Desulfotomaculum gibsoniae

Genome analysis ofstrain
GrollT a highly versatile Gram-positive sulfate-reducing
bacterium
Jan Kuever1, Michael Visser2, Claudia Loeffler3, Matthias Boll3, Petra Worm2, Diana Z.
Sousa2, Caroline M. Plugge2, Peter J. Schaap4, Gerard Muyzer5, Ines A.C. Pereira6, Sofiya
N. Parshina7, Lynne A. Goodwin8,9, Nikos C. Kyrpides8, Janine Detter9, Tanja Woyke8,
Patrick Chain8,9, Karen W. Davenport8,9, Manfred Rohde10, Stefan Spring11; Hans-Peter
Klenk11, Alfons J.M. Stams2,12
1Department of Microbiology, Bremen Institute for Materials Testing, Bremen, Germany
2Wageningen University, Laboratory of Microbiology, Wageningen, The Netherlands
3Albert-Ludwigs-University Freiburg, Institute of Biology II, Freiburg, Germany
4Wageningen University, Laboratory of Systems and Synthetic Biology, Wageningen,
The Netherlands
5Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynam-
ics, University of Amsterdam, Amsterdam, The Netherlands
6Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras,
Portugal
7Winogradsky Institute of Microbiology Russian Academy of Sciences, Moscow, Russia
8DOE Joint Genome Institute, Walnut Creek, California, USA
9Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA
10HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany
11Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany
12University of Minho, Centre of Biological Engineering, Braga, Portugal
Keywords: spore-forming anaerobes, sulfate reduction, autotrophic, anaerobic degrada-tion of aromatic compounds, complete oxidizer is a mesophilic member of the polyphyletic spore-forming genuswithin the familyhis bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of or-ganic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduc-tion of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the ab- sence of sulfate. It does not require any vitamins for growth. Here, we describe the fea-tures ofstrain GrollT together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequencedspp. to date. A total of 4,666 candidate pro-tein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA path-way, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transfor-mation and degradation of aromatic compounds, which are lacking in the other se-quencegenomes. Introduction
strain GrollT (DSM
CO2 coupled to sulfate reduction. The strain is 7213) is a mesophilic sulfate-reducing bacte- also able to grow autotrophically with H2/CO2 rium isolated from a freshwater ditch in Bre- and sulfate, and is able to ferment pyruvate and men, Northern Germany [1,2]. It grows with a crotonate. In the absence of sulfate, it grows wide range of substrates, including organic ac- slowly on H2/CO2, formate, and methoxylated ids, such as medium-chain fatty acids, short- aromatic compounds.does not re- chain fatty acids, and several aromatic com- quire vitamins for growth. pounds [1]. These substrates are degraded to The Genomic Standards Consortium Desulfotomaculum gibsoniae The genusis a heterogeneous The genus is divided group of anaerobic spore-forming sulfate- phylogenetically into different subgroups [1]. To reducing bacteria, with thermophilic, meso- get a thorough understanding of the evolution- philic, and psychrophilic members that grow at ary relationships of the different neutral or alkaline pH values [3]. Their cell wall subgroups and the physiolo- stains Gram-negative, but the ultrastructure of gy of the individual species, it is important to the cell wal is characteristic of Gram-positive have genome sequence information. Here, we bacteria [4]. They are physiologically very di- present a summary of the features o verse. In contrast to Gram-negative sulfate- strain GrollT, together with the de- reducing bacteria and closely related scription of the complete genomic sequencing very little is known about their physiology, but and annotation. A special emphasis is put on the members of this genus are known to play an im- ability of this strain to grow on a large variety of portant role in the carbon and sulfur cycle in di- aromatic compounds and the responsible genes, and its capacity for acetogenic growth in the ab- sence of sulfate. Figure 1. Neighbor joining tree based on 16S rRNA gene sequences showing the phylogenetic affiliations
ofand related species and highlighted to show the subgroups ofcluster 1is printed in bold type. The recently describe(cluster
1a), (cluster 1f)(cluster 1e), and
(cluster 1a) and the entire cluster 1g are not included in the tree. A set of
species were used as outgroup, but were pruned from the tree. Closed circles represent
bootstrap values between 75 and 100%. The scale bar represents 10% sequence difference.
Standards in Genomic Sciences Classification and features
is a member of the phylum
was shown with pyruvate, crotonate, formate, H2 hylogenetic analysis of the 16S + CO2, and methoxylated aromatic compounds as rRNA genes oshows that it clusters substrates. In the presence of an electron accep- icluster 1, subgroup b. (Fig- tor it can completely oxidize substrates to CO2. ure 1 [1]). Other species in this subgroup are Suitable electron acceptors are sulfate, thiosul- fate and sulfite. The cells oare straight or slightly curved rods (1.0-2.5 × 4-7 μm) with pointed ends (Figure 2). Spores of are spherical and located in the center is a mesophilic sulfate reducer, with of the cells, causing swelling. A summary of the an optimum growth temperature between 35- classification and general features o 37°C [1,2]. Fermentative and acetogenic growth is presented in Table 1. Figure 2. Scanning electron micrograph ofstrain GrollT. Genome sequencing and annotation
Genome project history
The genome project is listed in the Genome was selected for sequencing in the OnLine Database (GOLD) [18] as project DOE Joint Genome Institute Community Se- Gi07572, and the complete genome sequence is quencing Program 2009, proposal deposited in Genbank. Sequencing, finishing and 300132_795700 'Exploring the genetic and annotation of thegenome were per- physiological diversity of formed by the DOE Joint Genome Institute (JGI) species', because of its phylogenetic position in using state of the art sequencing technology one of thsubgroups and its [19]. A summary of the project information is ability to use aromatic compounds for growth. shown in Table 2. Desulfotomaculum gibsoniae
Table 1. Classification and general features ofstrain GrollT (DSM 7213) according to the MIGS rec-
ommendations [5].
Evidence
Property
Current classification Domain Bacteria Type strain Groll Negative with a Gram-positive cell wall Straight or slightly curved rods with point- Motile, but motility was lost during culti- Spherical and central, slightly swelling the Temperature range CO2 (autotrophic) and many organic compounds including aromatic com- Sulfate-dependent growth and fermenta-tive growth with pyruvate, crotonate, formate, H2 + CO2, and methoxylated aromatic Electron acceptor Sulfate, thiosulfate and sulfite Fresh water, mud, soil 0-35 g l-1, no addition of NaCl necessary Obligate anaerobe Biotic relationship Geographic location Grolland, Bremen, Germany Sample collection 60 cm (water), 1 cm sediment Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). Evidence codes are from the Gene Ontology project [17].
Standards in Genomic Sciences Table 2. Genome sequencing project information
Property
Finishing quality Finished Three genomic libraries: one Illumina shotgun library, one 454 standard library, and one paired end 454 library Sequencing platforms Illumina GAii, 454 Titanium 479 × Illumina; 27.2 × pyrosequence Gene calling method Prodigal, GenePRIMP Genbank Date of Release Source material identifier DSM 7213T Obtain insight into the phylogenetic and physiological diver-sity of Desulfotomacum species, Project relevance and genes for anaerobic degradation of aromatic compounds Growth conditions and DNA isolation
with parallel phrap (High Performance Software, strain GrollT, DSM 7213, was grown LLC). Possible mis-assemblies were corrected anaerobically in DSMZ medium 124a with gapResolution [22], Dupfinisher [24], or Grol Medium) [2,20] at sequencing cloned bridging PCR fragments with 35°C. DNA was isolated from 0.5-1 g of cell paste subcloning. Gaps between contigs were closed using Jetflex Genomic DNA Purification kit by editing in Consed, by PCR and by Bubble PCR (GENOMED 600100) following the standard pro- primer walks (J.-F. Chang, unpublished). A total tocol as recommended by the manufacturer. of 132 additional reactions were necessary to DNA quality was inspected according the guide- close some gaps and to raise the quality of the lines of the genome sequence laboratory. final contigs. Illumina reads were also used to correct potential base errors and increase con- Genome sequencing and assembly
sensus quality using a software Polisher devel- The genome was sequenced using a combination oped at JGI [25]. The error rate of the final ge- of Illumina and 454 sequencing platforms. Al nome sequence is less than 1 in 100,000. To- general aspects of library construction and se- gether, the combination of the Illumina and 454 quencing can be found at the JGI website [21]. sequencing platforms provided 506.2 × coverage Pyrosequencing reads were assembled using the of the genome. The final assembly is based on Newbler assembler (Roche). The initial Newbler 2,347 Mb of Illumina draft data and 133 Mb of assembly consisting of 139 contigs in one scaf- pyrosequence draft data. fold was converted into a phrap [22] assembly by making fake reads from the consensus, to col- Genome annotation
lect the read pairs in the 454 paired end library. Genes were identified using Prodigal [26] as part Illumina GAii sequencing data (2,432 Mb) was of the DOE-JGI genome annotation pipeline [27], assembled with Velvet [23] and the consensus followed by a round of manual curation using sequences were shredded into 1.5 kb over- the JGI GenePRIMP pipeline [28]. The predicted lapped fake reads and assembled together with CDSs were translated and used to search the Na- the 454 data. The 454 draft assembly was based tional Center for Biotechnology Information on 220 Mb 454 draft data and all of the 454 (NCBI) non-redundant database, UniProt, TIGR- paired end data. Newbler parameters are - Fam, Pfam, PRIAM, KEGG, COG, and InterPro da- consed -a 50 -l 350 -g -m -ml 21. The tabases. Additional gene prediction analysis and Phred/Phrap/Consed software package [22] functional annotation was performed within the was used for sequence assembly and quality as- Integrated Microbial Genomes - Expert Review sessment in the subsequent finishing process. (IMG-ER) platform [29]. After the shotgun stage, reads were assembled http://standardsingenomics.org Desulfotomaculum gibsoniae
Genome properties
The genome consists of one circular chromo-
function with the remaining annotated as hypo- some of 4,855,529 bp (45.49% GC content) and thetical proteins. The statistics of the genome includes no plasmids. A total of 4,762 genes are summarized in Table 3. 70.24% of the total were predicted, of which 4,666 are protein- genes were assigned to the COG functional cate- coding genes. In addition, 3,464 of protein cod- gories (Table 4 and Figure 3). ing genes (72.7%) were assigned to a putative Table 3. Genome statistics
Attribute
% of total
Genome size (bp) DNA coding region (bp) DNA G+C content (bp) Protein-coding genes Genes in paralog clusters Genes assigned to COGs Genes with signal peptides Genes with transmembrane helices Figure 3. Graphical map of the chromosome. From outside to the center: Genes on forward strand (color
by COG categories), genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs
red, other RNAs black), GC content (black), GC skew (purple/olive).
Standards in Genomic Sciences Table 4. Number of genes associated with the general COG functional categories
Description
RNA processing and modification Replication, recombination and repair Chromatin structure and dynamics Cell cycle control, mitosis and meiosis Nuclear structure Defense mechanisms Signal transduction mechanisms Cell wall/membrane biogenesis Extracellular structures Intracellular trafficking and secretion Posttranslational modification, protein turnover, chaperones Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metabolites biosynthesis, transport and catabolism General function prediction only Function unknown Insights into the genome
Degradation of aromatic compounds
strates m- and p-cresol since the genome of can grow on a large variety of aro- possesses one set of these genes and matic compounds (Figure 4) [1,2]. Other bacte- can only grow with p-cresol [34,35]. I ria capable of growth via anaerobic degradation m- and p-cresol are expected to be of aromatic compounds linked to nitrate reduc- converted to 3- or 4-hydroxybenzylsuccinate. tion, Fe(III) reduction, or sulfate reduction are The genes coding for enzymes involved in the much more restricted [30,31]. subsequent β-oxidation (bhsABCDEFGH), yield- In sulfate-reducing bacteria (e.g.
ing 3- or 4-hydroxybenzoyl-CoA, are also pre- methylated aromatic compounds such sent in two copies. In growth experiments tolu- as toluenes, xylenes or cresols are thought to be ene degradation was not observed for degraded via an initial fumarate addition to the [1,2]. The genome provides no oppos- methyl group followed by β-oxidation-like reac- ing information. All genes for the degradation of tions [32-34]. The genes putatively coding for the growth substrates phenylacetate and phenol the enzyme catalyzing the fumarate addition re- are present including the type of phenylphos- action (hbsABC) are present in two copies in the phate carboxylase typically found in strict an- genome ofThey might have differ- aerobes [36].
ent substrate specificities for the growth sub- Desulfotomaculum gibsoniae Figure 4. Degradation of aromatic compounds inbased on genomic data. Enzymes are highlight-
ed in red color. Possible encoding genes and their locus tags are shown below. Abbreviations:
BamBCDEFGHI, class II benzoyl-CoA reductase; BCL, benzoate CoA-ligase; BCT, succinyl-CoA:benzoate
CoA-transferase; BzdNOPQ, class I benzoyl-CoA reductase; BhsABCDEFGH, beta-oxidation of
hydroxybenzylsuccinate; DCH, cyclohexa-1,5-diene-CoA hydratase; HAD, 6-OH-cyclohex-1-ene-1-carbonyl-
CoA dehydrogenase; HbsABC, hydroxybenzylsuccinate synthase; OAH, 6-oxo-cyclohex-1-ene-1-carbonyl-
CoA hydrolase; PadBCD, phenylacetyl-CoA acceptor oxidoreductase; padEGHI, phenylglyoxylate
oxidoreductase; PadJ, phenylacetate CoA-ligase; PcmRST, 4-OH-benzoyl-CoA reductase; PPC,
phenylphosphate carboxylase; PpsAB, phenylphosphate synthase.
Standards in Genomic Sciences All genes encoding enzymes of the upper benzo- is catechol, a substrate metabolized yl-CoA degradation pathway were identified in only by a very limited number of anaerobic bac- he growth substrate benzoate is teria. The pathway of catechol metabolism via activated to benzoyl-CoA either via ATP- protocatechuate was outlined 20 years ago [2] dependent CoA-ligase (bcl) or succinyl-CoA de- and is now confirmed by the genome analysis. pendent CoA-transferase (bct) [37,38]. There are For the degradation of lignin monomers, the side two classes of dearomatizing benzoyl-CoA chains will be degraded and the methoxy-group reductases (BCRs) [39]. Class I are ATP- will be removed by o-demethylation. The genes dependent FeS enzymes composed of four dif- responsible for this mechanism are present in ferent subunits [40]. There are two subclasses of the genome (Desgi_0674 to Desgi_0676). The re- ATP-dependent BCRs of th and the sulting compounds can then be degraded by the type. ATP-independent class II BCRs pathways outlined in Figure 4. contain eight subunits and harbor a tungsten- Phylogenetic trees based on hbsA which is a containing cofactor in the active site [41]. The homolog to bssA (Figure 5A) and hbsC which is a ATP-independent class II BCR is characteristic of homolog tobssC (Figure 5B) show deeply strictly anaerobic aromatic compound degrading branching lineages for th bacteria [42]. Ithe genes of the genes and no clear affiliation to other catalytic subunit (bamB) of the class II BCR are sulfate-reducing bacteria except, in the case of present in six copies. All of the predicted seven the hbsC gene to alkane-oxidizing species. Inter- genes for the putative electron activating subu- estingly, similar genes were also found in the nits of class II BCR (bamCDEFGHI) were identi- fied in at least two copies and arranged next to each other. Surprisingly, genes of a class I BCR known to use benzoate or its hydroxyl deriva- with high similarity (47-68% amino acid identi- tives, whereas the only other species of this ge- ty) to class I BCRs of thetype nus, can grow very (bzdNOPQ) were found, but these were not lo- well on toluene [44-46]. Using the bamB and cated in a single transcriptional unit. It is unclear bamC genes for phylogenetic tree construction which of the putative BCR-encoding genes is (Figure 6A and Figure 6B), the picture is even used for benzoyl-CoA and/or 3-OH-benzoyl-CoA more heterogenous. The different genes are affil- reduction. The genes necessary to convert the iated with genes found in sulfate-reducing and product of BCRs, a cyclic conjugated dienoyl- other bacteria, hence a clear clustering cannot CoA, to 3-OH-pimelyl-CoA via modified β- be seen. Again, genome data provides some in- oxidation (dch, had, oah) are present in one copy teresting insights.is each. It is unclear whether these genes are also not described as a benzoate utilizing bacterium, involved in 3-OH-benzoyl-CoA degradation. One but seems to have some similar genes [47]. of the more unusual growth substrates of Figure 5A. Phylogenetic tree based on amino acid sequences of bssA and hbsA. The trees were calculat-ed with the "One-Click" mode of the online phylogenetic analysis program Phylogeny.fr [43]. Dots repre- sent bootstrap values between 75 and 100%. The sequences ofare printed in bold.sp. EbN1 andsp. FRC-32 are identical to Aromatoleum aromaticum and respectively. Desulfotomaculum gibsoniae Figure 5B. Phylogenetic tree based on amino acid sequences of bssC and hbsC. The trees were calculat-ed with the "One-Click" mode of the online phylogenetic analysis program Phylogeny.fr [43]. Dots repre- sent bootstrap values between 75 and 100%. The sequences ofare printed in bold.sp. EbN1 andsp. FRC-32 are identical to Aromatoleum aromaticum and respectively. Figure 6A. Phylogenetic tree based on amino acid sequences of five of the six homologs of bamB. The trees were calculated with the "One-Click" mode of the online phylogenetic analysis program Phyloge-ny.fr [43]. Dots represent bootstrap values between 75 and 100%. The sequences of are printed in bold,sp. FRC-32 is identical to Standards in Genomic Sciences Figure 6B. Phylogenetic tree based on amino acid sequences of six homologs of bamC. The trees were calculated with the "One-Click" mode of the online phylogenetic analysis program Phylogeny.fr [43]. Dots represent bootstrap values between 75 and 100%. The sequences ofare printed in bold,sp. FRC-32 is identical to
Complete substrate oxidation, autotrophic
The acetyl-CoA pathway idoes not growth and homoacetogenic growth
perform acetate oxidation, as described in The genome ofcontains putative [48], but facilitates complete oxida- genes that code for the enzymes of the complete tion of substrates leading to acetyl-CoA, auto- tricarboxylic acid (TCA) cycle: Citrate synthase, trophic growth on H2 + CO2 (or formate) in the Desgi_1296, 2412; aconitase, Desgi_1576; presence of sulfate as electron acceptor, and isocitrate dehydrogenase, Desgi_4665; 2- slow homoacetogenic growth on pyruvate, oxoacid:ferredoxin oxidoreductase, Desgi_0085- crotonate, formate, hydrogen plus carbon diox- 0088, 2095, 2585-2588, 3041-3044; succinyl- ide, and methoxylated aromatic compounds [1]. CoA synthetase, Desgi_1954-1955; succinate de- Three putative acetyl-CoA synthase encoding hydrogenase, Desgi_0077-0080, 3996-3998; genes can be found in thegenome fumarase, Desgi_0075, 1952-1953; malate dehy- (Figure 8). All three genes have a putative car- drogenase, Desgi_1960. These genes could be bon monoxide dehydrogenase catalytic subunit involved in the complete oxidation to CO2 encoding gene (cooS) downstream. However, oreover, the complete acetyl-CoA only Desgi_2051 is part of an operon structure pathway is also present in the genome of D. gib- containing other genes coding for enzymes in- soniae (Figure 7). Howeveris not volved in the acetyl-CoA pathway. able to grow on acetate with or without sulfate. Desulfotomaculum gibsoniae
C1 compound degradation
pha subunit contains a twin-arginine transloca- In addition to the three cooS genes downstream tion (tat) motif and genes encoding proteins of of the genes coding for the acetyl-CoA synthase, the Tat system; TatA (Desgi_1521) and TatC has two other cooS genes in its ge- (Desgi_1526) were found near the alpha subunit nome, Desgi_2753, and Desgi_3080. The latter coding gene. The second FDH (Desgi_2136- has a transcriptional regulator (Desgi_3081) 2139) might be a confurcating FDH. Desgi_2138 downstream and a ferredoxin (Desgi_3079) and shows similarity with the NADH binding 51kD a nitrite reductase (Desgi_3078) upstream. subunit of NADH:ubiquinone oxidoreductase Growth tests on CO have not yet been per- and Fe-S cluster binding motifs, which were formed. However, the presence of multiple cooS found in all subunits. genes with neighbor genes like ferredoxin and No methanol methyltransferase genes can be nitrate reductase, or genes coding for the acetyl- found in the genome ofwhich cor- CoA pathway indicates thatmay relates with the absence of growth on methanol [1]. Other methyltransferase genes that might can grow on formate coupled to sul- point to growth with methylated amines were fate reduction. In the genome, two putative not found, except for a possible dimethylamine formate dehydrogenases (FDHs) were found. methyltransferase beta subunit (Desgi_3904) One FDH (Desgi_1522-23) is translocated over and a cobalamin binding protein (Desgi_3903). the membrane and bound to a polysulfide However, another methyltransferase gene, reductase (NrfD)-like protein containing 10 mtbA, which is absent from the genome, is nec- trans-membrane helixes (Desgi_1524). The al- essary for growth with dimethylamine. Figure 7. Acetyl-CoA pathway inbased on genomic data. Enzymes are depicted in bold ital-
ic. Next to these enzymes are the possible encoding genes, and their locus tags. Genes with the locus tags
Desgi_2048 and Desgi_2050 putatively code for the small subunit and the large subunit of the iron-sulfur
protein, respectively. This protein is involved in transferring the methyl from tetrahydrofolate to acetyl-
CoA. Abbreviations: A-CoA S, acetyl-CoA synthetase; AcsA, carbon monoxide dehydrogenase; AcsB, ace-
tyl-CoA synthase; CFeSP, iron-sulfur protein; CH3, methyl; THF, tetrahydrofolate; MeTr, methyltransferase.
Standards in Genomic Sciences
Figure 8. Gene orientation of putative coding acetyl-CoA synthase and neighboring genes in the genome of Abbreviations: acsA, carbon monoxide dehydrogenase; acsB, acetyl-CoA synthase; acsE, methyl-
tetrahydrofolate methyltransferase; CFeSP, iron-sulfur protein; cooC, carbon monoxide dehydrogenase maturation
factor; Fe-S, Iron sulfur; hp, hypothetical protein; hyd, hydrogenase; metF, methylene-tetrahydrofolate reductase;
up, uncharacterized protein.

Propionate and butyrate oxidation
1489), which can also utilize butyrate (Figure The genome ocontains at least one 10A). Ianother gene cluster copy of genes putatively encoding enzymes in- (Desgi_1916-1925) is present which only lacks volved in propionate oxidation via the one gene coding for butyryl-CoA:acetate CoA- methylmalonyl-CoA pathway (Figure 9A). This transferase (Figure 11). Desgi_1918 and includes genes in a methylmalonyl-CoA (mmc) Desgi_1920-1925 have a similar organization to cluster (Desgi_1951-1961), which have a genetic genes found in(Dtox_1697-1703) organization similar to those seen D. kuznetzovii [51]. In addition to the genes encoding enzymes (Desku_1358-1369) and involved in butyrate β-oxidation, these clusters (Pth_1355-1368) [48-50]. contain genes for electron transfer flavoproteins However, a few differences were found. The ge- (Desgi_1920-1921 and Dtox_1698-1699) and for nome oflacks genes coding for Fe-S oxidoreductases (Desgi_1922 and methylmalonyl-CoA decarboxylase epsilon and Dtox_1700). Although Dtox_1700 is annotated as gamma subunits. Moreover, the mmc cluster of a cysteine-rich unknown protein, a protein blast contains a single gene encoding the of these ORFs against thegenome alpha subunit of succinyl-CoA synthase revealed 53.65% identity (Evalue = 0.0) with the (Desgi_1955), whereas the mmc clusters of D. putative Fe-S oxidoreductase encoded by kuznetzovii and contain Desgi_1922. Two genes encoding acyl-CoA two encoding genes. Bifurcating hydrogenases synthetases (Desgi_1916-1917) are present up- may be used to re-oxidize ferredoxin, which is stream of the acetyl-CoA dehydrogenase gene in generated by pyruvate:ferredoxin(Desgi_1918), but these are not oxidoreductase and NADH, which in turn is gen- found near this cluster inowev- erated from malate dehydrogenation for the er, these genes are present in the same gene formation of hydrogen. The membrane- cluster location in other butyrate-degrading sul- anchored extracellular formate dehydrogenases fate-reducing bacteria (SRB), namely and hydrogenases may be involved in generating a proton motive force for succinate reduction. (Desku_1226-1234) and Genes putatively coding for butyrate β-oxidation enzymes are also present in the genome o (B064DRAFT_00829-00837). Acyl-CoA ne complete cluster of genes puta- synthetases are most likely involved in the bio- tively encoding all the enzymes required to con- synthesis of coenzyme A [52]. Several other clus- vert butyrate is present (Desgi_4671-4675, Fig- ters of genes containing at least three genes en- ure 9B). Gene organization in this cluster is simi- coding enzymes involved in butyrate conversion lar to that found in D. reducens (Dred_1493- can be found in the genome of Desulfotomaculum gibsoniae Figure 9. (A) Propionate and (B) butyrate degradation pathways inbased on genomic data. The
two main gene clusters for propionate and butyrate degradation are indicated in red and blue colors. For bu-
tyrate degradation, additional gene clusters were found, shown in different colors. For the other steps in the
propionate degradation pathway, genes were found to be located in different places in the genome. Abbrevia-
tions: PCT, propionate CoA transferase; MCE, methylmalonyl-CoA epimerase; MCM, methylmalonyl-CoA
mutase; SCS, succinyl-CoA synthase; SDH, succinate dehydrogenase; FHT, fumarase (fumarate hydratase);
MDH, malate dehydrogenase; ODC, oxaloacetate decarboxylase; PFO, pyruvate: ferredoxin oxidoreductase;
PFL, pyruvate formate lyase; POT, propionyl-CoA:oxaloacetate transcarboxylase; BAT, butyryl-CoA: acetate-
CoA transferase; ACD, acyl-CoA dehydrogenase; ECH, enoyl-CoA hydratase; 3-HCD, 3-hydroxybutyryl-CoA
dehydrogenase; ACA, acetyl-CoA acetyltransferase.
Desulfotomaculum gibsoniae Grol, DSM 7213
Dred_1493- Dred_1489 Figure 10. Orthologous neighborhood genes for gene cluster Desgi_4671- 4675, encoding enzymes necessary
for butyrate degradation. Light blue – 3-hydroxybutyryl-CoA dehydrogenase (3-HCD); green – enoyl-CoA
hydratase (ECH); old pink – acetyl-CoA acetyltransferase (ACA); dark green – Acyl-CoA dehydrogenase (ACD);
red – butyryl-CoA: acetate CoA-transferase (BAT).
Standards in Genomic Sciences Desulfotomaculum gibsoniae Grol, DSM 7213
Desulfotomaculum acetoxidans, DSM 771
Desulfotomaculum alcoholivorax, DSM 16058
Desulfotomaculum kuznetsovii 17, DSM 6115
Desulfurispora thermophila, DSM 16022
Figure 11. Orthologous neighborhood genes for gene cluster Desgi_1916- 1925, encoding enzymes necessary for
butyrate degradation. Purple/pink – acyl-CoA synthetase; red – acyl-CoA dehydrogenase (ACD); orange/dark pur-
ple – electron transfer flavoprotein; yellow – Fe-S oxidoreductase; old pink – acetyl-CoA acetyltransferase (ACA);
light blue – 3-hydroxybutyryl-CoA dehydrogenase (3-HCD); green – enoyl-CoA hydratase (ECH).

Sulfate reduction
have a Gram-positive AprBA-like The genome contains single copies of the sulfate andfumaroxidans [55]. It adenyltransferase (Desgi_3703), adenosine-5´- seems that bothsp. and phosphosulfate (APS) reductase (Desgi_3701– have been the source of the 3702) and dissimilatory sulfite reductase (Desgi entire aps reductase/ QmoA complex for mem- 4661-4662) as are found in most of the other bers of the Gram-negative members of the genus [18-20]. A membrane- [55]. The genomes o bound pyrophosphatase (Desgi_4294) is used for energy regeneration as in other thermoconiculi have two different systems that spp. The QmoABC complex can be linked to the aps reductase. contains only the A and B subunit, the C subunit Ithe dsrAB (Desgi_4661–4662) is is lacking (Desgi_3699–3700). In all members of linked to the same truncated dsr operon coding the genusthe QmoAB is fol- only for dsrC and dsrMK (Desgi 4648–4649) as lowed by HdrCB (Desgi_3697–3698). This ar- in othespp [48,51,56]. rangement is identical to that seen in the closely related species "Desulforhudis audaxivator", has six [FeFe] and three [NiFe] negative and strain hydrogenases, suggesting a lower redundancy in NaphS2, which possess a Gram-positive AprBA the case of [FeFe] enzymes than other members [53]. Interestingly, the same organization is also of the genus. The [FeFe] hydrogenases include found in some phototrophic sulfur-oxidizing one membrane-associated protein (Desgi_0926- bacteria, such adentrificans, 0928) that contains a tat motif in the alpha sub- unit (Desgi_0926), suggesting an extracellular selenatireducens [54]. Other closely related localization; one monomeric hydrogenase Gram-positive SRB like (Desgi_0935) encoded close to the membrane- thermoconiculi and have a bound enzyme, which suggests the possibility of complete QmoABC system like all other SRB and co-regulation; two copies of trimeric NAD(P)- the Green Sulfur Bacteria, or have QmoAB linked dependent bifurcating hydrogenases to a Fe-S oxidoreductase/HdrD as seen in (Desgi_4669-4667 and Desgi_3197-3195); one spp. This latter modification is enzyme (Desgi_0771) that is part of a multi-gene also seen in other Gram-negative SRB, which cluster encoding two flavin-dependent Desulfotomaculum gibsoniae oxidoreductases that is also present in other lybdenum-iron cofactor biosynthesis protein spp., and one HsfB-type NifE, nitrogenase molybdenum-iron protein, al- hydrogenase (Desgi_3194) encoding a PAS- pha and beta chains, nitrogenase cofactor bio- sensing domain that is likely involved in sensing synthesis protein NifB; ferredoxin, iron only and regulation, and possibly with the bifurcating nitrogenase protein AnfO (AnfO_nitrog) Desgi_3195 hydrogenase. (Desgi_2428-2419) were detected within the The [NiFe] hydrogenases include one enzyme annotated genome sequence. Thus (Desgi_1398 – 1397) that may also be bound to probably has the capacity for nitrogen fixation. the membrane by a cytochrome b (Desgi_1402); However, the fixation of molecular nitrogen has one simple dimeric enzyme (Desgi_1231-1230); not been analyzed in this species so far. and one trimeric group 3 hydrogenase (Desgi_1166-1164), similar to methyl-viologen reducing hydrogenases from methanogens, and The work conducted by the U.S. Department of which is encoded next to a HdrA-like protein Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02- A cluster of nitrogenase genes, specifically genes 05CH11231, and was also supported by grants encoding nitrogenase iron protein, nitrogen reg- CW-TOP 700.55.343, ALW 819.02.014 of the ulatory protein PII, nitrogenase molybdenum- Netherlands Science Foundation (NWO), ERC iron protein alpha chain, nitrogenase molyb- (project 323009), and BE-Basic (project denum-iron protein beta chain, nitrogenase mo- References
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Réponses concernant le questionnaire sur l'hyperprolactinémie, MC Wimmer, interne de gynécologie-obstétrique CHU Rennes 1 « Evaluation des connaissances concernant l'hyperprolactinémie, dans la population des internes de gynécologie-obstétrique » 2 Quelques résultats et discussion Cette étude épidémiologique s'est intéressée aux connaissances des internes de gynécologie-obstétrique en France sur le thème de l'hyperprolactinémie. Le taux de réponse au questionnaire est de 17.7 % des internes. Ce taux de participation est proche d'études publiées dans ce domaine : 19 % concernant l'enseignement du siège en 2006 [15], 33.6 % concernant le formation sur la dystocie des épaules [16]. Une nouvelle relance devrait permettre d'améliorer le taux de réponse à notre questionnaire. Il est intéressant de noter une disparité au sein des régions de France, parmi les réponses. Le taux des internes de la région de Rennes est le plus élevé, traduisant qu'une motivation locale au remplissage du questionnaire peut être utile. Il serait intéressant de connaître les motifs de non remplissage du questionnaire. Le principal résultat de cette étude, est la mise en évidence de lacunes chez les internes de gynécologie obstétrique, concernant le sujet de l'hyperprolactinémie. En effet, la moyenne globale de 5,7 sur 20 (+/- 2.4). Les lacunes dominent dans le contexte de la grossesse que ce soit en physiologie ou lors d'un adénome à prolactine. Les connaissances pratiques semblent elles mieux maitrisées notamment sur la réalisation d'IRM hypophysaire en urgence lors d'une suspicion d'adénome à prolactine, sur la conduite thérapeutique en première intention, et sur les traitements hyperprolactinémiants. Près de 20 % des réponses enregistrées étaient des « ne sais pas », mettant en évidence leur clairvoyance sur leur manque de connaissances sur le sujet. Il est intéressant de noter que le cursus ne semble pas permettre une meilleure courbe d'apprentissage. En effet, les internes de fin de cursus n'ont pas obtenus de meilleurs résultats que les plus jeunes. En revanche, les internes à destinée médicale semblent avoir des connaissances plus importantes à l'inverse des internes mentionnant un cursus plus chirurgical ou orienté en obstétrique. Les résultats n'ont qu'une tendance significative due aux plus faible nombre de personne dans les groupes gynécologie médicale +/- PMA et échographie, DAN. Quelles peuvent être les principales hypothèses de ces mauvais résultats ? Les internes de gynécologie-obstétriques ne rencontrent probablement pas ou peu ces pathologies lors de leurs stages pratiques, car elles sont principalement gérées en consultation. Lors des cours théoriques, les thèmes de gynécologie endocrinienne sont peut- être abordés moins fréquemment que ceux de chirurgie ou d'obstétrique [11]. De plus, ces connaissances s'acquièrent plutôt dans des stages spécialisés (gynécologie-endocrinologie) qui ne peuvent s'intégrer dans la maquette de tous les internes. Une deuxième hypothèse est que les connaissances restent potentiellement mal intégrées et/ou mal utilisées. En 2015, Mesdag et collaborateurs ont réalisé un état des lieux sur l'enseignement en gynécologie-obstétrique en France [11]. L'offre d'enseignement est très variable sur le plan de la méthodologie employée, des formateurs et de la périodicité. L'accès est difficile de par l'éloignement géographique des lieux de stages, et des impératifs de service. Dans ce travail les étudiants auraient souhaités des cours plus axés sur des conduites à tenir en pratique, et des ateliers de formation aux gestes techniques. Cette étude concluait à la nécessité d'une réforme de l'enseignement avec comme pistes de travail l'uniformisation de l'enseignement en France, la possibilité d'un

Microsoft powerpoint - chm102chapter13a

The Rules for Boiling Points • The boiling points of compounds depend on how strongly they stick together: The more strongly they stick together, the higher the boilingpoint (the more heat it takes to rip them apart). • There are two main forces that hold molecules together: These require both positive hydrogens (from O-H or N-H bonds) and electron lone pairs (found mainly on O and N atoms)