2-Hydroxyacyl-CoA lyase catalyzes acyloin condensation for one-carbon bioconversion.

Despite the potential of biotechnological processes for one-carbon (C1) bioconversion, efficient biocatalysts required for their implementation are yet to be developed. To address intrinsic limitations of native C1 biocatalysts, here we report that 2-hydroxyacyl CoA lyase (HACL), an enzyme involved in mammalian α-oxidation, catalyzes the ligation of carbonyl-containing molecules of different chain lengths with formyl-coenzyme A (CoA) to produce C1-elongated 2-hydroxyacyl-CoAs. We discovered and characterized a prokaryotic variant of HACL and identified critical residues for this newfound activity, including those supporting the hypothesized thiamine pyrophosphate-dependent acyloin condensation mechanism. The use of formyl-CoA as a C1 donor provides kinetic advantages and enables C1 bioconversion to multi-carbon products, demonstrated here by engineering an Escherichia coli whole-cell biotransformation system for the synthesis of glycolate and 2-hydroxyisobutyrate from formaldehyde and formaldehyde plus acetone, respectively. Our work establishes a new approach for C1 bioconversion and the potential for HACL-based pathways to support synthetic methylotrophy.

Salix purpurea Stimulates the Expression of Specific Bacterial Xenobiotic Degradation Genes in a Soil Contaminated with Hydrocarbons.

The objectives of this study were to uncover Salix purpurea-microbe xenobiotic degradation systems that could be harnessed in rhizoremediation, and to identify microorganisms that are likely involved in these partnerships. To do so, we tested S. purpurea's ability to stimulate the expression of 10 marker microbial oxygenase genes in a soil contaminated with hydrocarbons. In what appeared to be a detoxification rhizosphere effect, transcripts encoding for alkane 1-monooxygenases, cytochrome P450 monooxygenases, laccase/polyphenol oxidases, and biphenyl 2,3-dioxygenase small subunits were significantly more abundant in the vicinity of the plant's roots than in bulk soil. This gene expression induction is consistent with willows' known rhizoremediation capabilities, and suggests the existence of S. purpurea-microbe systems that target many organic contaminants of interest (i.e. C4-C16 alkanes, fluoranthene, anthracene, benzo(a)pyrene, biphenyl, polychlorinated biphenyls). An enhanced expression of the 4 genes was also observed within the bacterial orders Actinomycetales, Rhodospirillales, Burkholderiales, Alteromonadales, Solirubrobacterales, Caulobacterales, and Rhizobiales, which suggest that members of these taxa are active participants in the exposed partnerships. Although the expression of the other 6 marker genes did not appear to be stimulated by the plant at the community level, signs of additional systems that rest on their expression by members of the orders Solirubrobacterales, Sphingomonadales, Actinomycetales, and Sphingobacteriales were observed. Our study presents the first transcriptomics-based identification of microbes whose xenobiotic degradation activity in soil appears stimulated by a plant. It paints a portrait that contrasts with the current views on these consortia's composition, and opens the door for the development of laboratory test models geared towards the identification of root exudate characteristics that limit the efficiency of current willow-based rhizoremediation applications.

Characterization of the reaction center bound tetraheme cytochrome of Rhodocyclus tenuis.

Properties of the tetrahemic reaction center bound cytochrome have been investigated by different techniques. The mid-point potentials of the four hemes were determined by redox titration. The best fit of the data was obtained with a (n = 1) Nernst curve by using the following values of the redox parameters: Em = +420 mV for the two high-potential hemes and Em = +110 and +60 mV for the two low-potential hemes. The mid-point potentials of the two high-potential hemes are the highest reported so far. The spectral properties of the four hemes in the alpha-band have been determined by absorption spectroscopy and measurements of light-induced difference spectra in membranes of Rhodocyclus tenuis. The two high potential hemes present very similar spectra centered at 557 nm. The absorption spectra of the two low-potential hemes are very similar, and their alpha-band centered around 551 nm. Spectral properties at 100 K and the linear dichroism of optical transitions allow the determination of the relative orientations of the hemes with respect to the membrane plane. The orientation patterns thus obtained corresponds to none of the arrangements described so far for reaction center bound cytochromes.

Photoinduced cyclic electron transfer in Rhodocyclus tenuis cells: participation of HiPIP or cyt c8 depending on the ambient redox potential.

We demonstrate the participation of a cytochrome c8 and a high-potential iron-sulfur protein (HiPIP) in the photoinduced electron transfer in whole cells of Rhodocyclus tenuis depending on the redox state or background continuous illumination. At high redox potentials (above 350 mV) or under a strong background illumination (5 W m-2), the cytochrome c8 acts as the physiological electron donor to the photo-oxidized high-potential hemes of the tetraheme cytochrome bound to the reaction center. For redox potentials ranging from 200 to 310 mV or under weak background illumination (1. 25 W m-2), the electron carrier is the HiPIP. The electron transfer between cyt c8 and HiPIP and the tetraheme cytochrome has half-times of 300 and 480 micros, respectively. A slow electrogenic phase of the membrane potential is linked to their rereduction. This phase is sensitive to a specific inhibitor of the cyt bc1 complex, indicating involvement of cyt c8 and HiPIP in the photoinduced cyclic electron transfer at these two redox conditions.

1H and 13C NMR assignments and structural aspects of a ferrocytochrome c-551 from the purple phototrophic bacterium Ectothiorhodospira halophila.

Two-dimensional nuclear magnetic resonance was used to assign the 1H and 13C resonances of ferrocytochrome c-551 from Ectothiorhodospira halophila, a halophilic phototrophic purple bacterium. This 78-residue protein belongs to a small subgroup of class I cytochromes c together with the analogous cytochromes c-551 from E. halochloris and E. abdelmalekii. A nearly complete assignment of 13C resonances was obtained at natural abundance using a gradient-enhanced 1H-detected heteronuclear single quantum coherence experiment (HSQC). This was found to be extremely useful for the unambigous assignment of side chain protons. The secondary structure of the protein was determined from analyses of short- and medium-range nuclear Overhauser enhancements (NOE), amide proton exchange and 13C alpha chemical shifts. Three helices could be identified which are well conserved among the class I cytochromes c. There is some evidence for two other regions of less well defined helical structure. From a preliminary analysis of long-range NOE it is shown that in the E. halophila cytochrome c-551 the general cytochrome c fold is well conserved, including the three conserved helices (residues 2-8, 41-50, 63-76), the regions around the heme ligands (Cys14-Ser15-Ser16-Cys17-His18, Met55) and the omega loop (residues 18-28). In addition, three variable segments of the protein are discussed in detail, one of those including a cis-proline, a feature so far unique in the cytochrome c family. Structural alignments of the E. halophila cytochrome c-551 with two other Pseudomonas cytochrome c5 homologs (Azotobacter vinelandii cytochrome c5 and Chlorobium limicola cytochrome c-555) are provided which are based on sequence similarities and secondary structure alignments.

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus.

The structural genes (hup) of the H2 uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68,108 dalton) and by alignment with the NH2 amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34,256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe-4S] feredoxins.

[ATP-dependent citrate lyase in the green phototrophic bacterium, Chlorobium limicola]. / ATF-zavisimaia tsitrat-liaza u zelenoi fototrofnoi bakterii Chlorobium limicola.

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum, strains 1C and L, grown autotrophically and in media with organic substances were shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA only when ATP, CoA and Mg2+ were added. These results suggest that C. limicola f. thiosulfatophilum produces ATP-linked citrate lyase (E. C. 4.1.3.8) which may play an important role in the carbon metabolism of this bacterium.

[Comparative study of NADP-reductase properties in two species of purple bacteria]. / Sravnenie svoist' NADP-reduktazy u dvukh vidow purpurnykh bakterii.

Unlike Rhodospirillum rubrum, the highly purified preparations of NADP-reductase Thiocapsa roseopersicina are capable of reduction of cytochrome c though they do not catalyse diaphorase reaction in the presence of methyl viologen or benzyl viologen and NADH. T. roseopersicina reductase has more high temperature optimum (50-65 degrees) and more high thermal stability (65 degrees) and it is capable to catalyse diaphorase and menadione-reductase reactions under more high pH values (11.0-12.0) than NADP-reductase of R. rubrum. NADP-reductase of T. roseopersicina is more stable under storing than the enzyme from R. rubrum: the semi-inactivation period of the enzyme when storing in Ar or the air is about 10 and 4 days, respectively, and it takes about three days for R. rubrum.

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria.

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine sythase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.

Ribulose 1,5-diphosphate carboxylase and Cholorobium thiosulfatophilum.

1. Cell-free extracts of the photosynthetic bacterium Cholorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase--the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO2 fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate--the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.