The isolation and partial characterization of a new restriction endonuclease from Providencia stuartii.

We describe the isolation of a new class 2 restriction endonuclease from Providencia stuartii 164. Using the procedure of osmotic shock treatment, we have partially purified this enzyme (Pst 1) and have begun preliminary work to characterize its specificity and requirements. Pst 1 requires Mg++ as the only cofactor and produces more than 18 cleavages in wild type lambda. We have determined the location of 7 of these cleavages by the use of deletion and insertion mutants of lambda.     

A site-specific endodeoxyribonuclease from Streptomyces albus CMI 52766 sharing site-specificity with Providencia stuartii endonuclease PstI.

A class II site-specific endodeoxyribonuclease (SalPI) was identified in cell-free extracts of Streptomyces albus CMI 52766 after high speed centrifugation and fractionation through Bio Gel AO.5M. SalPI cleaves lambda DNA into at least 18 fragments. Five cleavage sites were located in the linear lambda map by the use of double and triple restriction enzyme digests involving EcoRI, HindIII, SalGI and another new Streptomyces enzyme, SacI. The results were indistinguishable from those previously obtained for a Providencia stuartii enzyme, PstI, by Smith, Blattner & Davies (Nucleic Acids Res. 1976 3, 343). SalPI and PstI were shown by a double digest test to have the same site specificity. None of 34 phages tested was obviously restricted by S. albus CMI 52766, and correspondingly DNA from two of them was not cleaved in vitro by SalPI. DNA from Streptomyces phage that does not form plaques on S. albus CMI 52766, and plasmid SCP2 DNA from S. coelicolor A3 (2), were both cleaved.     

Fractionation of the Providencia stuartii cell envelope

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.     

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii

Abstract A new type II restriction endonuclease, named Ajo I, was detected in Acinetobacter johnsonii . The enzyme Ajo I, an isoschizomer of Pst I, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.     

Chlorhexidine resistance and the lipids of Providencia stuartii

The lipid composition of Providencia stuartii has been shown to resemble closely that of Proteus mirabilis. The ability of some Pv, stuartii strains to survive exposure to high concentrations of the antiseptic chlorhexidine could not be explained in terms of differences in lipid content between sensitive and resistant strains. In addition, resistance could not be attributed to either reduced adsorption of the antiseptic or to its gross enzymic degradation.     

Type II SsrI restriction endonuclease from Staphylococcus saprophyticus

The recognition sequence and cleavage site for restriction endonuclease SsrI have been determined, the latter being 5'-GTT decreases AAC-3'. The enzyme was isolated from Staphylococcus saprophyticus strain and may be used in DNA investigation instead of its isoshizomer HpaI.     

Adherence to uroepithelial cells of Providencia stuartii isolated from the catheterized urinary tract

The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (10(8) bacteria per 10(5) cells). Washed UEC, retained on 8 micron pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.     

Structure of the O-polysaccharide of Providencia stuartii O49

The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O47

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O47:H4, strain 3646/51. Studies by sugar and methylation analyses along with Smith degradation and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY and H-detected 1H,13C HSQC and HMBC experiments, showed that the polysaccharide has a branched hexasaccharide repeating unit with the following structure: [carbohydrate structure: see text]     

Characterization of a site-specific restriction endonuclease from Streptomyces aureofaciens.

A new type II sequence-specific restriction endonuclease, SauI, was isolated from Streptomyces aureofaciens IKA18/4. The purified enzyme was free of contaminating exonuclease and phosphatase activities. SauI cleaved lambda DNA at two sites, but did not cleave pBR322, simian virus 40, or phi X174 DNA. SauI recognized the septanucleotide sequence 5'-CCTNAGG-3' and cleaved at the position indicated by the arrow, producing a trinucleotide 5'-terminal extension.     

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.     

Characterization of the Escherichia coli X-ray endonuclease, endonuclease III

The X-ray endonuclease endonuclease III of Escherichia coli has been purified to apparent homogeneity by using the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The most purified fraction shows endonucleolytic activity against apurinic and apyrimidinic (AP) sites and a dose-dependent response to DNA that has been X irradiated, UV irradiated, or treated with OsO4. The endonuclease also nicks OsO4-treated DNA that has been subsequently treated with alkali to produce fragmented thymine residues and DNA treated with potassium permanganate. The enzyme does not incise the alkali-labile sites present in DNA X irradiated in vitro in the presence of hydroxyl radical scavengers. The most purified fractions exhibit two distinct activities, an AP endonuclease that cleaves on the 3' side of the damage leaving a 3'-OH and a 5'-PO4 and a DNA N-glycosylase that recognizes at least two substrates, thymine glycol residues and urea residues. The glycosylase activity is sensitive to N-ethylmaleimide while the AP endonuclease is not.     

Subunit assembly modulates the activities of the Type III restriction-modification enzyme PstII in vitro

We demonstrate that, like other Type III restriction endonuclease, PstII does not turnover such that a DNA substrate is only fully cleaved at a Res₂Mod₂-to-site ratio of ~1:1. However, unlike other Type III enzymes, the cleavage rate profiles varied with protein concentration: using 5 nM DNA and 25 nM PstII, approximately half of the DNA was cut at a fast rate while the remainder was cut 24 times more slowly; in comparison, with 100 nM PstII cleavage occurs at a single fast rate. The inclusion of the methyl donor S-adenosyl methionine does not alter the rates with 100 nM PstII but with 25 nM PstII the reaction stopped after completion of the initial fast cleavage phase owing to methylation. Concentration-dependent rates were also observed in methylation assays: at 100 nM PstII, a single slow rate was measured while at lower PstII concentrations both fast and slow rates were measured. We propose a model in which the intact Res₂Mod₂ complex favoured at high PstII concentrations is a fast endonuclease/slow methyltransferase while the various subassemblies which coexist at lower concentrations are fast methyltransferases. A potential role for disassembly in control of restriction activity in vivo is discussed.     

Characterization of aarA, a pleiotrophic negative regulator of the 2'-N-acetyltransferase in Providencia stuartii.

We have utilized transposon mutagenesis to obtain insertional mutations in Providencia stuartii that activate the chromosomal aac(2')-la gene. Two closely linked mini-Tn5Cm insertions were obtained in a locus designated aarA, and a single insertion was obtained in a separate locus, aarC. Nucleotide sequence analysis, complementation studies, and localization of the sites of mini-Tn5Cm insertion have allowed the identification of the aarA coding region. The deduced AarA protein had a molecular mass of 31,086 kDa and displayed characteristics of an integral membrane protein. A strain deleted for the aarA gene by allelic exchange showed at least a fourfold increase in the accumulation of aac(2')-la mRNA and an eightfold increase in aminoglycoside resistance. Mutations in aarA were pleiotrophic and also resulted in loss of pigmentation and a deficiency in cell separation during division.     

S-Adenosyl homocysteine and DNA ends stimulate promiscuous nuclease activities in the Type III restriction endonuclease EcoPI

In the absence of the methyl donor S-adenosyl methionine and under certain permissive reaction conditions, EcoPI shows non-specific endonuclease activity. We show here that the cofactor analogue S-adenosyl homocysteine promotes this promiscuous DNA cleavage. Additionally, an extensive exonuclease-like processing of the DNA is also observed that can even result in digestion of non-specific DNA in trans. We suggest a model for how DNA communication events initiating from non-specific sites, and in particular free DNA ends, could produce the observed cleavage patterns.     

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.     

Complete genome sequence of Providencia stuartii clinical isolate MRSN 2154

Here we present the complete genome sequence of Providencia stuartii MRSN 2154, isolated from an Afghan national. P. stuartii is a Gram-negative bacillus capable of causing infections in a wide variety of human tissues. Because Providencia readily acquires plasmids bearing drug resistance loci, it is of growing clinical significance.     

Reversal of the surface effects of chlorhexidine diacetate on cells of Providencia stuartii

Chlorhexidine diacetate (CHA) increased the hydrophobicity of the cell surface of cells of three strains of Providencia stuartii. Removal of at least some of the CHA from the cells by washing them with an appropriate antidote partially reversed the hydrophobicity-increasing action of the biguanide. The effects of other treatments on cell surface hydrophobicity were examined with these strains and, for comparison, with two strains each of Escherichia coli and Pseudomonas aeruginosa: ethyl-enediamine tetraacetic acid affected all strains, although not to the same extent, whereas thermal injury (55°C) produced marked changes only with the two E. coli strains.     

Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan.

The relationship between the acetylation of peptidoglycan and that of aminoglycosides in Providencia stuartii has been investigated both in vivo and in vitro. Adaptation of the assay for peptidoglycan N-->O-acetyltransferase permitted an investigation of the use of peptidoglycan as a source of acetate for the N acetylation of aminoglycosides by gentamicin N-acetyltransferase [EC 2.3.1.59; AAC(2')]. The peptidoglycan from cells of P. stuartii PR50 was prelabelled with 3H by growth in the presence of N-[acetyl-3H]glucosamine. Under these conditions, [3H]acetate was confirmed to be transferred to the C-6 position of peptidoglycan-bound N-acetylmuramyl residues. Isolated cells were subsequently incubated in the presence of various concentrations of gentamicin and tobramycin (0 to 5x MIC). Analysis of various cellular fractions from isolated cells and spent culture medium by the aminoglycoside-binding phosphocellulose paper assay revealed increasing levels of radioactivity associated with the filters used for whole-cell sonicates of cells treated with gentamicin up to 2 x MIC. Beyond this concentration, a decrease in radioactivity was observed, consistent with the onset of cell lysis. Similar results were obtained with tobramycin, but the increasing trend was less obvious. The transfer of radiolabel to either aminoglycoside was not observed with P. stuartii PR100, a strain that is devoid of AAC(2')-Ia. A high-performance anion-exchange chromatography-based method was established to further characterize the AAC(2')-Ia-catalyzed acetylation of aminoglycosides. The high-performance liquid chromatography (HPLC)-based method resolved a tobramycin preparation into two peaks, both of which were collected and confirmed by 1H nuclear magnetic resonance to be the antibiotic. Authentic standards of 2'-N-acetyltobramycin were prepared and were well separated from the parent antibiotic when subjected to the HPLC analysis. By applying this technique, the transfer of radiolabelled acetate from the cell wall polymer peptidoglycan to tobramycin was confirmed. In addition, isolated and purified AAC(2')-Ia was shown to catalyze in vitro the transfer of acetate from acetyl-coenzyme A, soluble fragments of peptidoglycan, and N-acetylglucosamine to tobramycin. These data further support the proposal that AAC(2')-Ia from P. stuartii may have a physiological role in its secondary metabolism and that its activity on aminoglycosides is simply fortuitous.     

SbvI restriction endonuclease from Streptococcus bovis

Restriction endonuclease Sbv I, an isoschizomer of Hae III, has been isolated from rumen amylolytic bacterium Streptococcus bovis II/1. Sbv I was purified from cell extract by phosphocellulose chromatography and heparin-Sepharose chromatography. The recognition sequence of Sbv I was identified by digestion of pBR322, pUC9 and Λ-DNA and comparing the cleavage patterns obtained with computer-derived data. Sbv I recognizes the 4-bp palindrome, 5'-GGCC-3' and cleaves DNA after the second G in the sequence, producing blunt ends.