INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1988, p. 201-206 Vol. 38. No. 2 0020-7713/88/040201-06$02.OO/O

Specificity of a Monoclonal Antibody for Alkaline Phosphatase in coli and Shigella Species M. 0. HUSSON,1*2*P. A. TRINELY2C. MIELCAREK,2 F. GAVINI,2 C. CARON,lq2D. IZARD,l AND H. LECLERC' Faculte' de Me'decine, Laboratoire de Bacte'riologie A, 59045 Lille Cedex, France' and Unite' Institut National de la Sante' et de la Recherche Me'dicale 146, Domaine dir CERTIA, 59650 Villeneuve-d'Ascq Cedex, France2

The specificity of monoclonal antibody 2E5 for the alkaline phosphatase of was studied against the alkaline phosphatases of 251 other bacterial strains. The organisms used included members of the six species of the genus Escherichia (E. coli, E. fergusonii, E. hermannii, E. blattae, E. vulneris, E. adecarboxylata), 41 species representing the family , and, in addition, Pseudomonas aeruginosa, Aeromonas spp., Plesiomonas shigelloides, Acinetobacter calcoaceticus, and Vibrio cholerae non-01. Three methods were used. An enzyme-linked immunosorbent assay was performed against 21U of alkaline phosphatase per ml; immunofluorescence against bacterial cells and Western blotting against periplasmic proteins were also used. All of our experiments demonstrated the high specificity of monoclonal antibody 2E5. This antibody recognized only E. coli (118 strains tested) and the four species of the genus Shigella (S. sonnei, S. flexneri, S. boydii, S. dysenteriae; 12 strains tested).

Since the description of hybridoma production by Kohler mouse immunized with purified Escherichia coli ATCC and Milstein in 1975 (14), many monoclonal antibodies 10536 alkaline phosphatase as described elsewhere (12). (MoAbs) have been produced against microorganisms. Some Specificity testing of MoAb 2E5. (i) ELISA. Activated of these are now used for the detection of , such as polyvinyl chloride microtitration plates (Nunc or Flow Lab- Chlamydia trachomatis (29), Legionella pneumophila (9), oratories) were coated with 100 pl of alkaline phosphatase (2 and group B Streptococcus (19). The success of such anti- IU/ml) in phosphate-buffered saline (PBS; 0.05 M, pH 9.6) bodies is correlated with their high antigenic affinity and by incubating the preparation at 4°C for 18 h. The plates especially with their specificity, as established by enzyme- were washed three times with 200 pl of this buffer and were linked immunosorbent assay (ELISA), immunofluores- saturated by using 2% bovine serum albumin in PBS in the cence, or immunoblotting against a great number of various wells at 37°C for 1 h. After three washes with 200 pl of PBS bacterial species. containing 0.05% Tween 20, the plates were covered and Escherichia coli alkaline phosphatase was chosen to im- stored at 4°C. The immune reaction was performed by munize BALB/c mice and to obtain specific antibodies. This adding 100 pl of MoAb 2E5 for 1 h at 37°C. Subsequently, enzyme protein is a dimer composed of two identical sub- the wells were washed three times with PBS containing units. It is localized in the periplasmic space and at the cell 0.05% Tween 20 and then incubated with 100 r~.lof horse- surface of the gram-negative cell envelope in association radish peroxidase-conjugated goat anti-mouse immunoglob- with lipopolysaccharides (8, 17). Studies of its immunologi- ulin (Institut Pasteur Production Laboratories), Peroxidase cal properties by immunoprecipitation or quantitative micro- activity was detected colorimetrically by adding 0.04% 0- complement fixation (6, 8) have demonstrated a protein phenylenediamine in citrate-phosphate buffer (pH 5) supple- sequence divergence between Escherichia coli and some mented with 0.006% hydrogen peroxide. The reaction was other species of Enterobacteriaceae. Recently, we produced developed in the dark for 15 min at room temperature and MoAbs against alkaline phosphatase of Escherichia coli (12). stopped by adding 30 pl of 1.3 M H2S0,. Absorbance at 490 In this paper, we describe the specificity of MoAb 2E5, nm was measured with a micro-ELISA plate photometer which was studied by using the three techniques described (Autoreader MR600; Dynatech Laboratories, Inc.). Assays above. were carried out in duplicate. Negative controls (serum of nonimmunized BALB/c mice) and positive controls (serum MATERIALS AND METHODS of immunized BALB/c mice) were included in each test. Reactions were considered positive when the mean of repli- Bacterial strains and alkaline phosphatase extraction. The cate titrations was two times higher than the mean of 251 bacterial strains included in this study are listed in Table negative controls. For some species (Escherichia hermannii, 1. Cells were grown at 30°C for 18 h in phosphate limiting Escherichia vulneris, Enterobacter cloacae, Proteus vul- medium to derepress alkaline phosphatase synthesis, as garis, Proteus penneri, Providencia rustigianii, Pvovidencia described by Garen and Levinthal (11). Bacteria were har- alcalifaciens, Yersinia pseudutuberculosis, Buttiauxella vested, and alkaline phosphatase was released with other agrestis) no enzymatic activity was found after heat treat- periplasmic proteins by heat treatment at 56°C in tris(hy- ment or sonication. In these cases, the ELISA was per- droxymethy1)aminomethane (Tris) hydrochloride buffer (pH formed by using periplasmic proteins (15 pg/ml). 8) as described by Tsuchido et al. (26). Alkaline phosphatase (ii) Immunofluorescence assay. Indirect immunofluores- activity was assayed with 0.2 mg of the substrate p-nitro- cence was performed with MoAb 2E5 and fluorescein iso- phenyl phosphate per ml in 1 M Tris buffer (pH 8) at 400 nm. thiocyanate-conjugated rabbit anti-mouse immunoglobulin MoAb 2E5 production. MoAb 2E5 was obtained from the (Institut Pasteur Production). The working dilutions of the fusion of SP20/Ag-14 myeloma cells and spleen cells from a MoAbs and the conjugate were determined by checkerboard titration. PBS (pH 7.6) was used to dilute the antibody and * Corresponding author. the conjugate. The MoAb and the conjugate were diluted

201 :!02 HUSSON ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Strains tested and cross-reactivities of MoAb 2E5 specific for Escherichia coli alkaline phosphatase Cross-reactivity as determined by: Bacterial strain(s)" ELISA against Immuno- enzymatic fluorescence of Western extract bacterial cells Escherichia spp. strains E. coli ATCC 10536 + + E. coli (38 environmental strains) + E. coli (59 medical strains) + E. coli (20 fecal strains) + E. fergusonii CDC 3296-73, CDC 3014-74 - E. fergusonii CDC 3458-74, CDC 1016-74, CDC 568-73 - E. hermannii ATCC 33650, CDC 899-73 E. hermannii CDC 2808-70, CDC 1933-74, CDC 2472-72 E. vulneris ATCC 33821, CDC 2898-73, CDC 3763-72, CDC 2954-71 E. blattae ATCC 29907 E. adecarboxylata ATCC 23216 Shigella spp. strains S. sonnei ATCC 9290, CUETM 77-73 + S. sonnei CFML 969 S. dysenteriae WRAIR 1617, CUETM 79-327 S.flexneri WRAIR 24570, CFML 970, ATCC 12661, CFML 17 S. boydii CDC 2406-51, WRAIR C10, CFML 398 Salmonella spp. strains S. typhimurium ATCC 23565 S. typhimurium ATCC 7823, CFML 1010 S. paratyphi CUETM 79-368 S. derby CFML 303, CFML 302 S. sohio CFML 305, CFML 317 S. enteritidis CFML 308 S. arizonae ATCC 7823 Levinea spp. strains L. rnalonatica CDC 25408 L. malonatica CUETM 77-12, CFML 359, CFML 416, CFML 875 L. amalonatica CDC 25406, CUETM 77-9 ICitrobacter freundii ATCC 8090 ICitrobacterfreundii CDC 460-61, CUETM 78-263, CUETM 78-280, CUETM 78-273, CFML 615, CFML 495, CFML 624 (Citrobacrerfreundii CU ETM 77-166, CUETM 77-152 Klebsiella spp. strains K. pneumoniae ATCC 13882 K. pneumoniae CUETM 77-130, CUETM 77-174, CUETM 78-146, CUETM 78-258, CUETM 77-180, CFML 509, CFML 220 K. oxytoca ATCC 13182, ATCC 77-146, CUETM 78-177, CUETM 78-182, CUETM 78-181, CUETM 78-178, CFML 685, CFML 173, CFML 48 Enterobacter spp. strains E. cloacae ATCC 13047 E. cloacae CUETM 77-121, CUETM 77-120, CUETM 77-126, CUETM 77-116, CUETM 77-125 E. amnigenus ATCC 33072 E. aerogenes ATCC 13048, CUETM 67-71 E. gergoviae ATCC 33028 E. sakazakii ATCC 29544 E. taylorae CDC 697-81 E. agglomerans ATCC 27155 Hafnia alvei CFML 877, CFML 880, CFML 884, CFML 883, CFML 165 Serratia spp. strains S. marcescens ATCC 16880 S. marcescens CUETM 78-210, CUETM 78-212, CUETM 78-248, CUETM 78-164, CFML 485, CFML 757 S. liquefaciens CUETM 78-243, CUETM 78-172, CUETM 78-244 S. liquefaciens CUETM 78-174, CUETM 78-228 S. fonticola ATCC 29844, CUETM 78-10 S. odorifera CUETM 83-88 Proteus spp. strains P. rnirabilis ATCC 29906 P. mirabilis CFML 699, CFML 1004 P. vulgaris ATCC 13315 P. penneri ATCC 33519

Continued on following page VOL. 38, 1988 SPECIFICITY OF MoAb 203

TABLE 1-Continued Cross-reactivity as determined by: Bacterial straints)" ELISA against Immuno- enzymatic fluorescence of extract bacterial cells blotting Morganella morganii ATCC 25830 Providencia spp. strains P. rustigianii ATCC 33673 P. stuartii ATCC 29914 P. alcalfaciens ATCC 9886 Yersinia spp. strains Y. enterocolitica CDC 135 Y. pseudotuberculosis ATCC 29833 Rahnella aquatilis ATCC 33071 Ewingella americana ATCC 33852 Buttiauxella agrestis ATCC 33320 Kluyvera spp. strains K. ascorbata ATCC 33434 K. cryocrescens ATCC 33435 Edwarsiella tarda CUETM 78-301 Cedecea spp. strains C. lapagei ATCC 33432 C. davisae ATCC 33431 Leminorella spp. strains L. richardii ATCC 33998 L. grimondii ATCC 33999 Pseudomonas aeruginosa ATCC 10145 Aeromonas spp. strains A. sobria CDC 9538-76 A. punctata ATCC 15468 A. hydrophila ATCC 7966 Acinetobacter lwofi ATCC 17986 Plesiomonas shigelloides ATCC 14029 Vibrio cholerae non-01 CUETM 77-100 ATCC, American Type Culture Collection, Rockville. Md.; CDC, Centers for Disease Control, Atlanta, Ga.; WRAIR, Walter Reed Army Institute of Research, Washington, D.C.; CUETM, Collection de I'unite Ecotoxicologie Microbienne, Villeneuve d' Ascq, France; CFML, Collection Faculte de Mkdecine de Lille, Lille, France. +, Positive; -, negative; (-), no alkaline phosphatase activity found, ELISA performed against periplasmic proteins extracted by heat treatment (26).

150. Portions (25 pl) of bacterial cultures were air dried and from the second gel to nitrocellulose paper (Bio-Rad Labo- cold fixed on glass slides. The slides were washed three ratories) by using the method of Towbin et al. (25). The times with PBS before the addition of 25 pI of MoAb 2E5 and nitrocellulose paper gel sandwich was electrophoresed over- were incubated at 37°C for 30 min in a moist chamber. After night at a constant current of 200 mA at 10°C. Immunochem- washing with PBS, 25 pl of conjugate was added to each ical staining was performed by first blocking the paper with sample, and the slides were incubated for an additional 30 2% casein in Tris-buffered saline (TBS; 20 mM Tris hydro- min. The slides were washed. Cover slips were mounted chloride, 500 mM NaCl, pH 7.5) for 45 min and then with glycerol-0.1 M Tris (pH 7.5) (9:l). Fluorescence was incubating it with MoAb 2E5 diluted 1:200 in 0.05% TBS- observed with a Leitz Orthoplan fluorescence microscope by Tween. The nitrocellulose paper was then washed with TBS using incident light (Leitz Laboratories). (three 15-min washes with 200 ml of buffer per wash) and (iii) Immunoblot analysis. Periplasmic proteins obtained by was subsequently stained for 3 h with horseradish peroxi- using the techniques described above were resolved by dase-conjugated goat anti-mouse immunoglobulin diluted electrophoresis on slab gels (1.5 mm thick), using the method 1500 in 0.05% TBS-Tween. The nitrocellulose paper was of Laemmli (15), with some modifications. A 7.5% acryl- washed as described above and developed in TBS containing amide (BDH) resolving gel and a 4% stacking gel were used. 0.1% (wthol) diaminobenzidine and 0.015% (vol/vol) hydro- The samples were prepared by mixing 1 part of protein (2 mg gen peroxide for 30 min. of protein per ml) with 1 part of the sample buffer (62.5 mM Tris hydrochloride [pH 6.81, 0.5% [wt/vol] bromophenol RESULTS AND DISCUSSION blue). Samples (20 pl) were applied to each gel lane. Elec- trophoresis was carried out at 20 mA per get (constant MoAb 2E5 was tested against 251 gram-negative bacteria current) until the bromophenol blue tracking dye entered the (Table l),including 118 Escherichia cofi strains isolated from separating gel, at which time the constant current was different sources (medical, fecal, environmental) and 16 increased to 30 mA per gel. When a Western blot was strains belonging to the five other species of the genus performed, two gels were run in parallel. In the first alkaline Escherichia (Escherichia fergusonii [5 strains], Escherichia phosphatase was revealed by p-nitrophenyl phosphate (10 hermannii [5 strains], Escherichia vulneris [4 strains], Esch- mg/ml), and then proteins were stained with Coomassie erichia adecarboxylata [l strain], and Escherichia blattae [ 1 brilliant blue and destained as described by Weber and strain]). We also tested members of 46 additional species Osborn (28). Proteins were transferred electrophoretically classified in the family Enterobacteriaceae and a few mem- FIG. 1. (A) Electrophoresis of native periplasmic proteins. Native periplasmic proteins of various members of the Enterobacteriaceae were obtained by heat treatment at 56°C in Tris hydrochloride buffer (pH 8) as described Tsuchido et al. (26). The strains were stained with Coomassie blue. Lane 1, Escherichia coli ATCC 10536; lane 2, Escherichia coli CUETM 85-110; lane 3, Escherichia coli CUETM 85-111; lane 4, Escherichia adecarboxylata ATCC 23216; lane 5, Escherichia blattae ATCC 29907; lane 6, Escherichia vulneris ATCC 33821; lane 7, Escherichia fergusonii CDC 3296-73; lane 8, Klebsiella oxytoca ATCC 13182; lane 9, Salmonella typhimurium ATCC 23565; lane 10, Levinea malonatica CDC 25408; lane 11, Citrobacter freundii ATCC 8090; lane 12, Shigella sonnei ATCC 9290; lane 13, Klebsiella pneumoniae ATCC 13882; lane 14, Enterobacter cloacae ATCC 13047; lane 15, Escherichia hermanii ATCC 33650; lane 16, Serratiu marcescens ATCC 16880; lane 17, Serratia liquefuciens CUETM 78-243; lane 18, Yersinia enterocolitica CDC 135. (B) Western electrophoresis blot showing interaction of MoAb 2E5 with periplasmic proteins of strains of Enterobacteriuceae. For lane contents see above. 204 VOL. 38. 1988 SPECIFICITY OF MoAb 205 bers of the genera Pseudomonas (Pseudomonas aerugi- fluid, blood cultures). Immunofluorescence and enzyme im- nosa), Acinetobacter, Aeromonas, Plesiomonas, and Vihrio munocapture assays are presently being developed in our (Vibrio cholerae non-01). laboratory for the detection of Escherichia coli alkaline When we studied the specificity of MoAb 2E5 by using the phosphatase in such specimens. indirect immunofluorescence technique and the ELISA, the only recognized species in the genus Escherichia was Esch- LITERATURE CITED erichia coli; all of the Escherichia coli strains, independent 1. Bhatti, A. R. 1973. Variation of alkaline phosphatase isoen- of ongin, gave positive reactions with MoAb 2E5. Thus, all zymes in Escherichia coli and Serratia marcescens. FEBS Lett. Escherichia coli strains produced alkaline phosphatase, and 32:81-83. all had at the surface of this enzyme protein the epitope 2. Brenner, D. J., B. R. Davis, A. G. Steigerwalt, C. F. Riddle, recognized by MoAb 2E5. This observation is consistent A. C. McWhorter, S. D. Allen, S. D. Farmer, Y. Saitoh, and with the results of studies of bacterial alkaline phosphatase G. R. Fanning. 1982. Atypical biogroups of Escherichia coli gene regulation (24, 30) in which alkaline phosphatase syn- found in clinical specimens and description of Escherichia thesis appeared independent of strain, source, strain se- hermannii sp. nov. J. Clin. Microbiol. 15703-713. rotypes, or virulence factors. A structural gene (phoA) and 3. Brenner, D. J., G. R. Fanning, G. V. Miklos, and A. G. three regulatory genes (phoB, phoM, phoR) for alkaline Steigerwalt. 1973. Polynucleotide sequence relatedness among Shigella species. Int. J. Syst. Bacteriol. 23:l-7. phosphatase have been described, and only phoB mutants 4. Brenner, D. J., A. C. McWhorter, J. K. Leete-Knudson, and and phoR phoM double mutants do not produce this enzyme A. G. Steigerwalt. 1982. Escherichia vulneris: a new species of protein (27). Such mutants were not observed among the 118 Enterobacteriaceae associated with human wounds. J. Clin. strains tested. Furthermore, alkaline phosphatase is antigen- Microbiol. 151133-1140. ically conserved in Escherichia coli (6). 5. Burgess, N. R. H., S. N. McDermott, and J. Whiting. 1973. Escherichia fergusonii (lo), the species most closely re- Aerobic bacteria occurring in the hind-gut of the cockroach, lated to Escherichia coli based on deoxyribonucleic acid Blatta orientalis. J. Hyg. 71:l-7. (DNA)-DNA hybridization data (relative binding ratio range, 6. Cocks, G. T., and A. C. Wilson. 1969. Immunological detection 49 to 63%), was not recognized. Negative reactions were of single amino acid substitutions in alkaline phosphatase. Escherichia blattae (5), Escherichia vul- Science 164:188-189. also obtained with 7. Cocks, G. T., and A. C. Wilson. 1972. Enzyme evolution in the neris (4), Escherichia hermannii (2), and Escherichia ade- Enterobacteriaceae. J. Bacteriol. 110:793-802. carboxylata, for which the creation of a new genus, Lecler- 8. Done, J., C. D. Slorey, J. P. Lake, and J. K. Pollak. 1965. The cia, has been proposed recently (13, 23); Escherichia cytochemical localization of alkaline phosphatase in Esche- adecarboxylata is more distantly related to Escherichia coli richia coli at the electron microscope level. Biochem. J. 96:27c- based on DNA-DNA hybridization data (relative binding 28c. ratio range, 30 to 40%). For all of the other species studied 9. Edelstein, P. H., K. B. Beer, J. C. Sturge, A. J. Watson, and except Shigella species the reactions were negative. This L. C. Goldstein. 1985. Clinical utility of a monoclonal direct interaction is not surprising. It is consistent with the pheno- fluorescent reagent specific for Legionella pneumophila: com- Esche- parative study with other reagents. J. Clin. Microbiol. 22:419- typic and genetic homology of these species with 421. richia coli. Using DNA-DNA hybridization, Brenner et al. 10. Farmer, J. J., 111, G. R. Fanning, B. R. Davis, C. M. O’Hara, C. (3) demonstrated that Shigella strains were indistinguishable Riddle, F. W. Hickman-Brenner, M. A. Asbury, V. A. Lowery, from Escherichia coli strains. and D. J. Brenner. 1984. Escherichia fergusonii and Enterobac- Since the epitope recognized by the antibody might be ter taylorae, two new species of Enterobacteriaceae isolated present at the surface of proteins other than alkaline phos- from clinical specimens. J. Clin. Microbiol. 21:77-81. phatase, immunostaining was performed against native peri- 11. Garen, A., and C. Levinthal. 1960. Purification and characteri- plasmic proteins resolved after polyacrylamide gel electro- zation of alkaline phosphatase. Biochim. Biophys. Acta 38:470- phoresis. The results of these experiments are summarized 483. 12. Husson, M. O., P. A. Trinel, D. Izard, C. Mielcarek, F. Gavini, in Table 1 and Fig. 1. Our data confirmed the specificity of and H. Leclerc. 1987. Antigenic specificity of Escherichia coli MoAb 2E5. This MoAb recognized only the alkaline phos- alkaline phosphatase studied with monoclonal antibodies: im- phatases of Escherichia coli and Shigella species. Thus, munological characterization of E. coli and Shigella strains. after immunoblotting, four bands were stained. These rep- Ann. Inst. Pasteur (Paris) 138:39-48. resented active dimers of four isoenzymes because they 13. Izard, D., J. Mergaert, F. Gavini, A. Beji, K. Kersters, J. De hydrolyzed p-nitrophenyl phosphate. Native alkaline phos- Ley, and H. Leclerc. 1985. Separation of Escherichia adecar- phatase behaves as several electrophoretically different mo- hoxylata from the “Erwinia herbicola-Enterobacter agglomer- lecular species when the material is subjected to starch or ans” complex and from the other Enterobacteriaceue by acid agar gel electrophoresis. Structural studies have focused on nucleic acid and protein electrophoretic techniques. Ann. Inst. Pasteur (Paris) 136B3151-168. three prevalent isoenzymes (1, 16, 22). The origin of isoen- 14. Kohler, G., and C. Milstein. 1975. Continuous culture of fused zymes 2 and 3 is the sequential cleavage of the N-terminal cells secreting antibody of predefined specificity. Nature (Lon- arginine residues of isoenzyme 1 by a protease (20, 22). don) 256:495497. However, under various conditions such as a different 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the growth medium, more than three bands have been observed assembly of the head of bacteriophage T4. Nature (London) in extracts (18, 22). This phenomenon was observed in our 227:680-685. study with Escherichia coli. The relative amounts of these 16. Levinthal, C., E. R. Signer, and K. Fetherolf. 1962. Reactivation different isoenzymes synthesized by Shigella sonnei were and hybridization of reduced alkaline phosphatase. Proc. Natl. lower. Acad. Sci. USA 48:1230-1237. 17. MacAlister, T. J., R. T. Irvin, and J. W. Costerton. 1977. Cell In conclusion, MoAb 2E5 is specific for the alkaline surface localized alkaline phosphatase of Escherichia coli as phosphatases of Escherichia coli and Shigella strains. This visualized by reaction product deposition and ferritin-labeled suggests that this MoAb may be used successfully to detect antibodies. J. Bacteriol. 130:318-328. Escherichia coli directly in water or food (since it is consid- 18. Nesmeyanova, M. A., 0. B. Motlokh, M. N. Kolot, and I. S. ered a fecal indicator) and in clinical specimens (urine, spinal Kulaev. 1981. Multiple forms of alkaline phosphatase from 206 HUSSON ET AL. INT. J. SYST.BACTERIOL.

Escherichia coli cells with repressed and derepressed biosyn- 243-249. thesis of the enzyme. J. Bacteriol. 146:453459. 25. Towbin, H., T. Staehlin, and I. Gordon. 1979. Electrophoretic 19. Rench, M. A., T. G. Metzger, and C. J. Baker. 1984. Detection transfer of proteins from polyacrylamide gels to nitrocellulose of group B streptococcal antigen in body fluids by a latex- sheets: procedure and some applications. Proc. Natl. Acad. Sci. coupled monoclonal antibody assay. J. Clin. Microbiol. 20:852- USA 76:435O-4353. 854. 26. Tsuchido, T., N. Katsvi, A. Takeuchi, M. Takano, and I. 20. Schlesinger, M. J., and L. Andersen. 1968. Multiple molecular Shibasaki. 1985. Destruction of the outer membrane permeabil- forms of the alkaline phosphatase of Escherichia coli. Ann. ity barrier of Escherichiu coli by heat treatment. Appl. Environ. N.Y. Acad. Sci. 151:159-170. Microbiol, 50:298-303. 21. Schlesinger, M. J., W. Bloch, and P. M. Kelley. 1975. Differ- 27. Wanner, B. L., and P. Latterell. 1980. Mutants affected in ences in the structure, function, and formation of two isozymes alkaline phosphatase expression: evidence for multiple regula- of Escherichia coli alkaline phosphatase, p. 333-342. In C. L. tors of the phosphate regulor in Escherichia coli. Genetics 96: Markert (ed.), Isozymes, vol. 1. Molecular structure. Academic 353-366. Press, Inc., New York. 28. Weber, K., and M. Osborn. 1969. The reliability of molecular 22. Schlesinger, M. J., and R. Olsen. 1968. Expression and localiza- weight determinations by dodecyl sulfate-polyacrylamide gel tion of Escherichia coli alkaline phosphatase synthesized in electrophoresis. J. Biol. Chem. 244:4406-4412. Salmonella typhimurium. J. Bacteriol. 96:1601-1605. 29. Williams, T., A. C. Maniar, R. C. Brunham, and G. W. 23. Tamura, K., R. Sakazaki, Y. Kosako, and E. Yoshizaki. 1986. Hammond. 1985. Identification of Chlamydia trachomatis by Leclercia adecarboxylara gen. nov., comb. nov., formerly direct immunofluorescence applied in specimen originating in known as Escherichia adecarboxylata. Curr. Microbiol. 13:179- remote area. J. Clin. Microbiol. 22:1053-1054. 184. 30. Willsky, G. R., and M. H. Malamy. 1976. Control of the 24. Tommassen, J., and B. Lugtenberg. 1982. Pho-regulon of Esch- synthesis of alkaline phosphatase and the phosphate-binding erichia coli K 12: a minireview. Ann. Inst. Pasteur (Paris) 133: protein in Escherichia coli. J. Bacteriol. 127595409.