RESEARCH ARTICLE Open Access The Genome Of Pelobacter Carbinolicus .
Aklujkar et al. BMC Genomics 2012, 0RESEARCH ARTICLEOpen AccessThe genome of Pelobacter carbinolicus revealssurprising metabolic capabilities andphysiological featuresMuktak Aklujkar1*, Shelley A Haveman1, Raymond DiDonato Jr1, Olga Chertkov2, Cliff S Han2, Miriam L Land3,Peter Brown1 and Derek R Lovley1AbstractBackground: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formatetransfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetateand is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced inorder to understand its metabolic capabilities and physiological features in comparison with its relatives,acetate-oxidizing Geobacter species.Results: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol,1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATPsynthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties andgenomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagineencoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way.Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can functioncatabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that functionin 2,3-butanediol production. Pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneckin both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis toproduction of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages,including three proteins with similarity to the geopilin of electroconductive nanowires.Conclusions: Several surprising metabolic capabilities and physiological features were predicted from the genomeof P. carbinolicus, suggesting that it is more versatile than anticipated.Keywords: Pelobacter, Genome, Metabolism, Physiology, Geobacter, 2,3-butanediolBackgroundPelobacter carbinolicus is a bacterial species isolated fromanoxic mud by anaerobic enrichment on the growth substrate 2,3-butanediol, an end product of fermentations [1].P. carbinolicus was assigned to the genus Pelobacter of theDeltaproteobacteria on the basis of its ability to consumefermentatively alcohols such as 2,3-butanediol, acetoinand ethanol, but not sugars, with acetate plus ethanoland/or hydrogen as the end products [2]. Subsequently,Pelobacter species, which cannot oxidize acetate, were* Correspondence: muktak@microbio.umass.edu1University of Massachusetts Amherst, Amherst, MA 01003, USAFull list of author information is available at the end of the articleshown to be phylogenetically distributed throughout theorder Desulfuromonadales [3,4], among species that growby oxidation of acetate with either S or Fe(III) but notsulfate as the electron acceptor. P. carbinolicus belongs tothe family Desulfuromonadaceae [4-7] and Pelobacter propionicus to Geobacteraceae. The complete genome sequence of P. carbinolicus has led to the discoveries that itexpresses c-type cytochromes [8] and that it utilizes Fe(III)as a terminal electron acceptor indirectly via reduction ofS [9]. In silico metabolic models have been constructedfor P. carbinolicus and P. propionicus [10], their genomeshave been compared to those of acetate-oxidizing, nonfermentative Geobacteraceae [11], and a shortage of 2012 Aklujkar et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.
Aklujkar et al. BMC Genomics 2012, 0Pcar R0021Pcar R0026Pcar R0031Pcar R0032Pcar R0043Pcar R0047Pcar LeutRNA-Leu, fragmentPcar R0061 mutant tRNA-AsnPcar R0062 AAAA-GGGGGG-AAAAAA-Page 2 of GCGGGGGGGGGUCCCCUCAAAAAAACCCCCCCCCCCCCCAAAAAAAC U C U G G G U A G C U C A A U G G G U - A G A G C A G C G U G C A C A G A A C A C G C C U U C C C U G G U A G C U C A G U U G G U - A G A G C A G G U G G C U G U U A A C C A C C C U ----------- G U C G C A G G U U C G A G U C C U G C U - - - - - - - C C A- G U C G C U G G U U C G A G U C C G G C C C G G G G A G C C A3’ACC5’ GU AC GC GC GrtcBrtcA-1rtcA-2U GG CGD G ADC U C G AGG A G CG D AU GCC G G C CUACG CG C U G GTCU UmutanttRNA-AsnrtcR5’C 3’U AC GU ARNA-bindingAGCGGG A G CAG D AtRNA-AsnPcar R0062Gt6AUCmutant tRNAPcar R0061AGCm7GCAACAAG1.000Fraction of proteins with 2asparagines1.000Fraction of proteinsCC GG CU AAUG C A G GTCU UCGG CG CCUQC G U C CUC U C G AGGU GG UD A Am7GG CU 405060708090Minimum number of asparagines0246810121416Minimum asparagine demand indexP. carbinolicusD. acetoxidansP. carbinolicusD. acetoxidansG. metallireducensG. sulfurreducensG. metallireducensG. sulfurreducensG. bemidjiensisG. bemidjiensisFigure 1 The mutant tRNA-Asn of P. carbinolicus and patterns of asparagine usage in proteins. The alignment (top) shows thatPcar R0061 lacks features typical of the six tRNA-Leu species; it is a mutated copy of tRNA-Asn. The cloverleaf diagrams of tRNA-Asn (left) andthe Pcar R0061 transcript (right) illustrate that base-pairing is retained through reciprocal mutations and unlike the extended CCA-30 end of tRNAAsn, the 30 end of a mature Pcar R0061 transcript is predicted to be recessed and possibly longer by two bases. The Pcar R0061 transcript maybe modified similarly to tRNA-Asn, except for the queuosine and threonylcarbamyl modifications of the anticodon loop. The operon diagrams(middle) show that Pcar R0061 is in one of two gene clusters encoding RNA 30-phosphate cyclases. The graphs (bottom) show that the P.carbinolicus genome encodes fewer proteins with either more than 50 asparagine residues (left) or an asparagine demand index above 7.0 (right),compared to other Desulfuromonadales.
Aklujkar et al. BMC Genomics 2012, 0histidyl-tRNA caused by the CRISPR locus has been proposed to account for the loss of some ancestral genes suchas multiheme c-type cytochromes by the P. carbinolicusgenome [12]. However, there are many features of the P.carbinolicus genome that these studies have notaddressed. The aim of this paper is to present these features as they pertain to current assumptions and questionsabout the physiology and metabolism of P. carbinolicus,from substrate uptake to enzymology to electron transferprocesses and outer surface features.Results and discussionContents of the P. carbinolicus genomeThe genome of P. carbinolicus was sequenced and theannotation was curated as detailed in the Methods section. The previous annotation consists of 3352 orfs, 33pseudogenes, and 63 structural RNA genes. During curation, 89 false orfs and one pseudogene were removed,five pseudogenes were reclassified as orfs and one orf asa pseudogene, 46 orfs and 31 pseudogenes were added,one tRNA gene was reclassified as a mutant tRNA gene,and 448 nucleotide sequence features including riboswitches, CRISPR spacers and multicopy sequences wereidentified. The current annotation consists of 3313 orfs,58 pseudogenes, 62 structural RNAs and 449 other nucleotide sequence features. The locations of multicopynucleotide sequences of the P. carbinolicus genome relative to genes, their coordinates and their alignments canbe found in the supplementary material (Additional file1: Table S1; Additional file 2: Figure S1).The mutant tRNAThe tRNA gene that had to be reclassified, Pcar R0061,was originally annotated as specific for leucine, but its sequence does not align with the six true tRNA-Leu genesof P. carbinolicus (Figure 1); it aligns with tRNA-Asn(Pcar R0062) except that the asparagine anticodon GUUhas mutated to a leucine anticodon CAG and a deletionof seven bases has buried the aminoacylation site (CCA30) within the acceptor stem. The deletion is expected tointerfere with 30 end trimming and aminoacylation of themutant tRNA and prevent the mistranslation of CUGleucine codons as asparagine. Another indication thatPcar R0061 may not function in protein translation isthat the universally conserved frameshift control baseU33 has mutated to A. Mutations at other positions inPcar R0061 are reciprocal (preserving base-pairing in thefolded transcript), indicating that the mutant tRNA maybe under selective pressure to maintain the cloverleaffold (Figure 1) for some function; it is not a pseudogene.The location of Pcar R0061 suggests a function in RNArepair or editing (Figure 1). On its 30 side, three genestranscribed in the same direction encode a stomatin-likemultimeric membrane protein (Pcar 2837), an RNA 30-Page 3 of 24phosphate cyclase (Pcar 2836 or rtcA-1) [13] and an RNA20,30-cyclic phosphate- -50-hydroxyl ligase (Pcar 2835 orrtcB) [14]. On the 50 side of Pcar R0061, transcribed divergently, is the transcriptional regulator of rtcAB (rtcRPcar 2838) [15]. Accordingly, the mutant tRNA may be either a substrate or a guide for the RNA-active enzymes.Another RNA 30-phosphate cyclase (Pcar 2495 or rtcA-2)and RNA-binding protein (Pcar 2498) may also participate.Asparagine metabolismThe mutant tRNA might also have a role in the synthesisof asparagine, for which no asparagine synthetase wasidentified in P. carbinolicus [10]. P. carbinolicus is predicted to convert oxaloacetate to aspartate using both anonspecific aminotransferase found in Geobacteraceae(Pcar 2772) and an aspartate-specific aminotransferase(Pcar 1573) with 30% sequence identity to that ofThermus thermophilus [16]. In T. thermophilus, whichlacks asparagine synthetase and asparaginyl-tRNA synthetase, aspartate is attached to tRNA-Asn by a nondiscriminating aspartyl-tRNA synthetase [17], thencorrected to asparaginyl-tRNA by the amidotransferasesystem (homologous to Pcar 2167-Pcar 2169). In contrast,P. carbinolicus possesses an asparaginyl-tRNA synthetase(Pcar 0586) and a discriminating aspartyl-tRNA synthetase(Pcar 1040) similar to those of Geobacteraceae, but nonon-discriminating aspartyl-tRNA synthetase. Therefore,either P. carbinolicus possesses an unidentified novel asparagine synthetase or its asparaginyl-tRNA synthetasecan be modulated to accommodate aspartate in lieu ofasparagine, with subsequent correction by the amidotransferase system. In the latter case, the role of the tRNAAsn-derived mutant tRNA might be to modulate theasparaginyl-tRNA synthetase homodimer by binding toone subunit in a manner that allows the other subunit toreact tRNA-Asn with aspartate.If asparaginyl-tRNA synthesis is difficult in P. carbinolicus, one would expect the P. carbinolicus genome to encode fewer proteins with numerous closely spacedasparagine residues than the genomes of other Desulfuromonadales. A similar expectation for a histidyl-tRNA synthesis defect was previously validated [12]. When the totalnumber of asparagine residues and the asparagine demandindex (defined as the number of asparagines divided bythe harmonic mean distance between them) were computed for every protein in P. carbinolicus and other Desulfuromonadales, the resulting patterns showed thatproteins with numerous and closely spaced asparagineresidues are in fact fewer in P. carbinolicus (Figure 1), as ifasparaginyl-tRNA is limiting.Three 2,3-butanediol dehydrogenasesThe following seven sections will focus on different growthsubstrates. The initial description of P. carbinolicus
Aklujkar et al. BMC Genomics 2012, 0Page 4 of 24established that it consumes all three stereoisomers of2,3-butanediol [1], whereas many other species are limited by the stereospecificities of their 2,3-butanedioldehydrogenases [18-20]. MDR family dehydrogenasesthat act on (R)-chiral hydroxyl groups interconvert(2R,3R)-2,3-butanediol with (R)-acetoin and/or meso-2,3butanediol with (S)-acetoin, while SDR family dehydrogenases that act on (S)-chiral hydroxyl groups interconvert(2S,3S)-2,3-butanediol with (S)-acetoin and/or meso-2,3butanediol with (R)-acetoin. Genome sequencing of P.carbinolicus revealed three 2,3-butanediol dehydrogenases(MDR budX Pcar 0330, SDR budY Pcar 0903, SDR budZPcar 2068), but the published studies have either notedonly one [11] or assigned them to only two stereoisomers[10]. The correct assignment of all three enzymes to theirsubstrates could have commercial value for the production of optically pure 2,3-butanediol [21,22]. The BudXprotein has 39% sequence identity to enzymes of Paenibacillus polymyxa [19,20,23-25] and Bacillus subtilis [26,27]that have higher activity with (2R,3R)-2,3-butanediol thanwith meso-2,3-butanediol. BudY and BudZ are mostclosely related to each other, and 40-47% identical tomeso-2,3-butanediol dehydrogenase of Klebsiella pneumoniae [28] and (2S,3S)-2,3-butanediol dehydrogenase ofCorynebacterium glutamicum [29]. The active site of theC. glutamicum enzyme excludes meso-2,3-butanediol andis formed by eleven amino acid residues [30], all of whichare conserved in BudY. Two of these residues are differentin the K. pneumoniae enzyme that excludes (2S,3S)-2,3butanediol [31], and two residues are different in BudZ(Table 1). Therefore, BudY may be tentatively annotatedas (2S,3S)-2,3-butanediol dehydrogenase and BudZ asmeso-2,3-butanediol dehydrogenase, assignments that willhave to be validated experimentally. P. carbinolicus doesnot grow on (2S,3S)-2,3-butanediol alone as it does withthe other stereoisomers (S. Haveman, unpublished), suggesting that BudY may not be expressed constitutively andBudZ may have a strong preference for meso-2,3butanediol.Expression of budX and budZ is upregulated duringgrowth of P. carbinolicus on racemic acetoin comparedto growth on ethanol [9]. This may mean that the twoenzymes act in concert to interconvert the stereoisomersof acetoin through meso-2,3-butanediol. The stereospecificity of acetoin dehydrogenase has not been determinedexperimentally, but Neisseria winogradskyi and Micrococcus ureae oxidize meso-2,3-butanediol through (S)acetoin only [18,20]. If acetoin dehydrogenase prefers(S)-acetoin, P. carbinolicus could use first BudZ to reduce the carbonyl group of (R)-acetoin to an (S)-chiralhydroxyl group in meso-2,3-butanediol, then BudX tooxidize the (R)-chiral hydroxyl group to a carbonylgroup in (S)-acetoin. Strains of P. carbinolicus growingon acetoin transiently accumulate meso-2,3-butanediolto a lesser extent than optically active 2,3-butanediol[32], consistent with conversion of (R)-acetoin via meso2,3-butanediol to (S)-acetoin for degradation while(2R,3R)-2,3-butanediol serves as an electron sink. Expression of budY does not change during growth on racemic acetoin [9], consistent with the prediction thatBudY has no activity on meso-2,3-butanediol. ThePcar 2067 gene on the 30 side of budZ, encoding anSDR family oxidoreductase, is also upregulated on acetoin and should be investigated for a possible role in acetoin/2,3-butanediol metabolism.The acetoin dehydrogenase gene clusterGenome sequencing revealed that the previouslysequenced acetoin dehydrogenase genes acoABCSL ofP. carbinolicus [33] are within a cluster of 28 genes(Additional file 3: Table S2) mostly upregulated duringgrowth on acetoin and all transcribed in the same direction [9]. The third gene of this cluster is budX(Pcar 0330) and the seventh gene (Pcar 3424) encodes aTable 1 Conservation of active site residues of C. glutamicum (2S,3S)-2,3-butanediol dehydrogenase in Pcar 0903,Pcar 2068 and Ppro 3110, compared to meso-2,3-butanediol dehydrogenase of K. pneumoniaeK. pneumoniaePcar 0903 BudYPcar 2068 BudZPpro Y161P184P182P194P187P191G185G183G195G188G192C. W192W190W202W195W199I195I193I205I198I202Mutations that could alter the substrate specificity from (2S,3S)-2,3-butanediol to meso-2,3-butanediol are indicated in italics.
Aklujkar et al. BMC Genomics 2012, 0Page 5 of 24small protein similar to the C-termini of BudY and BudZ(Figure 2), which might function as a modulator of 2,3butanediol metabolism. The Pcar 0329 gene on the 50 sideof budX encodes a multitransmembrane protein thatmight facilitate transport of acetoin and 2,3-butanediolacross the inner membrane, and Pcar 0334, the eighthgene of the cluster, encodes a possible modulator of transport, a protein of the DUF190 family distantly related tothe GlnK protein that controls the ammonium transportchannel. Five genes of the cluster (Pcar 0337, Pcar 0338,Pcar 0339, Pcar 0340, Pcar 0342) encode a partial set ofenzymes for biosynthesis of thiamin, a cofactor of acetoindehydrogenase; amidst them is the acoX gene of unknownfunction (Pcar 0341) that is typical of acetoin dehydrogenase gene clusters. The P. carbinolicus genome possessesseemingly redundant genes for each thiamin biosynthesis enzyme (Additional file 4: Table S3), and mostare quite divergent in sequence from their homologs inGeobacteraceae, while thiH has not been identified inany Geobacteraceae genome. P. carbinolicus has onlyone source of lipoate, the other cofactor of acetoin dehydrogenase: the lipB (Pcar 0350) gene product transfers an octanoyl group from an acyl carrier protein tothe enzyme and then the acoS (Pcar 0346) gene product converts it to a dihydrolipoyl group. P. carbinolicuslacks the lplA gene of Geobacteraceae to attach freeoctanoate or dihydrolipoate to enzymes, and althoughtheir lipB gene product sequences align well, acoS isvery different in sequence from its counterpart in Geobacteraceae, lipA. Altogether, the ancillary enzymes ofacetoin dehydrogenase appear to be a mosaic of genesof various origins.AcoR, the activator of acetoin dehydrogenase gene expression [34,35], has three counterparts in P. carbinolicusencoded by acoR-1 (Pcar 0336) in the acetoin dehydrogenase gene cluster, acoR-2 (Pcar 0902) next to budY, andacoR-3 (Pcar 1734) next to a gene encoding an oxidoreductase of the aldo/keto reductase family (Pcar 1733) thatshould be investigated for a possible function in acetoin/2,3-butanediol metabolism. The three AcoR proteins share52-73% sequence identity. Their multiplicity suggests thatcontrol of acetoin/2,3-butanediol metabolism in P. carbinolicus may be more complex than in other species. Indeed, our unpublished microarray data for P. carbinolicusgrowing by disproportionation of 2,3-butanediol to ethanol plus acetate indicate 4.5-fold and 9.7-fold upregulationof acoR-2 and acoR-3, respectively, compared to growthPcar 3424BudY (Pcar 0903)BudZ (Pcar 2068)K. pneumoniaeC. AEYFLCVVVVVSSASSCYYFYLLLLLVAVAASSSSSEQEEPDDDNDby oxidation of 2,3-butanediol to acetate, and 5.6-foldand 9.2-fold upregulation, respectively, compared togrowth by oxidation of ethanol to acetate. None of thethree acoR genes changes expression during growth onacetoin.Other gene products of the cluster (Pcar 0333,Pcar 0349, Pcar 0351) are predicted to act on acyl-CoAsubstrates (Additional file 3: Table S2), which is surprising because there is not a single acyl-CoA dehydrogenase, enoyl-CoA hydratase, or thiolase gene inP. carbinolicus. These enzymes might degrade a byproduct of acetoin dehydrogenase formed by accidentalaldol addition of acetyl-CoA to acetaldehyde (Figure 3).Consistent with this prediction, acetate is a minor product when P. carbinolicus oxidizes 1,3-butanediol to 3hydroxybutanoate [32].Metabolism of glycerol and 1,3-propanediolP. carbinolicus was initially described as unable to degrade glycerol [1], but some strains in pure culturedisproportionate glycerol to 1,3-propanediol plus 3hydroxypropanoate with acetate as a carbon source [36]and the type strain utilizes glycerol with Geobacter sulfurreducens as a syntrophic partner (Z. Summers, personal communication). Therefore, an attempt was madeto delineate the pathway of glycerol metabolism in P.carbinolicus based on its genome (Figure 4). The glycerol dehydratase (Pcar 1397) and activating enzyme(Pcar 1396) of P. carbinolicus are 57% and 38% identicalto characterized homologs in Clostridium butyricum[37], respectively. The C. butyricum enzyme dehydratesboth glycerol to 3-hydroxypropanal and 1,2-propanediol topropanal, consistent with utilization of 1,2-propanediolby a P. carbinolicus strain [32]. Oxidation of 3hydroxypropanal to 3-hydroxypropanoate may yield oneATP if 3-hydroxypropanoyl-CoA is an intermediate. P.carbinolicus possesses multiple predicted isozymes ofacetaldehyde dehydrogenase (Pcar 1246, Pcar 2758,Pcar 2851), phosphate acetyltransferase (Pcar 2542 andPcar 2850) and acetate kinase (Pcar 2543 and Pcar 0557)that could nonspecifically catalyze these reactions. The finalATP-yielding step might also be catalyzed by propanoatekinase (Pcar 2427) or butanoate kinase (Pcar 2852). Tooxidize 3-hydroxypropanoate to 3-oxopropanoate, acandidate alcohol dehydrogenase (Pcar 2506) isencoded next to the gene for the next enzyme, a decarboxylating AGGGGGGGGGTILMMVVVLVCLMYFS GNNNNIA N E G C F L R CIP H F Q SFigure 2 Alignment of the protein sequence of Pcar 3424, a hypothetical protein encoded near budX, with the C-termini ofBudY and BudZ of P. carbinolicus and their characterized homologs: meso-2,3-butanediol dehydrogenase of K. pneumoniae and(2S,3S)-2,3-butanediol dehydrogenase of C. glutamicum.F
Aklujkar et al. BMC Genomics 2012, 0Page 6 of 24O OHCH3-C-C-CH3H(S)-acetoin Pcar 0343 NAD CoAPcar 0344 Pcar 0345 Pcar 0347NADH H OOCH3-CHacetaldehydeOH CH3-C S-CoAacetyl-CoACH3OOCH3-C-CH2-C S-CoAHO-C-CH2-C oyl-CoANADPcar 0333?Pcar 0349?NADH H OOCH2-C-CH2-C S-CoA3-oxobutanoyl-CoAH2OPcar 0351?H OOCH3-COacetate CH3-C S-CoAacetyl-CoAFigure 3 Hypothesized roles of acyl-CoA-active genes of theacetoin dehydrogenase gene cluster in P. carbinolicus. Theproducts of acetoin dehydrogenase, acetaldehyde and acetyl-CoA,might accidentally undergo aldol addition before leaving the activesite, forming either isomer of 3-hydroxybutanoyl-CoA. Themethylmalonyl-CoA epimerase family protein (Pcar 0333) mightconvert the (R)-isomer to the (S)-isomer, which 3-hydroxyacyl-CoAdehydrogenase (Pcar 0349) can oxidize. A hydrolase/acyltransferaseencoded in the cluster (Pcar 0351) might split 3-oxobutanoyl-CoAinto acetate plus acetyl-CoA.dehydrogenase (Pcar 2505) with 41% sequence identityto that of B. subtilis [38]. This step produces acetylCoA, which yields one ATP upon conversion to acetate.Of the predicted energy yield of two ATP per glycerolmolecule in syntrophic culture, a part must be expendedto convert three NADH to hydrogen/formate molecules,which G. sulfurreducens consumes along with acetate.P. carbinolicus possesses a 1,3-propanediol dehydrogenase (Pcar 2510) that is 66% identical to the characterized K. pneumoniae enzyme [39]. Thus, the machinerymay be present for P. carbinolicus in pure culture toderive two ATP from fermentation of four glycerolmolecules to three molecules of 1,3-propanediol and oneof acetate (Figure 4). P. carbinolicus in syntrophic culturemay also oxidize 1,3-propanediol to yield two ATP, partof which it must expend to convert four NADH to fourhydrogen/formate molecules to transfer to a syntrophicpartner [32]. Nearby the 3-oxopropanoate dehydrogenasegene are genes for a hydrogenase (hndD-1 Pcar 2502)and an NADPH oxidoreductase subunit (Pcar 2503)similar to SfrB of Geobacteraceae [40] that together maydispose of electrons from glycerol and 1,3-propanediol.The glycerol dehydratase gene cluster and the 1,3propanediol dehydrogenase gene cluster share severalnotable features (Additional file 3: Table S2). ThePcar 1398 gene on the 50 side of the glycerol dehydratase genes and the Pcar 2509 gene on the 30 side of the1,3-propanediol dehydrogenase gene encode multitransmembrane proteins that share 47-54% sequence identitywith the predicted acetoin/2,3-butanediol channel(Pcar 0329). These two proteins may facilitate diffusionof glycerol and 1,3-propanediol, respectively. ThePcar 2508 gene encodes a DUF190 family protein thatmay modulate one or both channels. Three outer membrane proteins sharing 49-56% sequence identity areencoded by Pcar 1395 on the 30 side of the glyceroldehydratase genes, by Pcar 2512, which is transcribeddivergently from the 1,3-propanediol dehydrogenasegene, and by Pcar 3009. They may facilitate diffusionacross the outer membrane for glycerol, 1,3-propanediol,and acetoin/2,3-butanediol, respectively. Yet anothertriad of paralogous genes (Pcar 1394, Pcar 2515,Pcar 2884; 55-62% sequence identity) linked to thesegene clusters encodes radical SAM domain oxidoreductases whose substrates are unknown.Metabolism of 1,2-ethanediolP. carbinolicus can grow by disproportionation of 1,2ethanediol to ethanol plus acetate [1], yielding 0.5 ATP(Figure 4). However, its genome does not encode athree-subunit adenosylcobalamin-dependent diol dehydratase [41] to convert 1,2-ethanediol to acetaldehyde.The 1,2-ethanediol dehydratase of P. carbinolicus strainsseems to be more oxygen-sensitive [36]; it may be a glycyl radical enzyme encoded by Pcar 0937 (34% identityto glycerol dehydratase Pcar 1397), and Pcar 0943 (38%identity to Pcar 1396) may encode its activating enzyme.The intervening genes are of uncertain relevance to 1,2ethanediol metabolism (Additional file 3: Table S2). Thereactions of glycerol and 1,2-ethanediol metabolism aremissing from the published metabolic model of P. carbinolicus [10], which attributes a pyruvate formate-lyasefunction to both dehydratases on the basis of similarityto an Escherichia coli protein for which such a functioncould not be substantiated [42]. Experimental validation
Aklujkar et al. BMC Genomics 2012, 0Page 7 of 24NADH H H2-OHPcar 1397 3-hydroxypropanalPcar 1396glycerolNAD CoAPcar 25101,3-propanediolPcar 1246? Pcar 2758? Pcar 2851?NADH H OHPO42-CoAADPATPOOOHO-CH2-CH2-C O-P-O-HO-CH2-CH2-C S-CoAHO-CH2-CH2-C-O-Pcar 2543?O3-hydroxypropanoyl-CoA Pcar 2542?Pcar 2850?Pcar 0557?3-hydroxypropanoyl-phosphate Pcar 2427?Pcar 2852?3-hydroxypropanoateNADPcar 2506?OHO-CH2-CH2-OH NH -CH -CH -OH322Pcar 0937 Pcar 0943?ethanolamine or choline Pcar 0467Pcar 0491NH4 or (CH3)N HH2OethanolNAD CoAOCH3-CH2-OH3-oxopropanoateNAD CoAPcar 2505NADH CO2NADH H OCH3-C S-CoACH3-CHNAD NADH H Pcar 0251?Pcar 0255?Pcar 1594?Pcar 2847?Pcar 2848?Pcar 1246?Pcar 2758?Pcar 2851?acetaldehydeFd H2OFd2e (3) H Oacetyl-CoAPcar 0254?Pcar 2542Pcar 0456?Pcar 2850Pcar 0665?Pcar 2853? Pcar 2543OPcar 0557HPO42CoAOCH3-C )3N -CH2-CH2-OHNADH H ATPADPOacetyl-phosphateFigure 4 Metabolic pathways for oxidation of glycerol, 1,3-propanediol, 1,2-ethanediol, ethanolamine, choline and ethanol byP. carbinolicus. Each of these substrates is converted to acetaldehyde, which is either oxidized to acetyl-CoA to yield energy by substrate-levelphosphorylation or reduced to ethanol to dispose of NADH or oxidized to acetate to supply doubly reduced ferredoxin (Fd2e).of 1,2-ethanediol dehydratase function will surely provevaluable.Metabolism of ethanolamine and cholineDuplicate genes encoding ethanolamine ammonia-lyase(eutBC-1 Pcar 0491, eutBC-2 Pcar 0467, 70% identical insequence), each being an unusual fusion of the large andsmall subunits, were found in the genome of P. carbinolicus,strains of which grow by splitting ethanolamine or cholineinto ammonia or trimethylamine plus acetaldehyde, whichis disproportionated to ethanol plus acetate (Figure 4) [36].The duplication suggests that ethanolamine ammonialyase and choline trimethylamine-lyase may be distinctenzymes. The genes surrounding the two lyase genes arealso duplicates, and encode periplasmic substrate-bindingproteins (Pcar 0492, Pcar 0468) and multitransmembraneproteins (Pcar 0490, Pcar 0466) that may mediate uptakeof ethanolamine or choline, as well as proteins of unknown function (Pcar 0493, Pcar 0469) of which a thirdparalog (Pcar 1023) is encoded next to one of the twoammonium transporters. The ethanolamine ammonia-lyase gene cluster consists of 43 genes transcribed in thesame direction (Additional file 3: Table S2), and encodesone of the four predicted acetaldehyde:ferredoxin oxidoreductases (aorA-2 Pcar 0456).The genes between eutBC-1 and eutBC-2 have functions in biosynthesis of cobalamin, the cofactor of ethanolamine ammonia-lyase (Additional file 3: Table S2).Some of them are seemingly redundant with genes elsewhere in the genome (Additional file 4: Table S3), andsome have diverged considerably from their counterpartsin Geobacteraceae. Notably, there is
The location of Pcar_R0061 suggests a function in RNA repair or editing (Figure 1). On its 3 0 side, three genes transcribed in the same direction encode a stomatin-like multimeric membrane protein (Pcar_2837), an RNA 30-phosphate cyclase (Pcar_2836 or rtcA-1) [13] and an RNA 20,30-cyclic phosphate--5 0-hydroxyl ligase (Pcar_2835 or rtcB) [14].
May 02, 2018 · D. Program Evaluation ͟The organization has provided a description of the framework for how each program will be evaluated. The framework should include all the elements below: ͟The evaluation methods are cost-effective for the organization ͟Quantitative and qualitative data is being collected (at Basics tier, data collection must have begun)
Silat is a combative art of self-defense and survival rooted from Matay archipelago. It was traced at thé early of Langkasuka Kingdom (2nd century CE) till thé reign of Melaka (Malaysia) Sultanate era (13th century). Silat has now evolved to become part of social culture and tradition with thé appearance of a fine physical and spiritual .
On an exceptional basis, Member States may request UNESCO to provide thé candidates with access to thé platform so they can complète thé form by themselves. Thèse requests must be addressed to esd rize unesco. or by 15 A ril 2021 UNESCO will provide thé nomineewith accessto thé platform via their émail address.
̶The leading indicator of employee engagement is based on the quality of the relationship between employee and supervisor Empower your managers! ̶Help them understand the impact on the organization ̶Share important changes, plan options, tasks, and deadlines ̶Provide key messages and talking points ̶Prepare them to answer employee questions
Dr. Sunita Bharatwal** Dr. Pawan Garga*** Abstract Customer satisfaction is derived from thè functionalities and values, a product or Service can provide. The current study aims to segregate thè dimensions of ordine Service quality and gather insights on its impact on web shopping. The trends of purchases have
Chính Văn.- Còn đức Thế tôn thì tuệ giác cực kỳ trong sạch 8: hiện hành bất nhị 9, đạt đến vô tướng 10, đứng vào chỗ đứng của các đức Thế tôn 11, thể hiện tính bình đẳng của các Ngài, đến chỗ không còn chướng ngại 12, giáo pháp không thể khuynh đảo, tâm thức không bị cản trở, cái được
Amendments to the Louisiana Constitution of 1974 Article I Article II Article III Article IV Article V Article VI Article VII Article VIII Article IX Article X Article XI Article XII Article XIII Article XIV Article I: Declaration of Rights Election Ballot # Author Bill/Act # Amendment Sec. Votes for % For Votes Against %
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