not transcribed at this temperature. In this study, a strain carrying the papBAp-lac fusion was subjected to mini-Tn10 (mTn10) mutagenesis to isolate mutants that could initiate transcription of pilin at the nonpermissive temperature. Two classes of thermoregulatory mutants were identified in which the mTn10 mutation was linked to the mutant phenotype. Class I mutants displayed a phase variation phenotype at both 37 degrees C and 23 degrees C, whereas class II mutants displayed a uniform Lac+ colony phenotype at both temperatures. Preliminary analysis of these mutants showed that the mTn10 insertions in the class I mutants were chromosomally located, whereas the mTn10 insertions in the class II mutants were located within the papBAp-lac fusion phage. Southern blot analysis of the class I mutants demonstrated that mTn10 was present in the same 5.9-kilobase SalI DNA fragment in each mutant. Two of the class I mTn10 mutations were mapped to approximately 23.4 min on the E. coli K-12 chromosome. The locus defined by the class I mTn10 mutations was designated tcp, for thermoregulatory control of pap. Analysis of phase transition rates of the class I mutants showed that the phase-off (Lac-)----phase-on (Lac+) transition rates were higher than those observed with the nonmutant E. coli strain.

Phase-variation of pyelonephritis-associated pili in Escherichia coli: evidence for transcriptional regulation.

The regulation of pyelonephritis-associated pili (pap) pilin gene transcription has been examined using two operons (pap-17 and pap-21) isolated from the pyelonephritogenic Escherichia coli strain C1212. DNA sequence analysis and E. coli minicell analysis were used to map two genes (papB and papI) within the pilin regulatory regions of both pap-17 and pap-21, and the protein products of these genes were identified. Pilin transcription, initiated at the papBA promoter, was monitored by constructing single copy operon fusions with lacZYA in E. coli K-12. Inoculation of E. coli (pap'-lac) strains onto solid M9 minimal medium containing glycerol and the Lac indicator X-gal (M9-Glycerol) yielded both Lac+ and Lac- colony phenotypes. The Lac+ ("phase on') and Lac- ("phase off') phenotypes were heritable since reinoculation of M9-Glycerol with bacteria picked from Lac+ colonies gave rise to a much higher fraction of Lac+ colonies than reinoculation of M9-Glycerol with bacteria picked from Lac- colonies. Measurement of phase transition rates for E. coli (pap17'-lac) inoculated onto M9-Glycerol showed that the Lac(-)----Lac+ transition frequency (1.57 X 10(-4)/cell/generation) was reduced 35-fold when cells were inoculated onto minimal medium containing glucose (M9-Glucose). However, the Lac+----Lac-transition frequency obtained using M9-Glycerol (2.60 X 10(-2)/cell/generation) was 1.4-fold lower compared to results obtained with M9-Glucose. In contrast, lowering the incubation temperature of E. coli (pap17'-lac) cultures from 37 degrees C to 23 degrees C caused all cells to shift to the Lac- state.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and comparison of Escherichia coli strains from canine and human patients with urinary tract infections.

We analyzed Escherichia coli strains isolated from dogs with urinary tract infections (UTIs) in an attempt to determine if any of these strains were similar to E. coli isolated from humans with UTIs. Using genotypic and phenotypic traits, we identified four canine and six human E. coli UTI isolates that all appeared to be closely related or identical. All isolates shared similar DNA sequences for pyelonephritis-associated pili (pap), alpha-hemolysin (hly), and insertion sequence 5 (IS5), on the basis of Southern blot analysis. Similar outer membrane protein, pilin, and plasmid profiles were obtained for each of the isolates, although minor heterogeneity was observed. All of these isolates expressed a neuraminidase-sensitive binding phenotype in contrast to the majority of human isolates, which are known to express an adhesin that recognizes terminal digalactoside residues. Taken together, these results suggest that similar E. coli uropathogens may be capable of infecting both dogs and humans. To determine if the intestinal tracts of dogs were a reservoir for uropathogenic E. coli, eight paired rectal and urine pap+ E. coli strains were cultured from dogs with UTIs. By using the same genotypic and phenotypic criteria described above as a basis for strain identity, seven of eight urine-rectal pairs showed intrapair identity. However, each urine-rectal pair displayed a unique overall profile and could be distinguished from the other pairs. We conclude that the uropathogen colonizing the bladders of dog can also be the predominant strain colonizing the intestinal tracts.