Expression of an Acid Urease with Urethanase Activity in E. coli and Analysis of Urease Gene.

Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1 U/mg for urease and 0.6 U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4-7. And the recoveries of both urease and urethanase activities were more than 50% in 5-25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30 h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.

Biosorption of aluminum, cobalt, and copper ions by Providencia rettgeri isolated from wastewater.

Twenty-three bacterial isolates from polluted water and soil were screened for heavy metals resistance (i.e., Al(3+), Co(2+), and Cu(2+)). The most potent isolate was identified by morphological characteristics, biochemical tests and confirmed by API20E kits as Providencia rettgeri MAM-4. Removal of Al(3+) from aqueous solution by P. rettgeri is more efficient (∼fourfold) than that by B. cereus ATCC 11778 (a comparison strain) at concentration of 200 mg L(-1) Al(3+). P. rettgeri was able to remove Co(2+) more than B. cereus ATCC 11778 at concentration of 50 mg L(-1) Co(2+). Inoculation of P. rettgeri into clay enhanced significantly the removal of Al(3+), Co(2+), and Cu(2+). P. rettegri MI (mutant strain) was able to tolerate more Al(3+) than that of the parent strain. P. rettgeri was resistant to 7 out of 15 antibiotics tested. P. rettgeri MAM-4 isolated from wastewater had ability to remove Al(3+), Co(2+), and Cu(2+) efficiently from aqueous media; and enhanced significantly metal biosporption by clay. This study has revealed that P. rettgeri could be employed as an effective and economic technology for the removal such metal elements from polluted environment.

Molecular characterizations of cytolethal distending toxin produced by Providencia alcalifaciens strains isolated from patients with diarrhea.

Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides--CdtA, CdtB, and CdtC--encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206-215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G(2)/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.

Evolution of an incompatibility group IncA/C plasmid harboring blaCMY-16 and qnrA6 genes and its transfer through three clones of Providencia stuartii during a two-year outbreak in a Tunisian burn unit.

During a 2-year period in 2005 and 2006, 64 multidrug-resistant Providencia stuartii isolates, including 58 strains from 58 patients and 6 strains obtained from the same tracheal aspirator, were collected in a burn unit of a Tunisian hospital. They divided into four antibiotypes (ATB1 to ATB4) and three SmaI pulsotypes (PsA to PsC), including 49 strains belonging to clone PsA (48 of ATB1 and 1 of ATB4), 11 strains to clone PsB (7 of ATB2 and 4 of ATB3), and 4 strains to clone PsC (ATB3). All strains, except for the PsA/ATB4 isolate, were highly resistant to broad-spectrum cephalosporins due to the production of the plasmid-mediated CMY-16 ß-lactamase. In addition, the 15 strains of ATB2 and ATB3 exhibited decreased quinolone susceptibility associated with QnrA6. Most strains (ATB1 and ATB3) were gentamicin resistant, related to an AAC(6')-Ib' enzyme. All these genes were located on a conjugative plasmid belonging to the incompatibility group IncA/C(2) of 195, 175, or 100 kb. Despite differences in size and in number of resistance determinants, they derived from the same plasmid, as demonstrated by similar profiles in plasmid restriction analysis and strictly homologous sequences of repAIncA/C(2), unusual antibiotic resistance genes (e.g., aphA-6), and their genetic environments. Further investigation suggested that deletions, acquisition of the ISCR1 insertion sequence, and integron cassette mobility accounted for these variations. Thus, this outbreak was due to both the spread of three clonal strains and the dissemination of a single IncA/C(2) plasmid which underwent a remarkable evolution during the epidemic period.

Textile dye degradation by bacterial consortium and subsequent toxicological analysis of dye and dye metabolites using cytotoxicity, genotoxicity and oxidative stress studies.

The present study aims to evaluate Red HE3B degrading potential of developed microbial consortium SDS using two bacterial cultures viz. Providencia sp. SDS (PS) and Pseudomonas aeuroginosa strain BCH (PA) originally isolated from dye contaminated soil. Consortium was found to be much faster for decolorization and degradation of Red HE3B compared to the individual bacterial strain. The intensive metabolic activity of these strains led to 100% decolorization of Red HE3B (50 mg l(-1)) with in 1h. Significant induction of various dye decolorizing enzymes viz. veratryl alcohol oxidase, laccase, azoreductase and DCIP reductase compared to control, point out towards their involvement in overall decolorization and degradation process. Analytical studies like HPLC, FTIR and GC-MS were used to scrutinize the biodegradation process. Toxicological studies before and after microbial treatment was studied with respect to cytotoxicity, genotoxicity, oxidative stress, antioxidant enzyme status, protein oxidation and lipid peroxidation analysis using root cells of Allium cepa. Toxicity analysis with A. cepa signifies that dye Red HE3B exerts oxidative stress and subsequently toxic effect on the root cells where as biodegradation metabolites of the dye are relatively less toxic in nature. Phytotoxicity studies also indicated that microbial treatment favors detoxification of Red HE3B.

Enterocyte-like Caco-2 cells as a model for in vitro studies of diarrhoeagenic Providencia alcalifaciens invasion.

The entry of Providencia alcalifaciens into the enterocyte-like cell line Caco-2 compared to HEp-2 was studied. Of the 22 P. alcalifaciens strains, 13 and 21 were invasive for Caco-2 and HEp-2 cells, respectively. In contrast to HEp-2 cells, P. alcalifaciens was internalised by Caco-2 cells via receptor-mediated endocytosis. Tyrosine kinases play an important role in P. alcalifaciens uptake, also microfilaments and microtubules are engaged in this process. Inhibition of endosome acidification by ammonium chloride did not seem to have any significant effect on P. alcalifaciens invasion. Similarly to Shigella flexnerii, the invasion of Caco-2 cells by these bacteria occurred more effectively through the basolateral pole than through the apical surface of these cells. Plasmid DNA analysis showed the presence of plasmids of 5-172 kb in 13 strains regardless of their invasive ability. The presence of extracellular bacterial protein, most likely a kind of an invasin, is required for the invasion of Caco-2 and HEp-2 cells.

Demonstration and characterization of manganese superoxide dismutase of Providencia alcalifaciens.

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N-terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA-encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens.

Functional characterization of Escherichia coli GlpG and additional rhomboid proteins using an aarA mutant of Providencia stuartii.

The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the beta-lactam antibiotic cefotaxime.

ESBL-producing multidrug-resistant Providencia stuartii infections in a university hospital.

OBJECTIVES: To investigate the epidemiological and clinical findings of extended-spectrum beta-lactamase (ESBL)-producing Providencia stuartii infections in a large Italian university hospital. PATIENTS AND METHODS: All consecutive episodes of P. stuartii infection that occurred during 1999-2002 were included in the study. For each patient, we recorded the area of hospitalization and drug susceptibility of the P. stuartii strains. Patients with ESBL-producing P. stuartii infection were considered cases and those with non-ESBL-producing P. stuartii infection were used as controls. RESULTS: One hundred and sixteen (52%) out of 223 P. stuartii strains collected during the study period were found to be ESBL-producing. On the basis of PCR and DNA sequencing experiments, TEM-52 was identified in 87% of isolates and TEM-72 in 13%. All ESBL-producing P. stuartii infections were nosocomially acquired. The prevalence increased from 31% of P. stuartii infections in 1999 to 62% in 2002 (P = 0.04). All 116 strains were classified as ESBL-producing multidrug-resistant P. stuartii, since 88% of the isolates were cross-resistant to ciprofloxacin and amikacin and the other 12% were cross-resistant to ciprofloxacin and gentamicin. At logistic regression analysis, advanced age (P < 0.001), previous hospitalization (P < 0.01), neoplastic disease (P < 0.001) and previous antibiotic therapy (P < 0.001) were independent risk factors for the development of ESBL-producing infections. CONCLUSIONS: This 4 year surveillance of Providencia complaints clearly indicates that infections caused by ESBL-producing multidrug-resistant P. stuartii are an emerging problem.

A conserved mechanism for extracellular signaling in eukaryotes and prokaryotes.

Epidermal growth factor receptor (EGFr) is a key mediator of cell communication during animal development and homeostasis. In Drosophila, the signaling event is commonly regulated by the polytopic membrane protein Rhomboid (RHO), which mediates the proteolytic activation of EGFr ligands, allowing the secretion of the active signal. Until very recently, the biochemical function of RHO had remained elusive. It is now believed that Drosophila RHO is the founder member of a previously undescribed family of serine proteases, and that it could be directly responsible for the unusual, intramembranous cleavage of EGFr ligands. Here we show that the function of RHO is conserved in Gram-negative bacteria. AarA, a Providencia stuartii RHO-related protein, is active in Drosophila on the fly EGFr ligands. Vice versa, Drosophila RHO-1 can effectively rescue the bacterium's ability to produce or release the signal that activates density-dependent gene regulation (or quorum sensing). This study provides the first evidence that prokaryotic and eukaryotic RHOs could have a conserved role in cell communication and that their biochemical properties could be more similar than previously anticipated.

[Bactericidal activity and capacity for selection of mutants resistant to imipenmen and meropenem in strains of Proteus, Morganella and Providencia]. / Actividad bactericida y capacidad de selección de mutantes resistentes a imipenem y meropenem en cepas de Proteus, Morganella y Providencia.

BACKGROUND: To compare the bactericidal activity and frequency of mutation for meropenem and imipenem against Proteus mirabilis, Morganella morganii and Providencia rettgeri. MATERIAL AND METHODS: Minimum inhibitory concentrations (MIC) were determined by agar dilution method. Bactericidal activities were evaluated by killing curves method employing 4 and 16x MIC. One-step resistant mutant selection was performed by spreading more than 5 x 10(8) UFC/ml on cystine-lactose-electrolyte deficient agar containing 4 times the MIC of meropenem or imipenem. RESULTS: MIC were 8 to 16 times lower for meropenem. After 24 h, bactericidal activity was observed for meropenem at 4 and 16 x MIC against 76.7 and 100% of the strains in comparison to 26.7 and 83.3% with imipenem. After 24 h incubation with imipenem, re-growth occurred in 80 and 90% of P. mirabilis and M. morganii strains, respectively. Imipenem resistant mutants were selected from 3 strains of P. mirabilis. One of them was stable and MIC of meropenem and imipenem were 8 to 16-fold higher. CONCLUSIONS: From the laboratory point of view we consider that meropenem is more active against Proteeae because it was more potent in terms of inhibitory and bactericidal activity. In addition the risk to select for resistant mutants was significant with imipenem and P. mirabilis.

Mutations in aarE, the ubiA homolog of Providencia stuartii, result in high-level aminoglycoside resistance and reduced expression of the chromosomal aminoglycoside 2'-N-acetyltransferase.

The aarE1 allele was identified on the basis of the resulting phenotype of increased aminoglycoside resistance. The aarE1 mutation also resulted in a small-colony phenotype and decreased levels of aac(2')-Ia mRNA. The deduced AarE gene product displayed 61% amino acid identity to the Escherichia coli UbiA protein, an octaprenyltransferase required for the second step of ubiquinone biosynthesis. Complementation experiments in both Providencia stuartii and E. coli demonstrated that aarE and ubiA are functionally equivalent.

Loss of porins following carbapenem-resistance selection and adherence modification in enterobacteria.

The acquired resistance to the carbapenems is frequently joined to modified expression of porins or other outer membrane (OM) structures, thus bacterial adherence, that also depends on the presence of peculiar surface structures, might theoretically be influenced. In this study the ability to adhere to Hep-2 and I-407 eukaryotic cell monolayers was assayed for two susceptible strains of Serratia marcescens, one strain of Enterobacter cloacae and one of Providencia rettgeri in comparison with that of isogenic resistant mutants selected either by carbapenems or by cephalosporins. The mutants appeared slightly less adherent than the wild type strains, however, due to the high variability of this kind of assay, the differences observed in most cases could not be considered statistically significant. The data suggest that adherence, among the factors affecting the pathogenicity of the strains, remains probably unmodified in the resistant bacterial population possibly selected by a carbapenem treatment.

Proteus mirabilis urease: transcriptional regulation by UreR.

Proteus mirabilis urease catalyzes the hydrolysis of urea, initiating the formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Purified whole-cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences was probed with a 4.2-kb BglI fragment from within the urease operon. Autoradiographs of slot blots demonstrated 4.2- and 5.8-fold increases, respectively, in urease-specific RNA upon induction with urea. Structural and accessory genes necessary for urease activity, ureD, A, B, C, E, and F, were previously cloned and sequenced (B. D. Jones and H. L. T. Mobley, J. Bacteriol. 171:6414-6422, 1989). A 1.2-kb EcoRV-BamHI restriction fragment upstream of these sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame of 882 nucleotides, designated ureR, which is transcribed in the direction opposite that of the urease structural and accessory genes and encodes a 293-amino-acid polypeptide predicted to be 33,415 Da in size. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels of [35S]methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band with an apparent molecular size of 32 kDa. Fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence contains a potential helix-turn-helix motif and an associated AraC family signature and is similar to that predicted for a number of DNA-binding proteins, including E. coli proteins that regulate acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). These data suggest that UreR governs the inducibility of P. mirabilis urease.

Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli.

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.