Induction of urease by urea analogues in Evernia prunastri thallus

Urease activity in Evernia prunastri (L.) Ach. thallus is induced by incubation of lichen samples on 20 m M N,N-dimethylformamide and 20 m M N-formylurea or 40 m M thiourea although, in these two last cases, activity subsequently decreases again. The induction of enzyme activity is repressed by including 40 μ M cycloheximide in the medium. Filtration through Sepharose 6B of cell-free extracts from thalli incubated on 20 m M N,N-dimethylformamide shows a main peak of urease activity which has a molecular weight of about 560000 dalton. However, those extracts from thalli floated on 20 m M N-formylurea and 40 m M thiourea show several peaks of similar enzyme activity, which have molecular weights of about 1 100000, 670000, 260000 and 140000 dalton and 1 100000, 670000 and 140000 dalton respectively.
A time-course of urease activity could be related to the accumulation of lichen phenols in the thallus for samples incubated on N,N-dimethylformamide and thiourea.     

Regulation by repression of urease biosynthesis in Proteus rettgeri

Measuring the specific enzyme activity in cells of Proteus rettgeri it was shown that urease formation is controlled by repression through ammonia. Derepressed synthesis of the enzyme, as initiated by the absence of ammonia, required an external nitrogen source, which may not only be urea, but also nitrate, glutamate or nutrient broth. In contradiction to earlier reports the observations indicated that urea is not required for the synthesis of this enzyme, and that, therefore, urease is not an inducible enzyme in this microorganism.     

Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes

A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease.     

Site-directed mutagenesis of cysteine to threonine in Proteus vulgaris urease active site increases enzyme activity and stability

Cysteine-319 belongs to the flexible flap at the active site of Proteus vulgaris urease. Replacing this cysteine by threonine resulted in a 20-fold increase of specific activity. Temperature stability increased, susceptibility to inhibition by dipyridyl disulfide decreased, and pH optimum shifted from 8 to 6.9. K _m (35 to 12 mM) and V_max (47.4 to 1.8 mol min^–1) were substancially altered. Both variants of the enzyme were irreversibly inhibited by phenylmethanesulfonyl fluoride.     

乳粉中普通变形杆菌和奇异变形杆菌复合PCR-DHPLC检测技术的建立

应用复合PCR结合变性高效液相色谱(DHPLC)技术,建立乳粉中普通变形杆菌和奇异变形杆菌的高通量检测方法。根据普通变形杆菌的blaA和blaB基因及奇异变形杆菌的ureR基因序列分别设计特异性引物,复合PCR扩增的产物经DHPLC技术进行快速检测,并以37株参考菌株做特异性试验。试验结果表明,该方法具有很好的特异性,经复合PCR-DHPLC可同时检测乳粉中普通变形杆菌和奇异变形杆菌。该方法可以快速、准确、高通量检测普通变形杆菌和奇异变形杆菌,是乳粉中致病菌高通量检测的新技术。     

Nickel availability and urease expression in Proteus mirabilis

Cells of Proteus mirabilis, previously grown in nutrient broth (NB), exhibited an increase in urease activity during subsequent incubation in mineral medium even when protein biosynthesis was inhibited. During growth in NB, degradation of amino acids obviously led to the formation of nickel-complexing metabolites, and nickel ions were therefore inavailable for maximal expression of enzymatically active urease; this inhibition of urcase biosynthesis was overcome by the addition of nickel to the growth medium, and also by added glucose. Experiments concerning the incorporation of radioactive nickel into urease finally indicated that the observed increase in urease activity was caused by posttranslational insertion of nickel into preformed apourease.     

Induction of beta-lactamase in Proteus vulgaris

Various beta-lactam antibiotics, including monocyclic beta-lactams, induced the beta-lactamase of Proteus vulgaris; when clinical isolates were induced by benzylpenicillin, each strain produced a single beta-lactamase but the activity per milligram dry weight differed from strain to strain. The beta-lactamases of the P. vulgaris strains were heterogeneous with respect to their isoelectric points, but had almost the same specific activities, substrate specificities and Michaelis constants. The kinetics of beta-lactamase formation were investigated in three strains, each with a different beta-lactamase activity. Differential rates of enzyme synthesis and peak activity depended on the concentration of inducer. The plots of the reciprocals of the differential rates versus the reciprocals of the inducer concentrations were linear, and the maximum rate of enzyme synthesis and the concentration of the inducer giving half-maximum induction were determined from this double reciprocal plot. The maximum rates of enzyme synthesis were different in the three strains. The kinetic analysis of beta-lactamase formation revealed that the beta-lactamase activities in a single bacterial species were determined by differences in the rate of enzyme synthesis and not by differences in the properties of the enzyme.     

Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.