Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C]glycine and, to a lesser extent 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5-[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2-[1-14C]oxoglutarate was converted to 5-[5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and Characterization of a Temperate Bacteriophage Specific for Rhodopseudomonas spheroides

The isolation of a temperature phage specific for the photosynthetic bacterium Rhodopseudomonas spheroides is reported. This phage, Rphi-1, establishes a state of lysogeny and can be induced from the prophage state by exposure to mitomycin C or UV irradiation. Mutants of Rphi-1 which grow on a standard laboratory strain (2.4.1) of Rhodopseudomonas spheroides were isolated. Although the original Rphi-1 isolated was chloroform sensitive, the mutant which plates on strain 2.4.1 is chloroform resistant. Rphi-1 does not grow on closely related bacteria, such as Rhodopseudomonas palustris or Rhodopseudomonas capsulata. Rphi-1 mutants forms plaques with the same efficiency whether the plates are incubated under aerobic conditions in the dark or under anaerobic conditions in the light (phototropic conditions).     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

13C nuclear magnetic resonance studies on bacteriochlorophyll a biosynthesis in Rhodopseudomonas spheroides S

The 13C NMR spectra were analyzed in bacteriochlorophyll a and magnesium protoporphyrin methyl ester formed in Rhodopseudomonas spheroides S. in the presence of L-[1-13C]glutamate and [2-13C]glycine. After reassignment of three alpha-pyrrolic carbons (C-9, -14 and -16) of bacteriochlorophyll a, the spectra showed that C-2 of glycine was preferentially incorporated into the eight-carbon atoms in these tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA). C-2 of glycine was also incorporated specifically into methyl ester carbon of magnesium protoporphyrin IX methyl ester and methoxyl carbon of methoxycarbonyl group attached to isocyclic ring of bacteriochlorophyll a. No enrichment of these nine-carbon atoms was observed in the spectrum of bacteriochlorophyll formed in the presence of L-[1-13C]glutamate, showing exclusive operation of ALA synthase on bacteriochlorophyll biosynthesis.     

Ribonucleic Acid from Aerobically and Anaerobically Grown Rhodopseudomonas spheroides: Comparison by Hybridization to Chromosomal and Satellite Deoxyribonucleic Acid

Ribonucleic acid (RNA) species from aerobically and anaerobically grown Rhodopseudomonas spheroides were compared via hybridization to deoxyribonucleic acid (DNA). Both long-labeled and stable RNA bound to chromosomal DNA to the same extent, regardless of derivation. About 4% of the chromosomal DNA hybridized with total cell RNA and about 0.08% with stable RNA. About 4% of the mixed satellite DNA could be hybridized to total cell RNA from aerobic or anaerobic cultures, whereas essentially no stable RNA formed a hybrid with this DNA. Hybridization competition experiments with aerobic and anaerobic pulse-labeled RNA and chromosomal or satellite DNA demonstrated that no qualitative differences existed between the RNA species. It is concluded that identical species of RNA in the same relative amounts are synthesized by R. spheroides during aerobic or anaerobic growth on the same medium.     

Carotenoid biosynthesis in Rhodopseudomonas spheroides. S-Adenosylmethionine as the methylating agent in the biosynthesis of spheroidene and spheroidenone

[methyl-(14)C]Methionine and S-adenosyl[methyl-(14)C]methionine were incorporated into the methoxycarotenoids spheroidene and spheroidenone by Rhodopseudomonas spheroides. The incorporation was greatly enhanced in the presence of lysozyme. On degradation of labelled spheroidene by hydriodic acid, the (14)C label was recovered in methyl iodide. Degradation of spheroidenone by reduction and allylic dehydration and demethylation of the reduction product gave a mixture of unlabelled carotenoid hydrocarbons, including 3,4-didehydrolycopene and 3,4-didehydro-7',8'-dihydrolycopene. The label from [methyl-(14)C]methionine and S-adenosyl[methyl-(14)C]methionine was located specifically in the methoxy group of spheroidene and spheroidenone. The biosynthesis of methoxycarotenoids in Rps. spheroides involves methylation of the tertiary hydroxyl groups of intermediates with S-adenosylmethionine.     

Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides and Chromatium strain D

1. The anaerobic coproporphyrinogenase activity in an extract of Rhodopseudomonas spheroides is inhibited by 1,10-phenanthroline, alphaalpha'-bipyridyl, flavins, 2,4-dinitrophenol and 1,4-naphthaquinone. These compounds have no effect on the aerobic coproporphyrinogenase activity. 2. On removal of small-molecular-weight material from a crude extract, the anaerobic system becomes very unstable; it can be stabilized by adding succinate. Now nicotinamide nucleotides, in addition to Mg(2+), ATP and methionine, are required for protoporphyrin to be formed. 3. A mechanism for the anaerobic reaction is proposed, based on the cofactor requirements and the effect of inhibitors. 4. The enzyme responsible for aerobic activity has been partially purified and some of its properties are reported. 5. A crude extract of Chromatium strain D also exhibits coproporphyrinogenase activity under anaerobic conditions in the presence of S-adenosylmethionine or ATP plus methionine. The requirement for other cofactors is variable.     

A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg(2+) and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg(2+) caused the refolding of the initial products of unfolding and in the presence of Ni(2+) the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na(+)/Mg(2+) ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein-protein interactions in the structure of the R. spheroides ribosome.     

sn-Glycerol-3-phosphate acyltransferase activity in particulate preparations from anaerobic, light-grown cells of Rhodopseudomonas spheroides. Involvement of acyl thiolester derivatives of acyl carrier protein in the synthesis of complex lipids.

Crude particulate preparations obtained from anaerobic, light-grown cells of Rhodopseudomonas spheroides have been shown to possess a significant level of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity. In contrast to the enzyme from Escherichia coli, the R. spheroides glycerophosphate acyltransferase has a high specificity for acyl thiolester derivatives of acyl carrier protein (ACP) as acyl donors for the reaction. Only limited , nonlinear glycerophosphate incorporation into lipid occurs when acyl coenzyme A (CoA) derivatives are employed as acyl substrate. With oleyl-ACP as substrate, maximal enzyme activity was observed at 40 degrees, over a broad pH range (6.0 to 8.5) and did not require a divalent metal cation. The presence of dithiothreitol stimulated enzyme-activity 15 to 20%. When oleyl-ACP or palmityl-ACP was employed as sole acyl group donor, the major products recoverable from the reaction mixtures were lysophosphatidic acid, phosphatidic acid, and monoglyceride. Althouh oleyl-ACP and palmityl-ACP gave comparable maximal velocities in the initial acylation of glycerophosphate, the formation of phosphatidic acid occurred preferentially with the unsaturated acyl-ACP derivative.     

Bacteriophages of Rhodopseudomonas spheroides: Isolation and Characterization of a Rhodopseudomonas spheroides Bacteriophage

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.     

Magnesium protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Studies with whole cells

1. Whole cells of Rhodopseudomonas spheroides grown under semi-anaerobic conditions in the light incorporated magnesium into exogenous protoporphyrin when incubated with EDTA or the related chelators EGTA, N-(2-hydroxyethyl)-ethylenediamine-NN'N'- triacetate and trans-1,2-diaminocyclohexanetetra-acetate. 2. The reaction was demonstrated under anaerobic conditions in the light or at low oxygen partial pressure in the dark. Partial pressures of oxygen greater than 15% inhibited the reaction. 3. Cells grown under pure oxygen were completely inactive, but on adaptation to growth under low oxygen partial pressure (O(2)+N(2), 5:95) the development of activity paralleled the synthesis of bacteriochlorophyll. 4. The reaction with normal cells did not require protein synthesis, but cells that had lost their activity by being illuminated in Mg(2+)-deficient medium did not recover it in the absence of protein synthesis. 5. The product of the reaction was magnesium protoporphyrin monomethyl ester. 6. Evidence is presented that insertion of magnesium is obligatorily coupled with methylation and it is concluded that the reaction is dependent on a multienzyme complex.     

Control of magnesium–protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Role of light, oxygen, and electron and energy transfer

1. Magnesium-protoporphyrin chelatase activity, previously shown in whole cells of Rhodopseudomonas spheroides, could not be demonstrated in cell-free extracts prepared in different ways, although spheroplasts retained moderate activity. Slight activity was detected also in whole cells of Rhodospirillum rubrum. 2. The effects on the activity of the enzyme of inhibitors of electron and energy transfer were studied in whole cells of Rps. spheroides. Amytal, rotenone, azide and cyanide inhibited at low pO(2) in the dark but not under anaerobic conditions in the light. Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, as well as uncouplers and oligomycin, inhibited under all environmental conditions. 3. The effects on magnesium chelatase activity of intermediates of the tricarboxylic acid cycle, of thenoyltrifluoroacetone, of a number of artificial electron donors or acceptors, of various quinones and of the oxidation-reduction indicator dyes Benzyl Viologen and Methyl Viologen are described. 4. It was concluded that electron transport between a b-type and a c-type cytochrome as well as associated energy conservation and transformation reactions were essential for activity. There was also a specific requirement for ATP. 5. Exogenous protoporphyrin and magnesium protoporphyrin monomethyl ester were incorporated into bacteriochlorophyll or late precursors by whole cells. 6. Evidence is presented that the insertion of magnesium was the only step inhibited by oxygen in the biosynthetic pathway between protoporphyrin and bacteriochlorophyll.     

Uptake of C4 dicarboxylates and pyruvate by Rhodopseudomonas spheroides.

The uptake of C4 dicarboxylates by cells from exponential cultures of Rhodopseudomonas spheroides followed saturation kinetics at concentrations below 100 muM with Km values for succinate, malate, and fumarate of 2.7, 2.3, and 0.8, respectively. Corresponding Vmax values of 50, 52, and 67.5 nmol/min per mg of protein at 20 C were obtained. Each of these compounds interfered competitively with uptake of the others, and a common transport system appears to be involved. Fructose-grown cells took up C4 dicarboxylates only at very low rates, and pyruvate-grown cells took up C4 dicarboxylates at one-third the rates found with succinate-grown cultures. Malonate and maleate inhibited uptake less severely, and aspartate and alpha-ketoglutarate had no effect at 100-fold excess. Divalent metals stimulated uptake. Light or respiration was required for uptake, and entered materials were rapidly converted to other metabolities, notably amino acids. Pyruvate entry appeared to be mediated by several systems, of which only one could be resolved kinetically. This system had a Km of 13 muM and Vmax of 5.6 nmol/min per mg of protein at 20 C. A number of related mono- and dicarboxylates interfered with pyruvate uptake. The pyruvate uptake system was distinguishable from the C4 dicarboxylate system by the absence of divalent cation stimulation and by substrate and inhibitor specificity.     

Activation of δ-aminolaevulate synthetase in extracts of Rhodopseudomonas spheroides

1. The spontaneous activation of delta-aminolaevulate synthetase in extracts from Rhodopseudomonas spheroides grown semi-anaerobically in the light requires oxygen and does not take place anaerobically in the dark. 2. Activation is completely prevented by azide or cyanide and is partially inhibited by chlorpromazine. These compounds inhibit markedly the succinoxidase activity of extracts. 3. NADH delays activation, but when it has been oxidized by the extract activation begins at the normal rate and complete activation occurs. By contrast both the rate and the extent of activation are markedly decreased by even small amounts of carboxylic acids. 4. The inhibitory effects of succinate and citrate on activation can be prevented by malonate and fluorocitrate respectively. 5. These results suggest that for activation to occur some endogenous compound has to be oxidized via the electron transport chain. 6. Activation occurs under anaerobic conditions in the light, probably by photo-oxidation.     

The mechanism of the attachment of esterifying alcohol in bacteriochlorophyll a biosynthesis.

The mechanism through which the C-17(3) carboxy group of bacteriochlorophyllide a is esterified to produce bacteriochlorophyll aphytyl of Rhodopseudomonas spheroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum was studied by using 5-aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms. The latter species was prepared by an exchange reaction in which 5-aminolaevulinate hydrochloride was heated in H218O in an autoclave. A method for the determination of the 18O content of the C-1 oxygen atoms of 5-aminolaevulinate was developed. As a prelude to the mechanistic work, a systematic study was undertaken to establish the optimal conditions under which a significant proportion of the bacteriochlorophyll a of the two photosynthetic organisms originated from the exogenously added 5-aminolaevulinate. It was found that, when Rps. spheroides and Rsp. rubrum were grown in the presence of about 0.15mM- and 1.2mM-5-aminolaevulinate respectively, 30-40% of their chlorophyll was derived from the added precursor. In these conditions, 5-amino[1,4-18O3]laevulinate was incorporated into bacteriochlorophyll aphytyl and bacteriochlorophyll ageranylgeranyl by the relevant organisms. The samples of chlorophylls were then hydrolysed with alkali to obtain phytol and geranylgeraniol, which were converted into the corresponding trimethylsilyl derivatives and analysed by gas chromatography-mass spectrometry. The data were used to deduce that the alcohols contained 90-95% of the 18O originally present at each of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results it is suggested that the ester bond at C-17(3) is formed, not by a chlorophyllase type of enzymic reaction, but by a process involving the nucleophilic attack by the C-17(3) carboxylate group of the chlorophyllide on the activated form of an isoprenyl alcohol.     

Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia.

The anaerobic biodegradation of naphthalene (NAP) and phenanthrene (PHE) was investigated by using sediment collected from the Arthur Kill in New York/New Jersey harbor. The initial cultures were composed of 10% sediment and 90% mineral medium containing 20 mM sulfate. Complete loss of NAP and PHE (150 to 200 muM) was observed after 150 days of incubation. Upon refeeding, NAP and PHE were utilized within 14 days. The utilization of both compounds was inhibited in the presence of 20 mM molybdate. [14C]NAP and [14C]PHE were mineralized to 14CO2. The activities could be maintained and propagated by subculturing in mineral medium. In the presence of halogenated analogs, 2-naphthoate was detected in NAP-utilizing enrichments. The mass spectrum of the derivatized 2-napththoate from the enrichment supplemented with both [13C]bicarbonate and NAP indicates the incorporation of 13CO2 into NAP. In the PHE-utilizing enrichment, a metabolite was detected by both high-pressure liquid chromatography and gas chromatography-mass spectrometry analyses. The molecular ion and fragmentation pattern of its mass spectrum indicate that it was phenanthrenecarboxylic acid. The results obtained with [13C] bicarbonate indicate that 13CO2 was incorporated into PHE. It appears, therefore, that carboxylation is an initial key reaction for the anaerobic metabolism and NAP and PHE. To our knowledge, this is the first report providing evidence for intermediates of PAH degradation under anaerobic conditions.     

Challenge of psychrophilic anaerobic wastewater treatment

Psychrophilic anaerobic treatment is an attractive option for wastewaters that are discharged at moderate to low temperature. The expanded granular sludge bed (EGSB) reactor has been shown to be a feasible system for anaerobic treatment of mainly soluble and pre-acidified wastewater at temperatures of 5--10 degrees C. An organic loading rate (OLR) of 10--12 kg chemical oxygen demand (COD) per cubic meter reactor per day can be achieved at 10--12 degrees C with a removal efficiency of 90%. Further improvement might be obtained by a two-module system in series. Stabile methanogenesis was observed at temperatures as low as 4--5 degrees C. The specific activity of the mesophilic granular sludge was improved under psychrophilic conditions, which indicates that there was growth and enrichment of methanogens and acetogens in the anaerobic system. Anaerobic sewage treatment is a real challenge in moderate climates because sewage belongs to the 'complex' wastewater category and contains a high fraction of particulate COD. A two-step system consisting of either an anaerobic up-flow sludge bed (UASB) reactor combined with an EGSB reactor or an anaerobic filter (AF) combined with an anaerobic hybrid reactor (AH) is successful for anaerobic treatment of sewage at 13 degrees C with a total COD removal efficiency of 50% and 70%, respectively.     

Evaluation of biosynthetic pathways to delta-aminolevulinic acid in Propionibacterium shermanii based on biosynthesis of vitamin B12 from D-[1-13C]glucose

Analysis of the (13)C nuclear magnetic resonance (NMR) spectrum of (13)C-labeled vitamin B(12) biosynthesized from D-[1-(13)C]glucose by Propionibacterium shermanii provided evidence suggesting that delta-aminolevulinic acid (ALA) incorporated in the (13)C-labeled vitamin B(12) may have been synthesized via both the Shemin pathway and the C5 pathway under anaerobic conditions in the ratio of 1 < [(ratio of ALA biosynthesis from the Shemin pathway)/(that from the C5 pathway)] < 1.8. The D-ribose moiety of vitamin B(12) was labeled with (13)C at R-1, R-3, and R-5. The aminopropanol moiety of vitamin B(12) was labeled on Pr-1 and Pr-2, but not Pr-3.     

Changes in Ribonucleic Acid Turnover During Aerobic and Anaerobic Growth in Rhodopseudomonas spheroides

Differences in the rate and extent of degradation of ribonucleic acid (RNA) labeled by a 30-sec pulse in aerobically or anaerobically grown Rhodopseudomonas spheroides have been studied by using rifampin to block RNA synthesis. In anaerobic cultures, unstable RNA is degraded with a half-life of 1.25 to 2.0 min, and about 40% of the pulse-labeled RNA is stable. In aerobic cultures, the half-life of unstable RNA is increased to 2.5 to 4.0 min, and 50% of the RNA is stable. When aerobic cultures are transferred to anaerobic conditions, there is a rapid drop in half-life and in the proportion of stable RNA. When anaerobic cultures are made aerobic, the reverse changes occur after a lag of about 30 min. Addition of puromycin to either aerobic or anaerobic cultures caused the pulse-labeled RNA to be degraded at the same rate and to the same extent as the RNA in an anaerobic control culture. In contrast, addition of chloramphenicol enhanced the difference in RNA half-life and increased the proportion of stable RNA by about 10% in each case. It is concluded that there is a difference in the stability of an RNA component under aerobic and anaerobic conditions.