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Complete genome sequence of Segniliparus rotundus type strain (CDC 1076T)
Standards in Genomic Sciences volume 2, pages 203–211 (2010)
Segniliparus rotundus Butler 2005 is the type species of the genus Segniliparus, which is currently the only genus in the corynebacterial family Segniliparaceae. This family is of large interest because of a novel late-emerging genus-specific mycolate pattern. The type strain has been isolated from human sputum and is probably an opportunistic pathogen. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of the family Segniliparaceae. The 3,157,527 bp long genome with its 3,081 protein-coding and 52 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain CDC 1076T (= DSM 44985 = ATCC BAA-972 = JCM 13578) is the type strain of the species Segniliparus rotundus , which is the type species of the genus Segniliparus. Besides S. rotundus, the genus Segniliparus contains currently only one additional species: S. rugosus at present. Segniliparus is currently the only genus in the family Segniliparaceae. The generic name of the genus derives from the Latin word ‘segnis’, meaning ‘slow’, and the Greek word ‘liparos’, fat/fatty, meaning ‘one with slow fats’, to indicate the possession of slow reacting fatty acids, i.e., late eluting mycolic acids detected with HPLC . The species name is derived from the Latin word ‘rotundus’, rounded, referring to the smooth, round-domed colony forms . Strain CDC 1076T was isolated from human sputum in Tennessee, USA . Currently, only one additional strain of the species, CDC 413 (with identical 16S rRNA gene sequence), is known, which has been isolated from the human nasal region in Missouri, USA . The 16S rRNA gene sequence of the type strain for the second species in the genus, S. rugosus , differs by only 1.1% from that of strain CDC 1076T. S. rugosus strains have been isolated from patients with cystic fibrosis in Australia and most probably USA [2,3], suggesting that S. rotundus could also be an opportunistic pathogen. The next closest relatives of S. rotundus outside the genus are the members of the genus Rhodococcus, which share 93.3 to 94.8% 16S rRNA genes sequence similarity with strain CDC 1076T . Environmental screens and metagenomic surveys did not detected a single phylotype with more than 90–92% 16S rRNA gene sequence similarity, indicating a rather limited ecological distribution of the members of the genus Segniliparus (status February 2010). Here we present a summary classification and a set of features for S. rotundus CDC 1076T, together with the description of the complete genomic sequencing and annotation.
Classification and features
Figure 1 shows the phylogenetic neighborhood of for S. rotundus CDC 1076T in a 16S rRNA based tree. The sequence of the sole 16S rRNA gene in the genome is identical with the previously published 16S rRNA sequence generated from DSM 44985 (AY608918).
CDC 1076T cells are short rods with 0.4µm width by 1.0–1.3 µm length (Table 1 and Figure 2), forming round, smooth, dense and domed colonies . Occasionally, v-forms are produced, but no true branching, mycelium, or spores have been reported. The colonies are non-pigmented, non-photochromogenic and do not produce a diagnostic odor . It is negative for arylsulfatase after three days but positive after 14 days. Strain CDC 1076T does not grow on MacConkey agar, is weakly positive for iron uptake, Tween opacity and Tween hydrolysis, but negative for nitrate and tellurite reduction and for growth in lysozyme (21 days) . Strain CDC 1076T does not produce niacin and develops bubbles in the semi-quantitative catalase test . Using the API CORYNE test kit, strain CDC 1076T is positive for β-glucosidase and pyrazinamidase activities and negative for alkaline phosphatase, β-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase and pyrrolidonyl arylamidase activity at 33°C . Strain CDC 1076T is susceptible to amikacin, cefoxitan, clarithromycin, ciprofloxacin, doxycycline, imipenem and sulfamethoxazole at or below the respective MIC breakpoints but intermediate to tobramycin . Glucose, maltose, D-fructose and trehalose are used as carbon source for growth with acid production, but not adonitol, L-arabinose, cellobiose, dulcitol, i-erythritol, galactose, i-myo-inositol, lactose, mannose, melibiose, raffinose, L-rhamnose, salicin, D-mannitol, D-sorbitol and sodium citrate . Strain CDC 1076T hydrolyzes urea but not acetamide, adenine, casein, citrate, aesculin, hypoxanthine, tyrosine and xanthine .
The cell wall of strain CDC 1076T contains mycolic acids and meso-diaminopimelic acid . The mycolic acid HPLC pattern is a triple cluster of contiguous eluting peaks starting at approx. 6.0 min and ending with the last peak co-eluting with the internal standard. The TLC mycolic acid pattern reveals α+- and α-mycolates . The fatty acids composition of the strain is dominated by straight-chain saturated acids such as the taxon-specific C10:0 (21.0%), C16:0 (18.5%), C14:0 (15.3%), 10-methyl-C18:0 (7.4%, tuberculostearic acid), C20:0 (4.9%), C12:0 (2.4%), C18:0 (1.9%), with some by straight-chain desaturated acids, C18:1 cis (15.1%) and C16:1ω9t (9.7%); (personal communication with R.M. Kroppenstedt). Quinones are mainly MK 8(H4) and MK 8(H2) with some MK 8(H6) and traces of MK 9(H2) (R.M. Kroppenstedt, personal communication).
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project . The genome project is deposited in the Genome OnLine Database  and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
S. rotundus CDC 1076T, DSM 44985, was grown in DSMZ medium 645 (Middlebrook Medium)  at 28°C. DNA was isolated from 1–1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with lysis modification LALMP according to Wu et al. .
Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 technologies . An Illumina GAii shotgun library with reads of 443 Mb, a 454 Titanium draft library with average read length of 304 bases, and a paired-end 454 library with average insert size of 4 Kb were generated for this genome. All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. Illumina sequencing data was assembled with VELVET  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 183 Mb 454 data, and 454 paired-end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The initial assembly contained 26 contigs in one scaffold. We converted the initial 454 assembly into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired-end library. The Phred/Phrap/Consed software package (www.phrap.com) was used for sequence assembly and quality assessment  in the following finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution (unpublished, http://www.jgi.doe.gov/), Dupfinisher , or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 108 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed genome sequences had an error rate less than one in 100,000 bp.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology In-formation (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform .
The genome consists of a 3,157,527 bp long chromosome (Table 3 and Figure 3). Of the 3,133 genes predicted, 3,081 were protein-coding genes, and 52 RNAs; 75 pseudogenes were also identified. The majority of the protein-coding genes (63.0%) were assigned with a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
Butler WR, Floyd MM, Brown JM, Toney SR, Daneshvar MI, Cooksey RC, Carr J, Steigerwalt AG, Charles N. Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov. Int J Syst Evol Microbiol 2005; 55:1615–1624. PubMed doi:10.1099/ijs.0.63465-0
Butler WR, Sheils CA, Brown-Elliott BA, Charles N, Colin AA, Gant MJ, Goodill J, Hindman D, Toney SR, Wallace RJ, Jr., et al. First Isolations of Segniliparus rugosus from patients with cystic fibrosis. J Clin Microbiol 2007; 45:3449–3452. PubMed doi:10.1128/ICM.00765-07
Hansen T, Van Kerckhof J, Jelfs P, Wainwright C, Ryan P, Coulter C. Segniliparus rugosus infection, Australia. Emerg Infect Dis 2009; 15:611–613. PubMed doi:10.3201/eid1504.081479
Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 2007; 57:2259–2261. PubMed doi:10.1099/ijs.0.64915-0
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMed
Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452
Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642
Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci 2009; 5541:184–200. doi:10.1007/978-3-642-02008-7_13
Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM, Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2010; 38:D346–D354. PubMed doi:10.1093/nar/gkp848
Ivanova N, Sikorski J, Jando M, Lapidus A, Nolan M, Lucas S, Glavina Del Rio T, Tice H, Copeland A, Cheng JF, et al. Complete genome sequence of Gordonia bronchialis type strain (3410T). Stand Genomic Sci 2010; 2:19–28. doi:10.4056/sigs.611106
Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE, III, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998; 393:537–544. PubMed doi:10.1038/31159
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed doi:10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576
Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Springer, New York, 2001, p. 119–169.
Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 1997; 47:479–491; doi:10.1099/00207713-47-2-479.
Classification of bacteria and archaea in risk groups. www.baua.de TRBA 466.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene Ontology: tool for the unification of biology. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova N, Kunin V, Goodwin L, Wu M, Tindall BJ, et al. A phylogeny-driven genomic encyclopedia of Bacteria and Archaea. Nature 2009; 462:1056–1060. PubMed doi:10.1038/nature08656
List of growth media used at DSMZ: http://www.dsmz.de/microorganisms/media_list.php
Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed doi:10.1517/146224184.108.40.2063
Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829. PubMed doi:10.1101/gr.074492.107
Sims D, Brettin T, Detter J, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F, Lucas S, et al. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci 2009; 1:12–20. doi:10.4056/sigs.761
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Genomics 2010; 11:119.
Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods (In press).
Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed doi:10.1093/bioinformatics/btp393
This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-1 and SI 1352/1-2.
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Sikorski, J., Lapidus, A., Copeland, A. et al. Complete genome sequence of Segniliparus rotundus type strain (CDC 1076T). Stand in Genomic Sci 2, 203–211 (2010). https://doi.org/10.4056/sigs.791633
- novel mycolic acid
- opportunistic pathogen