Vol. 57, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1991, p. 320-3230099-2240/91010320-04$02.00/0Copyright © 1991, American Society for Microbiology

Identification of uidA Gene Sequences in3-D-Glucuronidase-Negative Escherichia coli

PETER FENG,1* ROSALIND LUM,2 AND GEORGE W. CHANG2Division of Microbiology, Food and Drug Administration, Washington, D.C. 20204,1 andDepartment of Nutritional Sciences, University of California, Berkeley, California 947202

Received 17 July 1990/Accepted 12 October 1990

A probe specific for the uidA gene of Escherichia coli hybridized with 112 of 116 E. coli isolates examined,including 31 Pl-D-glucuronidase-negative and 12 enterohemorrhagic E. coli serotype 0157:H7 isolates. Southernhybridizations confirmed the presence of a 900-bp Hinfl fragment from the uidA gene in all isolates examined,suggesting that uidA gene sequences are present in most E. coli.

A fluorogenic assay for the rapid identification of Esche-richia coli in food and water (6) is based on the presence ofthe enzyme ,B-D-glucuronidase (GUD) which cleaves thesubstrate 4-methylumbelliferyl-,-D-glucuronide (MUG) torelease a fluorogenic radical (6, 8). When the MUG com-pound is incorporated into conventional bacteriological me-dia, the presence of E. coli can be easily determined by thebluish fluorescence in the medium that is observed underlong-wave UV light (6, 8, 14). A modification of the fluoro-genic test used to identify E. coli in chilled or frozen foodswas recently adopted as official final action by the Associa-tion of Official Analytical Chemists (14).GUD is an inducible enzyme that is encoded by the uidA

gene in E. coli (2, 10, 15). About 94% of E. coli strains andsome Shigella spp. (44%) and Salmonella spp. (29%) appearto be the only members of the family Enterobacteriaceaethat produce GUD (6, 8, 14), except for the pathogenicenterohemorrhagic E. coli of serotype 0157:H7, which isMUG negative (5).The high MUG-positive rate reported for E. coli appears

to be constant regardless of the source of the isolates (16). Ina recent study (3), however, about 34% of the human fecalisolates of E. coli examined showed a negative reaction tothe MUG test. This figure is much higher than the 6%MUG-negative rate typically reported for E. coli (6, 8, 14).Therefore, to study the limitations of the MUG assay, asynthetic oligonucleotide probe was directed at the uidAgene of E. coli to examine the distribution of GUD enzymegenes in E. coli strains.An ABI 380B automated DNA synthesizer (Applied Bio-

systems, Foster City, Calif.) was used to synthesize anoligonucleotide probe (termed PF-15) to an 18-base sequenceof uidA (10). The PF-15 probe (5'-TACAGCGAAGAGGCAGTC-3') is specific for a 900-bp Hinfl fragment in theuidA gene (Fig. 1). The probe was purified by passagethrough a polyacrylamide gel and was labeled on the 5' endby using T4 polynucleotide kinase and [y-32P]ATP (12, 17).A collection of 62 MUG-positive and -negative E. coli

strains from environmental sources and 12 human isolates ofenterohemorrhagic E. coli serotype 0157:H7 were obtainedfrom P. A. Hartman (Iowa State University, Ames, Iowa).Another 42 MUG-positive and -negative E. coli were iso-

* Corresponding author.

lated from human fecal specimens (3). The identity of someisolates was reconfirmed by using API 20E biochemicalstrips (Analytab Products, Plainview, N.Y.); the 0157:H7serotypes were verified by the California Department ofHealth Services.Each isolate used in this study was first reexamined for

GUD enzyme activity by a stab in a tryptic soy agar mediumsupplemented with 50 ,ug of MUG per ml (Hach Co.,Loveland, Colo.). The plates were incubated at 35°C andmonitored for the appearance of fluorescence over a 48-hperiod. For colony hybridizations, pure cultures were repli-cated onto Whatman no. 541 cellulose filters (Whatman Ltd.,Clifton, N.J.) and lysed by microwave irradiation (4, 9).Filters were hybridized at 37°C overnight by using 106 cpmof labeled probe per ml of hybridization solution. Unboundprobes were removed by two 30-min washes at 52°C in 6xSSC (lx SSC contains 0.15 M NaCl plus 0.015 M sodiumcitrate [pH 7.0]) + 0.1% sodium dodecyl sulfate. The disso-ciation temperature for PF-15 was calculated to be 56°C (13).Hybridized filters were examined by autoradiography (Fig.2).

Results for both the PF-15 probe and the MUG assay areshown in Table 1. Of the 116 E. coli isolates examined, 112hybridized with the PF-15 probe, including 31 that wereMUG negative; four MUG-positive E. coli isolates failed tohybridize or were weakly reactive with PF-15. Bacteriaother than E. coli were negative by both MUG testing andcolony blotting. The most interesting observation was notedwith E. coli serogroup 0157:H7 isolates. These enterohem-orrhagic serotypes, present in about 2% of beef, pork, lamb,and poultry test samples, typically do not show GUDactivity (5, 8). In fact, the MUG-negative phenotype hasbeen used as a selection criterion for this pathogenic sero-type. However, in our study, serotype 0157:H7 reactedstrongly with the PF-15 probe, which is targeted to the uidAgene that encodes the GUD enzyme. These results suggestthat most if not all E. coli isolates carry sequences for theuidA gene regardless of GUD activity.To confirm that the PF-15 probe hybridized specifically to

the uidA gene of E. coli, chromosomal DNA was extractedfrom several MUG-positive and -negative isolates (18), di-gested with Hinfl, Southern transferred onto nitrocellulosepaper (19), and probed as described above. Southern hybrid-ization of DNA from selected isolates showed that PF-15

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NOTES 321

Hinf I Hinfl700 bp I 900 bp

Hinf I TABLE 1. Characterization of isolates by MUG assay andcolony blotting with PF-15 oligonucleotide probe

PF-15

FIG. 1. Map of Hinfl restriction sites (not to scale) of uidA gene,showing position of PF-15 probe.

reacted with a single 900-bp Hinfl fragment (Fig. 3). Thisprobe-reactive band was found in both MUG-positive and-negative isolates of E. coli, including an isolate of E. coliserotype 0157:H7. The identification of the 900-bp Hinflfragment by PF-15 is consistent with the predicted result onthe basis of the restriction map of the uidA gene (10). Theresults of characterizing 23 isolates with the probe aresummarized in Table 2. PF-15 did not hybridize with isolatesof Escherichia hermanii or a Pseudomonas sp., both ofwhich were MUG negative.

No. MUG PF-15 No.Bacteria tested reaction reaction observed

Escherichia coli 104 + + 69- + 31+ - 4

E. coli 0157:H7 12 - + 12E. hermanii 1 - - 1Shigella sp. 1 - - 1Pseudomonas sp. 1 - - 1

In the colony hybridization study, some isolates of E. coliwere MUG positive but were only weakly reactive ornonreactive with PF-15 on colony blots. These isolates werealso examined by Southern blot analysis. The resultingautoradiogram showed that although the PF-15-reactive900-bp Hinfl fragment was also present, the hybridization

1 2 3 4 5 6 7 8A

B

C

D

E

F

1 2 3 4 5 6 7 8G

H

J

IK

L7;

1 2 3 4 5 78

N

0

.9.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.P

R

FIG. 2. Colony hybridization of E. coli and other isolates withPF-15 probe. All colonies on the autoradiogram are E. coli exceptA3 (Shigella sp.), I1 (Pseudomonas sp.), and 15 (E. hermanii).Colonies H8, 04 to 08, and P1 to P6 are enterohemorrhagic E. coliserotype 0157:H7. The four corner positions of each colony filter,as well as positions L6, L7, and R7, are blanks.

1.35kb-*-1.07 kb-*- *a0 87kb-

0.6kb-*-

"I

J3 P3 15

11

I <

Aw

FIG. 3. Autoradiogram of Southern hybridization of PF-15 with

Hinfl-digested total DNA from selected MUG-positive and -nega-tive E. coli colonies. Isolates are designated according to gridpattern shown for colony blots in Fig. 2. Colony 15 is an isolate of E.

hermanii.

Hinf I

02 A6 D4 H302 A6 D4 H3

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TABLE 2. Summary of hybridization reactions of PF-15 probewith selected MUG-positive and -negative isolates

PF-15 reaction

Bacteria Isolatea MUGreaction Colony Southernblot analysis

Escherichia coli A6 - + +D4 + + +H3 - + +14 + _b +J3 + + +K8 + - +L3 + - +02 + + +Q5 + - +Q6 + - +

E. coli 0157:H7 H8 - + +04 - + +05 - + +07 - + +P1 - + +P3 - + +P6 - + +

E. hermanii IS -

Pseudomonas sp. I1 -

a Isolates are designated according to grid patterns used for colony blotsshown in Fig. 2.

b +, Weakly positive.

signals for most of these isolates were much weaker (Fig. 4).The weaker hybridization signals cannot be explained withcertainty; however, minor nucleotide sequence differencesin these isolates may affect PF-15 hybridization but not GUDenzyme activity. The weaker signal could account for thenegative or weakly positive results obtained for these iso-lates on colony blot hybridization. Probe characterizationsfor these isolates are summarized in Table 2.Colony blots and Southern analyses showed that both

MUG-positive and -negative E. coli hybridized well withPF-15, including E. coli serotype 0157:H7. The presence ofthe probe-reactive 900-bp Hinfl fragment in all of the isolatesexamined indicates that part of the genetic sequences of theuidA gene, which encodes for the GUD enzyme, is present inmost if not all E. coli isolates regardless of the GUDphenotype. This observation is consistent with the findingsreported by Bej et al., who used the polymerase chainreaction in their work (1). In that study, template DNA fromMUG-negative E. coli isolates and primers specific for theuidA gene were used in a polymerase chain reaction forpositive amplification of a uidA gene fragment. These resultscorrelate with our findings that uidA sequences are presentin MUG-negative E. coli isolates.

If most E. coli carry uidA gene sequences as suggested bythe hybridization data, then the absence of GUD enzymeactivity in some E. coli may be attributed to physiologicalfactors or genetic differences. GUD is an inducible enzyme,and there is evidence that its expression is affected bylactose-induced catabolite repression (7). Genetic mutationsin the regulatory or structural regions of the genome can alsoaffect uidA expression or the production of nonfunctionalenzymes. The latter effect is consistent with a report ofMUG-negative E. coli cell lysates coagglutinating with ananti-GUD antibody conjugated to staphylococcal cells (11).Restriction map analysis and sequencing of uidA genes fromboth MUG-positive and -negative isolates may explain theabsence of GUD enzyme activity in some E. coli.

FIG. 4. Autoradiogram of Southern hybridization of PF-15 withHinfI-digested DNA from E. coli isolates that were MUG positivebut that were negative or weakly positive on colony blots. Colony I1is a Pseudomonas sp.

We thank Paul A. Hartman for providing many of the isolatesused in this study and Cathy Powers and Sharon Abbott forserotyping some of the isolates. We also thank W. E. Hill and G. J.Jackson, Division of Microbiology, Food and Drug Administration,Washington, D.C., for critical reading of the manuscript.

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