Endonuclease from Proteus mirabilis

Two isoforms of nuclease displaying DNase and RNase activities were found in the culture liquid and periplasm of Proteus mirabilis. The enzyme was isolated from the periplasm and then purified to a functionally homogeneous state. The nuclease was equally potent in cleaving denatured and native DNAs by the endonuclease mechanism and was designated Pm endonuclease. The endonuclease was shown to be a temperature-dependent enzyme with a pH optimum of 10.4-10.6, requiring the presence of bivalent metal ions and inhibited by citrate and ethylenediaminetetraacetate.     

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.     

Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Phosphatase from Proteus mirabilis

Cell-free preparations of Proteus mirabilis contained a phosphatase (EC 3.1.3.1), whose activity surpassed that of alkaline phosphatase from Escherichia coli. Phosphatase was also found in the culture liquid of P. mirabilis. The composition of proteins displaying enzyme activity was assayed by polyacrylamide gel electrophoresis. Enzyme synthesis was studied at various stages of bacterial growth. Biosynthesis of phosphatase in P. mirabilis (similarly to that in other bacteria) was shown to be induced under conditions of inorganic phosphate deficiency in the medium.     

Effects of Inhibition and Restoration of Protein Synthesis on the Replication of the R Factor Rts1 in Proteus mirabilis

The effect of inhibition of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density gradient centrifugation. Only 12% of the copies of Rts1 were found to replicate during amino acid starvation, whereas there was a 30% increase in the amount of P. mirabilis chromosomal deoxyribonucleic acid (DNA) during the same period. Essentially the same amount of Rts1 and host chromosome replication was observed when chloramphenicol was used to inhibit protein synthesis. The replication of Rts1 DNA was also examined in experiments in which cultures were starved for amino acids in (14)N-labeled medium and then transferred to (15)N-labeled medium containing the required amino acids. These experiments showed that Rts1 replication took place throughout the first generation in (15)N-labeled medium and that each copy of Rts1 was replicated one time during the first generation of chromosomal DNA synthesis in (15)N-medium.     

Regulation of phenylalanine oxidase synthesis in Proteus mirabilis.

Cells of Proteus mirabilis could oxidize L-phenylalanine to phenylpyruvate only when grown in the presence of a number of amino acids, particularly, L-alanine, L-asparagine, L-glutamate, and L-glutamine. Production of phenylalanine oxidase was slowly lost upon growth in a minimal medium containing ammonium ions as a nitrogen source but was reversed by the addition of casein hydrolysate. Oxidase activity as well as a phenylalanine-dichlorophenolindophenol (DCIP) reductase activity increased in P. mirabilis only during cell multiplication. Both rifampin and nalidixic acid caused inhibition of oxidase synthesis. A phenylalanine-active transport was found to be operative when bacteria were grown in the absence of added amino acids. After anaerobic growth, cells of P. mirabilis had lost their ability to carry the phenylalanine oxidase reaction when assayed in the presence of air, and nitrate could not be used as an electron acceptor for the oxidation of phenylalanine. However, some phenylalanine-dichlorophenolindophenol reductase activity was still present in anaerobic bacteria at the early stage of cell multiplication.     

Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions.

Inactivation of RecBCD nuclease (exonuclease V) and SbcB nuclease (exonuclease I) in Escherichia coli K-12 diverts most of plasmid replication activity from circular monomer production to the synthesis of linear multimers. Linear multimer synthesis has been demonstrated in plasmids of diverse origins and copy numbers, including E. coli minichromosomes. The effect of dnaA, dnaB, recF, and recJ mutations on the rate of linear multimer synthesis in sbcB cells after gam inactivation of RecBCD nuclease was investigated. Results are consistent with the hypothesis that homologous recombination, but not activities at the plasmid origin of replication, is involved in initiation of linear multimer synthesis.     

Extracellular nucleases of Lysobacter enzymogenes: production of the enzymes and purification and characterization of an endonuclease

Lysobacter enzymogenes produced a nonspecific extracellular nuclease and an extracellular RNAase when grown in tryptone broth. Both enzyme activities appeared after the exponential growth phase of the organism. The addition of RNA to the medium specifically inhibited the production of the nuclease and the addition of phosphate prevented the synthesis of the RNAase. DNA had no effect on the enzyme production. The Lysobacter nuclease was purified 274-fold and its molecular weight was estimated to be between 22 000 and 28 000. Freshly purified nuclease showed one major protein band and one major activity band on polyacrylamide gels, whereas two major bands were seen after prolonged storage of the enzyme. The nuclease was most active at pH 8.0 and required Mg2+ or Mn2+. Little activity was obtained in the presence of Ca2+. The enzyme degraded double-stranded DNA more rapidly than single-stranded DNA or RNA and was essentially inactive with poly(A) or poly(C) as the substrate. Extensive hydrolysis of double-stranded DNA by the enzyme yielded oligodeoxyribonucleotides with terminal 5'-phosphate groups. The Lysobacter RNAase appeared to have a molecular weight approximately twice that of the nuclease and was specific for ribonucleotide polymers.     

Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) containing an amide of D-galacturonic acid with L-alanine

The structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) was determined by chemical analyses along with one- and two-dimensional (1)H and (13)C NMR spectroscopy. The polysaccharide was found to contain an amide of D-galacturonic acid with L-alanine and based on the uniqueness of the O-polysaccharide structure and serological data, it was suggested to classify P. mirabilis OF into a new separate Proteus serogroup, O74. A weak cross-reactivity of P. mirabilis OF and P. mirabilis O5 was observed and accounted for by a similarity of their O-repeating units. The following structure of the polysaccharide of P. mirabilis OF was established: [chemical structure: see text]     

A decrease in the level of unscheduled DNA synthesis in preparations of irradiated rat hepatocyte nuclei under the effect of polyamines

The results obtained show the possibility of polyamine (spermine and spermidine) utilization to stabilize the nuclear chromatin and to protect it against nuclease degradation in the course of isolation of liver cell nuclei. Besides, the inhibition of the unscheduled DNA synthesis induced by nuclease incision and by gamma- or UV-radiation was demonstrated in the presence of polyamines.     

Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.

A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylated P. mirabilis peptidoglycan, the non-O-acetylated peptidoglycans from Escherichia coli and Bacillus subtilis, and the peptidoglycan monosaccharide precursors N-acetylglucosamine and N-acetylmuramic acid dipeptide. The monoclonal antibody did not react with D-alanine or lipopolysaccharide isolated from P. mirabilis. Based on this evidence, the binding epitope on the P. mirabilis peptidoglycan is predicted to be linear and to comprise the glycan backbone, including both the N- and O-acetyl moieties. Monoclonal antibody PmPG5-3 was used to localize the O acetylation of the P. mirabilis peptidoglycan by immunoelectron microscopy. Murein sacculi of P. mirabilis were heavily and randomly labelled with the immunogold, whereas very little labelling and no labelling were observed on the sacculi isolated from de-O-acetylated P. mirabilis and E. coli, respectively. Based on the apparent pattern of immunogold labelling, a physiological role for peptidoglycan O acetylation in P. mirabilis is proposed.     

Mannose-resistant Proteus-like and P. mirabilis fimbriae have specific and additive roles in P. mirabilis urinary tract infections

Proteus mirabilis is an important uropathogen that can cause complicated urinary tract infections (UTI). It produces several types of fimbriae, including mannose-resistant Proteus-like (MR/P) fimbriae and P. mirabilis fimbriae (PMF). Previously, we determined that these fimbriae affect the ability of P. mirabilis to colonize the urinary tract. The objective of this study was to analyse the effect of the simultaneous lack of P. mirabilis MR/P and PMF fimbriae in UTI pathogenesis. A double mutant lacking both fimbriae was generated by allelic replacement mutagenesis. This mutant was characterized genetically and phenotypically, and tested using an in vitro uroepithelial cell adhesion assay and the ascending UTI murine model. In vitro adhesion to uroepithelial cells by the P. mirabilis pmfA/mrpA-D mutant was reduced when compared with the wild-type, although no significant differences were observed when it was compared with the single mrpA-D and pmfA mutants. However, in vivo assays showed that colonization of kidneys and bladders by the P. mirabilis pmfA/mrpA-D mutant was significantly reduced when compared with the wild-type and both single mutants. These results indicate that, although redundancy can occur, MR/P and PMF fimbriae have specific and additive roles in P. mirabilis UTI.     

The DNase activity of an endoplasmic reticulum nuclease and its effect on DNA synthesis in vitro

An endoplasmic reticulum nuclease which was isolated previously in this laboratory from rat liver ( Kouidou et al. (1981) Eur.J. Bioch . 120, 9-14) was found to degrade linear and circular single stranded DNA but not double stranded DNA. The DNA fragments resulting from this cleavage were longer than 20 nucleotides. In addition the nuclease was found to improve the efficiency of DNA template used by DNA polymerase I in DNA synthesis in vitro. The results were the same whether incubation of the template with the nuclease was prior to addition of DNA polymerase I or simultaneously with polymerization. When nuclease was added after the completion of polymerization by DNA polymerase I it was ineffective unless the product was denatured. These data further corroborate the observation that double stranded DNA is not cleaved by this enzyme.     

Variation in expression of sex factor genes in the Proteus-Providencia group relative to Escherichia coli.

Several instances of anomalous expression of genes introduced from Escherichia coli K-12 into Proteus mirabilis have been described. It is shown here that control of sex pilus synthesis directed by the F-like R factor R1 and its depressed derivatives R1-16 (O-C) and R1-19 (i-minus) is also anomalous in P. mirabilis. Piliation in cells bearing the depressed plasmids is expressed at a lower level than in E. coli K-12, and repression is absent in R1-carrying cells. Preliminary results show a similar effect in Providencia. In Proteus morganii, a similarly reduced level of piliation in R1-16-+ or R1-19-+ cultures is observed, but an intermediate level of repression occurs in R1-+ cultures. Less extensive data suggest that expression of the sex factor genes of an R factor of the N incompatibility group differs far less between E. coli and P. mirabilis hosts. Possible bases for these effects are discussed.     

The distribution of DNA repair synthesis in chromatin and its rearrangement following damage with N-acetoxy-2-acetylaminofluorene.

The distribution of DNA repair synthesis in the chromatin of confluent human diploid fibroblasts damaged with N-acetoxy-2-acetylaminofluorene has been studied. Kinetic analysis of staphylococcal nuclease digestion data revealed that initially most of the repair synthesis occurred in nuclease sensitive regions of chromatin. Continuous labeling experiments and pulse chase experiments indicated that with time much of the 3H dThd initially incorporated into nuclease sensitive regions during repair appeared in nuclease resistant regions. Agarose gel electrophoresis was used to demonstrate that these resistant regions were core DNA. In agreement with previous findings [Smerdon, M.J. and Lieberman, M.W., (1978), Proc. Nat. Acad. Sci. USA, in press], studies of the time course of this rearrangement and of repair synthesis revealed similar time dependences and suggested a relationship between rates of repair synthesis and chromatin rearrangement.     

Nuclease biosynthesis and growth of Serratia marcescens in the presence of 2-(p-aminobenzenesulfonamide)-thiazole

The biosynthesis of nuclease in Serratia marcescens has been studied under the conditions of purine synthesis inhibition with 2-(p-aminobenzenesulfonamide)-thiazole. The addition of this sulfonamide to S. marcescens at different growth stages is found to inhibit both culture growth and nuclease synthesis.