Standards in Genomic Sciences (2009) 1: 159-165 DOI:10.4056/sigs.2494 Complete genome sequence of Anaerococcus prevotii type strain (PC1T) Kurt LaButti1, Rüdiger Pukall2, Katja Steenblock2, Tijana Glavina Del Rio1, Hope Tice1, Alex Copeland1, Jan-Fang Cheng1, Susan Lucas1, Feng Chen1, Matt Nolan1, David Bruce1,3, Lynne Goodwin1,3, Sam Pitluck1, Natalia Ivanova1, Konstantinos Mavromatis1, Galina Ovchinnikova1, Amrita Pati1, Amy Chen4, Krishna Palaniappan4, Miriam Land1,5, Loren Hauser1,5, YunJuan Chang1,5, Cynthia D. Jeffries1,5, Patrick Chain1,6, Elizabeth Saunders1,3, Thomas Brettin1,3, John C. Detter1,3, Cliff Han1,3, Markus Göker2, Jim Bristow1, Jonathan A. Eisen1,7, Victor Markowitz4, Philip Hugenholtz1, Nikos C Kyrpides1, Hans-Peter Klenk2, and Alla Lapidus1* 1 DOE Joint Genome Institute, Walnut Creek, California, USA 2 DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany 3 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 4 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 5 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA 6 Lawrence Livermore National Laboratory, Livermore, California, USA 7 University of California Davis Genome Center, Davis, California, USA *Corresponding author: Alla Lapidus Keywords: Firmicutes, Clostridiales, ‘Peptostreptococcaceae’, Gram-positive, coccoid, human oral microflora, skin, non-motile, non-sporulating, anaerobic Anaerococcus prevotii (Foubert and Douglas 1948) Ezaki et al. 2001 is the type species of the genus, and is of phylogenetic interest because of its arguable assignment to the provisionally arranged family ‘Peptostreptococcaceae’. A. prevotii is an obligate anaerobic coccus, usually arranged in clumps or tetrads. The strain, whose genome is described here, was originally isolated from human plasma; other strains of the species were also isolated from clinical specimen. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the genus. Next to Finegoldia magna, A. prevotii is only the second species from the family ‘Peptostreptococcaceae’ for which a complete genome sequence is described. The 1,998,633 bp long genome (chromosome and one plasmid) with its 1852 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project. Introduction Anaerococcus  prevotii   strain   PC1T  (=   DSM  20548   =   ATCC   9321   =   JCM   6508)   is   the   type   strain   of   the   species   and   the   type   species   of   the   genus   [1].   Six   strains   of   the   species   were   characterized   by   Fou-­‐ bert  and  Douglas  in  1948,  originally  designated   as   ‘Micrococcus   prevotii’,   but   subsequently   placed   in   the  genus  Peptococcus  [2].  Based  on  a  comparative   study   published   by  Ezaki  et   al.   [3],  the  type   strain   of   P.   prevotii   was   then   transferred   to   the   genus   ‘Peptostreptococcus’   and   later   on   assigned   to   the   novel   genus   Anaerococcus   as   A.   prevotii   [1].   The   organism   is   a   Gram-­‐positive,   anaerobic,   indole-­‐ negative  coccus.   The  major  metabolic  end  product   from   metabolism   of   peptone-­‐yeast-­‐glucose   (PYG)   is   butyric   acid.   A.   prevotii   was   provisionally   as-­‐ signed   to   the   arranged   family   ‘Peptostreptococ-­ caceae’   within   the   order   Clostridiales,   also   desig-­‐ nated  as   Family  XI  Incertae   sedis  [4].   Here   we   pre-­‐ sent  a   summary   classification  and  a   set  of   features   for   A.   prevotii   strain   PC1T   together   with   the   de-­‐ The Genomic Standards Consortium Anaerococcus prevotii type strain PC1T scription   of   the   complete   genomic  sequencing   and   annotation. Classification and features Within  the  last  few  years,   several   changes   occurred   in   the   classi`ication   of   the   anaerobic   Gram-­‐positive   cocci.   There   are   currently  `ive   genera   of   anaerobic   Gram-­‐positive   cocci   which   may   be   isolated   from   humans   (Peptostreptococcus,   Peptoniphilus,   Parvi-­ monas,   Finegoldia,   and   Anaerococcus).   Members   of   the   species   A.   prevotii   are   frequently   recovered   from   human   clinical  specimens  such  as  vaginal  dis-­‐ charges  and   ovarian,   peritoneal,   sacral   or  lung   ab-­‐ scesses.   In  particular,  A.   prevotii  was   also  described   as  a  common  isolate  of  the  normal  `lora   of   skin,  the   oral   cavity   and   the   gut   [3].   Historically   the   Gram-­‐ positive   anaerobic  cocci   were   identi`ied   mainly  by   using  phenotypic  traits,  but  as  shown  by  Song  et  al.,   this  often  led  to  the  misidenti`ication  of  A.  vaginalis   strains,   which   were   mistakenly   assigned   to   A.   prevotii   or  A.  tetradius  [5].   Currently  Genbank   does   not   contain   any16S   rRNA   sequences   from   culti-­‐ vated  strains  that   can   be   clearly  linked   to  the   spe-­‐ cies  A.  prevotii  with   over  95%  gene   sequence   simi-­‐ larity.  Recently,  the  temporal  diversity  of  the  human   skin   microbiome   was   analyzed   using   16S   rRNA   gene   phylotyping.   It   is   noteworthy   that   several   clones   originated   from   different   skin   sites   (gluteal   crease,   occiput,   umbilicus,   popliteal   fossa,   volar   forearm).   These   isolates   were   taken   from   two   pa-­‐ tients  and  showed   close   relationships  to  A.  prevotii   [6].   No   closely   related   isolates   or   uncultivated   clones   with   more   than   84%   16S   rRNA   gene   se-­‐ quence   identity   are   recorded   from   global   ocean   screenings   and   environmental  samples   (except  for   human  skin). Figure  1   shows  the   phylogenetic   neighborhood  of  A.   prevotii  strain   PC1T   in  a  16S   rRNA   based   tree.   The   four  16S   rRNA  gene  copies  in  the  genome   of  strain   PC1T  differ  by  up  to  15  nucleotides  from   each  other,   and  by  up   to   9  nucleotides  from  the  previously  pub-­‐ lished   16S   rRNA   sequence   generated   from   strain   CCUG   41932   (AF542232).   The   difference   between   the   genome   data   and   the   reported   16S   rRNA  gene   sequence   is  most  likely  due   to  sequencing  errors  in   the  previously  reported  sequence  data.   Figure 1. Phylogenetic tree highlighting the position of A. prevotii PC1T relative to all type strains of the genus Anaerococcus and the type strains of all other genera within the family ‘Peptostreptococcaceae’ inferred from 1,302 aligned characters [7,8] of the 16S rRNA sequence under the maximum likelihood criterion [9]. Rooting was done with the type species of the genus Clostridium. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [10] are shown in blue, published genomes in bold. A.   prevotii   PC1T   cells   are   Gram-­‐positive   and   non-­‐ motile(Table  1).  Cells  grown  in  PYG  broth  are  0.6-­‐0.9   µm   in   diameter   and   occur   in   pairs,   tetrads   or   ir-­‐ regular   clumps  or  short  chains  (Figure  2).   Colonies   range   from   0.5   to   2   mm   in   diameter   on   Columbia   blood   agar.   Optimum   temperature   for   growth   is   37°C.   Strain   PC1T   metabolizes  peptones  and  amino   acids   and   the   major   metabolic   end   product   from   PYG  medium  is  butyric  acid.  Most  species  of  the  ge-­‐ nus  Anaerococcus   ferment  carbohydrates  weakly.  A.   160 prevotii   is   proteolytic.   α-­‐Glucosidase,   α   –galactosi-­‐ dase,   ß-­‐glucuronidase   and   pyroglutamyl   arylami-­‐ dase   activities  are   detectable   [19,20].  Production   of   urease  may   vary  among  strains  of  the  species.   Most   strains   produce   ammonia   from   threonine   and   ser-­‐ ine   [3]   by  deamination  of   the   amino   acids  to  pyru-­‐ vate.   A.   prevotii   is   resistant   to  sodium   polyanethol   sulfonate   [21],   but   susceptible   to   the   penicillins   [19]. Standards in Genomic Sciences Labuti, et al. Chemotaxonomy Cell  wall  amino  acid   analysis  of   strain  PC1T  yielded   peptidoglycan   type   A4αʹ′,   composed   of   L-­‐Lys-­‐D-­‐Glu   [22],  type  A12.2  according   to   the  DSMZ  catalogue  of   strains.   Cell   wall   sugars   are   glucose,   glucosamine   and  galactose  [22].  Major  cellular  fatty  acid  compo-­‐ sition   of   the   type   strain   was   analyzed   by   Lambert   and   Arm`ield   in   1979   [23]   and   by   Ezaki   et   al.   in   1983   [3],   but   the   results   of   these   studies   are   con-­‐ tradictory.   No   other   chemotaxonomic   data   are   available  at  present. 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   pro-­‐ ject.   The   genome   project   is   deposited   in   the   Ge-­‐ nomes   OnLine   Database   [10]  and   the   complete   ge-­‐ nome   sequence   has   been   deposited   in   GenBank.   Sequence,  `inishing   and   annotation  were  performed   by   the   DOE   Joint   Genome   Institute   (JGI).   A   sum-­‐ mary  of  the  project  information  is  shown  in  Table  2 Table 1. Classification and general features of A. prevotii PC1T in accordance with the MIGS recommendations [11] Evidence MIGS ID Property Term code Domain Bacteria TAS [12] Phylum Firmicutes TAS [13] Class Clostridia TAS [4] Order Clostridiales TAS [14] Current classification Family ‘Peptostreptococcaceae’ TAS [4] Genus Anaerococcus TAS [1] Species Anaerococcus prevotii TAS [1] MIGS-22 MIGS-6 MIGS-15 MIGS-14 MIGS-4 MIGS-5 MIGS-4.1 MIGS-4.2 MIGS-4.3 MIGS-4.4 Gram stain Cell shape Motility Sporulation Temperature range Optimum temperature Type strain PC1 positive coccoid nonmotile nonsporulating mesophile 37°C TAS [1,3] TAS [15] TAS [15] TAS [15] TAS [15] TAS [15] TAS [15] Salinity Oxygen requirement growth in PYG +6% NaCl anaerobic TAS [16] TAS [15] Carbon source unknown Energy source Habitat Biotic relationship Pathogenicity Biosafety level Isolation Geographic location Sample collection time peptones human mouth, skin and vaginal microflora free living opportunistic infections 2 human plasma not reported not reported Latitude – Longitude Depth Altitude not reported not reported not reported TAS [1,3] TAS [3,10] NAS TAS [10] TAS [17] TAS [3] Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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). These evidence codes are available from the Gene Ontology project [18]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. . http://standardsingenomics.org 161 Anaerococcus prevotii type strain PC1T Table 2. Genome sequencing project information MIGS ID Property Term MIGS-31 Finishing quality MIGS-28 Libraries used Finished Three genomic libraries: two Sanger libraries - 8 kb pMCL200 and fosmid pcc1Fos – and one 454 pyrosequence standard library MIGS-29 Sequencing platforms ABI3730, 454 GS FLX, Illumina GA MIGS-31.2 Sequencing coverage 6.8 Sanger; 42.3 pyrosequence MIGS-30 Assemblers Newbler version 1.1.02.15, Arachne MIGS-32 Gene calling method Genbank IDs Prodigal, GenePRIMP CP001708 (chromosome) CP001709 (plasmid pAPRE01) 4/28/2009 (chromosome) 4/28/2009 (plasmid pAPRE01) Genbank Dates of Release MIGS-13 GOLD ID Gc01089 NCBI project ID 29533 Database: IMG-GEBA 2501533213 Source identifier DSM 20548 Project relevance Tree of Life, GEBA Figure 2. Scanning electron micrograph of A. prevotii PC1T (M. Rohde, HZI Braunschweig) Growth condi tions and DNA isolation A.   prevotii  strain   PC1T,   DSM   20548,   was   grown   an-­‐ aerobically  in  DSMZ  medium   104  [24]  at  37°C.  DNA   was  isolated  from  1-­‐1.5  g  of   cell  paste   using  Qiagen   Genomic   500   DNA   Kit   (Qiagen,   Hilden,   Germany)   following   the   instructions   given   by   the   manufac-­‐ turer,   but   with   a   modi`ied   protocol   for   cell   lysis,   LALMP,  according  to  Wu  et  al.  [25]. Genome sequencing and assembly The   genome  was  sequenced  using  a   combination  of   Sanger,   454  and  Illumina   sequencing   platforms.   All   162 general   aspects   of   library   construction   and   se-­‐ quencing   can   be   found   at   the   JGI   web   site.   Reads   produced   by  454   Pyrosequencing   were   assembled   using   the   Newbler   assembler   version   1.1.02.15   (Roche).   Large   Newbler   contigs   were   broken   into   2,196   overlapping   fragments   of   1,000   bp   and   en-­‐ tered   into   the   assembly   as   pseudo-­‐reads.   The   se-­‐ quences   were   assigned   quality   scores   based   on   Newbler  consensus   q-­‐scores   with   modi`ications  to   account   for   overlap   redundancy   and   to   adjust   in-­‐ `lated   q-­‐scores.   A  hybrid  454/Sanger  assembly  was   made   using   the   Arachne   assembler.   Possible   mis-­‐ assemblies  were   corrected   and   gaps  between   con-­‐ tigs   were  closed  by  custom   primer  walks  from  sub-­‐ clones   or  PCR  products.   A  total  of  66  Sanger   `inish-­‐ ing   reads  were   produced.  Illumina  reads  were   used   to   improve  the   `inal  consensus  quality  using   an   in-­‐ house   developed   tool   (the   Polisher).   The   `inal   as-­‐ sembly   consisted   of   18,576   Sanger   and   464,157   Roche/454   reads.   The   error   rate   of   the   completed   genome   sequence   is   less   than   1   in   100,000.   To-­‐ gether  all  sequence  types  provided  49.1  coverage  of   the  genome. Genome annotation Genes  were   identi`ied   using  Prodigal  [26]  as  part  of   the  Oak  Ridge   National  Laboratory  genome   annota-­‐ tion  pipeline,  followed   by   a   round   of   manual  cura-­‐ tion   using   the   JGI   GenePRIMP   pipeline   [27].   The   predicted  CDSs  were   translated  and   used  to  search   the   National   Center   for  Biotechnology  Information   (NCBI)   nonredundant   database,  UniProt,   TIGRFam,   Standards in Genomic Sciences Labuti, et al. Pfam,   PRIAM,   KEGG,   COG,   and   InterPro   databases.   Additional   gene   prediction   analysis   and   functional   annotation   was   performed   within   the   Integrated   Microbial  Genomes  (IMG-­‐ER)  platform  [28]. Genome properties The  genome  is  1,998,633  bp  long  (chromosome  and   one  circular  plasmid)  with   a  35.6%  GC   content  (Ta-­‐ ble   3).   Of   the   1,913   genes   predicted,   1,852   were   protein   coding  genes,   and   61  were   RNAs.   A   total   of   46   pseudogenes  were   also  identi`ied,  with  73.1%  of   the   genes   being   assigned   a   putative   function.   The   remaining   genes   were   annotated   as   hypothetical   proteins.   The   distribution   of   genes  into   COGs   func-­‐ tional  categories  is  presented  in  Figure  3  and  Table  4. Table 3. Genome Statistics Attribute Value % of Total Genome size (bp) 1,998,633 100.00% DNA Coding region (bp) 1,815,671 90.85% 712,291 35.64% DNA G+C content (bp) Number of replicons 2 Extrachromosomal elements 1 Total genes 1913 100.00% RNA genes 61 3.19% rRNA operons Protein-coding genes 4 1852 96.81% 46 2.405% 1399 73.13% Genes in paralog clusters 231 12.08% Genes assigned to COGs 1421 74.28% Genes assigned Pfam domains 1428 74.65% Genes with signal peptides 337 17.62% Genes with transmembrane helices 467 24.41% Pseudo genes Genes with function prediction CRISPR repeats 0 Figure 3. Graphical circular map of the genome. 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, sRNAs red, other RNAs black), GC content, GC skew. http://standardsingenomics.org 163 Anaerococcus prevotii type strain PC1T Table 4. Number of genes associated with the 21 general COG functional categories Code J A K L B D Y V T M N Z W U O C G E F H I P Q R S - Value 133 % 7.2 0 118 105 1 20 0 55 43 69 5 0 0 19 61 84 144 107 61 56 37 102 9 84 118 431 0.0 6.4 5.7 0.1 1.1 0.0 3.0 2.3 3.7 0.3 0.0 0.0 1.0 3.3 4.5 7.8 5.8 3.3 3.0 2.0 5.5 0.5 4.5 6.4 23.3 Description Translation, ribosomal structure and biogenesis RNA processing and modification Transcription 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 Cell motility Cytoskeleton 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 Not in COGs Acknowledgements We   would  like  to   gratefully  acknowledge  the  help  of  Su-­‐ sanne   Schneider  (DSMZ)  for  DNA   extraction  and  quality   analysis.   This  work  was  performed  under  the  auspices  of   the  US  Department  of  Energy  Of`ice  of   Science,  Biological   and   Environmental   Research  Program,   and   by   the   Uni-­‐ versity  of  California,  Lawrence  Berkeley  National  Labo ratory   under   contract   No.   DE-­‐AC02-­‐05CH11231,   Law-­‐ rence  Livermore  National   Laboratory   under  Contract  No.   DE-­‐AC52-­‐07NA27344,   and  Los   Alamos   National   Labora-­‐ tory  under  contract   No.  DE-­‐AC02-­‐06NA25396,  as  well   as   German  Research  Foundation  (DFG)  INST  599/1-­‐1. 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