The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

Molecular masses of cAMP-binding proteins in yeasts

Abstract The cAMP-binding proteins of different yeasts were photoaffinity labeled using 8- N ₃-[³²P]cAMP, and the M _r values of the labeled proteins estimated by SDS-polyacrylamide gel electrophoresis. The M _r values of the cAMP-binding proteins may be grouped into two size classes: (A) M _r of about 50 000 represented by Saccharomyces cerevisiae and S. uvarum , and (B) M _r of about 60 000 represented by Kluyveromyces fragilis, K. lactis, K. marxianus, S. globosus and S. rouxii .     

Derivatives of Rhodobacter capsulatus strain AD2 cured of their endogenous plasmid are unable to utilize nitrate

Abstract Derivatives of Rhodobacter capsulatus AD2 unable to grow with nitrate as sole N source were isolated after conjugation with a plasmid containing a cloned 3.4 kb Hin dIII fragment from the endogenous plasmid of this strain. These derivatives lacked the M _r 74 × 10⁶ plasmid found in the wild-type, and failed to revert to growth on nitrate. Cultures of the plasmid-cured strains also lacked dissimilatory nitrate reductase activity, suggesting that genes required for both assimilatory and dissimilatory nitrate reduction are located on the endogenous plasmid.     

Purification and some properties of the methyl-CoM reductase of Methanothrix soehngenii

Abstract The methyl-CoM reductase from Methanothrix soehngenii was purified 18-fold to apparent homogeneity with 50% recovery in three steps. The native molecular mass of the enzyme estimated by gel-fitration was 280 kDa. SDS-polyacrylamide gel electrophoresis revealed three protein bands corresponding to M _r 63 900, 41 700 and 30 400 Da. The methyl-coenzyme M reductase constitutes up to 10% of the soluble cell protein. The enzyme has K _m apparent values of 23 μM and 2 mM for N -7-mercaptoheptanoylthreonine phosphate (HS- HTP = component B ) and methyl-coenzyme M (CH₃CoM) respectively. At the optimum pH of 7.0 60 nmol of methane were formed per min per mg protein.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

Peptidase N of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa possesses a peptidase N activity analogous to those described in Escherichia coli and Salmonella typhimurium . This activity resides in a protein with an M _r value of 85 000. Part of this peptidase activity appears to be associated with the cytoplasmic membrane. The K _M value for this peptidase bound to the cytoplasmic membrane is in the range of 0.5 mM.     

Purification of aspartate transcarbamoylase from Pseudomonas syringae

Abstract The aspartate transcarbamoylase (ATCase) from Pseudomonas syringae has been purified. The purified enzyme was shown by SDS-PAGE to give two bands. Unambiguous results from N-terminal sequencing suggested that each band represented a homogeneous polypeptide. The M _r (relative molecular mass) of the polypeptides was estimated to be 47 kDa and 34 kDa. The M _r of the holoenzyme determined by gel filtration and electrophoretic migration in polyacrylamide gradient gels under non-denaturing conditions was estimated at approximately 490 kDa. These findings suggest a subunit structure different from any previously described for a bacterial ATCase.     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Intracellular precursors of endo-β-1,4-glucanase in Trichoderma reesei

Abstract Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting was employed to detect intracellular precursors of endo-β-1,4-glucanases (EGs) in Trichoderma reesei QM9414 under conditions of de novo induction by sophorose and de novo carbon catabolite derepression by lactose. Secretion of EGs was always preceded by intracellular accumulation of lower M _r precursors, which became processed to larger M _r forms immediately prior to their extracellular appearance. Treatment of the larger M _r forms with α-mannosidase converted them to forms with the same M _r as the smaller forms, whereas Endo H treatment was without effect. These results are consistent with a requirement of O -linked glycosylation for secretion of EGs by T. reesei .     

The involvement of thioredoxin and thioredoxin reductase in the dimethyl sulphoxide reductase system of Saccharomyces cerevisiae

Abstract Dimethyl sulphoxide (DMSO) reductase activity in crude extracts of Saccharomyces cerevisiae NCYC240 was stimulated by addition of thioredoxin, but not by addition of thioredoxin reductase. The activity was partially purified. DEAE-cellulose could be used to separate thioredoxin and its reductase (which bound to the column) from the terminal DMSO-reductase protein (which failed to bind). The highly unstable purified terminal reductase so obtained required both thioredoxin and thioredoxin reductase to reconstitute activity with either dithiothreitol (DTT) or NADPH as electron donor. Partially purified terminal reductase had an M _r of about 15000.     

Evidence for the location of OprM in the Pseudomonas aeruginosa outer membrane

Abstract OprM with a M _r of 49 K is associated with the multidrug resistance of Pseudomonas aeruginosa . Detergent fractionation of bacterial cells has demonstrated that OprM is located in the outer membrane from which it sediments with the other major outer membrane proteins. In this study we have determined the location of OprM as the P. aeruginosa outer membrane. Western immunoblots of cell fractions, obtained by sucrose density gradient centrifugation of whole cell lysates, were probed with an OprM-specific murine polyclonal antiserum.     

Structural and antigenic differences between two types of meningococcal pili

Abstract Strains of meningococci, which were shown to be pilated by electron microscopy, could be divided into two groups on the basis of antigenicity and subunit M _r. Strains from group 1 which reacted with monoclonal antibodies directed against gonococcal pili, had pili with subunit M _r similar to that of gonococci which could be detected by radioimmune precipitation or electroblotting. Strains from group 2 failed to react with the monoclonal antibodies and had pili with lower subunit M _r which could only be detected by radioimmune precipitation using polyclonal antipilus antiserum and not by electroblotting.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Properties of a soluble thiosulfate sulfur transferase (rhodanese) of the marine methanogen Methanosarcina frisia

Abstract: Cell-free extracts of Methanosarcina frisia contain high thiosulfate sulfur transferase (TST) (rhodanese), slight thiosulfate reductase but no thiosulfate: acceptor oxidoreductase activity. Neither adenylylsulfate reductase nor sulfite: acceptor oxidoreductase activity could be detected. TST is an acidic protein with an M _r of 25 000 and was enriched by ion-exchange chromatography and gel filtration. The enzyme has a temperature optimum at 60°C and a pH optimum at pH 11. The K _m values for thiosulfate and cyanide are 0.53 mM and 1.57 mM, respectively. Low concentrations of cysteine, glutathione, dithioerythritol, and dihydrolipoate increase the activity of the enzyme while unphysiological concentrations of these effectors cause a decrease. Sulfite and N -bromosuccinimide inhibit the energy activity extremely.     

Intergeneric protoplast fusion in lactic acid bacteria

Abstract Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of M _r 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The M _r 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the M _r 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the M _r 130 000 and 28 000 proteins are structurally different.     

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Thioredoxin and related proteins in procaryotes

Abstract Thioredoxin is a small ( M _r 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S₂) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)₂] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)₂ serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)₂ is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S₂ by light and ferrodoxin; Trx-(SH)₂ is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control.
Thioredoxin-negative mutants ( trxA ) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.