Two restriction endonucleases from Bacillus globiggi.

The sites of action of the restriction enzyme Bgl II on lambda DNA are mapped. This enzyme recognises the sequence 5' ...AGATCT...3' and makes staggered cuts producing sticky ends. In lambda DNA, the second A in this sequence is methylated about 50% of the time by a bacterial methylase absent in E. coli dam. In contrast to Bgl II, Bgl I makes many cuts in lambda DNA and produces 5' terminals which are not substrates for polynucleotide kinase.     

Purification and properties of a site-specific restriction endonuclease AaaI from Acetobacter aceti subsp. aceti No. 1023

Abstract A type II restriction endonuclease, named Aaa I, was purified from Acetobacter aceti subsp. aceti No. 1023. The optimum pH and temperature were determined to be 8.5 and 37°C, respectively. The enzyme activity was stimulated by the addition of either NaCl or KCl and their optimum concentrations were 100 mM for both cations. Aaa I recognized the hexanucleotide sequence and cleaved it at the positions indicated by the arrows. Aaa I was an isoschizomer of Xma III from Xanthomonas malvacaerum and Eco 52I from Escherichia coli .     

Oxaloacetate decarboxylase from Acetobacter aceti

An oxaloacetate (OAA) decarboxylase (EC 4.1.1.3) has been purified 72-fold from Acetobacter aceti cells grown on ethanol, and its molecular weight was estimated to be about 80,000 by gel filtration. Several properties distinguished this enzyme from the OAA decarboxylase from A. xylinum:

1. It was not a constitutive enzyme; the activity was 6- to 20-fold higher in cells grown on a C₂ substrate (acetate or ethanol) than in cells grown on a C₃ compound (pyruvate or propionate).
2. The optimum pH was 7.5; a value of 5.6 was reported for the enzyme from A. xylinum.
3. The enzyme did not need a divalent cation and was not inhibited by EDTA.
4. The K _mvalue for OAA was found to be 0.22 mM. It was not affected by the addition of nicotinamide adenine dinucleotide.
5. The enzyme activity was neither inhibited by acetate nor by L-malate.

In addition, the OAA decarboxylase from A. aceti was insensitive to monovalent cations, avidin or acetyl coenzyme A.     

Phosphoenolpyruvate carboxylase from Acetobacter aceti

In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K _m values for PEP and NaHCO₃ were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K _m for Mn²⁺, Co²⁺ and Mg²⁺ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg²⁺. Mn²⁺ and Co²⁺ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K _a 0.18 mM) or propionyl CoA (K _a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

Type II restriction endonucleases from Bacillus sphaericus

Abstract The galactophilic lectin of the bacterium Pseudomonas aeruginosa (PA-I) was used for mitogenic stimulation of peripheral bloodlymphocytes from cancer-bearing patients and healthy subjects. This lectin, which preferentially stimulates sialidase-treated lymphocytes, was shown to be useful in the detection of an impairment in the mitogenic response of the patients' lymphocytes. Its efficiency was at least as that of the Phaseolus vulgaris lectin (PHA), which is widely used for the diagnosis and prognosis of deficient immunocompetence states.     

Pyruvate,orthophosphate dikinase from Acetobacter aceti

A pyruvate, orthophosphate dikinase (EC 2.7.9.1) has been isolated from Acetobacter aceti grown on pyruvate as the only source of carbon and energy. The enzyme was purified 65-fold, and its molecular weight was determined to be about 330,000 by gel filtration.The optimum pH was 8.0 in the forward direction [phosphoenolpyruvate (PEP) formation] and 7.1 for the backward reaction (pyruvate production). In both directions Mg²⁺ was required (forward K _m 1.70 mM; reverse K _m 0.87 mM) and no other divalent cation was able to replace it. The K _m values for pyruvate, ATP, and P_i were 27 M, 0.20 mM, and 0.83 mM, respectively, in the forward direction. The K _m values for PEP, AMP, and PP_i were 0.13 mM, 6 M, and 62 M, respectively, for the reverse reaction. The substrate-product pairs pyruvate-PEP, ATP-AMP, P_i-PP_i were competitive inhibitors to each other in both directions. These product inhibition studies suggest for the enzyme from A. aceti nonclassical three-site Tri (Uni Uni) Ping-Pong kinetics.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - SDS sodium dodecyl sulphate - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione     

Alanine racemase from the acidophile Acetobacter aceti

Acetobacter aceti converts ethanol to acetic acid, and survives acetic acid exposure by tolerating cytoplasmic acidification. Alanine racemase (Alr) is a pyridoxal 5' phosphate (PLP) -dependent enzyme that catalyzes the interconversion of the d- and l-isomers of alanine and has a basic pH optimum. Since d-alanine is essential for peptidoglycan biosynthesis, Alr must somehow function in the acidic cytoplasm of A. aceti. We report the partial purification of native A. aceti Alr (AaAlr) and evidence that it is a rather stable enzyme. The C-terminus of AaAlr has a strong resemblance to the ssrA-encoded protein degradation signal, which thwarted initial protein expression experiments. High-activity AaAlr forms lacking a protease recognition sequence were expressed in Escherichia coli and purified. Biophysical and enzymological experiments confirm that AaAlr is intrinsically acid-resistant, yet has the catalytic properties of an ordinary Alr.     

Site-specific restriction endonucleases in Bacillus licheniformis

Abstract We systematically studied site-specific restriction endonucleases in Bacillus licheniformis strains and detected endonuclease activity in 25 of 217 strains tested. Three different activities were obtained. One of these activities detected in 21 strains was the most representative within the species and produced a banding pattern, after digestion of A DNA, identical to that seen with Cla I. Two other strains isolated from soil samples from China and USA were found to produce a DNA-cleaving enzyme with the same recognition sequence as Bsa I. One producer strain, isolated from a Peruvian soil sample, showed to possess a mixture of two isoschizomers, Cla I and Bsa I. Finally, one strain produced an endonuclease activity, not previously described in B. licheniformis , that showed the same recognition sites as Bsu 361.     

The relaxation of specificity of BanI restriction endonuclease from Bacillus aneurinolyticus IAM 1077

The restriction endonuclease BanI from Bacillus aneurinolyticus IAM 1077, which recognizes 5′-GGPyPuCC-3′ and cleaves between G and G within this sequence, has decreased substrate specificity at high nuclease concentrations. The relaxation of its specificity was enhanced during modified reactions: digestion of pBR322 DNA or lambda DNA in the presence of high glycerol and dimethyl-sulfoxide (DMSO) produced additional fragments in addition to the inherent fragments. Therefore, it is required to check the reaction conditions carefully for generation of inherent fragments.     

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Φ×174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Φ×174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg²⁺ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 °C (pH 8.0) with 100 mM NaCl and 10 mM Mg²⁺ concentrations.     

BpaI and BpnI: novel type II restriction endonucleases from Bacillus pasteurii and Bacillus pantothenticus

Two novel type II restriction endonucleases, designated as BpaI and BpnI, were isolated from Bacillus pasteurii strain1761 and Bacillus pantothenticus strain1639, respectively. They were partially purified and SDS-PAGE indicated Mr values of 28 and 67 kDa for BpaI, 28 and 48 kDa for BpnI. The partially purified endonucleases hydrolyzed DNA into discrete fragments: pUC18 (2.6 kb for BpaI; 1.8 and 0.8 kb for BpnI), pBR322 (2.5 and 1.8 kb for BpaI; 2.6 and 1.7 kb for BpnI) and phix174 DNA (3.2 and 2.1 kb for BpaI; 4 and 1.3 kb for BpnI).     

Characterization of the acetyl-CoA synthetase of Acetobacter aceti.

The acetate activating system of Acetobacter aceti has been studied. The enzyme responsible, acetyl-CoA synthetase, has been purified about 500-fold from crude cell extracts and was approximately 85% pure as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The purified enzyme showed optimal activity at pH 7.6 in both Tris-HCL and potassium phosphate buffers. In its purest form, the enzyme was stable at 4 degrees-C but denatured upon freezing. The Km values for CoA, ATP and acetate were found to be 0.104 mM, 0.36 mM and 0.25 mM respectively; propionate and acrylate were also activated by the enzyme but not butyrate, isobutyrate or valerate. GTP, UTP, CTP and ADP could not replace ATP in the reaction, and cysteine or pantetheine failed to replace CoA. The cationic requirements were studied and of the divalent cations tested, only Mn2+ could significantly replace Mg2+ in the reaction; K+ and NH4+ stimulated enzyme activity but inhibited at high concentrations; Na+ was a poor activator, but did not inhibit at higher concentrations. The effect of a number of glucose and other metabolites on enzyme activity has been tested.     

The restriction endonucleases in Bacillus amyloliquefaciens N strain. Substrate specificities.

Two species of restriction endonuclease were isolated by gel filtration and DEAE-cellulose chromatography from a cell-free extract of Bacillus amyloliquefaciens (B. subtilits) N strain; a lower molecular weight endonuclease (endonuclease R.BamNI) and a higher molecular-weight one (endonuclease R.BamNx). Both of them required only Mg2+ for their activities. Endonuclease R.BamNx introduced a larger number of site-specific scissions in Excherchia coli phage lambda DNA that endonuclease R.BamNI did. Endonuclease R.BamNx cleaved Bacillus phage phi 105C DNA at the specific sites which are classified into two groups: one type of sites is modified by B. amyloliquefaciens H strain in vivo while the other is not affected. It was also active on DNA'S OF E. coli phage T7, lambdadvl, Simian virus 40 (SV40) and colicinogenic factor ColEI and was inactive on DNAs of Bacillus phages phi 29 and M2. Endonuclease R.BamHI isolated from H strain by Wilson and Young. This endonuclease was active on DNAs of phage lambda, lambdadvl and SV40, adn was inactive on DNAs of phages phi 105C, phi 29, M2 and T7, and ColEI DNA.     

Two new restriction endonucleases from Proteus vulgaris.

Two novel sequence-specific endonucleases have been isolated from Proteus vulgaris, ATCC 13315. PvuI recognizes the sequence: 5' C G A T decrease C G 3' 3' G C increase T A G C 5' and PvuII recognizes the sequence: 5' C A G decrease C T G 3' 3' G T C increase G A C 5' and cleave as indicated by the arrow (decrease). PvuI is an isoschizomer of XorII, RshI, and XniI. No enzyme with the specificity of PvuII has been described previously.     

Two unique restriction endonucleases from Neisseria lactamica.

Two new site-specific endonucleases, N1a III and N1a IV, have been isolated from Neisseria lactamica. N1a III recognizes the sequence, CATG, and cleaves 3' of the sequence to produce a four base 3' extension. N1a IV recognizes the sequence, GGNNCC, and cleaves between the two N's to produce blunt ended fragments.     

A new restriction endonuclease from Acetobacter pasteurianus.

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.