Organohalide-respiring Desulfoluna species isolated from marine environments.

The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12 Cofactors for Catalysis.

The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt-free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single-crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized "entatic" states, thereby promoting the activation of B12 -cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B12 .

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.

Refined experimental annotation reveals conserved corrinoid autotrophy in chloroform-respiring Dehalobacter isolates.

Two novel chlorinated alkane-respiring Dehalobacter restrictus strains CF and DCA were isolated from the same enrichment culture, ACT-3, and characterized. The closed genomes of these highly similar sister strains were previously assembled from metagenomic sequence data and annotated. The isolation of the strains enabled experimental verification of predicted annotations, particularly focusing on irregularities or predicted gaps in central metabolic pathways and cofactor biosynthesis. Similar to D. restrictus strain PER-K23, strains CF and DCA require arginine, histidine and threonine for growth, although the corresponding biosynthesis pathways are predicted to be functional. Using strain CF to experimentally verify annotations, we determined that the predicted defective serine biosynthesis pathway can be rescued with a promiscuous serine hydroxymethyltransferase. Strain CF grew without added thiamine although the thiamine biosynthesis pathway is predicted to be absent; intracellular thiamine diphosphate, the cofactor of carboxylases in central metabolism, was not detected in cell extracts. Thus, strain CF may use amino acids to replenish central metabolites, portending entangled metabolite exchanges in ACT-3. Consistent with annotation, strain CF possesses a functional corrinoid biosynthesis pathway, demonstrated by increasing corrinoid content during growth and guided cobalamin biosynthesis in corrinoid-free medium. Chloroform toxicity to corrinoid-producing methanogens and acetogens may drive the conservation of corrinoid autotrophy in Dehalobacter strains. Heme detection in strain CF cell extracts suggests the 'archaeal' heme biosynthesis pathway also functions in anaerobic Firmicutes. This study reinforces the importance of incorporating enzyme promiscuity and cofactor availability in genome-scale functional predictions and identifies essential nutrient interdependencies in anaerobic dechlorinating microbial communities.

Anaerobic biosynthesis of the lower ligand of vitamin B12.

Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.

Insights into organohalide respiration and the versatile catabolism of Sulfurospirillum multivorans gained from comparative genomics and physiological studies.

Sulfurospirillum multivorans, a free-living ε-proteobacterium, is among the best studied organisms capable of organohalide respiration. It is able to use several halogenated ethenes as terminal electron acceptor. In this report, the complete genome sequence of S. multivorans including a comparison with genome sequences of two related non-dehalogenating species, Sulfurospirillum deleyianum and Sulfurospirillum barnesii, is described. The 3.2 Mbp genome of S. multivorans revealed a ∼ 50 kbp gene region encoding proteins required for organohalide respiration and corrinoid cofactor biosynthesis. This region includes genes for components not detected before in organohalide-respiring organisms. A transcript analysis of genes coding for some of these proteins indicates the involvement of a putative quinol dehydrogenase in organohalide respiration. The presence of genes encoding a variety of oxidoreductases reflects the organism's metabolic versatility. This was confirmed by growth studies with different electron acceptors including perchlorate and several sulfur-containing compounds. A comparison with other ε-proteobacteria indicates horizontal acquisition of many genes in the S. multivorans genome, which might be the basis of the bacterium's catabolic flexibility.

Exogenous 5,6-dimethylbenzimidazole caused production of a non-functional tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans.

Corrinoid-dependent reductive dehalogenation is mediated by phylogenetically diverse anaerobic bacteria that either synthesize corrinoids de novo or are dependent on corrinoid salvaging from the environment. The tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-negative Epsilonproteobacterium Sulfurospirillum multivorans harbours a norpseudo-B12 as corrinoid cofactor. Norpseudo-B12 differs from coenzyme B12 in the nucleotide loop structure. Adenine instead of 5,6-dimethylbenzimidazole (DMB) serves as lower ligand base of the central cobalt ion, and the nucleotide loop of norpseudo-B12 lacks a methyl group at position 176. In this study, S. multivorans was grown anaerobically with PCE in the presence of DMB. At a DMB concentration of 25 µM, the adenine moiety in the nucleotide loop of norpseudo-B12 was quantitatively replaced by DMB. The formation of the DMB-containing nor-B12 severely affected PCE-dependent growth and the PceA activity. In DMB-treated cells processing of the cytoplasmic PceA precursor was impeded, a result pointing to retarded cofactor incorporation. PceA enriched from cells cultivated with DMB contained nor-B12 . Nor-B12 purified from cells grown in the presence of DMB mediated the abiotic reductive dehalogenation of trichloroacetate to dichloroacetate at a 25-fold lower rate in comparison with norpseudo-B12 , a fact underpinning the relevance of norpseudo-B12 as efficient catalyst for reductive dehalogenation in general.

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics.

Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring bacterium, which displays extremely narrow metabolic capabilities. It grows only via coupling energy conservation to anaerobic respiration of tetra- and trichloroethene with hydrogen as sole electron donor. Dehalobacter restrictus represents the paradigmatic member of the genus Dehalobacter, which in recent years has turned out to be a major player in the bioremediation of an increasing number of organohalides, both in situ and in laboratory studies. The recent elucidation of the D. restrictus genome revealed a rather elaborate genome with predicted pathways that were not suspected from its restricted metabolism, such as a complete corrinoid biosynthetic pathway, the Wood-Ljungdahl (WL) pathway for CO2 fixation, abundant transcriptional regulators and several types of hydrogenases. However, one important feature of the genome is the presence of 25 reductive dehalogenase genes, from which so far only one, pceA, has been characterized on genetic and biochemical levels. This study describes a multi-level functional genomics approach on D. restrictus across three different growth phases. A global proteomic analysis allowed consideration of general metabolic pathways relevant to organohalide respiration, whereas the dedicated genomic and transcriptomic analysis focused on the diversity, composition and expression of genes associated with reductive dehalogenases.

The CbiB protein of Salmonella enterica is an integral membrane protein involved in the last step of the de novo corrin ring biosynthetic pathway.

We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B12) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but l-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.

Structural characterization of novel cobalt corrinoids synthesized by enzymes of the vitamin B12 anaerobic pathway.

Investigation on the use of the oxidized form (factor 3 (3a)) of the trimethylated intermediate (precorrin 3 (2)) as a substrate for the enzymes of the anaerobic pathway to vitamin B12 led to the synthesis of three pairs of novel cobalt corrinoids. The products were made with the aid of the Salmonella typhimurium enzymes CbiH, CbiF, CbiG, and CbiT, were synthesized in several 13C labeled versions, and were isolated as methylesters after esterification. Structures were determined by detailed NMR and MS analyses. Each set of products was obtained in the decarboxylated (RMe) and non-decarboxylated (R=CH2COOCH3) forms (at the C-12 position of the porphyrinoid).