Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms

The cross-feeding of microbial products derived from 14C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [14C]bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of 14C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of 14C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized 14C-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of 14C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Membranes of bacteria and mechanism of action of the antibiotic gramicidin S]

The cyclopeptide antibiotic gramicidin S taken at a concentration of 100--200 mkg/mg membrane protein rapidly increases the permeability of M. lysodeikticus protoplast membranes for substrates of respiratory chain and exogenous cytochromes c. Prolonged incubation of gramicidin S with protoplasts results in their lysis which is more fast at low temperatures. In contrast to natural gramicidin, a derivative of gramicidin S with acetylated amino groups does not inhibit either the micrococcus membrane dehydrogenase or the whole of respiratory chain and does not affect the osmotic barrier of protoplasts. Aliphatic diamines (at concentrations up to 0.1 M) and Ca2+ ions (10(-2) M) do not affect the functioning of the respiratory chain in isolated micrococcus membranes. Another derivative of the antibiotic with an increased distance of loaded amino groups from the cyclopeptide framework (diglycyl gramicidin S) affects the membrane in a way similar to that of natural gramicidin. Washing of gramicidin-treated membranes with NaCl enhances the inhibitory effect of the antibiotic on membrane enzymes. The data obtained suggest that in addition to ionic interactions some hydrophobic interactions also occur during gramicidin S binding to the bacterial membrane, probably at the expense of a hydrophobic peptide ring. It is assumed that gramicidin S, similar to Ca2+ and some other membranotropic agents provides for phase separation of negatively charged phospholipids from other groups of phospholipids, manifesting itself in an appearance of "frozen" sites on the membrane which destroys its barrier properties. This is due to the formation of ionic bonds of negatively charged phospholipids. Simultaneously, unlike Ca2+, gramicidin S, when interacting with membrane proteins, prevents their redistribution in more liquid parts of the membrane, which results in a situation when the respiratory enzymes become surrounded by alkyl chains with restricted motion.     

Possibility of reversing the action of the membranotropic antibiotic, gramicidin S, on bacteria

The study on the possibility of eliminating gramicidin S from the bacterial cells which had adsorbed it showed that a part of the labeled antibiotic bound by the bacteria may be washed out with buffer or salines. When the cells which had adsorbed gramicidin S were treated with lecithin emulsion, a significant part of the bound antibiotic was transferred to the lecithin liposomes. This turned the gramicidin S effect to the cells: significant but not complete reduction of the membrane barrier properties and dehydrogenase reactivation. Elimination of gramicidin S also reduced the colony forming capacity in a part of the cells.     

Conformation of gramicidin S

A molecular conformation of Gramicidin S was derived on the basis of conformational calculations taking into account the available experimental data. The conformation is characterized by a dyad axis which relates the two chemically equivalent halves of the molecule and contains four hydrogen bonds; other structural features agree with experimental results. X Ray Crystallographic evidences for the relative position of the Ornithine residues is also reported which supports an important feature of the structure of Gramicidin S.     

Dependence of in vivo inactivation kinetics of gramicidin S synthetase on cell physiology

Gramicidin S synthetase, the enzyme complex catalyzing the biosynthesis of the antibiotic gramicidin S in Bacillus brevis, is subject to O(2)-dependent in vivo inactivation during exponential aerobic growth after reaching a peak in specific activity. The five amino acid substrates of the synthetase are capable of stabilizing its activity to varying degrees in whole cells shaken aerobically. Depending on the time of cell harvesting before, during, or after the peak in intracellular gramicidin S synthetase specific activity, the enzyme has a long, medium, or short half-life, respectively. The kinetic profiles of gramicidin S synthetase in B. brevis cells indicate that both the kinetics of synthetase loss and the degree of its amino-acid-mediated stabilization are a strong function of the cells' physiological development.     

Change in lipid-protein interactions in the membranes of bacteria exposed to gramicidin S]

Effect of cyclopeptide antibiotic gramicidin S on some enzymes and physical state of isolated Micrococcus lysodeikticus membranes is studied. Malate and lactate dehydrogenases were monotonously inhibited under the increase of gramicidin S concentration, while the activity of NADH-dehydrogenase firstly decreased and then reversed to the initial level under further increase of gramicidin S concentration. The oxygen uptake under oxidation of NADH and malate with membranes almost completely inhibited by the antibiotic, while the activity of ascorbate-TMPD-oxidase activity slightly inhibited by the same concentration of gramicidin. The addition of Triton X-100 completely eliminated the inhibitory effect of gramicidin on malate dehydrogenase. The introduction into the membrane of spine probes (2,2,6,6-tetramethyl-4-palmitoylamidopiperidine-1-oxile and 2(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxyazolidinyloxile) revealed that gramicidin caused the condensation of membrane lipid component. It is suggested that ionic interaction of gramicidin S with membrane phospholipids brings to "a freezing" of lipids which is a direct cause of impairing the activity of membrane respiration enzymes and the change of their position in the lipid matrix, thus inhibiting energy-producing processes in cell.     

Biosynthesis of gramicidin S with the aid of dipeptides by gramicidin S synthetase.

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.     

Oxidative determination of 14C-labeled 2-oxo acids

A simple and rapid assay for the determination of 1-14C- or U-14C-labeled 2-oxo acids is described. It is based on the selective and complete oxidation of the carboxyl group to 14CO2. Preceding purification procedures are not necessary. In rat hindlimb perfusion studies, the procedure was used to develop an indirect method for the estimation of the intracellular dilution of [1-14C]pyruvate and to determine the relationship between the transamination and decarboxylation rates of leucine in the perfused tissue by the use of tracer doses of L-[1-14C]leucine.     

Synthesis and utility of 14C-labeled nicotinamide cofactors

A new method for the synthesis of the reduced form of beta-nicotinamide [U-14C]adenine dinucleotide 2(')-phosphate([Ad-14C]NADPH) is presented. The present synthesis results in a radioactive material with a specific activity that is greater than 220 mCi/mmol. This method could easily be adapted for syntheses of 14C-labeled NADH, NADP(+), or any nicotinamide cofactors with radiolabels in other positions. Since these cofactors are so ubiquitous, the use and applications of such labeled material has broad implications. The utility of the labeled cofactor for determination of substrates for nicotinamide-dependent enzymes in the nano- to femtomole scale, in alternative enzymatic assays, and in kinetic isotope effect studies is discussed.     

Preparation of 14C-labeled tuberculin purified protein derivative

(14)C-labeled tuberculin purified protein derivative ((14)C-tuberculin PPD) has been prepared from culture filtrates of Mycobacterium tuberculosis var. hominis grown in a culture medium containing uniformly labeled (14)C-amino acids. With a mixture of (14)C-amino acids (an acid hydrolysate of (14)C-Chlorella protein) in the medium, the recoveries of (14)C in the final product were higher than with (14)C-labeled-l-glutamic acid. (14)C-tuberculin PPD was separated into tuberculoprotein and nucleic acid by paper electrophoresis. The specific radioactivity of tuberculoprotein was substantially greater than that of the nucleic acid. (14)C-tuberculin PPD is advocated as a means to measure the adsorption of tuberculin to glass or other surfaces. It could also prove useful as a means to study the structure and mode of action of tuberculin.     

The cell wall of Bacillus brevis producing gramicidin S, a cyclodecapeptide antibiotic

Gramicidin S is tritiated in Bacillus brevis G.-B. intact cells by activated tritium atoms (which is indicative of its surface localisation). The cell wall protein is tritiated very weakly, which shows that it is screened, apparently, by gramicidin adsorbed on its surface. The cell wall protein is not a glycoprotein and its interaction with exogenous gramicidin S causes cell aggregation. As follows from the Rf value after the chromatographic separation of B. brevis lipids, the reaction of staining, and the data of H-NMR spectroscopy for the fraction of phospholipids, the main membrane phospholipid is an anionic acetylated phosphatidyl ethanolamine.     

Flow scintillation counting of 14C-labeled microalgal photosynthetic pigments

Photopigment radiolabcling, a useful method for measuring thein situ carbon-specific growth rates of microalgae, is basedon the determination of synthesis rates of chemosystematic (i.e.specific for microalgal phylogenetic groups) chlorophylls andcarotenoids using photosynthetically assimilated ¹⁴C as a radiotracer.The reliability of this method depends on accurate measurementsof the ¹⁴C-specific activity of individual photopigments. Typically,photopigments are separated by high-performance liquid chromatography(HPLC) with fraction collection of individual peaks, followedby further purification and standard scintillation counting.To simplify analyses, we evaluated in-line flow scintillationcounting to determine its applicability and reliability formeasuring the activity of radio-labeled photopigments. Incubationswere conducted using both pure cultures and natural phyto-planktonsamples. The radiochemical purity of photopigments was determinedby extract acidification (10% HC1) to transform chlorophyllsinto degradation products. Purity was also checked by comparingabsorbance spectra with purified standards. Although ¹⁴C-labeledcolorless compounds are a common feature in radiograms, thesecompounds do not co-elute with photopigments using our HPLCprotocol. Flow scintillation counting, coupled with a highlyselective HPLC protocol, provides an efficient, reliable andfeasible alternative to fraction collection/repurification methodsfor measuring the ¹⁴C-specific activity of microalgal photosyntheticpigments.