Oxidizable Carbon Sources Research Articles

Microbial prospection of an Amazonian blackwater lake and whole-genome sequencing of bacteria capable of polyhydroxyalkanoate synthesis

Biopolymers are driving the plastic industry to the next generation of environmentally friendly bioproducts, considering green chemistry principles and contemporary economic concepts, such as environmental, social, and governance (ESG) criteria. Hence, microbial biopolymers arise in this context. Resulting from a natural carbon and energy storage process, polyhydroxyalkanoates are the raw material for a range of products based on plastic, with the advantage of being biodegradable in a short period of time. Discovering new biopolymers with different properties, carbon sources and PHA-related enzymes will facilitate market development as well as competition with petrochemical polymers. This work reports the experimental findings of PHA production and genomic data for two bacteria, Ralstonia pickettii and Aquitalea sp., isolated from a blackwater lake located in the ecological reserve of Tupe, Iranduba, AM, Brazil. They were able to produce PHB from carbon sources related to sugar, and R. pickettii also produced PHB from soybean oil and lignin derivatives. Whole-genome sequencing of these isolates enabled the identification of the genetic background to use other oxidizable carbon sources, such as lactic and malonic acids, amino acids, and lignin. Bacteria possibly capable of polyhydroxyalkanoate synthesis from lignin were investigated by genomics and chemistry of biomaterials. Aquitalea sp and Ralstonia pickettii isolated from a blackwater lake rich humic substances, showed great potential to be developed for this purpose. Canonical lignin degradation pathways, such as protocatechuate 4,5-dioxygenase-dependent pathway, CoA-dependent non-β-oxidation pathway of ferulic acid and β-ketoadipate pathway were mapped in their genomes. In addition, traces of P(3HB) were found when using vanillic and gallic acids as sole carbon source, which indicates that the downstream β-ketoadipate pathway is functional.

In Vivo-In Vitro Comparative Toxicology of Cadmium Sulphide Quantum Dots in the Model Organism Saccharomyces cerevisiae.

The aim of this work was to use the yeast Saccharomyces cerevisiae as a tool for toxicogenomic studies of Engineered Nanomaterials (ENMs) risk assessment, in particular focusing on cadmium based quantum dots (CdS QDs). This model has been exploited for its peculiar features: a short replication time, growth on both fermentable and oxidizable carbon sources, and for the contextual availability of genome wide information in the form of genetic maps, DNA microarray, and collections of barcoded mutants. The comparison of the whole genome analysis with the microarray experiments (99.9% coverage) and with the phenotypic analysis of 4688 barcoded haploid mutants (80.2% coverage), shed light on the genes involved in the response to CdS QDs, both in vivo and in vitro. The results have clarified the mechanisms involved in the exposure to CdS QDs, and whether these ENMs and Cd2+ exploited different pathways of response, in particular related to oxidative stress and to the maintenance of mitochondrial integrity and function. Saccharomyces cerevisiae remains a versatile and robust alternative for organismal toxicological studies, with a high level of heuristic insights into the toxicology of more complex eukaryotes, including mammals.

Inorganic polyphosphates in mitochondria

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (approximately 15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7-10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca2+/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P(i) from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.

Regulation of the Ubiquinone (Coenzyme Q) Biosynthetic Genes ubiCA in Escherichia coli

Ubiquinone (Coenzyme Q) is an essential component of bacterial respiratory chains. The first committed step in the biosynthetic pathway is the formation of 4-hydroxybenzoate from chorismate by the enzyme chorismate pyruvate-lyase encoded by the ubiC gene. The 4-hydroxybenzoate is prenylated by 4-hydroxybenzoate octaprenyltransferase encoded by the ubiA gene. The two genes are linked at 91.5 min in the Escherichia coli chromosome. To study the regulation, operon fusions were constructed between these two genes and the lacZ gene. The fusions were introduced into the chromosome as a single copy at the lambda attachment site. Expression of beta-galactosidase was determined in strains carrying the operon fusions ubiC'-lacZ(+) ubiCA'-lacZ(+), and ubiA'-lacZ(+). In glycerol media, the highest level of expression was observed with the operon fusion ubiC'-lacZ(+). Compared with the ubiC'-lacZ(+), the ubiCA'-lacZ(+) operon fusion showed 26% of the activity while the ubiA'-lacZ(+) operon fusion had an activity of 1%. Thus, the ubiC gene is regulated by the upstream promoter while the ubiA gene lacks its own promoter. The effect of fermentable and oxidizable carbon sources on the expression of ubiC'-lacZ(+) was determined. The expression was low in the case of a fermentable carbon source, glucose, while in the presence of oxidizable carbon sources the expression increased 2- to 3-fold. When the expression of ubiC'-lacZ(+) and ubiCA'-lacZ(+) operon fusions were compared under a wide variety of conditions, the levels of beta-galactosidase varied coordinately, suggesting that the ubiCA genes are organized into an operon. The variations in transcription of the operon under different nutritional conditions and in the regulatory mutants, arcA, fnr, and narXL are presented.

Aer and Tsr guide Escherichia coli in spatial gradients of oxidizable substrates.

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an 'all-transducer-knockout' strain of E. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).

Influence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids

The effect of yeast extract and its less complex substituents on the rate of aerobic dechlorination of 2-chlorobenzoic acid (2-ClBZOH) and 2,5-dichlorobenzoic acid (2,5-Cl2BZOH) by Pseudomonas sp. CPE2 strain, and of 3-chlorobenzoic acid (3-ClBZOH), 4-chlorobenzoic acid (4-ClBZOH) and 3,4-dichlorobenzoic acid (3,4-Cl2BZOH) by Alcaligenes sp. CPE3 strain were investigated. Yeast extract at 50 mg/l increased the average dechlorination rate of 200 mg/l of 4-ClBZOH, 2,5-Cl2BZOH, 3,4-Cl2BZOH, 3-ClBZOH and 2-ClBZOH by about 75%, 70%, 55%, 7%, and 1%, respectively. However, in the presence of yeast extract the specific dechlorination activity of CPE2 and CPE3 cells (per unit biomass) was always lower than without yeast extract, although it increased significantly during the exponential growth phase. When a mixed vitamin solution or a mixed trace element solution was used instead of yeast extract the rate of 4-ClBZOH dechlorination increased by 30%-35%, whereas the rate of 2,5-Cl2BZOH and 3,4-Cl2BZOH dechlorination increased by only 2%-10%. The presence of vitamins or trace elements also resulted in a specific dechlorination activity that was generally higher than that observed for the same cells grown solely on chlorobenzoic acid. The results of this work indicate that yeast extract, a complex mixture of readily oxidizable carbon sources, vitamins, and trace elements, enhances the growth and the dechlorination activity of CPE2 and CPE3 cells, thus resulting in an overall increase in the rate of chlorobenzoic acid utilization and dechlorination.

Regulatory features of protein synthesis in isolated mitochondria from Artemia embryos.

Optimal conditions were developed for measuring rates of protein synthesis in isolated mitochondria from encysted embryos of Artemia franciscana to 1) identify the required chemical constituents, 2) assess the influence of extramitochondrial pH on protein synthesis, and 3) investigate potential mechanisms coordinating nuclear and mitochondrial gene expression. Isolation procedures resulted in intact, highly coupled mitochondria [respiratory control ratio = 6.48 +/- 0.43 (SE), n = 21]. Requirements for maximal rates of protein synthesis, measured as incorporation of [3H]leucine (60 microM), included an oxidizable carbon source (10 mM succinate), adenine nucleotides (1.5 mM ADP), phosphate (10 mM), K+ (125 mM), Mg2+ (10 mM), amino acids (0.3 mM of each), sucrose or trehalose (500 mM), EGTA (1 mM), and bovine serum albumin (1 mg/ml). Rates were linear for 60 min at 25 degrees C (r = 0.99). Fluorography of translated products revealed 13 peptides. Previous research has shown that anoxia-induced acidification of intracellular pH (pHi) results in suppression of protein biosynthesis, as judged by cytochrome-c oxidase synthesis. In the present study, mitochondrial protein synthesis was acutely sensitive to external pH, with 80% inhibition observed by lowering pH from 7.5 to 6.8. Thus acidification of pHi may serve as one intracellular signal contributing to a coordinated suppression of both cytoplasmic and mitochondrial protein synthesis during transitions from active to anoxia-induced quiescent states.

IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.

Molybdate transport by Bradyrhizobium japonicum bacteroids.

Bacteroid suspensions of Bradyrhizobium japonicum USDA 136 isolated from soybeans grown in Mo-deficient conditions were able to transport molybdate at a nearly constant rate for up to 1 min. The apparent Km for molybdate was 0.1 microM, and the Vmax was about 5 pmol/min per mg (dry weight) of bacteroid. Supplementation of bacteroid suspensions with oxidizable carbon sources did not markedly increase molybdate uptake rates. Anaerobically isolated bacteroids accumulated twice as much Mo in 1 h as aerobically isolated cells did, but the first 5 min of molybdate uptake was not dependent on the isolation condition with respect to O2. Respiratory inhibitors such as cyanide, azide, and hydroxylamine did not appreciably affect molybdate uptake, even at concentrations that inhibited O2 uptake. The uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the ionophores nigericin and monensin significantly inhibited molybdate uptake. The electrogenic ionophores valinomycin and gramicidin stimulated molybdate uptake. Rapid pH shift experiments indicated that molybdate transport depends on a transmembrane proton gradient (delta pH), and it is probably transported electroneutrally as H2MoO4. Most of the 99MoO4(2-) taken up was not exchangeable with a 100-fold excess of unlabeled MoO4(2-). Tungstate was a competitive inhibitor of molybdate uptake, with a Ki of 0.034 microM, and vanadate inhibited molybdate uptake slightly.

The pho1 mutation. A frameshift, and its compensation, producing altered forms of physiologically efficient ATPase in yeast mitochondria.

The pho1 mutation belongs to the OL12 gene on mitochondrial DNA of Saccharomyces cerevisiae, which codes for a membrane factor subunit of the mitochondrial ATPase (apparent molecular mass 20 kDa). We analysed the ATPase complex from the pho1 mutant and from three revertants, after immunoprecipitation from mitochondrial extracts, by dodecyl sulphate/acrylamide gel electrophoresis. In two revertants the OL12 gene product appeared as an abundant slower migrating peptide, while in the pho mutant, two bands appeared in very low amounts. For the third revertant, a strong band appeared at the normal level. Sequencing of the OL12 gene from these strains gave the following results: the pho1 mutation is a frameshift, arising by insertion of an extra thymidine into a group of three. Two of the revertants contain the same group of four thymidines, but genetic compensation of the frameshift occurs 24 base pairs downstream by the loss of four bases, implying a deficit of one codon. The third revertant has recovered the normal three-thymidine sequence. There is excellent correlation between the modified sequences and electrophoretic migration of the peptide product. Owing to the leakiness of the pho1 phenotype (reduced but not nil growth rate on oxidizable carbon sources, 5-10% highly oligomycin-sensitive ATPase complex, low amounts of OL12 gene product peptides), some translational correction of the frameshift is bound to occur. Based on these results, the compatibility of abnormal ATPase architecture with modified energetic efficiency is discussed.

Relationship between growth inhibition and mitochondrial function in petite-negative yeasts. I. Effects of antibiotics and dyes upon pathogenic and non-pathogenic Candida species.

Antibiotics and dyes which preclude growth of Saccharomyces cerevisiae in media containing oxidizable carbon sources arrested the growth of Candida albicans, Candida tropicalis and Candida utilis even in glucose medium. The growth in the presence of sub-inhibitory concentrations of the various antibiotics and dyes determined a reduction in the cell survival but with no accumulation of respiratory deficient mutants. Under these culture conditions, the total respiration declined leaving a residual antimycin A-resistant--hydroxamate-sensitive O2 uptake, and the amount of the respiratory cytochromes aa3 and b synthesized was reduced. SDS gel electrophoresis of soluble proteins prepared from the antibiotic-treated cells showed some bands in the MW range 92-100 K, which became faint after the cells were grown in the presence of some mitochondrial inhibitors. The ultrastructural analysis of these cells evidenced disappearance of the mitochondrial cristae and their replacement by unfolded membranes. The data obtained suggest that the petite negative trait of Candida could depend on the non-viability or on the very low viability of those cells which have lost their mitochondrial function.

The energized membrane and cellular autolysis in Bacillus subtilis

Lysis of exponential cultures of B. subtilis follows the addition of reagents that dissipate either the electrical or pH gradients of cellular membranes. Stationary-phase cells or cultures that have been inhibited in division by macromolecular-synthesis inhibitors also lyse when uncoupling agents or ionophores are added to the growth medium. Autolysis occurs after brief starvation for a carbon source. Protoplasts are unaffected by azide or other lysis-inducing agents. Electron-donating agents, such as phenazine methosulfate and ascorbate, are effective in retarding autolysis. The addition of an oxidizable carbon source to starved and lysing cultures prevents their autolysis. These results suggest that cellular lysis in B. subtilis and energized membrane are tightly coupled. The fluorescence intensity and the wavelength of maximal fluorescence of 8-anilino-1-naphthalene sulfonic acid, when added to bacterial suspensions, appear to be qualitatively related to the rate of cell lysis. Analyses show that ATP limitations are probably not involved in the elicitation of lysis by ionophores, uncoupling agents or starvation. Measurements of protonmotive forces in the lysis-prone cells suggest that a threshold force of more than 85 mV may be required to maintain cellular integrity. Lipoteichoic acids, polyelectrolytes such as dextran sulfate or phospholipids do not modify the rate of cellular lysis when added to suspensions containing azide or other reagents that eliminate transmembrane protonmotive forces. We interpret the results to suggest that the in vivo control of autolysin activity in B. subtilis is related to the energized membrane.

Genetic and biochemical requirements for chemotaxis to L-proline in Escherichia coli.

Chemotaxis to L-proline was examined by the capillary assay, using a set of Escherichia coli strains bearing well-defined defects in the enzymes of proline transport and utilization. Aspartate taxis was measured as a constitutive, control activity whose receptor and transducer requirements are known. Proline chemotaxis showed a pattern of induction more analogous to that of proline dehydrogenase than of that of proline transport, but chemotaxis to proline was eliminated by mutations eliminating either or both of these activities. No response to proline was observed in the absence of a proline concentration gradient or when succinate was provided as an oxidizable carbon source. These data suggest that the chemotactic response to proline results from a direct impact of proline oxidation on the energy metabolism of the cell.

Respiratory Control in Escherichia coli K 12

Contrary to the generl view point that respiratory control does not exist in Escherichia coli, we have been able to demonstrate this phenomenon in this micro‐organism. E. coli cells growing aerobically on an oxidizable carbon source respire at a high rate, which cannot be increased by the addition of uncouplers of oxidative phosphorylation. Upon removal of the carbon source, the rate of respiration decreases progressively to reach, after 10 min, less that 1/20 of its previous value. Under these conditions the addition of an uncoupler increases the residual respiration by a factor of 3 to 9. This increase is due to the release of respiratory control. Release of respiratory control cannot be observed upon addition of ADP which does not penetrate across the membrane. Respiratory control can also be shown in the presence of non‐limiting respiratory substrate provided that it is not a carbon source. This was shown by measuring the rate of oxidation of ascorbate and phenazine methosulfate.NADH oxidase activity of membrane vesicles obtained by high pressure disintegration was increased by a factor of about 1.5 upon the addition of uncouplers and also upon addition of ADP plus phosphate. In contrast, membrane vesicles depleted from BF1 ATPase (a soluble bacterial coupling factor presenting ATPase activity) did not exhibit respiratory control. It could be restored either by rebinding of BF1 ATPase or by addition of dicyclohexylcarbodiimide, an ATPase inhibitor.

An oligomycin-resistant adenosine triphosphatase and its effects on cellular growth, mitochondrial oxidative phosphorylation and respiratory proton translocation in Saccharomyces cerevisiae.

Mutations at the OLI 1 or OLI 2 loci of mitochondria DNA in Saccharomyces cerevisiae are associated with a diminished growth rate in nutritionally suboptimal cultures supplemented with an oxidizable carbon source. In the case of mutant OR146(OLI1) there is a 35% loss of mitochondrial protein during fractionation in vitro, suggesting that the mutationally altered adenosine triphosphatase(ATPase) confers some instability on the mitochondrial membrane. The possibility is discussed that this reflects an unstable mitchondrial population in vivo, leading the observed growth deficiency. Mitochondria from mutant OR146 at the OLI 1 locus show a relatively oligomycin-resistant State-3 respiration, but the same ADP/O and respiratory-control quotients as the isonuclear wild-type. A slightly lowered Qo2 with NADH-linked substrates was observed and is discussed. For both strains the apparent H+/O ratios were close to 4 with pyruvate, ethanol and alpha-oxoglutarate, but consistently lower with succinate and citrate. For each substrate a characteristic t 1/2 (time for half-decay of the transmembrane pH differential) range was found, consistent with the view that the substrates effecitvely carry the protons back across the membrane. As expected, H+/O ratios were independent of t 1/2 for all substrates, with the exception of alpha-oxoglutarate in the case of the wild-type, where an inverse correlation was found. The lack of this correlation in the case of the mutant was the only apparent difference in the translocation parameters observed. A hypothesis relating this to the functioning of the oligomycin-resistant ATPase is proposed.

Respiration-linked ATP formation by an “oxidative phosphorylation mutant” of yeast

The “oxidative phosphorylation mutant” op 1 is unable to grow on oxidizable carbon sources, although it is capable of high rates of respiration. The characteristics of the adenine nucleotide pool from respiring and aerobically fermenting whole cells of this mutant ( op 1/ op 1) and of a normal strain ( OP 1/ op 1) were the following: The ease of extraction of adenine nucleotides from whole cells was dependent on culture conditions and genotype. The method was standardized to give reproducibly high extraction yields. The op 1 mutant is able to build up a high and stable steady-state concentration of ATP at the expense of respiratory energy. The level of ATP is at the most 20 to 30% lower than in the normal strain. The respiration of this mutant thus cannot be considered as uncoupled. The steady-state concentration of AMP is five times higher in respiring op 1 cells and, consequently, the “energy charge” of respiring mutant cells is significantly lowered, even when ATP levels are similar to normal. Aerobically fermenting op 1 cells also have a higher AMP level than the normal strain, but the difference is only 2-fold and does not significantly affect the “energy charge.” The op 1 mutant is capable of synthesizing mitochondrial ATPase at a normal high rate, provided that glucose repression is eliminated.