A. H. Stouthamer

A theoretical study on the amount of ATP required for synthesis of microbial cell material

The amount of ATP required for the formation of microbial cells growing under various conditions was calculated. It was assumed that the chemical composition of the cell was the same under all these conditions. The analysis of the chemical composition of microbial cells of Morowitz (1968) was taken as a base. It was assumed that 4 moles of ATP are required for the incorporation of one mole of amino acid into protein. The amount of ATP required on account of the instability and frequent regeneration of messenger RNA was calculated from data in the literature pertaining to the relative rates of synthesis of the various classes of RNA molecules in the cell. An estimate is given of the amount of ATP required for transport processes. For this purpose it was assumed that 0.5 mole of ATP is necessary for the uptake of 1 g-ion of potassium or ammonium, and 1 mole of ATP for the uptake of 1 mole of phosphate, amino acid, acetate, malate etc. The results of the calculations show that from preformed monomers (glucose, amino acids and nucleic acid bases) 31.9 g cells can be formed per g-mole of ATP when acetyl-CoA is formed from glucose. When acetyl-CoA cannot be formed from glucose and must be formed from acetate, YATPMAX is only 26.4. For growth with glucose and inorganic salts a YATPMAX value of 28.8 was found. Addition of amino acids was without effect on YATPMAX but addition of nucleic acid bases increased the YATPMAX value to that for cells growing with preformed monomers. Under these conditions 15–20% of the total ATP required for cell formation is used for transport processes. Much lower YATPMAX values are found for growth with malate, lactate or acetate and inorganic salts. During growth on these substrates a greater part of the ATP required for cell formation is used for transport processes. The calculated figures are very close to the experimental values found.The interrelations between YATPMAX and YATP, the specific growth rate (μ), the maintenance coefficient (me) and the P/O rate are given. From a review of the literature evidence is presented that these parameters may vary under different growth conditions. It is concluded that in previous studies on the relation between ATP production and formation of cell material these effects have unjustly been neglected.

Molar growth yields and fermentation balances of Lactobacillus casei L3 in batch cultures and in continuous cultures.

SUMMARY: Fermentation balances determined for different substrates in batch and continuous cultures of Lactobacillus casei revealed two pathways of pyruvate conversion by this organism, a reduction to lactate and the phosphoroclastic cleavage. Pyruvate formed anaerobically from mannitol and citrate was split by the phosphoroclastic enzyme. Lactate was the main fermentation product formed during aerobic growth on mannitol and anaerobic and aerobic growth on glucose. In glucose-limited continuous cultures pyruvate conversion was dependent on the dilution rate. At low dilution rates glucose was fermented exclusively to acetate, ethanol and formate. At high rates only small amounts of acetate, ethanol and formate were formed and lactate production was maximal. Lactate dehydrogenase of L. casei had an absolute requirement for fructose-1,6-diphosphate and manganous ions. The specific activity of lactate dehydrogenase did not differ significantly at different dilution rates. It was concluded that the intracellular level of fructose-1,6-diphosphate controlled the pathway of pyruvate conversion. In batch cultures Y ATP values were between 18.2 and 20.9. No evidence for oxidative phosphorylation was found. In continuous cultures YATP values varied from 18.7 at low dilution rates to 23.5 at high dilution rates. From the dependence of YATP on the dilution rate, a maintenance coefficient of 1.52 x 10-3 was calculated. The Y ATP value corrected for energy of maintenance was 24.3. The possibility that the molar growth yields were erroneously high because of assimilation of growth substrate into intracellular polysaccharides, or because of energy yield from components of the medium other than the added energy source, was excluded.

A theoretical evaluation of growth yields of yeasts

AbstractGrowth yields ofSaccharomyces cerevisiae andCandida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower inS. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors:-S. cerevisiae had a lower protein content thanC. utilis;- uptake of glucose byC. utilis requires energy whereas inS. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation inC. utilis is 35% higher than inS. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth ofS. cerevisiae on glucose was 16 g biomass·mol ATP-1 In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways:- via the assumption that the gap is independent of the growth substrate (i.e. afixed amount of ATP bridges the difference between the theoretical and experimental values).- alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.

Determination of the efficiency of oxidative phosphorylation in continuous cultures of Aerobacter aerogenes.

For anaerobic glucose-limited chemostat cultures of Aerobacter aerogenes a values of 14.0 g/mole was found for YATPmaxand a value of 6.8 mmoles ATP/g dry weight/hr for the maintenance coefficient. Both values are much lower than those previously determined for tryptophan-limited anaerobic chemostat cultures.It is concluded that generally the largest part of the maintenance energy is not used for true maintenance processes.For aerobic glucose-limited chemostat cultures two phases could be differentiated. Acetate production started at μ values higher than 0.53. The slopes of the curves relating the specific rates of glucose- and oxygen consumption with μ became higher and lower respectively above the μ value of 0.53. Using the YATP values obtained in the anaerobic experiment a P/O ratio of about 1.3 could be calculated for glucose- and tryptophan-limited chemostat cultures. In sulfate-limited chemostat cultures acetate was produced at all growth rates. At high growth rates also pyruvate and α-ketoglutarate were produced. With the YATP values obtained in the anaerobic experiment a P/O ratio of about 0.4 was calculated for sulfate-limited chemostat cultures.

Factors determining the degree of anaerobiosis of Bifidobacterium strains

SummaryThe degree of sensitivity of twelve Bifidobacterium (Lactobacillus bifidus) strains to O2 was determined by measuring the size of the inhibition zones obtained when the bacteria were grown in deep agar cultures under air, and by measuring growth in aerated cultures. The size of the inhibition zones varied from 1 to 23 mm. Growth in aerated cultures differed markedly for the strains investigated. No strain grew on agar plates under aerobic conditions.The small inhibition zone of three Bifidobacterium strains might be explained by the presence of a weak catalase activity, which removes traces of H2O2 possibly formed. It is also possible that the NADH oxidase of these strains does not form H2O2 at all. Most probably, the lack of growth on an agar medium results from the fact that these strains only grow below a certain oxidation-reduction potential.One strain, which was rather insensitive to O2, formed a small amount of H2O2 from NADH oxidation. The absence of H2O2 in aerated liquid cultures and cell suspensions of this strain, which lacked catalase and NAD peroxidase activity, must be explained by removal of the traces of H2O2 formed, by an unknown peroxidase system or by a chemical reaction with pyruvate formed during glucose fermentation.For two strains, which were moderately sensitive to O2, accumulation of H2O2 seems to be the principal reason for anaerobiosis. H2O2 turned out to inactivate specifically fructose-6-phosphate phosphoketolase, a key enzyme of the fermentation pathway of bifidobacteria.In the culture medium of two strains, which were extremely sensitive to O2, no H2O2 could be detected after aeration. During anaerobic growth of these strains, the oxidation-reduction potential of the culture decreased so much that neutral red was decolourized. Cell suspensions of these strains only fermented glucose when cysteine was added. It was concluded that these strains required a low oxidation-reduction potential for growth and fermentation.

Generation of ATP during cytochrome-linked anaerobic electron transport in propionic acid bacteria.

Summary: Enzyme determinations in bacteria-free extracts and dual-wavelength experiments with membrane suspensions established that Propionibacterium freudenreichii converted glycerol into triose phosphate via glycerol kinase and NAD-independent glycerol 1-phosphate dehydrogenase which is closely linked to cytochrome b. Glycerol 1-phosphate dehydrogenase uses fumarate as a final hydrogen acceptor. The enzyme system catalysing fumarate reduction with glycerol 1-phosphate as a hydrogen donor, is membrane bound and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO). Fumarate reduction with reduced benzyl-viologen is not inhibited by HOQNO. Cytochrome b is therefore probably involved in the anaerobic electron transport from glycerol 1-phosphate to fumarate. Molar growth yields and fermentation balances were determined for P. freudenreichii and P. pentosaceum growing on glucose, fructose, glycerol and lactate and ATP yields (mol of ATP formed/mol of substrate fermented) were calculated assuming that 1 mol ATP is formed in the electron transport from glycerol 1-phosphate and lactate to fumarate, and that 2 mol ATP are formed in the electron transport from NADH to fumarate. Mean Y ATT values (g dry wt bacteria/mol ATP) were 15·2 and 12·9 for P. freudenreichii, and 16·4 and 11·8 for P. pentosaceum each growing on complex or synthetic medium respectively. The observation that for each strain YATP values were constant for the same medium, supported our assumptions on energy generation in propionic acid bacteria.

Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans

Methanol dehydrogenase of Paracoccus denitrificans was shown to be very similar to the enzyme of Pseudomonas sp, M. 27. The Km value for methanol with excess activator (ammonium ions) is 35 μM. The pH optimum for enzyme activity with 2,6-dichlorophe-nolindophenol as electronacceptor was at 9.0 A CO-binding type of cytochrome c was present only in cells grown with methanol as carbon and energy source.It has been shown that methanol-oxidation involves electron-transport via cytochrome c and an a-type cytochrome to oxygen. Antimycin A did not inhibit this electron transport and 90% inhibition was obtained by 375 μM potassium cyanide. Electron transport from endogenous substrates is possible via cytochrome b and possibly cytochrome o to oxygen. Potassium cyanide inhibited 90% of the electron transport via this pathway at a concentration of 1.42 mM. Measurement of respiration-driven proton translocation proved that during oxidation of methanol to formaldehyde by oxygen one mole of adenosine triphosphate is synthesized in the site 3 region of the electron transport chain. The → H+/O value found confirmed the → H+/site ratio of 3–4 found in heterotrophic grown cells. During electron transport from endogenous substrates to oxygen there is a possible synthesis of 3 moles of adenosine triphosphate.In heterotrophically grown cells electron transfer to oxygen follows almost only the branch of the respiratory chain containing cytochrome o. In methanol-grown cells the pathway via the a-type cytochrome seems more important.

Influence of oxygen on growth, cytochrome synthesis and fermentation pattern in propionic acid bacteria.

Summary: The influence of oxygen on growth and cytochrome synthesis was investigated in four species of Propionibacterium. After anaerobic growth all species contained cytochrome b, cytochrome a or a 1, cytochrome a 2 and a carbon monoxide-binding pigment. Cytochrome b functions in an electron transport system from lactate to fumarate since the lactate-fumarate dismutation reaction, unlike lactate oxidation via methylene blue, was strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO). No species grew on agar plates under aerobic conditions but all grew in aerated media, though at different rates and with diminished synthesis of all cytochromes. In Propionibacterium freudenreichii and P. shermanii, which grew slower under aerobic conditions than P. rubrum and P. pentosaceum, cytochrome synthesis was more repressed by oxygen. Large amounts of pyruvate accumulated under aerobic conditions whereas acetate, propionate and succinate were not formed. The bacteria are therefore dependent on oxidative phosphorylation for energy under aerobic conditions. For P. freudenreichii an Y 0 value (g dry wt bacteria/g-atom oxygen taken up) of 15 was measured, suggesting a P/O value of 1 to 2. Absence of cytochrome synthesis resulting in the loss of oxidative phosphorylation may explain the absence of aerobic growth on agar plates.

Growth yields and the efficiency of oxidative phosphorylation during autotrophic growth of Paracoccus denitrificans on methanol and formate.

Paracoccus denitrificans was grown aerobically in chemostat or pH-auxostat cultures with methanol or formate as the limiting carbon source. The maximum growth yields (expressed in g biomass/mole), determined in methanol-limited cultures, on methanol, oxygen and carbon-dioxide were 13.4, 15.5 and 24.8 respectively. The maximum growth yields, determined in formate-limited cultures, on formate and oxygen were 2.9 and 10.5 respectively. Elemental analysis of these cells yielded a cell formula of C6H10.8N1.5O2.9. With the help of the known elemental cell composition an equation for material balances could be drawn up. The calculated yields on oxygen and CO2 (YO2 and YCO2) were in good agreement with the experimental values.Autotrophically grown cells on methanol possess three active energy conserving sites in the electron-transport chain. The overall P/O ratio (the amount of adenosine triphosphate (ATP) synthesized per atom oxygen consumed) is calculated to be 2.05 with the matching YATPmaxvalue of 3.8, which is lower than the theoretical value of 6.5. With only 2 phosphorylation sites impossible YATPmaxvalues would result.Autotrophic growth on formate yielded cells in the possesion of only two active energy conserving sites, most probably sites 1 and 2. Formate crosses the cytoplasmic membrane as free acid, which is equivalent to co-transport with one proton. Therefore transport of formate is energy requiring. With →H+/site ratios of 2 or 3 respectively 0.5 or 0.33 moles of ATP are necessary to transport 1 mole of formate. Consequently the P/O ratio in formate-grown cells is respectively 1.5 or 1.67 with the matching YATPmaxvalues of 3.1 or 3.5. Therefore during autotrophic growth on methanol and formate the same amount of adenosine triphosphate is necessary to synthesize 1 g of biomass. The 95% confidence intervals of the YATPmaxvalues overlap completely. The found YATPmaxis 50–60% of the theoretical value as was found for heterotrophic growth of Paracoccus denitrificans. We propose that metabolism in Paracoccus denitrificans is regulated on the level of phosphorylation in the site 1 and the site 3 region of the electron-transport chain.