L-malyl-coenzyme A/beta-methylmalyl-coenzyme A lyase is involved in acetate assimilation of the isocitrate lyase-negative bacterium Rhodobacter capsulatus

Cell extracts of Rhodobacter capsulatus grown on acetate contained an apparent malate synthase activity but lacked isocitrate lyase activity. Therefore, R. capsulatus cannot use the glyoxylate cycle for acetate assimilation, and a different pathway must exist. It is shown that the apparent malate synthase activity is due to the combination of a malyl-coenzyme A (CoA) lyase and a malyl-CoA-hydrolyzing enzyme. Malyl-CoA lyase activity was 20-fold up-regulated in acetate-grown cells versus glucose-grown cells. Malyl-CoA lyase was purified 250-fold with a recovery of 6%. The enzyme catalyzed not only the reversible condensation of glyoxylate and acetyl-CoA to L-malyl-CoA but also the reversible condensation of glyoxylate and propionyl-CoA to beta-methylmalyl-CoA. Enzyme activity was stimulated by divalent ions with preference for Mn(2+) and was inhibited by EDTA. The N-terminal amino acid sequence was determined, and a corresponding gene coding for a 34.2-kDa protein was identified and designated mcl1. The native molecular mass of the purified protein was 195 +/- 20 kDa, indicating a homohexameric composition. A homologous mcl1 gene was found in the genomes of the isocitrate lyase-negative bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum in similar genomic environments. For Streptomyces coelicolor and Methylobacterium extorquens, mcl1 homologs are located within gene clusters implicated in acetate metabolism. We therefore propose that L-malyl-CoA/beta-methylmalyl-CoA lyase encoded by mcl1 is involved in acetate assimilation by R. capsulatus and possibly other glyoxylate cycle-negative bacteria.     

Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides

Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and propionyl-CoA. In a second part glyoxylate and propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.     

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus,a bifunctional enzyme involved in autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.     

(2S)-Methylsuccinyl-CoA dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA assimilation

Many organic substrates are metabolized via acetyl-coenzyme A (CoA) and enter central carbon metabolism at the level of this compound. We recently described the outlines of the ethylmalonyl-CoA pathway, a new acetyl-CoA assimilation strategy that operates in a number of bacteria such as Rhodobacter sphaeroides , Methylobacterium extorquens and streptomycetes and replaces the glyoxylate cycle. This new pathway involves a unique central reaction sequence catalysed by characteristic enzymes. Here, we identified and characterized (2 S )-methylsuccinyl-CoA dehydrogenase from R. sphaeroides , a flavin adenine dinucleotide-containing enzyme that catalyses the last unknown step in the central part of the ethylmalonyl-CoA pathway, the oxidation of (2 S )-methylsuccinyl-CoA to mesaconyl-(C1)-CoA. This enzyme is highly specific for its substrate and forms a distinct subgroup within the superfamily of flavin-dependent acyl-CoA dehydrogenases. Homology modelling and comparative sequence analyses with well-studied members of this superfamily identified amino acids that may contribute to the narrow substrate specificity of (2 S )-methylsuccinyl-CoA dehydrogenase. The central part of the ethylmalonyl-CoA pathway was reconstituted in vitro using four recombinant enzymes. By this work, the ethylmalonyl-CoA pathway and its stereochemical course have been completely solved. This allowed defining the minimum set of enzymes necessary for its operation and to screen for further organisms following this acetyl-CoA assimilation strategy.     

Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.     

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

ABSTRACT: BACKGROUND: Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. RESULTS: Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of crystalline cellulose and ionic liquid-pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. CONCLUSIONS: T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.     

Structural and spectroscopic properties of a reaction center complex from the chlorosome-lacking filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii

The photochemical reaction center (RC) complex of Roseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemes c, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation from Chloroflexus aurantiacus. Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested that R. castenholzii had diverged from C. aurantiacus very early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. Although R. castenholzii is phylogenetically related to C. aurantiacus, the arrangement of its puf genes, which code for the light-harvesting proteins and the RC subunits, was different from that in C. aurantiacus and similar to that in purple bacteria. The genes are found in the order pufB, -A, -L, -M, and -C, with the pufL and pufM genes forming one continuous open reading frame. Since the photosynthetic apparatus and genes of R. castenholzii have intermediate characteristics between those of purple bacteria and C. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.     

Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential

The Cerrado is the second largest Brazilian biome, yet little is known about its wild fauna, flora and microbiota. This work aimed to identify epiphytic bacteria present in fruits native to three different regions of the Cerrado and to select cellulase-producing bacteria. Culture-dependent and culture-independent (PCR-DGGE) methods were used to characterize the microbiota from 32 native Cerrado fruits, and the selection of cellulase-producing bacteria was performed by a semi-quantitative test on carboxymethylcellulose agar medium. Analysis of the 16S rRNA gene sequences of 69 profile representatives showed that the isolates belonged to 29 bacterial genera (Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Streptomyces, Enterobacter, Microbacterium, Aerococcus, Bradyrhizobium, Methylobacterium, Erwinia, Pantoea, Acidithiobacillus, Ochrobactrum, Stenotrophomonas, Curtobacterium, Clostridium, Lactobacillus, Xanthomonas, Delftia, Klebsiella, Enterococcus, Burkholderia, Escherichia, Streptococcus, Citrobacter and Achromobacter). Species in the genera Methylobacterium, Stenotrophomonas, Clostridium, Pantoea and Enterobacter were detected by both culture-dependent and culture-independent methods. The species Lactobacillus fermentum, Acinetobacter sp. and Methylomonas methanica were detected only by PCR-DGGE. Additionally, 30 % (178 isolates) of the bacteria tested were able to produce cellulase. The best producers belonged to the genera Bacillus, Streptomyces, Paenibacillus, Enterobacter and Burkholderia, indicating that this ecosystem could be an attractive source for the study of novel enzymes.     

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.     

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes.     

Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase.

The Rhodobacter sphaeroides pgsA gene (pgsARs), encoding phosphatidylglycerophosphate synthase (PgsARs), was cloned, sequenced, and expressed in both R. sphaeroides and Escherichia coli. As in E. coli, pgsARs is located immediately downstream of the uvrC gene. Comparison of the deduced amino acid sequences revealed 41% identity and 69% similarity to the pgsA gene of E. coli, with similar homology to the products of the putative pgsA genes of several other bacteria. Comparison of the amino acid sequences of a number of enzymes involved in CDP-diacylglycerol-dependent phosphatidyltransfer identified a highly conserved region also found in PgsARs. The pgsARs gene carried on multicopy plasmids was expressed in R. sphaeroides under the direction of its own promoter, the R. sphaeroides rrnB promoter, and the E. coli lac promoter, and this resulted in significant overproduction of PgsARs activity. Expression of PgsARs activity in E. coli occurred only with the E. coli lac promoter. PgsARs could functionally replace the E. coli enzyme in both a point mutant and a null mutant of E. coli pgsA. Overexpression of PgsARs in either E. coli or R. sphaeroides did not have dramatic effects on the phospholipid composition of the cells, suggesting regulation of the activity of this enzyme in both organisms.     

Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria

Abstract Fifty different polyhydroxyalkanoic acid (PHA)-accumulating bacterial strains were investigated for the occurrence of phasin proteins bound to PHA granules and related to the GA24 protein of Alcaligenes eutrophus H16, by isolating PHA granules and Western blot analysis of granule-associated proteins employing antibodies raised against the GA24 protein. It could be demonstrated that the PHA granules of many poly(3-hydroxybutyrate)-accumulating bacteria exhibited ja similar protein pattern, and a predominant protein of 24 ± 2 kDa occurred in the granules of A. eutrophus strains A7, CH34, JMP222, N9A and TF93 exhibiting N-terminal amino acid sequences identical to that of the GA24 protein. Proteins bound to the granules of A. latus, Burkholderia caryophvli B. cepacia B. solanacearum, Pseudomonas glathei. Rhodobacter sphaeroides and Telluria mixta also gave positive immunoreactions. Granule-associated proteins of small size also; occurred in various strains of the Gram-positive bacteria Bacillus megaterium and R. ruher as well as in the Gram-negative bacteria Azotohacter sp., Chromatium vinosum, Comamonas acidovorans, Methylobacterium sp., Mycoplana ruhra, Paracoccus denitrificans, Pseudomonas sp., Rhodospirillum ruhrum, Rubrivivax gelatinosus and Thiocystis violacea ; however, they gave no immunoreaction. This study clearly demonstrated that phasins are wide-spread if not essential in PHA-accümulating bacteria.     

Newly isolated Streptomyces spp. as enantioselective biocatalysts: hydrolysis of 1,2-O-isopropylidene glycerol racemic esters

AIMS: To identify microbial strains with esterase activity able to enantioselectively hydrolyse esters of (R,S)-1,2-O-isopropylidene glycerol. METHODS AND RESULTS: The microbial hydrolysis of various racemic esters of 1,2-O-isopropylidene glycerol (IPG) was attempted by screening among Streptomyces spp. previously selected on the basis of their carboxylesterase activity. The best results were observed in the hydrolysis of butyrate ester and two strains appeared promising as they showed opposite enantioselectivity: Streptomyces sp. 90852 gave predominantly (S)-IPG, while strain 90930 mostly gave the R-alcohol. Streptomyces sp. 90930 was identified as Streptomyces violaceusniger, whereas Streptomyces sp. 90852 is a new species belonging to the Streptomyces violaceus taxon. The carboxylesterase belonging to strain 90852 gave a maximum value of enantiomeric ratio (E) of 14-16. This strain was lyophilized and used as dry mycelium for catalysing the synthesis of isopropylidene glycerol butyrate in heptane showing reaction rate and enantioselectivity (E = 6.6) lower than what observed for the hydrolysis. CONCLUSIONS: A new esterase with enantioselective activity towards (R,S)-IPG butyrate has been selected. The best enantioselectivity is similar or even better than the highest reported value in the literature with commercial enzymes. The enzyme is produced by a new species belonging to the S. violaceus taxon. SIGNIFICANCE AND IMPACT OF THE STUDY: New esterases from streptomycetes can be employed for the enantioselective hydrolysis of chiral esters derived from primary alcohols, not efficiently resolved with commercial enzymes.     

Distribution of an L-isoaspartyl protein methyltransferase in eubacteria.

A protein carboxyl methyltransferase (EC 2.1.1.77) that recognizes age-damaged proteins for potential repair or degradation reactions has been found in all vertebrate tissues and cells examined to date. This enzyme catalyzes the transfer of methyl groups from S-adenosylmethionine to the carboxyl groups of D-aspartyl or L-isoaspartyl residues that are formed spontaneously from normal L-aspartyl and L-asparaginyl residues. A similar methyltransferase has been found in two bacterial species, Escherichia coli and Salmonella typhimurium, suggesting that this enzyme performs an essential function in all cells. In this study, we show that this enzyme is present in cytosolic extracts of six additional members of the alpha and gamma subdivisions of the purple bacteria: Pseudomonas aeruginosa (gamma), Rhodobacter sphaeroides (alpha), and the gamma enteric species Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Serratia marcescens. DNA probes from the E. coli methyltransferase gene hybridized only to the chromosomal DNA of the enteric species. Interestingly, no activity was found in the plant pathogen Erwinia chrysanthemi, a member of the enteric family, nor in Rhizobium meliloti or Rhodopseudomonas palustris, two members of the alpha subdivision. Additionally, we could not detect activity in the four gram-positive species Bacillus subtilis, B. stearothermophilus, Lactobacillus casei, and Streptomyces griseus. The absence of enzyme activity was not due to the presence of inhibitors in the extracts. These results suggest that many cells may not have the enzymatic machinery to recognize abnormal aspartyl residues by methylation reactions. Since the nonenzymatic degradation reactions that generate these residues occur in all cells, other pathways may be present in nature to ensure that these types of altered proteins do not accumulate and interfere with normal cellular physiology.     

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4.

The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.     

Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate

3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed by using recombinant propionyl coenzyme A (propionyl-CoA) synthase from Chloroflexus aurantiacus. Using this assay, we demonstrate that R. sphaeroides can utilize 3-hydroxypropionate as the sole carbon source and energy source. We establish that acetyl-CoA is not the exclusive entry point for 3-hydroxypropionate into the central carbon metabolism and that the reductive conversion of 3-hydroxypropionate to propionyl-CoA is a necessary route for the assimilation of this molecule by R. sphaeroides. Our conclusion is based on the following findings: (i) crotonyl-CoA carboxylase/reductase, a key enzyme of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, was not essential for growth with 3-hydroxypropionate, as demonstrated by mutant analyses and enzyme activity measurements; (ii) the reductive conversion of 3-hydroxypropionate or acrylate to propionyl-CoA was detected in cell extracts of R. sphaeroides grown with 3-hydroxypropionate, and both activities were upregulated compared to the activities of succinate-grown cells; and (iii) the inactivation of acuI, encoding a candidate acrylyl-CoA reductase, resulted in a 3-hydroxypropionate-negative growth phenotype.     

Enzymes of the citramalate cycle in <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis>

Rhodospirillum rubrum is among the bacteria that can assimilate acetate in the absence of isocitrate lyase, the key enzyme of glyoxylate shunt. Previously we have suggested the functioning of a new anaplerotic cycle of acetate assimilation in this bacterium: citramalate cycle, where acetyl-CoA is oxidized to glyoxylate. This work has demonstrated the presence of all the key enzymes of this cycle in R. rubrum extracts: citramalate synthase catalyzing condensation of acetyl-CoA and pyruvate with the formation of citramalate, mesaconase forming mesaconate from L-citramalate, and the enzymes catalyzing transformation of propionyl-CoA + glyoxylate 3-methylmalyl-CoA ? mesaconyl-CoA. At the same time, R. rubrum synthesizes crotonyl-CoA carboxylase/reductase, which is the key enzyme of ethylmalonyl-CoA pathway discovered recently in Rhodobacter sphaeroides. Physiological differences between the citramalate cycle and the ethylmalonyl-CoA pathway are discussed.     

Characterization of two new facultative methanotrophs

Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Neither organism had such membranes when grown heterotrophically. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. The enzymes of this pathway were high in specific activity in cells grown on methane and were at low levels in cells grown either on heterotrophic substrates or on heterotrophic substrates plus methane. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.     

Enzyme production by genetically engineered Streptomyces strains: influence of culture conditions

Production of various extracellular enzymes (the beta-lactamases from Streptomyces albus G, Streptomyces cacaoi, Actinomadura R39, and the DD-carboxypeptidase from Streptomyces R61) by genetically engineered Streptomyces lividans TK24 in Lennox broth medium reached a maximum after 36 to 48 h. Subsequently, the enzyme activity drastically decreased probably due to an increased pH value and the production of an inactivator by Streptomyces lividans. Protease activity did not seem to play a major role. The increased pH and inactivator synthesis are related to amino acid catabolism and generally result in cellularlysis. The use of a medium where the catabolism of amino acids was made less likely by the presence of glucose and NH(4)Cl and by buffering at pH 7.4 considerably inproved the yield. Furthermore, the water activity of the medium seemed to be an important parameter for the production of extracellular proteins by genetically engineered Streptomyces. Better production was observed when the water activity was decreased to 0.96-0.98 by addition of sucrose.Under those conditions, the concentration of extracellular enzyme reached about 0.3 g (1 g in the best case)/L of culture supernantant.     

Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter sphaeroides

Aims:  To investigate the surviving capability of Rhodobacter sphaeroides under phototrophic conditions in the presence of high cobalt concentration and its influence on the photosynthetic apparatus biosynthesis.
Methods and Results:  Cells from R. sphaeroides strain R 26·1 were grown anaerobically in a medium containing 5·0 mmol l⁻¹ cobalt ions and in a control medium. Metal toxicity was investigated comparing the soluble proteome of Co²⁺-exposed cells and cells grown in control medium by two-dimensional gel electrophoretic analysis. Significant changes in the expression level were detected for 43 proteins, the majority (35) being up-regulated. The enzyme porphobilinogen deaminase (PBGD) was found down-regulated and its activity was investigated.
Conclusions:  The up-regulated enzymes mainly belong to the general category of proteins and DNA degradation enzymes, suggesting that part of the catabolic reaction products can rescue bacterial growth in photosynthetically impaired cells. Furthermore, the down-regulation of PBGD strongly indicates that this key enzyme of the tetrapyrrole and bacteriochlorophyll synthesis is directly involved in the metabolic response.
Significance and Impact of the Study:  Data and experiments show that the cobalt detrimental effect on the photosynthetic growth of R. sphaeroides is associated with an impaired expression and functioning of PBGD.