Whole-cell Suspensions Research Articles

The quantum requirement for H2 production by anoxygenic phototrophic bacteria

Cultures from three groups of phototrophic bacteria (green sulphur bacteria, purple non-sulphur bacteria and purple sulphur bacteria) were investigated in respect of the quantum requirement for H2 production (QH2). Rates of H2 formation were determined by means of a Clark-type H2 electrode with dense suspensions of whole cells and malate, acetate or sulphide as electron donor. At low light intensities (0–3 W·m−2 of monochromatic light) the minimum quantum requirement was 8.6 quanta per H2 with Chlorobium vibrioforme, 7.5 with Rhodospirillum rubrum, and 23.2 with Ectothiorhodospira shaposhnikovii. The physiological efficiency, defined as the measured QH2 compared to the theoretical value calculated from the energy requirement of the physiological processes involved, was 94%, 88%, or 28%, respectively. With increasing light intensities the quantum requirement also increased. Various hydrogenase inhibitors either inhibited both H2 uptake and production (Cu2+, NO), or affected neither of these activities (CO, C2H2, N2O, ethylenedinitrilotetraacetate). An uptake hydrogenase-deficient Hup−-mutant of R. rubrum had higher rates of net H2 production but a similar quantum requirement. The energetic efficiency of H2 production by various biological and artificial systems is discussed.

Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris.

The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium-contaminated waters and waste streams.

Expression of an inaccessible P1.7 subtype epitope on meningococcal class 1 proteins

Dot-blot analysis of whole-cell suspensions of meningococci showed that 81% of B:15:P1.16 strains from patients reacted with a monoclonal antibody (MAb) against subtype P1.7. The remaining strains, which did not react on dot-blots or in ELISA, demonstrated the P1.7 subtype epitope on immunoblots after denaturation of the cells with sodium dodecyl sulphate. The monomeric class 1 proteins of the two P1.16 subtype variants had slightly different mol. wts, but bound the P1.7 antibody equally well. These results were explained by a deletion of three codons in the gene encoding the first variable region of the P1.16 class 1 protein. The deletion accounted for the non-exposure of the P1.7 epitope on native cells. Other patient strains, with subtypes P1.3, P1.9 or without any known subtype, also showed a binding site for the P1.7 MAb, which became available only after denaturation. Demonstration of inaccessible epitopes may have consequences for subtype designations and vaccine development.

Transformation of vitamin D3 to 1 alpha,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains.

To enzymatically synthesize active metabolites of vitamin D3, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D3 to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] via 25-hydroxyvitamin D3 [25(OH)D3]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D3 to 25(OH)D3 and 1 alpha,25(OH)2D3 in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D3/l culture and 0.17 mg 1 alpha,25(OH)2D3/l culture. A related compound, vitamin D2, could be also converted to 25-hydroxyvitamin D2 and 1 alpha,25-dihydroxyvitamin D2 using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D3 in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D3 and at C-1 of 25(OH)D3 originates from cytochrome P-450.

Transformation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains

To enzymatically synthesize vitamin D derivatives, we screened about 300 Streptomyces sp. strains. Streptomyces sclerotialus FERM BP-1370 and Streptomyces roseoporus FERM BP-1574 were found to have the ability to convert 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, respectively, to 1 alpha, 25-dihydroxyvitamin D3. The average rates of 1 alpha hydroxylation of 25-hydroxyvitamin D3 were 6.9 micrograms liter-1 min-1 with FERM BP-1370 and 7.0 micrograms liter-1 min-1 with FERM BP-1574. The specific cytochrome P-450 inhibitors carbon monoxide, SKF-525-A, and metyrapone inhibited the hydroxylation of 1 alpha- and 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by FERM BP-1370 and FERM BP-1574. The cytochromes P-450 of these strains were detected by reduced CO difference spectra in the whole-cell suspensions. The appearance of cytochrome P-450 suggests that the cytochromes P-450 of FERM BP-1370 and FERM BP-1574 carry out the hydroxylation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3.

Identification of structuralnifgenes in an iron-oxidising moderate thermophile

Abstract The presence of nucleotides which are homologous to the nif HDK genes of Thiobacillus ferrooxidans was demonstrated in total DNA preparations of NMW-6, an iron-oxidising bacterium of the Thiobacillus -like group of moderate thermophiles. Acetylene reduction assays did not indicate active nitrogenase in suspensions of whole cells, despite varying the headspace gas composition, cell density, temperature, carbon and nitrogen content of the assay medium, and the temperature and nutritional conditions used to raise the biomass. Growth and iron oxidation by NMW-6 were observed in medium containing no added nitrogen at 37°C. Analysis of freshly prepared medium by HPLC indicated the presence of sufficient NH 4 , absorbed from the atmosphere, to support growth.

Acetate catabolism in the dissimilatory iron-reducing isolate GS-15

Acetate-grown GS-15 whole-cell suspensions were disrupted with detergent and assayed for enzymes associated with acetate catabolism. Carbon monoxide dehydrogenase and formate dehydrogenase were not observed in GS-15. Catabolic levels of acetokinase and phosphotransacetylase were observed. Enzyme activities of the citric acid cycle, i.e., isocitrate dehydrogenase, 2-oxoglutarate sythase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were observed.

Virulence and lienotoxicity of Bordetella bronchiseptica in mice

Whole-cell suspensions (WCSs) and cell-free sonicated extracts (SEs) of seven Bordetella bronchiseptica strains were studied for lethality and lienotoxicity in mice. Lethality was assessed after intravenous and intracerebral inoculation, and lienotoxicity by splenic atrophy after intravenous inoculation. The strains represented phase I isolates with or without cytotoxin production, their phase III subcultures and a phase IV variant. The lethality and lienotoxicity of the SEs were in close positive correlation with cytotoxin production. The WCSs of all phase I strains were lethal, irrespective of their cytotoxin- and lienotoxin-producing ability. The only difference was that cytotoxic phase I strains caused splenic atrophy while the noncytotoxic phase I strain induced splenic hypertrophy in the surviving mice. The WCSs of phase III and IV variants were non-lethal and caused splenic hypertrophy even though all but one of them showed some cyto- and lienotoxic activity when their SEs were tested. The results indicate that B. bronchiseptica possesses two different mouse lethal factors: one seems to be identical with the cytotoxin, the other is associated with cell integrity and viability and, presumably, propagation in vivo. It also follows from the results that only the SEs are suitable for accurate determination of the lienotoxin-producing ability of B. bronchiseptica.

Characterization of Egg Yolk Antibodies for Detection and Quantification of Selenomonas ruminantium by Using an Enzyme-Linked Immunosorbent Assay

The specificity of polyclonal antibodies prepared against strains of Selenomonas ruminantium, the effect of assay conditions, and quantification of individual strains in mixed-cell suspensions of selenomonad strains were examined in this study. Whole-cell suspensions were prepared with pure cultures of S. ruminantium PC18, HD(4), GA192, and D. Each cell suspension was injected into a Leghorn laying hen, and polyclonal antibodies were harvested from eggs laid in week 3 or 7 following initial immunization. Antibodies made to the S. ruminantium strains readily discerned the homologous strain from the heterologous strains. Cross-reactivity among antibodies and the heterologous S. ruminantium strains ranged from 5 to 26%. Among non-S. ruminantium species, cross-reactivity of S. ruminantium antibodies was greatest with Selenomonas sputigena (3 to 34%) and Succinivibrio dextrinosolvens (0 to 37%). Antibodies made to strains GA192 and D were used to quantify a mixture of the two strains. Both antibodies responded to graded concentrations of the homologous antigen in the biculture mixtures in accord with the change in the direct cell counts for each strain (strain D, R = 0.92; strain GA192, R = 0.90). This enzyme-linked immunosorbent assay enabled concurrent and accurate quantification of two strains of S. ruminantium subsp. ruminantium in a mixed-cell suspension with a precision of much less than 1 order of magnitude.

Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures

An assay was characterized that facilitated per-cell estimation of cytochrome P-450-dependent monooxygenase activities in whole-cell suspensions and cultures of murine epidermal keratinocytes (MEKs). 7-Ethoxycoumarin O-deethylase (7-ECD), 7-ethoxyresorufin O-deethylase (7-ERD), and 7-pentoxyresorufin O-deethylase (7-PRD) activities were monitored by fluorescent detection of their products. MEKs were made permeable by a freeze-thaw cycle, and xenobiotic metabolism occurred in situ. Analyses of cultured MEKs were made with the cells attached to the culture dishes. Product formation was proportional with MEK cell number and assay time and was dependent upon a NADPH-generating system. The three monooxygenase activities were inhibited to various degrees, in a dose-dependent manner, by the P-450 inhibitors α-naphthoflavone and metyrapone. The number of MEKs obtained from a single mouse was sufficient for multiple analyses. The assay was also used to determine monooxygenase activities in whole-cell suspensions of rat hepatocytes. Consitutive per hepatocyte 7-ECD, 7-PRD, and 7-ERD activities were 357-, 96-, and 1926-fold greater, respectively, than the activities measured in suspensions of dorsal MEKs.

Deoxyribonucleic Acid Ploidy and Cell Cycle Analysis of Colorectal Carcinoma by Flow Cytometry: A Prospective Study of 137 Cases Using Fresh Whole Cell Suspensions.

Several retrospective studies suggest that abnormal deoxyribonucleic acid (DNA) content in colorectal carcinoma correlates with adverse clinical outcome. Many of these studies have used naked nuclei retrieved from formalin-fixed, paraffin-embedded tissues for flow cytometry. The purpose of this study was to prospectively analyze 137 colorectal carcinomas using fresh whole-cell suspensions for flow cytometry and to determine whether abnormal DNA content (DNA aneuploidy or tumors with high proliferative activity) correlates with Dukes' stage, histologic grade, lymphocytic infiltration of the tumor, tumor fibrosis, extramural venous spread, or tumor size. Cell suspensions for flow cytometry were prepared by enzyme disaggregation with collagenase XI, DNase, and trypsin. Satisfactory DNA histograms were obtained from 132 of the 137 samples. The mean coefficients of variance for the G1/G0 of the external 2C control, internal 2C populations, and aneuploid populations were 2.5, 3.5, and 3.5, respectively. The mean percentage of viable cells was 97%. Of 132 cases, 102 (77%) demonstrated abnormal DNA histograms, of which 77 (58%) showed DNA aneuploidy. Abnormal DNA histograms of DNA aneuploidy did not correlate with Dukes' stage. Tumors of higher histologic grade were more likely to demonstrate DNA aneuploidy, however, these differences did not reach statistical significance. The authors conclude that (1) satisfactory DNA histograms can be obtained with the use of a fresh, whole-cell technique; (2) abnormal DNA histograms did not statistically correlate with standard clinical, grading, or staging parameters; and (3) carcinomas of high histologic grade showed an increased proportion of aneuploid DNA histograms, but this trend did not reach statistical significance.

Use of extracts versus whole-cell bacterial suspensions in the identification of Staphylococcus aureus beta-lactamase variants

We previously have shown that extracts of S. aureus isolates which produce the recognized serotypes of staphylococcal beta-lactamase (A, B, C, D) differ in the rates at which they hydrolyze selected cephalosporins, exhibiting substrate profiles which are distinctive for each serotype. In an effort to simplify the methods employed in identifying the different staphylococcal beta-lactamases, we evaluated whether distinctive substrate profiles could be obtained by using whole-cell suspensions of 115 beta-lactamase-producing isolates of S. aureus. Compared with extracts from the same strains, the whole-cell bacterial suspensions not only were simpler to prepare but enabled beta-lactamase typing of a higher proportion of the evaluated strains (86 versus 97%, respectively). Furthermore, the use of whole-cell bacterial suspensions enabled the simultaneous quantitation of the beta-lactamase activity exhibited by each strain. Additionally, by comparing the quantitative activity of beta-lactamase-induced and -uninduced preparations of the same strain, induction ratios (i.e., induced/uninduced activity) could be derived, yielding information regarding the regulation of beta-lactamase production by each strain. We believe that the utilization of whole-cell methods, such as those employed in this study, will facilitate the investigation of qualitative and quantitative differences in beta-lactamase production among clinical and reference isolates of S. aureus.

Electron transport in Heliobacterium chlorum wholecells studied by electroluminescence and absorbance difference spectroscopy

An electric field pulse induced luminescence signal was observed in suspensions of whole cells of the photosynthetic green bacterium Heliobacterium chlorum . This signal is associated with electron transport in the reaction center. The dependence of the electroluminescence on the delay between the flashand the pulse and the absorbance kinetics at 800 and 553 nm revealed the following properties of electron transport in whole cells: (1) most of P-798 + was rereduced by cytochrome c -553 in two phases with half-times of 0.11 and 0.7 ms and equalamplitudes (this is considerably faster than the timesreported for membrane fragments); (2) if reduced cytochrome c -553 is not available, P-798 + reacts back with a reduced acceptor in 20 ms; (3) oxidized cytochrome c -553 is rereduced in about 20 ms, presumably by an external donor, because it does not take place after resuspension of the cells in Tris buffer. In membrane fragments, spheroplasts and cells stored at −20°C, cytochrome oxidation is inhibited and no electroluminescence can be observed.

Direct evidence for the oxidation of palmitic acid by host-grown Mycobacterium leprae

Oxidation of palmitic acid by whole-cell suspensions of Mycobacterium leprae free from host tissues was investigated using manometric techniques. After a lag period of about 6–8 h, M. leprae suspensions catalysed an active oxidation of palmitic acid, and the oxidative process (oxygen uptake) was quite sensitive to rotenone, atabrine, amytal, antimycin A and cyanide. The spectrophotometric observations indicated that the M. leprae cytochrome system, under anaerobic conditions, was reduced in the presence of palmitic acid which was completely oxidized by oxygen. These data provide direct evidence that M. leprae cells are capable of oxidizing palmitic acid, and that oxidation is mediated by the electron transport system using oxygen as the terminal electron acceptor.

Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): direct evidence by cDNA transfection analysis.

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein expressed by most acute lymphoblastic leukemias, certain other lymphoid malignancies with an immature phenotype, and normal lymphoid progenitors. A computer search against the most recent GenBank release (no. 56) indicates that human CALLA cDNA encodes a protein nearly identical to the rat and rabbit neutral endopeptidase 24.11 ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopeptidase, which has been shown to inactivate a variety of peptide hormones including enkephalin, chemotactic peptide, substance P, neurotensin, oxytocin, bradykinin, and angiotensins I and II, had not been identified in lymphoid cells. To determine whether CALLA cDNA derived from human acute lymphoblastic leukemia cells (Nalm-6 cell line) encodes functional neutral endopeptidase activity, we generated CALLA+ stable transfectants in the CALLA- murine myeloma cell line J558 and analyzed them for enzymatic activity in a fluorometric assay based upon cleavage of the substrate glutaryl-Ala-Ala-Phe 4-methoxy-2-naphthylamide at the Ala-Phe bond. Total lysates as well as whole-cell suspensions of the Nalm-6 line and of the CALLA+ transfectants, but not of the CALLA- J558 cells, possessed neutral endopeptidase activity. This enzymatic activity was associated with the cellular membrane fraction and was abrogated by the specific neutral endopeptidase inhibitor phosphoramidon. The unequivocal identification of CALLA as a functional neutral endopeptidase provides insight into its potential role in both normal and malignant lymphoid function.

Role of the 92.5-kilodalton outer membrane protein of Campylobacter jejuni in serological reactions.

Outer membrane proteins from 12 strains of Campylobacter jejuni were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against Formalin-treated cell suspensions of three of the strains. The 92.5-kilodalton protein was confirmed to be a minor outer membrane protein and exhibited serological heterogeneity. Serological specificities of the proteins, however, were found not to correlate with serotypic specificities of the strains, as demonstrated by slide agglutination tests with whole-cell suspensions. The results suggested that the 92.5-kilodalton proteins were not likely to be participating antigenic determinants in the immunological reactions that give rise to agglutination of live-cell suspensions in the slide test.

Utilization and metabolism of NAD by Haemophilus parainfluenzae.

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.

Role of Photosynthetic Reactions in the Activity of Carbonic Anhydrase in Synechococcus sp. (UTEX 2380) in the Light

The role of the photosystems in the exchange of (18)O between species of inorganic carbon and water was studied in suspensions of the cyanobacterium Synechococcus sp. (UTEX 2380) using membrane-inlet mass spectrometry. This (18)O exchange is caused by the hydration-dehydration cycle of CO(2) and is catalyzed by carbonic anhydrase. We observed the complex (18)O exchange kinetics including dark-light-dark transients in suspensions of whole cells and found these to be identical to the (18)O exchange kinetics of physiologically fully active spheroplast preparations. There was no enhancement effect of inorganic nitrogen on inorganic carbon accumulation. Membrane preparations exhibited no uptake of inorganic carbon and very little carbonic anhydrase activity, although these membranes were photosynthetically fully competent. DCMU, the inhibitor of photosystem II, eliminated almost entirely the (18)O exchange activity of whole cells in the light. But this effect of DCMU could be reversed by addition of the electron donor couple 3,6-diaminodurene/ascorbate, suggesting the involvement of photosystem I in the events leading to (18)O exchange. Iodoacetamide, an inhibitor of CO(2) fixation, enhanced the (18)O exchange in whole cell suspensions and inhibited neither the uptake of inorganic carbon nor the dehydration of bicarbonate in the light. The proton carrier carbonylcyanide m-chlorophenylhydrazone and the inhibitors diethylstilbestrol and N,N' -dicyclohexyl carbodiimide affecting the membrane potential, totally abolished (18)O exchange in the light. From (18) O-labeled inorganic carbon experiments we conclude that one of the roles of photosystem I is to provide the active uptake of inorganic carbon into the cells, where carbonic anhydrase catalyzes the interconversion between CO(2) and HCO(3) (-) resulting in the (18)O exchange from inorganic carbon to water.

Photomechanical transduction in Amoeba proteus: An action spectrum

An action spectrum for the light-induced stop reaction of amoeboid movement in Amoeba proteus has been determined. The amoebae show a sensitivity in the blue with a peak between 440 and 470 nm. This agrees with the older qualitative observations (M. R. Harrington and E. Leaming, Am. J. Physiol., 3 (1900) 9 - 16; S. O. Mast, J. Exp. Zool., 9 (1910) 265 - 277) but no positive phototaxis was found (A. A. Schaeffer, Biol. Bull., 32 (1917) 45 - 74). Absorption spectra of suspensions of whole cells have an absorption band with a similar spectral peak. The response follows the Bunsen-Roscoe law and the sensitivity appears to be greater at low oxygen tension. The photomechanical link in this response, i.e. conversion of a light stimulus to a change in motion, suggests a primitive mechanism of avoidance that may have evolutionary implications (R. M. Eakin, Evol. Biol., 2 (1968) 194 - 242; L. von Salvini-Plawen and E. Mayr, Evol. Biol., 10 (1977) 207 - 263).