The novel genes, cbbQ and cbbO, located downstream from the RubisCO genes of Pseudomonas hydrogenothermophila, affect the conformational states and activity of RubisCO

The cbbQ and cbbO genes are located downstream from the RubisCO genes (cbbLS) in the thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila. Recombinant RubisCO enzymes were purified from E. coli cells which were transformed with plasmids expressing cbbLS, cbbLSQ, cbbLSO, or cbbLSQO. Co-expression of cbbQ and/or cbbO with cbbLS made the maximal rates of carboxylation (Vmax) of the recombinant RubisCOs about two-fold higher than that of the enzyme derived from only cbbLS. The RubisCOs with high Vmax also had a high stability when undergoing ultrasonic treatment. The results of the circular dichroism spectra and the 8-anilino-1-naphthalenesulfonate binding assay indicated that these recombinant RubisCOs were conformationally different to each other.     

Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.     

Maximum activity of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the product of the rbcX gene

Filamentous cyanobacteria of the genus Anabaena contain a unique open reading frame, rbcX, which is juxtaposed and cotranscribed with the genes (rbcL and rbcS) encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Plasmid constructions containing the genes from Anabaena sp. strain CA were prepared, and expression studies in Escherichia coli indicated that the product of the rbcX gene mimicked the ability of chaperonin proteins to facilitate the proper folding of recombinant RubisCO proteins. The purified recombinant Anabaena sp. strain CA RubisCO, much like the RubisCO enzymes from other cyanobacteria, was shown not to undergo inhibition of activity during a time course experiment, and the properties of this chaperoned recombinant protein appear to be consistent with those of the enzyme isolated from the native organism.     

Rapid degradation of cucumber cotyledon lipoxygenase

The lipoxygenase activity from cucumber cotyledons grown with their embryonic axis was separated into two fractions having M(r)s of 90,000 and 96,000, respectively, by hydrophobic chromatography. However, from de-embryonated cucumber cotyledons, only one form of lipoxygenase having a M(r) of 90,000 was purified. The three lipoxygenases could not be distinguished from each other either immunologically or by their enzymatic properties. Furthermore, peptide maps of the 90,000 and 96,000-lipoxygenases were identical. In a crude homogenate of cucumber cotyledons, the 96,000-lipoxygenase was rapidly degraded to the 90,000-form. Thus, it was inferred that the 90,000-lipoxygenase was probably the 96,000-form which had lost a peptide fragment of 6,000. It is suggested that there is a specific proteolytic activity for the degradation of 96,000-lipoxygenase. Estimation of changes in the proteolytic activity during seedling growth suggests that the activity at least partly contributes to the rapid in vivo degradation of cucumber cotyledon lipoxygenase.     

Cloning and sequencing of a form II ribulose-1,5-biphosphate carboxylase/oxygenase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila

The bacterial symbiont of the hydrothermal vent tubeworm fixes carbon via the Calvin-Benson cycle and has been shown previously to express a form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene cbbM, which encodes this enzyme, has been cloned and sequenced. The gene has the highest identity with the cbbM gene from Rhodospirillum rubrum, and analysis of the inferred amino acid sequence reveals that all active-site residues are conserved. This is the first form II RubisCO cloned and sequenced from a chemoautotrophic symbiont and from a deep-sea organism.     

Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost

A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2, 6-O-dimethyl)-beta-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75 degreesC) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GlcNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc)3. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl2, and (GlcNAc)2 inhibited the activities of all three enzymes, while MnCl2 and CaCl2 slightly activated all of the enzymes.     

Co-existence of leukoderma with features of Dowling-Degos disease: reticulate acropigmentation of Kitamura spectrum in five unrelated patients

Flavobacterium rigense strain PR2, a broad-spectrum mercury-resistant bacterium abundantly present in soil exhibited multiple metal resistance properties. Mercury resistance was due to the sequential action of two mercury-detoxicating enzymes, organomercurial lyase and mercuric reductase. The levels of these enzyme activities were determined using different mercury compounds as inducers and substrates. Mercuric reductase was partially purified from the bacterium and the physicochemical properties of the enzyme were studied. The effect of several enzyme inhibitors and heavy metal ions on the enzyme activity was also studied.     

Recombinations of subunits of Phaseolus vulgaris isolectins

Phaseolus vulgaris phytohemagglutinin is formed in vivo by the combination of erythrocyte (E)-reactive and lymphocyte (L)-reactive subunits into five tetrameric isolectins:L4,L3E1, L2E2, L1E3, and E4. Evidence for phytohemagglutinin subunit structure is obtained by in vitro dissociation of native isolectins in 6 M guanidine HCl followed by removal of dissociating agents to allow subunit recombination. Dissociation and recombination of L4 yielded a single protein, electrophoretically indistinguishable from the native L4. Similar treatment of E4 also yielded a single protein indistinguishable from native E4. Treatment of L3E1, L2E2, L1E3, or a mixture of L4 and E4, yielded five distinct proteins electrophoretically similar to all five native phytohemagglutinin isolectins. Milligram quantities of all five recombinant isolectins were prepared either from L2E2 or a mixture of L4 and L1E3 proportioned to yield equimolar quantitives of the two subunits on dissociation. The recombinant isolectins were purified by affinity and SP-Sephadex ion exchange chromatography. Electrophoretic and chromatographic properties and the erythroagglutinating and mitogenic activities of recombinant isolectins were essentially identical with the native isolectins. The inclusion of 125I-labeled L4 in the dissociation results in a distribution of 125I-labeled L subunit among the purified recombinant isolectins proportional to their proposed subunit structures.     

Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.     

Purification and characterization of beta-N-acetylglucosaminidase from Alteromonas sp. strain O-7

beta-N-Acetylglucosaminidase (EC 3.2.1.30) was purified from the outer membrane of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (GlcNAcase A) was purified by successive column chromatographies. The purified enzyme was found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass and pI of GlcNAcase A were 92kDa and 4.9, respectively. The optimum pH and temperature were 6.0-7.0 and 45 degrees C, respectively. GlcNAcase A was stable up to 40 degrees C at pH 7.0, and hydrolyzed N-acetylchitooligosaccharides from dimer to hexamer. The amino-terminal 16 amino acid residues of GlcNAcase A were sequenced.     

Expression of glnB and a glnB-like gene (glnK) in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant of Rhodobacter sphaeroides

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.     

Planned missing data designs in psychological research.

The authors describe 2 efficiency (planned missing data) designs for measurement: the 3-form design and the 2-method measurement design. The 3-form design, a kind of matrix sampling, allows researchers to leverage limited resources to collect data for 33% more survey questions than can be answered by any 1 respondent. Power tables for estimating correlation effects illustrate the benefit of this design. The 2-method measurement design involves a relatively cheap, less valid measure of a construct and an expensive, more valid measure of the same construct. The cost effectiveness of this design stems from the fact that few cases have both measures, and many cases have just the cheap measure. With 3 brief simulations involving structural equation models, the authors show that compared with the same-cost complete cases design, a 2-method measurement design yields lower standard errors and a higher effective sample size for testing important study parameters. With a large cost differential between cheap and expensive measures and small effect sizes, the benefits of the design can be enormous. Strategies for using these 2 designs are suggested. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inactivation of Listeria monocytogenes in milk by pulsed electric field

Pasteurized whole, 2%, and skim milk were inoculated with Listeria monocytogenes Scott A and treated with high-voltage pulsed electric field (PEF). The effects of milk composition (fat content) and PEF parameters (electric field strength, treatment time, and treatment temperature) on the inactivation of the bacterium were studied. No significant differences were observed in the inactivation of L. monocytogenes Scott A in three types of milk by PEF treatment. With treatment at 25 degrees C, 1- to 3-log reductions of L. monocytogenes were observed. PEF lethal effect was a function of field strength and treatment time. Higher field strength or longer treatment time resulted in a greater reduction of viable cells. A 4-log reduction of the bacterium was obtained by increasing the treatment temperature to 50 degrees C. Results indicate that the use of a high-voltage PEF is a promising technology for inactivation of foodborne pathogens.     

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.     

alpha-L-rhamnosidase of Sphingomonas sp. R1 producing an unusual exopolysaccharide of sphingan

A soil bacterium with alpha-L-rhamnosidase was isolated from a cumulative mixed culture containing a polysaccharide of gellan as a carbon source and identified to be Sphingomonas paucimobilis, known as a potent producer of gellan. The isolate (designated Sphingomonas sp. R1) produced an unusual exopolysaccharide of sphingan (denoted HWR1) distinct from gellan. The rhamnose in gellan was replaced with mannose in HWR1. The bacterium had a peculiar cell surface covered with many complicated plaits. alpha-L-Rhamnosidase purified from Sphingomonas sp. R1 grown in the presence of naringin was a monomer with a molecular mass of 110 kDa and most active at pH 8.0 and 50 degrees C. The enzyme required divalent metal ions for the activity and released L-rhamnose from various rhamnosyl glycosides.     

L-serine and L-threonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups

L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-94]. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids. L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM. L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate. L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P. asaccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.     

2-Ketocyclohexanecarboxyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palustris

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.     

D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.     

Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement

Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.