Genome analysis of the steroid-degrading denitrifying Denitratisoma oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3

Genome analysis of the steroid-degrading denitrifying Denitratisoma oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3 | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Genome analysis of the steroid-degrading denitrifying Denitratisoma oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3 Yi-Lung Chen , View ORCID Profile Sean Ting-Shyang Wei , Yin-Ru Chiang doi: https://doi.org/10.1101/710707 Yi-Lung Chen a Biodiversity Research Center , Academia Sinica, Taipei 115, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sean Ting-Shyang Wei a Biodiversity Research Center , Academia Sinica, Taipei 115, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sean Ting-Shyang Wei Yin-Ru Chiang a Biodiversity Research Center , Academia Sinica, Taipei 115, Taiwan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: yinru915{at}gate.sinica.edu.tw Abstract Full Text Info/History Metrics Preview PDF Abstract Steroid hormones (androgens and estrogens) are crucial for development, reproduction, and communication of multicellular eukaryotes. The ubiquitous distribution and persistence of steroid hormones in our ecosystems have become an environmental issue due to the adverse effects on wildlife and humans upon long-term exposure. Microbial degradation is critical for the removal of steroid hormones from ecosystems. The aerobic degradation pathways for androgens and estrogens and the anaerobic degradation pathway for androgen have been studied into some details; however, the mechanism for anaerobic estrogen degradation remains completely unknown. Here, we presented the circular genomes of D. oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3, two betaproteobacteria capable of anaerobic estrogen degradation. We identified the genes involved in steroid transformation and in the anaerobic 2,3- seco pathway in both genomes. Additionally, the comparative genomic analysis revealed that genes exclusively represented in estrogen-degrading anaerobes might play a role in anaerobic estrogen catabolism. Introduction Steroid sex hormones, including androgens and estrogens, play essential roles in the physiology, development, reproduction, and behaviors of vertebrates. The occurrence and persistence of steroid sex hormones in our environments, especially in aquatic ecosystems, result in interruption for animal physiology and behavior. Lambert et al (2015) showed that for amphibian, long-term exposure to estrogens even at extremely low concentration lead to a female-dominated frog population. Moreover, estrogens not only act as endocrine disruptor but have also been classified as Group 1 carcinogens by the World Health Organization ( http://monographs.iarc.fr/ENG/Classification/latest_classif.php ). The ability to produce steroid sex hormones is only conserved in eukaryotes, but interestingly, bacteria appear to be the major steroid degraders in the biosphere ( Holert et al., 2018 ), and adopt various catabolic pathways to degrade these recalcitrant compounds depending on the oxygen availability ( Chen et al ., 2017 ; Casabon et al ., 2017 ). In general, under aerobic condition, bacteria adopt the 9,10-seco pathway ( Bergstrand et al ., 2016 ) and the 4,5-seco pathway ( Chen et al ., 2017 , 2018 ) to degrade androgens and estrogens, respectively; under anaerobic condition, denitrifying bacteria degrade androgens through the 2,3-seco pathway ( Wang et al ., 2013 ; Yang et al ., 2016 ). To date, only betaproteobacterial Denitratisoma oestradiolicum DSM 16959 ( Fahrbach et al ., 2006 ) and gammaproteobacterial Steroidobacter denitrificans DSM 18526 are capable of anaerobic estrogen degradation ( Fahrbach et al ., 2008 ); however, their anaerobic catabolic mechanism remains unclear. In this study, we sequenced and annotated the genome of two estrogen-degrading anaerobes–D. oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3–from a municipal wastewater treatment plant. The comparative genomic analysis showed that these two betaproteobacteria harbor genes involved in anaerobic degradation for steroidal ABCD rings. Moreover, the genes only identified in three estrogen-degrading anaerobes might play important roles in estrogens catabolism. Material and Methods Genome sequencing, assembling and annotation Genomic DNA of strain DSM 16959 and strain DHT3 were extracted using the Easy Tissue & Cell Genomic DNA Purification Kit (GeneMark, Taiwan). The workflow for genome sequencing and bioinfomatic analysis is availabe in Figure 1 . For strain DHT3, purified genomic DNA was sequenced on two platforms: Illumina HiSeq 2500 (Illumina Inc., San Diego, CA, USA) and PacBio RSII (Pacific Biosciences, CA, USA). Two Illumina TruSeq ® DNA PCR-Free libraries with fragment size around 170 bp (fragment library) and 555 bp (jumping library) were prepared for Illumina paired-end sequencing (2x 125bp). Subsequently, under the default settings, the adapter sequences were removed using cutadapt (v1.4.2; Martin, 2011 ) and then low-quality bases were trimmed by Seqtk (v1.2-r94; https://github.com/lh3/seqtk ). After these two trimming steps, the sequences longer than 35 nucleotides were included for succeeding analysis. For Pacific Biosciences (PacBio) platform, one SMRT cell was used for sequencing. Reads acquired from PacBio were assembled de novo using RS_HGAP_assembly.3 protocol included in SMART Portal (version 2.3.0). These assembled contigs were further corrected by the Illumina reads using bowtie2 (version 2.2.3; Langmead and Salzberg, 2012 ) for alignment, and Samtools (Version: 0.1.19-44428cd; Li et al ., 2009 ) and bcftools ( https://github.com/samtools/bcftools ) to extract consensus sequence with default setting. The genome of the strain DSM 16959 was only sequenced on Illumina HiSeq 2500 platform. Two Illumina TruSeq ® DNA PCR-Free libraries (fragment library and jumping library) were also prepared. Same bioinformatic analysis processes were applied as to strain DHT3 except for adopting AllPaths-LG ( Gnerre et al ., 2011 ) on strain DHT3 genome assemble. Download figure Open in new tab Figure 1. Genome sequencing and bioinfomatic workflow for the strain DHT3 and its functional annotation. The whole genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline ( Tatusova et al ., 2016 ) and the protein-coding genes were classified into COG category by the eggNOG-mapper ( Huerta-Cepas et al ., 2017 ). For constructing the metabolic pathway in silico , the KEGG BlastKOALA was applied ( Kanehisa et al ., 2016 ). Comparative genomic analysis The bacterial comparative genomic analysis was based on the gene orthologue shared between the genomes of 6 steroid-degrading aerobes and anaerobes, including Sphingomonas sp. strain KC8 ( Chen et al ., 2017 ), Sterolibacterium denitrificans DSM 13999 ( Warnke et al ., 2017 ), Thauera terpenica strain 58Eu ( Foss and Harder, 1998 ), strain DHT3 (this study), Denitratisoma oestradiolicum DSM 16959 (this study) and Steroidobacter denitrificans DSM 18526 ( Yang et al ., 2016 ). Their phylogeny and physiological features on steroid degradation, as well as genome accession numbers are summarized in the Table 1 . Their total protein sequences of coding region were uploaded to the web server, OrthoVenn2 ( Xu et al ., 2019 ) for comparing and annotating the gene content based on their orthology under the parameters: e-value: 1e-15 and inflation value: 1.5. View this table: View inline View popup Download powerpoint Table 1. Steroid degradation capacity under aerobic and anaerobic conditions of six different steroid degraders. +: growth, −: no growth, ND: not determined. Results and Discussion The phylogenetic analysis showed that strain DHT3 displayed highest 16S rRNA gene similarity (97.5 %) to D. oestradiolicum DSM 16959 ( Fahrbach et al ., 2006 ), suggesting that this strain belongs to the genus Denitratisoma. Therefore, this microorganism is named as Denitratisoma sp. strain DHT3 in this study. Genome of Denitratisoma sp. strain DHT3 In the present study, we obtain the high-quality circular genome of the strain DHT3, which was sequenced by two sequencing technologies. The Illumina and PacBio sequencing systems generated 11,617,819 reads (read length 125×2 bp) and 68,673 reads (read length 15,254-bp), respectively. After the quality trimming, the total read length from high-throughput sequencing was ~2,503 Mbp. Through PacBio sequencing, 19 contigs with N 50 as 3.7 Mbps were obtained. After correction by the Illumina reads, one chromosome with 3.7 Mbps was revealed with 223-fold coverage of the genome ( Table 2 ). This genome sequence is available in NCBI with the accession number of CP020914 . View this table: View inline View popup Download powerpoint Table 2. Summary of the quality trimming and assembly results. ND: not determined. Based on NCBI annotation service, strain DHT3 chromosome is 3,655,661 bp with a G+C content of 64.9 %. Up to 3,246 protein coding genes, three rRNA operons (5S, 16S and 23S), 51 tRNA for all 21 amino acids, including selenocysteine, 1 CRISPR array and 71 pseudogenes are identified. Through eggNOG-mapper analysis, 2,917 protein-coding genes are classified into COG categories and the code S (function unknown) is the most abundant group (644 genes). This result implies the physiological traits and gene functions of the DHT3 are yet to be fully explored. Bacterial steroid degradation requires coenzyme A (CoA) for the activation of the recalcitrant structures through β-oxidation reactions ( Warnke et al ., 2017 ; Casabon et al ., 2017 ; Wu et al., 2019 ). We identified several genes involved in this β-oxidation (B9N43_01490 ~ 01520, 03830 and 04285). In the strain DHT3 genome, we noted the gene cluster (B9N43_4420~4465) for 3-(7a-methyl-1,5-dioxooctahydro-1H-inden-4-yl)propanoic acid (HIP) catabolism (namely the steroid C/D-rings degradation) We also identified the genes involved in the steroid A/B-rings degradation through the 2,3-seco pathway, including the gene cluster encoding the 1-testosterone hydratase/dehydrogenase (B9N43_01910~1920) as well as the steroid dehydrogenase genes involved in steroid A-ring degradation [e.g., B9N43_03425 and _11350 (encoding putative 3α-hydroxysteroid dehydrogenase); B9N43_04410 (encoding putative 3-oxosteroid Δ 1 -dehydrogenase); B9N43 _16370 (coding for putative 3β-hydroxysteroid dehydrogenase); and B9N43_15155 (encoding putative 3-oxosteroid Δ 4 -dehydrogenase)]. Moreover, 4 sets of gene clusters encoding the putative steroid C25 dehydrogenase (B9N43_01670~01680; _5455~5470; _11160~11175; and _15060~15070) were identified. This molybdoeznyme is known to mediate anaerobic hydroxylation reactions on the tertiary carbons of the side chain of various sterols ( Warnke et al., 2017 ) but is not reportedly involved in the steroidal core-ring degradation. The functions of these steroid C25 dehydrogenase genes remain further investigation. Surprisingly, the DHT3 genome lacks the complete gene sets for glycolysis and TCA cycle (6-phophofructokinas and isocitrate dehydrogenase are absent for this central carbohydrate metabolism). It also lacks genes encoding proteins involved in pentose phosphate pathways. However, the genes for glyoxylate cycle (B9N43_03845, 03880, 03890, 05130 and 15570) and propanol-CoA metabolism (B9N43_05915, 06245, 11225, 11235 and 11240), might be able to compensate the potential deficiency of oxaloacetate, linking fatty acid degradation processes to part of the TCA cycle for producing ATP, FADH2 and NADH. Unlike the versatile anaerobic androgen degrader, Comamonas testosteroni , strain DHT3 does not have the complete genes for aerobic degradation of aromatics. The ammonia can be acquired by the dissimilatory nitrate reduction (B9N43_01365, 01370, 07440, 07445, 07455, 14760 and 14775). For anaerobic growth, nitric oxide could be produced due to nitrate reduction and denitrification (B9N43_07235, 07440, 07445, 07455, 08060, 09155, 13500, 14760 and 14775), but gaseous nitrous oxide and nitrogen are not expected since the DHT3 genome lacks the gene of nitric oxide reductase subunit C. Molybdenum is an essential cofactor of the enzymes involving denitrification and the genes for molybdate transport system are identified (B9N43_04295 ~ 04305). The strain DHT3 is not able to use sulfate as terminal electron acceptor due to the lacking of anaerobic sulfur reduction and sulfate transport system in the genome. Vitamins are essential biomolecules required for cellular metabolism. Among which, cobamides such as cobalamin are involved in biosynthesis of methionine and fatty acids in organisms among all the three domains of life ( Fang et al ., 2017 ). Although strain DHT3 possesses complete genes in the lower pathway for cobamide assembly from cobyric acid, the aliphatic side-chain bridge, and lower axial ligand (B9N43_07700, 08460, 10030, 10270 ~ 10280, 10430, 10480, 11085, 11090, 11100, 14110 ~ 14120 and 16605), strain DHT3 genome lacks many genes for de novo biosynthesis of cobyric acid via either the aerobic biosynthetic pathway or the anaerobic biosynthetic pathway. Nevertheless, complete set of biosynthetic genes for biotin (from pimeloyl-CoA to biotin; B9N43_03065, 03075, 03090 and 10790), pantothenate (B9N43_04665, 04670, 07415, 07420, 07425 and 08335), and p-aminobenzoic acid (B9N43_04985, 06085, 07405, 07565, 11365, 13845 and 14435) were identified. Genome of Denitratisoma oestradiolicum DSM 16959 After quality trimming, 13,678,478 reads were generated by the Illumina sequencing systems. The bacterial genome was assembled de novo in silico using ALLPATHS-LG, resulting in 65 contigs (>1,000 bp) with an N 50 length of 170,890 bp. The genome was also annotated using the NCBI Prokaryotic Genome Annotation Pipeline and deposited as accession number of NCXS00000000. The assumed genome size of DSM 16959 is 4,144,705 bp with a G+C content of 62.0 %. Up to 3,680 protein coding genes, 5 rRNA operons (5S, 16S and 23S), 53 tRNA and 47 pseudogenes were identified. Reported as an estrogen-denitrifying bacterium ( Fahrbach et al ., 2006 ), several steroid degradation genes were identified in DSM 16959 genome ( Table 3 ). View this table: View inline View popup Download powerpoint Table 3. Putative genes involved in the steroid degradation of three denitrifying bacteria, Denitratisoma sp. strain DHT3, D. oestradiolicum DSM 16959 and Steroidobacter denitrificans DSM 18526. Number in each parenthesis indicates total number of the involved genes. Comparative genomic analysis To further mine genes involved in steroid anaerobic catabolism, 6 bacterial genomes of different steroid degraders were chosen for this analysis. Up to 689 homologous gene clusters are shared among these genomes ( Figure 2 ), including the genes involved in the 2,3-seco pathway and in steroid C/D-rings degradation. The latter implies that HIP might be the common metabolite in either aerobic or anaerobic degradation pathways. Based on their physiological characteristics and genomic difference analysis, we found that there are 45 homologous gene clusters were only identified in these estrogen denitrifying bacteria (strain DSM 18526, DSM 16959 and DHT3), and 41 of them are single copy genes. Strikingly, 10 of these shared genes are located in two confined area in each of the genome, and most of their function are unknown through this analysis (Table S1; cluster_name: cluster0020 ~ 0028). As a result, these 10 genes might be the key to unveiling the biochemical pathway of anaerobic estrogen degradation (Table S1 and Table 3 ). Download figure Open in new tab Figure 2. Venn diagram of shared orthologus clusters (A) among the six aerobic and anaerobic steroid degraders, and (B) among three estrogen-degrading denitrifies. Footnotes The authors declare no conflict of interest. References ↵ Bergstrand , L. H. , Cardenas , E. , Holert , J. , Van Hamme , J. D. , and Mohn , W. W. ( 2016 ). Delineation of steroid-degrading microorganisms through comparative genomic analysis . MBio 7 , e00166 – 16 . doi: 10.1128/mBio.00166-16 . OpenUrl CrossRef ↵ Casabon I , Snieckus V , Crowe AM , Rogalski JC , Brown KL , Foster LJ , et al. ( 2017 ). 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Nucleic Acids Res 47 : W52 – W58 . OpenUrl ↵ Yang F-C , Chen Y-L , Tang S-L , Yu C-P , Wang P-H , Ismail W , et al. ( 2016 ). Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment . Isme J 10 : 1967 – 1983 . OpenUrl CrossRef Back to top Previous Next Posted July 22, 2019. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Genome analysis of the steroid-degrading denitrifying Denitratisoma oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3 Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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Share Genome analysis of the steroid-degrading denitrifying Denitratisoma oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3 Yi-Lung Chen , Sean Ting-Shyang Wei , Yin-Ru Chiang bioRxiv 710707; doi: https://doi.org/10.1101/710707 Share This Article: Copy Citation Tools Genome analysis of the steroid-degrading denitrifying Denitratisoma oestradiolicum DSM 16959 and Denitratisoma sp. strain DHT3 Yi-Lung Chen , Sean Ting-Shyang Wei , Yin-Ru Chiang bioRxiv 710707; doi: https://doi.org/10.1101/710707 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7970) Biochemistry (18639) Bioengineering (14770) Bioinformatics (44164) Biophysics (22465) Cancer Biology (19598) Cell Biology (26759) Clinical Trials (138) Developmental Biology (13904) Ecology (20894) Epidemiology (2067) Evolutionary Biology (25324) Genetics (16100) Genomics (23404) Immunology (18610) Microbiology (42249) Molecular Biology (17950) Neuroscience (92902) Paleontology (693) Pathology (2970) Pharmacology and Toxicology (5063) Physiology (8068) Plant Biology (15913) Scientific Communication and Education (2092) Synthetic Biology (4538) Systems Biology (10190) Zoology (2376) window.__CF$cv$params={r:'a37ed92a2b594fa7',t:'MTc4ODg3OTg5NA==',u:'01a0818d04ff7fb283935d80c227c3da',ut:'q0dOSaXqQb7yvGhkrF5iP9PsB_QqRZAlk0WgQo.Yyhc-1788879897-1.2.1.1-Qt.KM57mlYKJHlW2VB_aeAuuIQm9aTY08.kcKHzw3ParTM8.qMO4ft.SfrnSALKHawTnzDjyrzU8Bm6hgWu23O_HP7Cbtau0Y2nrjx0FCSI',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();