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A bank of over 4,200 lacZ protein fusions in Shigellaflexneri 2a was screened for fusions to temperatureregulated promoters. One mutant, BS260, was completely noninvasive on HeLa cells and mapped to a region on the 220-kb virulence plasmid in which we had previously localized several avirulent temperature-regulated operon fusions (A. E. Hromockyj and A. T. Maurelli, Infect. Immun. 57:2963-2970, 1989). The phenotype of BS260 was similar to that of the previously identified mxi (membrane expression of invasion plasmid antigens) mutants, since it made wild-type intracellular levels of the invasion plasmid antigens (Ipa) but was deficient in the surface expression of IpaB and IpaC. Six kilobases of DNA upstream of the BS260 fusion end joint were cloned, but no temperature-regulated promoter was found, whereas the fusion end joint clone of the noninvasive mxi operon fusion mutant BS226 contained a temperature-regulated promoter. The locus defined by BS260 was designated mxiA, and that defined by BS226 was designated mxiB. Closer analysis of the mxi4 and mxiB phenotypes by a cell-free enzyme-linked immunosorbent assay revealed that the mutants failed to excrete IpaB and IpaC into the culture medium, whereas wild-type cells actively released these antigens. Excretion of the ipa polypeptides from wild-type bacteria was confirmed by Western blot analysis of culture supernatants. Protease protection experiments revealed that wild-type S. flexneri 2a actually had much lower levels of surface-exposed IpaB and IpaC relative to those in the total antigen pool. In addition, examination of cellular fractions showed that, although there was no IpaB or IpaC in the outer membrane of BS260 and BS226, the antigens did accumulate in the cytoplasmic membrane. A 76-kDa temperature-regulated polypeptide in wild-type S. flexneri was identified as the putative mxi4 gene product. These results strongly suggest that IpaB and IpaC represent truly excreted proteins of S. flexneri and that the mxi4 and mxiB loci on the plasmid code * Corresponding author. plasmid-associated, temperature-regulated proteins are involved in the initial steps in pathogenesis of this microorganism (7, 8). In a previous report from this laboratory, the random mutagenesis of S. flexneri 2a with AplacMu53 to create operon fusions to temperature-regulated genes was described. Three temperature-regulated operon fusion mutants were isolated that were avirulent and deficient in surface expression of IpaB and IpaC while still making wild-type intracellular levels of the antigens (12). All three of these mutations map outside the ipa coding region and represent novel virulence genes on the invasion plasmid.

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-Ilb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II-producing strains of E. coli also suggested that additional variants of LT-II exist.More than a decade ago, the heat-labile enterotoxins of Escherichia coli and Vibrio cholerae were recognized to be a family of related protein toxins with similarities in structure, mode of action, and immunochemistry (12,13 Recently, new heat-labile enterotoxins were discovered (17,19,21), and the V. cholerae-E. coli enterotoxin family was divided into two distinct antigenic groups (36). Cholera toxin (CT) (12) and the type I E. coli heat-labile enterotoxins (LT-I), including the antigenic variants LTh-I and LTp-I (22), belong to serotype I, and antiserum to any one of them will neutralize the other type I toxins. In contrast, type II E. coli enterotoxins (LT-II) are not neutralized by antisera against type I toxins, but they are neutralized by antiserum to the prototype 21,36). Two antigenic variants of type II enterotoxin, designated LT-IIa and LT-IIb, were characterized (19, 21). Both consist of A and B subunits which are similar in size to the subunits of CT and LT-I. The ADP-ribosyltransferase activity of these LT-II toxins is similar to that of CT and LT-I (8; P. P. Chang, S.-C. Tsai, R. Adamik, B. C. Kunz, J. Moss, E. M. Twiddy, and R. K. Holmes, submitted for publication), but the gangliosidebinding specificities of LT-IIa and LT-IIb are different from those of CT and LT-I and from each other (14,19,21 (20). The plasmids pBR322, p...

Proteus mirabilis urease catalyzes the hydrolysis of urea to CO 2 and NH 3 , resulting in urinary stone formation in individuals with complicated urinary tract infections. UreR, a member of the AraC family, activates transcription of the genes encoding urease enzyme subunits and accessory proteins, ureDABCEFG, as well as its own transcription in the presence of urea. Based on sequence homology with AraC, we hypothesized that UreR contains both a dimerization domain and a DNA-binding domain. A translational fusion of the leucine zipper dimerization domain (amino acids 302 to 350) of C/EBP and the C-terminal half of UreR (amino acids 164 to 293) activated transcription from the ureD promoter (p ureD ) and bound to a 60-bp fragment containing p ureD , as analyzed by gel shift. These results were consistent with the DNA-binding specificity residing in the C-terminal half of UreR and dimerization being required for activity. To localize the dimerization domain of UreR, a translational fusion of the DNA-binding domain of the LexA repressor (amino acids 1 to 87) and the N-terminal half of UreR (amino acids 1 to 182) was constructed and found to repress transcription from p sulA -lacZ (sulA is repressed by LexA) and bind to the sulA operator site, as analyzed by gel shift. Since LexA binds this site only as a dimer, the UreR 1-182 -LexA 1-87 fusion also must dimerize to bind p sulA . Indeed, purified UreR-Myc-His eluted from a gel filtration column as a dimer. Therefore, we conclude that the dimerization domain of UreR is located within the N-terminal half of UreR. UreR contains three leucines that mimic the leucines that contribute to dimerization of AraC. Mutagenesis of Leu147, Leu148, or L158 alone did not significantly affect UreR function. In contrast, mutagenesis of both Leu147 and Leu148 or all three Leu residues resulted in a 85 or 94% decrease, respectively, in UreR function in the presence of urea (P < 0.001). On the contrary, His102 and His175 mutations of UreR resulted in constitutive induction in the absence of urea. We conclude that a dimerization domain resides in the N-terminal half of the polypeptide, that Leu residues may contribute to this function, and that sequences within the C-terminal half of UreR are responsible for DNA binding to the urease promoter regions. Selected His residues also contribute significantly to UreR function.Proteus mirabilis infects the urinary tract of humans and is most commonly responsible for causing disease in individuals with structural abnormalities of the urinary tract or in patients who undergo long-term catheterization (16). Cystitis, acute pyelonephritis, and urinary stone formation are all possible consequences of P. mirabilis infection (17).P. mirabilis produces a urea-inducible urease, a high-molecular-weight, multimeric, cytoplasmic nickel metalloenzyme.