RNA-activated protein cleavage with a CRISPR-associated endopeptidase.

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.

The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.

High CO2 levels drive the TCA cycle backwards towards autotrophy.

It has recently been shown that in anaerobic microorganisms the tricarboxylic acid (TCA) cycle, including the seemingly irreversible citrate synthase reaction, can be reversed and used for autotrophic fixation of carbon1,2. This reversed oxidative TCA cycle requires ferredoxin-dependent 2-oxoglutarate synthase instead of the NAD-dependent dehydrogenase as well as extremely high levels of citrate synthase (more than 7% of the proteins in the cell). In this pathway, citrate synthase replaces ATP-citrate lyase of the reductive TCA cycle, which leads to the spending of one ATP-equivalent less per one turn of the cycle. Here we show, using the thermophilic sulfur-reducing deltaproteobacterium Hippea maritima, that this route is driven by high partial pressures of CO2. These high partial pressures are especially important for the removal of the product acetyl coenzyme A (acetyl-CoA) through reductive carboxylation to pyruvate, which is catalysed by pyruvate synthase. The reversed oxidative TCA cycle may have been functioning in autotrophic CO2 fixation in a primordial atmosphere that is assumed to have been rich in CO2.

The 5,6,7,8-Tetrahydro-2-Naphthoyl-Coenzyme A Reductase Reaction in the Anaerobic Degradation of Naphthalene and Identification of Downstream Metabolites.

Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-coenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD+-dependent ß-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent ß-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a ß'-hydroxy-ß-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate cis-2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene.IMPORTANCE Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via in vitro enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of ß-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi.org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.

Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes.

CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.

The class II benzoyl-coenzyme A reductase complex from the sulfate-reducing Desulfosarcina cetonica.

Benzoyl-CoA reductases (BCRs) catalyse a key reaction in the anaerobic degradation pathways of monocyclic aromatic substrates, the dearomatization of benzoyl-CoA (BzCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at the negative redox potential limit of diffusible enzymatic substrate/product couples (E°' = -622 mV). A 1-MDa class II BCR complex composed of the BamBCDEGHI subunits has so far only been isolated from the Fe(III)-respiring Geobacter metallireducens. It is supposed to drive endergonic benzene ring reduction at an active site W-pterin cofactor by flavin-based electron bifurcation. Here, we identified multiple copies of putative genes encoding the structural components of a class II BCR in sulfate reducing, Fe(III)-respiring and syntrophic bacteria. A soluble 950 kDa Bam[(BC)2 DEFGHI]2 complex was isolated from extracts of Desulfosarcina cetonica cells grown with benzoate/sulfate. Metal and cofactor analyses together with the identification of conserved binding motifs gave rise to 4 W-pterins, two selenocysteines, six flavin adenine dinucleotides, four Zn, and 48 FeS clusters. The complex exhibited 1,5-dienoyl-CoA-, NADPH- and ferredoxin-dependent oxidoreductase activities. Our results indicate that high-molecular class II BCR metalloenzyme machineries are remarkably conserved in strictly anaerobic bacteria with regard to subunit architecture and cofactor content, but their subcellular localization and electron acceptor preference may differ as a result of adaptations to variable energy metabolisms.

Discovered by genomics: putative reductive dehalogenases with N-terminus transmembrane helixes.

Attempts for bioremediation of toxic organohalogens resulted in the identification of organohalide-respiring bacteria harbouring reductive dehalogenases (RDases) enzymes. RDases consist of the catalytic subunit (RdhA, encoded by rdhA) that does not have membrane-integral domains, and a small putative membrane anchor (RdhB, encoded by rdhB) that (presumably) locates the A subunit to the outside of the cytoplasmic membrane. Recent genomic studies identified a putative rdh gene in an uncultured deltaproteobacterial genome that was not accompanied by an rdhB gene, but contained transmembrane helixes in N-terminus. Therefore, rather than having a separate membrane anchor protein, this putative RDase is likely a hybrid of RdhA and RdhB, and directly connected to the membrane with transmembrane helixes. However, functionality of the hybrid putative RDase remains unknown. Further analysis showed that the hybrid putative rdh genes are present in the genomes of pure cultures and uncultured members of Bacteriodetes and Deltaproteobacteria, but also in the genomes of the candidate divisions. The encoded hybrid putative RDases have cytoplasmic or exoplasmic C-terminus localization, and cluster phylogenetically separately from the existing RDase groups. With increasing availability of (meta)genomes, more diverse and likely novel rdh genes are expected, but questions regarding their functionality and ecological roles remain open.

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids.

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

Metabolic reconstruction of the genome of candidate Desulfatiglans TRIP_1 and identification of key candidate enzymes for anaerobic phenanthrene degradation.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed pollutants. As oxygen is rapidly depleted in water-saturated PAH-contaminated sites, anaerobic microorganisms are crucial for their consumption. Here, we report the metabolic pathway for anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture (TRIP) obtained from a natural asphalt lake. The dominant organism of this culture belongs to the Desulfobacteraceae family of Deltaproteobacteria and genome-resolved metagenomics led to the reconstruction of its genome along with a handful of genomes from lower abundance bacteria. Proteogenomic analyses confirmed metabolic capabilities for dissimilatory sulfate reduction and indicated the presence of the Embden-Meyerhof-Parnas pathway, a complete tricarboxylic acid cycle as well as a complete Wood-Ljungdahl pathway. Genes encoding enzymes putatively involved in the degradation of phenanthrene were identified. This includes two gene clusters encoding a multisubunit carboxylase complex likely involved in the activation of phenanthrene, as well as genes encoding reductases potentially involved in subsequent ring dearomatization and reduction steps. The predicted metabolic pathways were corroborated by transcriptome and proteome analyses, and provide the first insights into the metabolic pathway responsible for the anaerobic degradation of three-ringed PAHs.

Enzymes involved in the anaerobic degradation of phenol by the sulfate-reducing bacterium Desulfatiglans anilini.

BACKGROUND: The sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet. RESULTS: The pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsß (phenylphosphate synthase beta subunit), ppcß (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors. CONCLUSIONS: The genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

Two enzymes of the acetone degradation pathway of Desulfococcus biacutus: coenzyme B12 -dependent 2-hydroxyisobutyryl-CoA mutase and 3-hydroxybutyryl-CoA dehydrogenase.

Degradation of acetone by the sulfate-reducing bacterium Desulfococcus biacutus involves an acetone-activation reaction different from that used by aerobic or nitrate-reducing bacteria, because the small energy budget of sulfate-reducing bacteria does not allow for major expenditures into ATP-consuming carboxylation reactions. In the present study, an inducible coenzyme B12 -dependent conversion of 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA was demonstrated in cell-free extracts of acetone-grown D. biacutus cells, together with a NAD+ -dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA. Genes encoding two mutase subunits and a dehydrogenase, which were found previously to be strongly induced during growth with acetone, were heterologously expressed in E. coli. The activities of the purified recombinant proteins matched with the inducible activities observed in cell-free extracts of acetone-grown D. biacutus: proteins (IMG locus tags) DebiaDRAFT_04573 and 04574 constituted a B12 -dependent 2-hydroxyisobutyryl-CoA/3-hydroxybutyryl-CoA mutase, and DebiaDRAFT_04571 was a 3-hydroxybutyryl-CoA dehydrogenase. Hence, these enzymes play key roles in the degradation of acetone and define an involvement of CoA esters in the pathway. Further, the involvement of 2-hydroxyisobutyryl-CoA strongly indicates that the carbonyl-C2 of acetone is added, most likely, to formyl-CoA through a TDP-dependent enzyme that is co-induced in acetone-grown cells and is encoded in the same gene cluster as the identified mutase and dehydrogenase.

Reversibility of citrate synthase allows autotrophic growth of a thermophilic bacterium.

Biological inorganic carbon fixation proceeds through a number of fundamentally different autotrophic pathways that are defined by specific key enzymatic reactions. Detection of the enzymatic genes in (meta)genomes is widely used to estimate the contribution of individual organisms or communities to primary production. Here we show that the sulfur-reducing anaerobic deltaproteobacterium Desulfurella acetivorans is capable of both acetate oxidation and autotrophic carbon fixation, with the tricarboxylic acid cycle operating either in the oxidative or reductive direction, respectively. Under autotrophic conditions, the enzyme citrate synthase cleaves citrate adenosine triphosphate independently into acetyl coenzyme A and oxaloacetate, a reaction that has been regarded as impossible under physiological conditions. Because this overlooked, energetically efficient carbon fixation pathway lacks key enzymes, it may function unnoticed in many organisms, making bioinformatical predictions difficult, if not impossible.

Detection of Diazotrophy in the Acetylene-Fermenting Anaerobe Pelobacter sp. Strain SFB93.

Acetylene (C2H2) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C2H2 block assays of N2O reductase, and Pelobacter acetylenicus was shown to grow on C2H2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N2ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N2ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N2ase. Diazotrophic growth was observed under N2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N2ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days.IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants.

Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila.

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. In the sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration.

HqiA, a novel quorum-quenching enzyme which expands the AHL lactonase family.

The screening of a metagenomic library of 250,000 clones generated from a hypersaline soil (Spain) allowed us to identify a single positive clone which confers the ability to degrade N-acyl homoserine lactones (AHLs). The sequencing of the fosmid revealed a 42,318 bp environmental insert characterized by 46 ORFs. The subcloning of these ORFs demonstrated that a single gene (hqiA) allowed AHL degradation. Enzymatic analysis using purified HqiA and HPLC/MS revealed that this protein has lactonase activity on a broad range of AHLs. The introduction of hqiA in the plant pathogen Pectobacterium carotovorum efficiently interfered with both the synthesis of AHLs and quorum-sensing regulated functions, such as swarming motility and the production of maceration enzymes. Bioinformatic analyses highlighted that HqiA showed no sequence homology with the known prototypic AHL lactonases or acylases, thus expanding the AHL-degrading enzymes with a new family related to the cysteine hydrolase (CHase) group. The complete sequence analysis of the fosmid showed that 31 ORFs out of the 46 identified were related to Deltaproteobacteria, whilst many intercalated ORFs presented high homology with other taxa. In this sense, hqiA appeared to be assigned to the Hyphomonas genus (Alphaproteobacteria), suggesting that horizontal gene transfer had occurred.

Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus.

BACKGROUND: The strictly anaerobic, sulfate-reducing bacterium Desulfococcus biacutus can utilize acetone as sole carbon and energy source for growth. Whereas in aerobic and nitrate-reducing bacteria acetone is activated by carboxylation with CO2 to acetoacetate, D. biacutus involves CO as a cosubstrate for acetone activation through a different, so far unknown pathway. Proteomic studies indicated that, among others, a predicted medium-chain dehydrogenase/reductase (MDR) superfamily, zinc-dependent alcohol dehydrogenase (locus tag DebiaDRAFT_04514) is specifically and highly produced during growth with acetone. RESULTS: The MDR gene DebiaDRAFT_04514 was cloned and overexpressed in E. coli. The purified recombinant protein required zinc as cofactor, and accepted NADH/NAD+ but not NADPH/NADP+ as electron donor/acceptor. The pH optimum was at pH 8, and the temperature optimum at 45 °C. Highest specific activities were observed for reduction of C3 - C5-aldehydes with NADH, such as propanal to propanol (380 ± 15 mU mg-1 protein), butanal to butanol (300 ± 24 mU mg-1), and 3-hydroxybutanal to 1,3-butanediol (248 ± 60 mU mg-1), however, the enzyme also oxidized 3-hydroxybutanal with NAD+ to acetoacetaldehyde (83 ± 18 mU mg-1). CONCLUSION: The enzyme might play a key role in acetone degradation by D. biacutus, for example as a bifunctional 3-hydroxybutanal dehydrogenase/reductase. Its recombinant production may represent an important step in the elucidation of the complete degradation pathway.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UNLABELLED: Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE: Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Fermentative Cyclohexane Carboxylate Formation in Syntrophus aciditrophicus.

Short-chain fatty acids such as acetic, propionic, butyric or lactic acids are typical primary fermentation products in the anaerobic feeding chain. Fifteen years ago, a novel fermentation type was discovered in the obligately anaerobic Deltaproteobacterium Syntrophus aciditrophicus. During fermentative growth with crotonate and/or benzoate, acetate is formed in the oxidative branch and cyclohexane carboxylate in the reductive branch. In both cases cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) is a central intermediate that is either formed by a class II benzoyl-CoA reductase (fermentation of benzoate) or by reverse reactions of the benzoyl-CoA degradation pathway (fermentation of crotonate). Here, we summarize the current knowledge of the enzymology involved in fermentations yielding cyclohexane carboxylate as an excreted product. The characteristic enzymes involved are two acyl-CoA dehydrogenases specifically acting on Ch1,5CoA and cyclohex-1-ene-1-carboxyl-CoA. Both enzymes are also employed during the syntrophic growth of S. aciditrophicus with cyclohexane carboxylate as the carbon source in coculture with a methanogen. An investigation of anabolic pathways in S. aciditrophicus revealed a rather unusual pathway for glutamate synthesis involving a Re-citrate synthase. Future work has to address the unresolved question concerning which components are involved in reoxidation of the NADH formed in the oxidative branch of the unique cyclohexane carboxylate fermentation pathway in S. aciditrophicus.

Transcriptional response of Desulfatibacillum alkenivorans AK-01 to growth on alkanes: insights from RT-qPCR and microarray analyses.

Microbial transformation of n-alkanes in anaerobic ecosystems plays a pivotal role in biogeochemical carbon cycling and bioremediation, but the requisite genetic machinery is not well elucidated.Desulfatibacillum alkenivorans AK-01 utilizes n-alkanes (C13 to C18) and contains two genomic loci encoding alkylsuccinate synthase (ASS) gene clusters. ASS catalyzes alkane addition to fumarate to form methylalkylsuccinic acids. We hypothesized that the genes in the two clusters would be differentially expressed depending on the alkane substrate utilized for growth. RT-qPCR was used to investigate ass-gene expression across AK-01's known substrate range, and microarray-based transcriptomic analysis served to investigate whole-cell responses to growth on n-hexadecane versus hexadecanoate. RT-qPCR revealed induction of ass gene cluster 1 during growth on all tested alkane substrates, and the transcriptional start sites in cluster 1 were determined via 5'RACE. Induction of ass gene cluster 2 was not observed under the tested conditions. Transcriptomic analysis indicated that the upregulation of genes potentially involved in methylalkylsuccinate metabolism, including methylmalonyl-CoA mutase and a putative carboxyl transferase. These findings provide new directions for studying the transcriptional regulation of genes involved in alkane addition to fumarate, fumarate recycling and the processing of methylalkylsuccinates with regard to isolates, enrichment cultures and ecological datasets.

Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP).

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.