Mechanisms of Inactivation by High-Voltage Atmospheric Cold Plasma Differ for Escherichia coli and Staphylococcus aureus

Atmospheric cold plasma (ACP) is a promising nonthermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen-containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. Identification of ROS and an understanding of their role are important for advancing ACP applications for a range of complex microbiological issues. In this study, the inactivation efficacy of in-package high-voltage (80 kV [root mean square]) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed in which reactive species were found to either react primarily with the cell envelope or damage intracellular components. Escherichia coli was inactivated mainly by cell leakage and low-level DNA damage. Conversely, Staphylococcus aureus was mainly inactivated by intracellular damage, with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on the intracellular ROS levels generated.     

Homoserine kinase from Escherichia coli K12.

Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.     

Preparation of tritiated lipopolysaccharides from Escherichia coli K12

Tritiated lipopolysaccharide (LPS) from E. coli K12 was prepared by coupling [3H]ethanolamine to the LPS core residue ketodeoxyoctonate (KDO) via activation of its carboxylic function with N-hydroxysuccinimide or N-hydroxy-sulfosuccinimide. Specific activities of 1.5 microCi/mg and 9 microCi/mg were obtained, respectively. Experiments comparing the activity of native and derivatized LPS suggested that the preparation of the radiolabelled LPS did not alter the structural properties of E. coli K12 LPS. This probe will be useful for studying the interactions between LPS and proteins.     

A specific polyadenylase from Escherichia coli K12.

A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12. The enzyme was purified about 850 times to practically electrophoretic homogeneity. It was free of poly(A) polymerase activity, as well as of the well known E. coli RNAases I and II. It is stimulated by bivalent cations like Mg2+ and Mn2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease. The enzyme does not degrade any other ribohomopolymer or RNA.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

YbiV from Escherichia coli K12 is a HAD phosphatase

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized YbiV as a HAD phosphatase. The crystal structure of YbiV reveals a two-domain protein, one with the characteristic HAD hydrolase fold, the other an inserted alpha/beta fold. In an effort to understand the mechanism, we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3), which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate-binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate YbiV may be a sugar phosphatase, which is supported by biochemical assays that measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.     

Termination troubles in Escherichia coli K12

In this issue of Molecular Microbiology, Schaub and Hayes report that, compared with other enterobacteria, Escherichia coli K12 carries two mutations - one in the prfB gene encoding the release factor RF2, and the other in the rpsG gene encoding r-protein S7 - that together concur in compromising translation termination at the essential rpsG gene. As a consequence, the growth of E. coli K12 is very sensitive to a further mutation (rluD(-) ) that depresses RF2 activity, whereas the growth of its close relative, E. coli B, is not. We tentatively discuss how the K12-specific mutations in RF2 and S7 might have occurred and why inefficient translation termination at rpsG inhibits growth. The work of Schaub and Hayes illustrates the fact that, due probably to its long history in the laboratory, E. coli K12 has accumulated mutations that sometimes limit its value as a model for studying basic steps in prokaryotic gene expression.     

Proline excretion by Escherichia coli K12

Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion.     

Purification and characterization of adenylate cyclase from Escherichia coli K12

Adenylate cyclase of Escherichia coli K12 has been purified 17,000-fold to near homogeneity from a 5-fold overproducing strain. One major band of Mr = 92,000 and several minor bands are seen on sodium dodecyl sulfate-polyacrylamide electrophoresis of the purest fractions. Identification of the enzyme with the 92,000-Da protein is based on the correlation of this band with activity when highly purified enzyme is eluted from ADP-sepharose columns. The native enzyme has a molecular weight of 95,000 determined by gel filtration, showing that the enzyme is active as a monomer. The purest enzyme has a specific activity of 700 nmol min-1 mg-1, indicating a turnover number of about 100 min-1. Our data indicate that there are only about 15 molecules of the enzyme in wild type cells of E. coli. In crude extracts, over 80% of the activity is soluble after centrifugation at 100,000 x g, indicating the enzyme is soluble or, at most, loosely membrane bound. The enzyme is only moderately stable in crude extracts and becomes more unstable as purification proceeds. Activity is stabilized by ATP, or at -20 degrees C as an ammonium sulfate precipitate or in 50% glycerol. The enzyme has an absolute requirement for divalent cations. Maximum activity with Mg2+ is reached at 30 mM. Mn2+ is a good substitute; Co2+ activates well at low concentrations but becomes inhibitory at high concentrations; and Ca2+ is a potent inhibitor in the presence of Mg2+. The isoelectric point of the enzyme is 6.1, and its pH optimum is 8.5. The enzyme is inhibited by its substrate, with a Km of about 1 mM and a Ki of about 1.5 mM, and is noncompetitively inhibited by PPi, ADP, GTP, and a number of other compounds. The data suggest that dissociation of PPi from the first enzyme-product complex is the rate-limiting step in the reaction. Activation of the enzyme, inferred to occur in vivo, could be produced by a postulated regulatory effector which speeds release of PPi from the enzyme-product complex.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

Purification and characterization of catalase HPII from Escherichia coli K12

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90,000 and 92,000. Only a single 92,000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532,000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4-11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.     

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.     

Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12

Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor. When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme. In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism. Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction. The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide. We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.