Methanogenic bacteria, which are presently identified on the basis of cell morphology and substrate conversion to CH,, can be differentiated from nonmethanogens and identified in pure or mixed culture on the basis of their autofluorescence under ultraviolet illumination.
Selenomonas ruminantium, a strictly anaerobic runinal bacterium, was grown at various dilution rates (D = 0.05, 0.25, and 0.35 h-1) under glucose-limited continuous culture conditions. Suspensions ofwashed cells prepared anaerobically in mineral buffer were subjected to nutrient starvation (24 to 36 h; 39°C; N2 atmosphere). Regardless of growth rate, viability declined logarithmically, and within about 2.5 h, about 50% of the populations were nonviable. After 24 h of starvation, the numbers of viable cells appeared to be inversely related to growth rate, the highest levels occurring with. the slowest grown population. Cell dry weight, carbohydrate, protein, ribonucleic acid (RNA), and deoxyribonucleic acid declined logarithmically during starvation, and the decline rates of each were generally greater with cells grown at higher D values. Both cellular carbohydrate and RNA declined substantially during the first 12 h of starvation. Most of the cellular RNA that disappeared was found in the suspending buffer as lowmolecular-weight, orcinol-positive materials. During growth, S. ruminantium made a variety of fermentation acids from glucose, but during starvation, acetate was the only acid made from catabolism of cellular material. Addition of glucose or vitamins to starving cell suspensions did not decrease loss of viability, whereas a starvation in the spent culture medium resulted in a slight decrease in the rate of viability loss. Overall, the data indicate that S. ruminantium strain D has very little survival capacity under the conditions tested compared with other bacterial species that have been studied.
The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)
Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h-1. Washed cell suspensions were subjected to long-term nutrient starvation
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