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A Zn(II)-translocating P-type ATPase from Proteus mirabilis.

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis gene was expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.

Adenosine Triphosphatases↗

Structure and serological characterization of an Nepsilon-[(R)-1-carboxyethyl]-L-lysine-containing O-chain of the lipopolysaccharide of Proteus mirabilis O13.

In this paper we present the structure and describe serological properties of the O-specific polysaccharide of Proteus mirabilis O13 lipopolysaccharide, which contains a unique component: an amide of D-galacturonic acid (D-GalA) with an unusual amino acid, Nepsilon-[(R)-1-carboxyethyl]-L-lysine (alaninolysine, AlaLys). Selective chemical degradations of either GalA or AlaLys resulted in the loss of the serological reactivity of the polysaccharide with anti-O serum against P. mirabilis O13. Neither synthetic stereoisomers of AlaLys nor the isolated amide of GalA with AlaLys inhibited the reaction of the O-antiserum with the homologous lipopolysaccharide. The O-antiserum did not cross-react with the lipopolysaccharide of Providencia alcalifaciens O23 containing an amide of D-glucuronic acid with AlaLys. These data showed that both uronic acid and amino acid components of the amide play an important role in manifesting the P. mirabilis O13-specificity, but the full specific epitope also includes another O-specific polysaccharide component(s). A cross-reactivity of anti-O13 serum with some other P. mirabilis strains was observed and attributed to a common heat-stable antigen(s) different from the lipopolysaccharide.

Antibodies, Bacterial↗

Galectin-3-mediated adherence of Proteus mirabilis to Madin-Darby canine kidney cells.

Proteus mirabilis is an important cause of urinary tract infections (UTIs) and can result in acute pyelonephritis. Proteus mirabilis expresses several, morphologically distinct, fimbrial species, and previous studies have shown that the nonagglutinating fimbriae (NAF) mediate bacterial adherence to a number of cell lines, including Madin-Darby canine kidney (MDCK) cells. Immunoblot overlay analysis of the plasma membrane fraction from MDCK cells with purified NAF revealed a 34-kDa band, which has been analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Database search identified galectin-3 as a potential protein candidate. Immunocytochemical assay of MDCK cells with a galectin-3-specific monoclonal antibody, anti-Mac-2, confirmed its presence on the plasma membrane extracellular surface. Preincubation of P. mirabilis with anti-Mac-2 monoclonal antibodies, specific for galectin-3, resulted in the inhibition of bacterial binding to MDCK cells. These data suggest a role for galectin-3, interacting with appropriately glycosylated surface receptors and P. mirabilis fimbriae, as a mediator of bacterial adherence in vitro.

Animals↗

Immunochemical studies on the O-antigens of Proteus mirabilis O23 and Proteus vulgaris O23.

Analysis by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy demonstrated that the O-specific polysaccharides of Proteus mirabilis PrK 42/57 and P. vulgaris PrK 43/57 are structurally similar to that of P. vulgaris PrK 44/57 and different from the polysaccharide of P. mirabilis PrK 41/57 studied earlier. The lipopolysaccharides of these strains were tested using enzyme immunosorbent assay, passive hemolysis and Western blot with O-antisera against P. mirabilis 42/57 and P. vulgaris 43/57 and 44/57, as well as with cross-absorbed O-antisera. The chemical and serological data revealed the basis for combining the four strains into Proteus serogroup O23 and division of this serogroup to three subgroups, one for P. vulgaris 43/57 and 44/57 and two others for P. mirabilis 41/57 and 42/57.

Animals↗

Selective IgA immune unresponsiveness to Proteus mirabilis fumarate reductase A-chain in rheumatoid arthritis.

OBJECTIVE: To determine if selective immune unresponsiveness to microbial antigens is associated with predisposition to rheumatoid arthritis (RA). METHODS: Proteins from Proteus mirabilis lysate were isolated by SDS-PAGE and examined by Western blotting for antibody responses in sera from patients with RA compared to healthy subjects and patients with psoriatic arthritis (PsA). RESULTS: Although RA patients had marked IgA immune responses to many P. mirabilis proteins compared to healthy subjects, selective unresponsiveness was found in RA to a 66 kDa protein identified as fumarate reductase A-chain (FRD-A) by mass spectroscopy. This was confirmed in Western blots with recombinant FRD-A from P. mirabilis. IgA unresponsiveness to FRD-A was found in 21/59 (35.6%) RA patients compared to 7/63 (11.1%) healthy individuals (p < 0.01) and 6/52 (11.5%) patients with PsA (p < 0.01). IgA unresponsiveness to FRD-A was present in 20/46 (43.5%) RA patients with IgA rheumatoid factors (RF) compared to 1/13 (7.7%) without RF (p < 0.025). CONCLUSION: Our results identify a selective hole in the IgA immune repertoire for P. mirabilis FRD-A in a subset of IgA RF-positive patients with RA.

Adult↗

Expression of the Proteus mirabilis lipoprotein gene in Escherichia coli. Existence of tandem promoters.

The Ipp gene from Proteus mirabilis was cloned onto pBR322 and expressed in Escherichia coli. The P. mirabilis lpp gene is unique in that it has two tandem promoters transcribing two mRNAs that differ in length by approximately 70 nucleotides at their 5'-ends. The two mRNAs thus encode the identical lipoprotein. The P. mirabilis prolipoprotein has a 19-amino acid signal peptide and a 59-amino acid lipoprotein sequence. In spite of the substantial differences in the amino acid sequence from the E. coli prolipoprotein, the P. mirabilis prolipoprotein is normally modified and processed in E. coli, and the resultant lipoprotein is assembled in the E. coli outer membrane as is the E. coli lipoprotein.

Amino Acid Sequence↗

Different binding specificities of P. mirabilis compared to E. coli.

The target cell specificities of P. mirabilis and E. coli strains were compared using human urinary tract epithelial cells and erythrocytes from human, guinea pig, sheep, ox and horse blood. P. mirabilis strains agglutinated the erythrocytes both in mannose-resistant and mannose-sensitive patterns. No relation was, however, found between hemagglutination and adhesion to uroepithelial cells. This is in contrast to E. coli where uroepithelial cell attachment is paralleled by mannose-resistant agglutination o human erythrocytes. The difference in receptor recognition between P, mirabilis and E. coli was further indicated by the fact that P. mirabilis strains attaching to human uroepithelial cells did not agglutinate guinea pig red cells coated with globotetraosylceramide, acting as a receptor for E. coli on human uroepithelial cells erythrocytes.

Animals↗

Experimental induction of Proteus mirabilis cystitis in the pony and evaluation of therapy with trimethoprim-sulfadiazine.

Proteus mirabilis cystitis was induced in 9 ponies by chemically eroding the bladder mucosa before the organism was inoculated. Comparisons were made in the treatment of P mirabilis cystitis between ponies treated daily for 13 days with a trimethoprim-sulfadiazine (TMP-SDZ) paste and both positive and negative controls. Urine cultures from ponies treated with TMP-SDZ became negative for P mirabilis between days 3 and 9 after the start of the treatment, whereas positive controls remained infected until day 13. Urine cultures from all ponies were negative for P mirabilis on day 28. Urine concentrations of TMP and SDZ were relatively high after day 1 of therapy.

Administration, Oral↗

Papillary necrosis in vitro: a scanning electron microscopic comparison of escherichia coli and Proteus mirabilis infection.

The effect of bacterial infection on excised renal papillae as a model for papillary necrosis and subsequent calcification was investigated. Sterile rat renal papillae were placed in 25 ml aliquots of filter sterilized human urine and then inoculated with one ml suspensions of sterilized human urine and then inoculated with one ml suspensions of sterile saline as a control, or 1 x 108/ml Escherichia coli or proteus mirabilis. After incubation at 37 degrees C for periods of 8 hr, 24 hr, 48 hr, 72 hr, 1 wk, 2 wk and 3 wk, urinary pH was measured, bacterial culture performed and the renal papillae were recovered and examined by scanning electron microscopy (SEM) and energy dispersive spectrophotometry (EDS). In the case of Proteus mirabilis, the sequence of events noted included bacterial-papillary interactions consisting of cell desquamation and strand formation, despite infrequent bacterial attachment. After 10 hr, a rapid, urease induced pH rise resulted in calcium salt deposition on the papillae surface. Organism death was apparent after 72 hr. Escherichia coli infected papillae demonstrated similar cell surface changes after a 8 hr as seen in P. mirabilis; however, frequent evidence of bacterial attachment and penetration was apparent. Bacterial attachment was a prominent feature throughout the incubation period with E. coli. After one week, rare areas of degenerating cells and bacteria with increased calcium levels as compared to surrounding areas were noted by EDS analysis. Urinary pH was stable throughout the incubation period. This study suggests varied roles for the organisms most associated with infection induced papillary necrosis (E coli) and papillary necrosis with subsequent stone formation (P. mirabilis). A role for bacterial calcification in the absence of bacterial urease activity by E. coli is also suggested.

Animals↗

Purification and properties of the alpha2beta2 complex of tryptophan synthetase of Proteus mirabilis.

A procedure is described for the purification of the tryptophan synthetase alpha2beta2 complex from cell extracts of Proteus mirabilis. A 30-fold purification was achieved with an overall yield of about 23% and a specific activity of 1,600. The complex can be dissociated and the subunits isolated in a pure form. The complex can be reconstituted from the isolated subunits to regain the initial activity. The alpha and beta2 subunits of the tryptophan synthetase complex of P. mirabilis are not significantly different from those of Escherichia coli and other enteric bacteria as to their physical properties, amino acid compositions, and enzymic properties. Complementation studies indicate that the alpha subunit of P. mirabilis hybridizes well with the beta2 subunit from E. coli. Similarly, the beta2 subunit of P. mirabilis readily complexes with the alpha subunits from E. coli, Salmonella typhimurium, and Serratia marcescens. The hybrids formed are all effective in catalyzing the conversion of indoleglycerol phosphate plus serine into tryptophan and glyceraldehyde 3-phosphate. However, these hybrids have reduced or no activity in the other reactions, namely, the condensation of indole and serine to form tryptophan or the aldolytic cleavage of indoleglycerol phosphate.

Escherichia coli↗

Inhibition of growth of Proteus mirabilis and Escherichia coli in urine in response to fasting and vegetarian diet.

It has recently been shown that serum antibody levels against Proteus mirabilis decreased in patients with rheumatoid arthritis who improved clinically during treatment with 7-10 days of fasting followed by a one-year vegetarian diet. As P. mirabilis is commonly implicated in urinary tract infections, this study was carried out to examine whether fasting and vegetarian diet may influence the growth of P. mirabilis and Escherichia coli in urine. Urine samples were collected from 22 patients who were referred to a health farm for various reasons. The dietary regimen recommended by the health farm consisted of fasting for 7 to 10 days followed by a vegan diet. The growth of both bacteria in urine samples collected after 8 days was significantly slower than in samples collected at baseline. In urine samples collected after 18 days growth was also reduced, although not significantly for E. coli. Our results show that dietary manipulation may reduce the ability of urine to support the growth of P. mirabilis and E. coli.

Adult↗

Ectoderm exerts the driving force for gastrulation in the sand dollar Scaphechinus mirabilis.

How the ectodermal layer relates to the invagination processes was examined in the sand dollar Scaphechinus mirabilis. When the turgor pressure of blastocoele was increased, invagination was completely blocked. In contrast, an increase in turgor pressure did not affect elongation of the gut rudiment in the regular echinoid Hemicentrotus pulcherrimus. Rhodamine-phalloidin staining showed that the distribution of actin filaments was different between two species of embryos. In S. mirabilis gastrulating embryos, abundant actin filaments were seen at the basal cortex of ectoderm in addition to archenteron cells, while the intense signal was restricted to the archenteron in H. pulcherrimus. To investigate whether actin filaments contained in the ectodermal layer exert the force of invagination, a small part of the ectodermal layer was aspirated with a micropipette. If S. mirabilis embryos were aspirated from the onset of gastrulation, invagination did not occur at all, irrespective of the suction site. Even after the archenteron had invaginated to one-half of its full length, further elongation of the archenteron was severely blocked by suction of the lateral ectoderm. In contrast, suction of the ectodermal layer did not affect the elongation processes in H. pulcherrimus. These results strongly suggest that the ectodermal layer, especially in the vegetal half, exerts the driving force of invagination in S. mirabilis.

Actin Cytoskeleton↗

A first assessment of genetic variation in Welwitschia mirabilis Hook.

Welwitschia mirabilis is a monotypic member of the family Welwitchiaceae which, along with Ephedra and Gnetum species, comprises the gymnospermous order Gnetales. While the monophyly of this order is now widely accepted, the relationship of the Gnetales to other seed plants is still contentious. Despite the unique phylogenetic position of W. mirabilis and its extraordinary physiological and anatomical adaptations, little is known about the plant's phylogeny or its current distribution in isolated locations throughout the Namib Desert. As a preliminary step in the design of an more extensive phylogeographic study, we analyzed 37 random amplified polymorphic DNA (RAPD) loci from 59 plants distributed among five sites separated by distances of 6-440 km. Cluster analysis and analysis of molecular variance (AMOVA) revealed significant levels of variation within and between populations and little evidence of inbreeding. Genetic differences between populations reflect the geographic distances separating them. Three of the populations formed discernable genetic clusters, suggesting that little gene flow occurs between populations separated by > or = 18 km. In contrast, gene flow is occurring between two populations separated by only 6 km, supporting previous observations that pollen dispersal is primarily local and that seeds are not readily wind-born over the large distances separating most W. mirabilis populations. As a working hypothesis, we propose that W. mirabilis had a continuous distribution across its current range as much as 105 million years ago, and that as a consequence of subsequent drying trends and physical disturbance, populations became progressively isolated, accounting for their current distribution.

Biological Evolution↗

High levels of genetic variation exist in Aspergillus niger populations infecting Welwitschia mirabilis hook.

Aspergillus niger is an asexual, haploid fungus which infects the seeds of Namibia's national plant, Welwitschia mirabilis, severely affecting plant viability. We used 31 randomly amplified polymorphic DNA markers to assess genetic variation among 89 A. niger isolates collected from three W. mirabilis populations in the Namib Desert. While all isolates belonged to the same vegetative compatibility group, 84% were unique genotypes, and estimates of genotypic evenness and Simpson's index of diversity approached 1.0 in the three populations. Analysis of molecular variance revealed that 78% of the total variation sampled was among isolates from individual W. mirabilis plants. Lower, but significant, amounts of variation detected among isolates from different plants (12%) and different sites (10%) also indicated some site- and plant-level genetic differentiation. Total gene diversity (H(T) = 0.264) was mostly attributable to diversity within populations (H(S) = 0.217); the relatively low level of genetic differentiation among the sites (G(ST) = 0.141) suggests that gene flow is occurring among the three distant sites. Although sexual reproduction has never been observed in this fungus, parasexuality is a well-known phenomenon in laboratory strains. We thus attribute the high levels of genetic variation to parasexuality and/or wind-facilitated gene flow from an as of yet undocumented broader host range of the fungus on other desert vegetation. Given the apparent ease of transmission, high levels of genetic diversity, and potentially broad host range, A. niger infections of W. mirabilis may be extremely difficult to control or prevent.

Aspergillus niger↗

Purification of two DD-carboxypeptidases/transpeptidases with different penicillin sensitivities from Proteus mirabilis.

Two membrane-bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD-carboxypeptidase and transpeptidase (named DD-carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X-100, affinity chromatography on matrix-bound ampicillin, and preparative isoelectric focusing in the presence of detergent. Purified enzymes H and L were, respectively, penicillin-binding proteins 4 and 5 among seven major penicillin-binding proteins present in P. mirabilis. The enzymes differed in the following properties. Enzyme H had an Mr of 49,000; isoelectric point at pH 8.2; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme-antibiotic complex EI* (half-life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of EI*; it functioned as an endopeptidase on peptide-crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43 000; isoelectric point at pH 5.9; low sensitivity to benzylpenicillin and low stability of EI* (half-life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase. The properties of enzyme L were identical with those of the single active DD-carboxypeptidase found previously in the spheroplast L-form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L-form and synthesis of peptide-crosslinked peptidoglycan, depends on the continuing fuction of enzyme L as a DD-carboxypeptidase and transpeptidase in the presence of the antibiotic.

Carboxypeptidases↗

Growth regulation in the gobiid teleost, Gillichthys mirabilis: roles of growth hormone, hepatic growth hormone receptors and insulin-like growth factor-I.

Studies of the teleost Gillichthys mirabilis were undertaken to assess the role of GH in regulating hepatic GH receptors, insulin-like growth factor-I (IGF-I) activity and cartilage growth. Hypophysectomized G. mirabilis were injected with saline vehicle or with 10, 100 or 1000 ng tilapia GH (tGH)/g every other day for 2-3 weeks. Growth, judged by increased body weight and length, was inhibited by hypophysectomy and stimulated by increasing tGH doses. Hepatic GH receptors, measured by 125I-labelled tGH binding, were decreased 50% by hypophysectomy. An additional dose-dependent reduction in binding was observed 24 h after tGH injection, but MgCl2 stripping of membranes suggested receptor occupation by exogenous tGH. IGF-I activity, measured by 35SO4 incorporation into oral cartilage explants in vitro, was decreased 50% by hypophysectomy and increased to 200% of intact control levels after injection of 1000 ng tGH/g. In a second experiment, 35SO4 incorporation by oral cartilage from hypophysectomized fish injected with 10, 100 or 1000 ng tGH/g was stimulated to intact control levels. Effects of feeding and tGH injection on in-vitro responsiveness of oral cartilage to recombinant bovine IGF-I (rbIGF-I; 10-1000 ng/ml) were also assessed. Oral cartilage from fed fish showed a parabolic dose-response curve, whereas oral cartilage from starved fish had a lower basal rate of 35SO4 incorporation and a linear dose-response relationship. Oral cartilage from hypophysectomized G. mirabilis showed a significantly attenuated response to rbIGF-I which was restored by tGH injection, suggesting that the GH status of the animal is important for sensitivity of target tissue to IGF-I. Because of its similarity to other vertebrate systems, G. mirabilis presents a good teleost model of growth regulation and of the functions and interactions of GH and IGF-I.

Animals↗

[F' transfer from Escherichia coli to Proteus mirabilis].

The task of this work was the establishment of an effective transfer system for F'-plasmids from Escherichia coli to Proteus mirabilis. It is shown that cells of PG VI act as recipients in crosses with E. coli F' strains but with a low transfer rate of the plasmid. The presumption that a restriction -- modification system in P. mirabilis was the only reason for the low transfer could not be confirmed. An indirect selection method was developed to isolate P. mirabilis cells which are better recipients. Conjugation experiments showed that the isolated mutants had a better recipient capacity (increase of about 100). This is true not only for the transfer of a F'-plasmid but also for a R-plasmid. The stability of these plasmids in the mutant cells, however, was much lower than in the wild type.

Conjugation, Genetic↗

DNA repair in Proteus mirabilis. III.Survival, dimer excision, and UV reactivation in comparison with Escherichia coli K12.

Measurements of UV sensitivity of wildtype cells (wt) and UV senistive mutants of E. coli and P. mirabilis suggest that the increased sensitivity of P. mirabilis (wt) is due either to incomplete repair of DNA lesions or to additive lethality probably as a result of UV induction of defective phage(s) present in P. mitabilis (Taubeneck, 1967). Direct estimates of the rate of pyrimidine dimer excision and a comparison of the UV reactivation capacity of E. coli and P. mirabilis for the temperate phages lambda and pi 1, respectively, support this conclusion.

Cell Survival↗