Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.

Urease is an inducible virulence factor of uropathogenic Proteus mirabilis. Although eight contiguous genes necessary for urease activity have been cloned and sequenced, the transcriptional organization and regulation of specific genes within the Proteus gene cluster has not been investigated in detail. The first gene, ureR, is located 400 bp upstream and is oriented in the direction opposite the other seven genes, ureDABCEFG. The structural subunits of urease are encoded by ureABC. Previously, UreR was shown to contain a putative helix-turn-helix DNA-binding motif 30 residues upstream of a consensus sequence which is a signature for the AraC family of positive regulators; this polypeptide is homologous to other DNA-binding regulatory proteins. Nested deletions of ureR linked to either ureD-lacZ or ureA-lacZ operon fusions demonstrated that an intact ureR is required for urea-induced synthesis of LacZ from either ureA or ureD and identified a urea-regulated promoter in the ureR-ureD intergenic region. However, lacZ operon fusions to fragments encompassing putative promoter regions upstream of ureA and ureF demonstrated that no urea-regulated promoters occur upstream of these open reading frames; regions upstream of ureR, ureE, and ureG were not tested. These data suggest that UreR acts as a positive regulator in the presence of urea, activating transcription of urease structural and accessory genes via sequences upstream of ureD. To address the role of the nonstructural regulatory and accessory genes, we constructed deletion, cassette, and linker insertion mutations throughout the ure gene cluster and determined the effect of these mutations on production and regulation of urease activity in Escherichia coli. Mutations were obtained, with locations determine by DNA sequencing, in all genes except ureA and ureE. In each case, the mutation resulted in a urease-negative phenotype.

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.

Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.

The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (10(8) bacteria per 10(5) cells). Washed UEC, retained on 8 micron pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.

Weekly urine specimens from 51 long-term catheterized patients yielded 699 isolates of Providencia stuartii. Urease-positive strains represented 23.7% (166) of the isolates, sucrose-positive strains represented 24.5% (171), and lactose-utilizing strains represented 0.7% (5). Urease and sucrose traits were transferred by conjugation to Escherichia coli via an 82-kilobase plasmid; lactose fermentation was transferred by a 150-kilobase plasmid.

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.

The cell surface of Bacillus subtilis contains several peptidoglycan-associated polypeptides. Cell walls were labeled with 125I or 35S, and the products were digested with lysozyme. When the digests were chromatographed on Sephacryl S-200, peaks of radioactivity corresponding to molecular weights of 240,000, 125,000, 20,000, 17,000, and 15,000 were observed. The walls solubilized by lysozyme were also subjected to sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis, and radioactive bands corresponding to apparent molecular weights of 24,000, 22,000, and 19,000 were found. Isoelectric focusing of the digests revealed the presence of a component having an isoelectric point of 3.7, and, possibly, of minor components having isoelectric points of 4.7 and 6.1. Proteases, including trypsin, subtilisin, and pronase, removed some of the radioactivity from [35S]-labeled walls. Significant proportions of label from [35S]walls were solubilized by the peptide-bond-breaking agents cyanogen bromide and N-bromosuccinimide. Small proportions of radioactivity were released from labeled walls by hydroxylamine and trichloroacetic acid. Direct, amino acid analyses of the walls showed the presence of several amino acids not commonly regarded as constituents of peptidoglycan. Cell walls from a protease-deficient mutant, and from a wall preparation enriched in cell poles, contained similar proportions of amino acids. In addition, wall preparations from an autolysin-deficient mutant, and walls from protease hyper-producing strains, contained amino acids that could not be removed by rigorous extraction-procedures. The results suggest that the cell walls of Bacillus subtilis contain tightly, or covalently, bound protein molecules or polypeptides that are refractory to removal by denaturants.

The concept that growth of the cell envelope could serve the role of segregating daughter chromosomes into daughter cells was proposed by Jacob et al. [1] in 1963. Although the genetic data were derived through study of the Gram-negative Escherichia coli, the morphological data were derived with the Gram-positive Bacillus subtilis. Their Replicon Model (RM) proposed that DNA was replicated from a chromosomal origin, and that membrane growth took place in the region between the duplicated origins. As originally stated, the model became less tenable as an explanation for DNA segregation when fluidity of the cell membrane became generally evident [21. The model could be saved by assuming that the wall served the role originally assigned to the membrane [3]. Recent reviews have tacitly assumed that the DNA is bound to the envelope and is segregated by "surface growth" [4,5] in which surface is assumed to be capable of supporting the segregation movement. But even that proposal became untenable when the diffuse nature of the wall growth in Gram-positive rods became clear both theoretically [6,7] and experimentally [see below]. Logically, growth can only take place in the region between the chromosomal-origin attachment sites if wall growth is to serve as the sole process for DNA segregation. Without some additional mechanism, sooner or later, a very asymmetric cell division would occur, yielding cells with extra or absent chromosomes. Finally, the role of mesosome-DNA attachment sites, which served an essential role in the original model has become very much clouded [8,9]. All the alternatives so far proposed for the RM still assume zonal growth for the cylindrical portion of the rod [4,5]. We now propose a Renovated Replicon Model (RRM) which leads to accurate segregation of the chromosomes and equipartitioning of cell contents. The model does not invoke the incorrect notion of an unique zone or zones of growth of the wall in the region between the initially adjacent origin attachment sites. The RRM also explains the high precision of the cell division process in dividing the cytoplasm evenly between daughter Cells [10]. A recent report by the Amsterdam group using E. coli revealed that the coefficient of variation for cell length at division of strain B / rA was 5% and that of strain B / r K was 9% in a minimal growth medium [11]. Moreover, Trueba and Woldringh [12] found that the shorter of two sisters is usually wider, implying that cell division in this organism is even more precise than the length measurement of cells by themselves would imply. In B. subtilis, Burdett and Higgins [13] reported that in 94% of the cells "the septum was within 0.15/~m of the calculated center of the cell" in cells ranging in length from 2 to 5/~m. Dr.

Lipoteichoic acids from Bacillus subtilis 168 and its gta mutants were partially purified and characterized. The teichoic acids contain glycerol residues and phosphorus in equimolar ratios, and fatty acids. Some contaminating protein and nucleic acids were also present in the preparations. Gel electrophoresis of the preparations revealed the presence of common proteins, suggestive of functional protein-lipoteichoic acid complexes in bacterial membranes. All of the lipoteichoic acids were partially d-glucosylated, as evidenced by enzymic analysis. The d-gIucose residues were in the α anomeric configuration, as shown by precipitation with concanàvalin A. The data suggested that some, but not all, of the pathways leading to the d-glucosylation of the cell-wall teichoic acid are functional in the d-glucosylation of the membrane teichoic acids. The lipoteichoic acids were complexed with methylated bovine serum albumin, and injected into rabbits, in order to elicit a humoral immune-response. Antibodies were directed against poly(glycerol phosphate) and not against the d-glucosyl residues of the lipoteichoic acids. The antibody preparations cross-reacted with all of the membrane teichoic acids. Soluble, cell-wall teichoic acid was a potent inhibitor of passive hemmagglutination in a system containing lipoteichoic acid-sensitized, sheep red-blood cells and anti-lipoteichoic acid antiserum.