The influence of growth conditions on the synthesis of molybdenum cofactor in Proteus mirabilis

Cell-free extracts of Proteus mirabilis were able to reconstitute NADPH-dependent assimilatory nitrate reductase in crude extracts of the Neurospora crassa mutant strain nit-1, lacking molybdenum cofactor. Molybdenum cofactor was formed in the cytoplasm of the bacterium even in the presence of oxygen during growth though under these conditions no molybdo enzymes are formed. As a consequence no cofactor could be released by acid treatment from membranes of cells grown aerobically. The amount of cofactor released from membranes of cells grown anaerobically under various conditions was proportional to the amount of molybdo enzymes formed. During growth in the presence of tungstate a cofactor, which lacks molybdenum, was found in the cytoplasm. For detection of this so-called demolybdo cofactor the presence of molybdate during reconstitution was essential. Moreover, the cytoplasmic cofactor pool in cells grown in the presence of tungstate appeared to be two to three times higher than in cells grown under similar conditions without tungstate. After anaerobic growth in the presence of tungstate, the inactive demolybdo reductases were shown to contain partly no cofactor and partly a demolybdo cofactor. The P. mirabilis chlorate resistant mutant S 556 did not contain molybdenum cofactor. In two other chl-mutants the cofactor activity was the same as in the wild type.     

Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.     

Biosynthesis of Proteus mirabilis Nuclease

The culture liquid and periplasm of Proteus mirabilis contained nuclease, an enzyme with DNase and RNase activities. The nuclease was most actively synthesized in the early exponential and stationary growth phases. Nuclease synthesis was regulated by nucleic acids (induction by substrate) and inorganic phosphate (end-product inhibition). The synthesis and secretion of nuclease by P. mirabilis was induced by mitomycin C, an inducer of the SOS functions of cells. This suggests the involvement of SOS-response proteins in the regulation of nuclease synthesis.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

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.     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.     

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between two HQNO inhibition sites, or more probably in a Q- or b-cycle.Abbreviation HQNO= 2-n-heptyl-4-hydroxy-quinoline-N-oxide     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Cold adapted features of Vibrio salmonicida catalase: characterisation and comparison to the mesophilic counterpart from Proteus mirabilis

The gene encoding catalase from the psychrophilic marine bacterium Vibrio salmonicida LFI1238 was identified, cloned and expressed in the catalase-deficient Escherichia coli UM2. Recombinant catalase from V. salmonicida (VSC) was purified to apparent homogeneity as a tetramer with a molecular mass of 235 kDa. VSC contained 67% heme b and 25% protoporphyrin IX. VSC was able to bind NADPH, react with cyanide and form compounds I and II as other monofunctional small subunit heme catalases. Amino acid sequence alignment of VSC and catalase from the mesophilic Proteus mirabilis (PMC) revealed 71% identity. As for cold adapted enzymes in general, VSC possessed a lower temperature optimum and higher catalytic efficiency (k _cat/K _m) compared to PMC. VSC have higher affinity for hydrogen peroxide (apparent K _m) at all temperatures. For VSC the turnover rate (k _cat) is slightly lower while the catalytic efficiency is slightly higher compared to PMC over the temperature range measured, except at 4°C. Moreover, the catalytic efficiency of VSC and PMC is almost temperature independent, except at 4°C where PMC has a twofold lower efficiency compared to VSC. This may indicate that VSC has evolved to maintain a high efficiency at low temperatures.     

Structure of the O-polysaccharide of Proteus mirabilis OC (CCUG 10702) from a new proposed Proteus serogroup O75

A neutral O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis OC (CCUG 10702) and studied by sugar and methylation analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text]. Based on the unique structure of the O-polysaccharide and serological data, we propose classifying P. mirabilis OC (CCUG 10702) into a new separate Proteus serogroup O75. A weak cross-reaction of O-antiserum against P. mirabilis OC with the lipopolysaccharide of P. mirabilis O49 was accounted for by a similarity in the O-polysaccharide structures.     

Efficiency of hydrogen photoproduction by photosystem I-enriched subchloroplast vesicles combined with Proteus mirabilis cells. Effects of some exogenous electron donors

High rates of hydrogen photoproduction are obtained when glutaraldehyde-fixed Photosystem I-enriched vesicles (Photosystem II-depleted) are added to hydrogenase-containing cells of Proteus mirabilis in the presence of the mediator methylviologen and a suitable electron donating system. This donor system includes ascorbate, dithioerythritol (DTE) and the mediator tetramethylphenylene-diamine (TMPD) and reduces the photosynthetic electron transfer chain at the level of plastocyanin. Both DTE and ascorbate are required for hydrogen photoproduction, DTE being the ultimate electron donor and ascorbate only having a catalytic function. Whereas the aerobic photoreduction of methylviologen is similar in the presence of DTE, ascorbate or both, under anaerobic conditions only combination of both compounds results in a high and stable amount of reduced methylviologen that can be utilized by the hydrogenase. It is concluded that oxidation reactions of reduced methylviologen, competing with the hydrogenase, rather than methylviologen photoreduction, limit hydrogen photoproduction in the presence of either DTE or ascorbate. These oxidation reactions are suggested to involve back reactions to the oxidized form(s) of ascorbate and DTE but backflow to the photosynthetic electron transfer chain (i.e. cyclic electron transfer) can not be excluded.Abbreviations Tes N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid - DTE 1,4-dithioerythritol - TMPD, N,N,N N-tetramethyl-p-phenylenediamine - DCMU 3-(3, 4-dichlorophenyl)-1, 1,-dimethylureum - EDAC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide - DNP-INT 2-iodo-6-isopropyl-3-methyl-2, 4, 4-trinitrodiphenyl ether - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone - PS photosystem - Chl chlorophyll     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10701 (OB) classified into a new Proteus serogroup, O74

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus mirabilis CCUG 10701 (OB) and studied by chemical analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: --> 3)-beta-D-GlcpNAc6Ac-(1 --> 2)-beta-D-GalpA4Ac-(1--> 3)-alpha-D-GalpNAc-(1 --> 4)-alpha-D-GalpA-(1 -->, where the degree of O-acetylation at position 6 of GlcNAc is approximately 50% and at position 4 of beta-GalA approximately 60%. Based on the unique structure of the O-polysaccharide and serological data, it is proposed to classify P. mirabilis CCUG 10701 (OB) into a new Proteus serogroup, O74.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus mirabilis 2002

The structure of the O-polysaccharide of the lipopolysaccharide of Proteus mirabilis 2002 was elucidated by chemical methods and 1H and 13C NMR spectroscopy. It was found that the polysaccharide consists of branched pentasaccharide repeating units having the following structure: [structure in text]. The O-polysaccharide of P. mirabilis 2002 has a common tetrasaccharide fragment with that of P. mirabilis 52/57 from serogroup O29, and the lipopolysaccharides of the two strains are serologically related. Therefore, based on the structural and serological data, we propose to classify P. mirabilis 2002 into the Proteus O29 serogroup as a subgroup O29a,29b.     

Structural and serological studies of the O-antigen of Proteus mirabilis O-9

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with chemical methods: [chemical structure: see text] where the degree of O-acetylation is approximately 70%. Immunochemical studies using rabbit polyclonal anti-Proteus mirabilis O-9 serum showed the importance of the O-acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti-P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.     

Structure of the O-polysaccharide of a serologically separate strain of Proteus mirabilis, TG 332, from a new proposed Proteus serogroup O50

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis TG 332 strain. The following structure of the O-polysaccharide was determined by chemical methods along with NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments: [see equation in text]. The O-polysaccharide studied has a unique structure among Proteus O-antigens. Accordingly, P. mirabilis TG 332 is serologically separate, and we propose to classify this strain into a new Proteus serogroup, O50. The nature of minor epitopes that provide a cross-reactivity of P. mirabilis TG 332 O-antiserum with the LPS of P. mirabilis O30 and Proteus penneri 34 (O60) is discussed.     

Proteus mirabilis interkingdom swarming signals attract blow flies

Flies transport specific bacteria with their larvae that provide a wider range of nutrients for those bacteria. Our hypothesis was that this symbiotic interaction may depend on interkingdom signaling. We obtained Proteus mirabilis from the salivary glands of the blow fly Lucilia sericata; this strain swarmed significantly and produced a strong odor that attracts blow flies. To identify the putative interkingdom signals for the bacterium and flies, we reasoned that as swarming is used by this bacterium to cover the food resource and requires bacterial signaling, the same bacterial signals used for swarming may be used to communicate with blow flies. Using transposon mutagenesis, we identified six novel genes for swarming (ureR, fis, hybG, zapB, fadE and PROSTU_03490), then, confirming our hypothesis, we discovered that fly attractants, lactic acid, phenol, NaOH, KOH and ammonia, restore swarming for cells with the swarming mutations. Hence, compounds produced by the bacterium that attract flies also are utilized for swarming. In addition, bacteria with the swarming mutation rfaL attracted fewer blow flies and reduced the number of eggs laid by the flies. Therefore, we have identified several interkingdom signals between P. mirabilis and blow flies.     

Structure of the neutral O-polysaccharide and biological activities of the lipopolysaccharide of Proteus mirabilis O20

Mild acid degradation of the lipopolysaccharide (LPS) of Proteus mirabilis O20 resulted in depolymerisation of the O-polysaccharide to give a repeating-unit pentasaccharide. A polysaccharide was obtained by O-deacylation of the LPS followed by nitrous acid deamination. The derived pentasaccharide and polysaccharide were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the repeating unit of the O-polysaccharide was established: [Carbohydrate structure: see text]. As opposite to most other P. mirabilis O-polysaccharides studied, that of P. mirabilis O20 is neutral. A week serological cross-reactivity was observed between anti-P. mirabilis O20 serum and LPS of a number of Proteus serogroups with known O-polysaccharide structure. The ability of LPS of P. mirabilis O20 to activate the serine protease cascade was tested in Limulus amoebocyte lysate and in human blood plasma and compared with that of P. mirabilis O14a,14c having an acidic O-polysaccharide. The LPS of P. mirabilis O20 was found to be less active in both assays than the LPS of P. mirabilis O14a,14c and, therefore, the structurally variable O-polysaccharide may influenced the biological activity of the conserved lipid A moiety of the LPS.     

Structure of a highly phosphorylated O-polysaccharide of Proteus mirabilis O41

A highly phosphorylated O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O41 followed by GPC. The initial and dephosphorylated polysaccharides and phosphorylated products from two sequential Smith degradations were studied by (1)H, (13)C and (31)P NMR spectroscopy and ESI-MS. The O-polysaccharide was found to have a tetrasaccharide repeating unit containing one ribitol phosphate (presumably d-Rib-ol-5-P) and two ethanolamine phosphate (Etn-P) groups, one of which is present in the stoichiometric amount and the other in a nonstoichiometric amount. The following structure of the O-polysaccharide was established:     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.