The effect of carbon dioxide on the production of an extracellular nuclease of Staphylococcus aureus

The production of the extracellular nuclease secreted by Staphylococcus aureus (Foggi strain) was studied in a fermentor in an attempt to improve yield and allow large-scale production of the enzyme. In shake flask cultures, 600 units/mL of the enzyme were produced routinely. However, only 450 units/mL of the enzyme at best were obtained in a small-scale fermentor (3 L). The supplementation of the air supply to the fermentor with carbon dioxide [20% (v/v)] increased levels of enzyme in the culture medium to 770 units/mL. Subsequently, this result was reproduced in larger fermentors (10 and 150 L). The possible mechanisms of the effect of carbon dioxide upon the growth of Staphylococcus aureus (Foggi) and the production of the enzyme are discussed.     

Heat Stable Nuclease in Mastitic Milk

The production of a heat stable nuclease (deoxyribonuclease) by Staphylococcus aureus was first reported by Cunningham et al. (1956). The detection of the enzyme was simplified by Lachica et al. (1971), who developed an agar diffusion test based on the metachromatic properties of toluidine blue.     

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater GWA groundwater aquifer     

Effect of Anaerobiosis on Staphylococcal Nuclease Production

Five strains of Staphylococcus aureus were examined quantitatively for the production of nuclease under aerobic and anaerobic conditions. Hydrolysis of deoxyribonucleic acid and ribonucleic acid was detected by measuring the release of acidsoluble nucleotides spectrophotometrically. We found that the enzyme was produced anaerobically, as well as aerobically, and that anaerobiosis had no effect on production of this enzyme when other conditions, such as pH, were held constant.     

Electron Microscopy During Release and Purification of Mesosomal Vesicles and Protoplast Membranes from Staphylococcus aureus

The mesosomes of log-phase Staphylococcus aureus ATCC 6538P and Staphylococcus aureus phage-type 80/81, as seen in situ in ultrathin sections, were of the vesicular type. The constituent vesicles ranged from 35 to 50 nm in diameter when the glutaraldehyde-osmium-uranium-lead sequence of fixation and staining was used. During protoplasting in hypertonic buffer containing a muralytic enzyme, vesicles of the same size were extruded and required magnesium ion to maintain structural integrity. The vesicles, purified from the protoplasting supernatant medium by density gradient centrifugation, maintained size and configuration in a homogeneous preparation. Cytoplasmic membranes, produced by osmotic shock and nuclease treatment of protoplasts, were similarly concentrated in gradients. However, they were not free of membrane-associated ribosomes nor of mesosomal vesicles except when prepared in the absence of magnesium.     

Enzymatic Detection of the Growth of Staphylococcus aureus in Foods

A specific method has been developed for the extraction and measurement of staphylococcal nuclease in foods in which Staphylococcus aureus has grown. The method was used to compare staphylococcal growth with nuclease production in foods under varying conditions of temperature, aerobiosis, and competition from other microorganisms. It was concluded that the nuclease is produced under any conditions that permit growth of S. aureus, and little or no interference with the test was encountered either from mixed, natural populations or from a variety of pure, laboratory cultures. Nuclease and enterotoxin A production were shown to vary in synchrony for the 234 (Casman) strain of S. aureus, and the sensitivity of the enzymatic detection of nuclease was comparable to the sensitivity of serological detection of enterotoxin A. It was found that 15 min at 121 C was required to reduce the nuclease activity in slurries of contaminated ham below the level present in the unheated slurry. The extraordinary heat resistance of the nuclease permits its detection even in foods heated subsequent to the growth of S. aureus. The nuclease analysis requires about 3 hr to complete and requires no unusual equipment or reagents.     

Rapid qualitative method for detecting staphylococcal nuclease in foods.

A rapid method for the detection of heat-stable staphylococcal nuclease in foods is described. The procedure consists of an acid precipitation, boiling, and centrifugation followed by enzyme detection in an agar plate containing deoxyribonucleic acid. To test the efficacy of the procedure, purified Staphylococcus aureus nuclease was added to various foods and recovery experiments were performed. Additionally, foods were inoculated and incubated with S. aureus, and the staphylococcal counts were compared with nuclease activity. The results indicate that the procedure possesses merit for a rapid method that can be incorporated into quality control programs. The procedure requires approximately 2.5 h, and it will detect nuclease levels as low as 10 ng/g of food.     

Halophilic Nuclease from a Moderately Halophilic Micrococcus varians

The moderately halophilic bacterium Micrococcus varians, isolated from soy sauce mash, produced extracellular nuclease when cultivated aerobically in media containing 1 to 4 M NaCl or KCl. The enzyme, purified to an electrophoretically homogeneous state, had both ribonuclease and deoxyribonuclease activities. The nuclease had maximal activity in the presence of 2.9 M NaCl or 2.1 M KCl at 40 C. The enzymatic activity was lost by dialysis against low-salt buffer, whereas when the inactivated enzyme was dialyzed against 3.4 M NaCl buffer as much as 77% of the initial activity could be restored.     

Purification of an alkaline nuclease from Physarum polycephalum.

An alkaline nuclease was purified from microplasmodia of Physarum polycephalum. The nuclease, active on denatured DNA and RNA and free of contamination by other nucleolytic activities, appeared to be a zinc-metallo protein. The enzyme was only active under conditions, where Zn2+ were retained in the enzyme. Loss of zinc occurred by the chelating action of EDTA, EGTA or ampholines, by acid of highly alkaline pH conditions or by high ionic strength. The addition of ZnCl2 to compensate losses, restored all activity, while all other divalent cations caused inhibition. The nuclease, with a molecular weight of 32 000, was stable at neutral pH at high temperatures with a half-life of 20 min at 80 degrees C. It was inhibited by any salt of buffer concentration above the level of zero ionic strength and showed a special sensitivity towards phosphate ions. The possible similarity of this enzyme to nuclease S1 from Aspergillus oryzae is pointed out.     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.     

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.     

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum.

The gene for staphylococcal nuclease (SNase), an extracellular enzyme of Staphylococcus aureus, was introduced into Corynebacterium glutamicum. The heterologous gene was expressed in this host organism, and SNase was efficiently exported to the culture medium. Amino-terminal sequencing of SNase secreted by C. glutamicum revealed that the signal peptide was apparently cleaved off at precisely the same position as in the original host, S. aureus. As with S. aureus, a second smaller form of SNase (A form), whose appearance is presumably the result of a secondary processing step, was found in the culture medium of the recombinant C. glutamicum strain. The A form was one residue shorter than the mature nuclease A produced by S. aureus. Variation of the sodium chloride concentration in the growth medium had a marked influence on the location and the processing of SNase by C. glutamicum. In a complex growth medium containing 4% sodium chloride, SNase was exclusively located in the supernatant, but a significant amount of the enzyme remained cell associated if the strain was grown in a low-salt medium. Also, high salt concentrations seemed to inhibit processing of the high-molecular-weight form of SNase (B form) to the smaller A form. Similarities and differences in the export and modes of processing of SNase by three different, nonrelated gram-positive host organisms are discussed. Finally, a versatile Escherichia coli-C. glutamicum tac-lacIq expression shuttle vector was constructed. With this vector, it was possible to achieve isopropyl-beta-D-galactopyranoside (IPTG)-inducible overexpression and secretion of SNase in C. glutamicum, whereby the expression level was dependent on the concentration of the inducer.     

Staphylococcal Nuclease in Udder Secretions of Cows with Acute Mastitis

High concentrations of nuclease produced by Staphylococcus aureus were demonstrated within a few hours by direct examination of udder secretions from cows with a severe staphylococcal mastitis. A positive correlation between the nuclease concentration and the severity of the mastitis was found. The cows with high nuclease concentrations were generally young individuals and/or in the first 2 weeks of the lactation period. Most had a low titre of antibodies against staphylococcal nuclease. Two-thirds of the cows in which high nuclease concentrations were demonstrated were culled or died because of the mastitis attack. The role which nuclease plays for staphylococcal virulence is discussed, and it is concluded that nuclease contributes to the pathogenicity of S. aureus.     

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization.

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.     

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.     

Secretion and processing of staphylococcal nuclease by Bacillus subtilis.

We have studied the secretion and processing of Staphylococcus aureus nuclease in Bacillus subtilis. We show that the initial species of nuclease found in the cell supernatants during short-term radioactive labeling (pulse-chase) had a molecular weight of approximately 18,800 and comigrated in a sodium dodecyl sulfate-polyacrylamide gel with staphylococcal nuclease B. This nuclease B form was processed to the mature nuclease A extracellularly by a phenylmethylsulfonyl fluoride-sensitive protease. The nuclease B-processing site is a consensus signal peptidase site, and the processing of nuclease B was coupled to secretion as judged by pulse-chase experiments. The nuclease A was shown by microsequencing of the N terminus to be 2 amino acid residues shorter than the nuclease A described for S. aureus Foggi. The nuclease B form was still the first species found in the culture supernatant after removal of the N-terminal 26 amino acids of the native 60-amino-acid signal peptide. However, removal of the N-terminal 72 amino acids abolishes secretion of any nuclease form and leads to the intracellular accumulation of nuclease.     

Two thermostable nucleases coexisted in Staphylococcus aureus: evidence from mutagenesis and in vitro expression

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1 ), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2 ). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus , the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli , and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1 -deleted mutant of S. aureus strain RN4220 (termed RNΔ nuc1 ) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1 -deleted strain still showed functional activity after 30 min at 121 °C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus .     

Inhibition of the catalytic properties of Staphylococcus aureus nuclease by monoclonal antibodies

Monoclonal antibodies (Mab) specific for Staphylococcus aureus nuclease (nuclease) were examined for their capacity to inhibit the enzyme-mediated cleavage of DNA. Within a panel of 22 anti-nuclease Mab produced by hybridoma cell lines derived from SJL/J, A/J or BALB/c mice, only five were capable of modifying nuclease activity. Of the five, only one protected DNA from enzymatic degradation whereas the others reduced the rate of the enzymatic reaction. When mixed together, partially inactivating Mabs were frequently more efficient inhibitors than when used individually. It was shown by competitive binding assay that nuclease could be bound simultaneously to more than one Mab. Mixtures of five inactivating Mabs were able to completely block the nuclease activity. Although the actual mechanism for Mab nuclease inactivation is not known, the present data are consistent with simple steric hindrance for the formation of the DNA-nuclease complex by bulky Mab molecules bound to epitopes close to, but distinct from, nuclease catalytic sites. A mathematical model for Mab binding and inactivation of nuclease, taking into account multiple binding events for one or two Mabs interacting with nuclease, was used to derive affinities and maximum reductions of the enzymatic rate (details on the derivation of the equations and on the hypotheses of the model are given in an appendix). This analysis showed that the observed cooperative effects were dependent on the formation of multi-molecular complexes in which nuclease is bound simultaneously to two (or more) different Mabs. It also shows that the formation of cyclic complexes, if allowed, might result in very high apparent affinities. Since in screening of hybridoma fusions, the probability of finding such pairs of monoclonal antibodies would be low, this phenomenon may explain the fact that no Mab, or mixture of Mabs, matched the polyclonal antisera in capacity to block nuclease enzymatic activity.Abbreviations Nuclease Staphylococcus aureus, Foggi Strain, nuclease - Ig immunoglobulin, Mab(s)monoclonal antibody(ies) - ELISA enzyme linked immunosorbent assay - RIA radioimmunoassay     

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.