Open Access

Complete genome sequence of Sphaerobacter thermophilus type strain (S 6022T)

  • Amrita Pati
  • , Kurt LaButti
  • , Rüdiger Pukall
  • , Matt Nolan
  • , Tijana Glavina Del Rio
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Susan Lucas
  • , Feng Chen
  • , Alex Copeland
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Sam Pitluck
  • , David Bruce
  • , Lynne Goodwin
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Amy Chen
  • , Krishna Palaniappan
  • , Patrick Chain,
  • , Thomas Brettin,
  • , Johannes Sikorski
  • , Manfred Rohde
  • , Markus Göker
  • , Jim Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Alla Lapidus
Corresponding author

DOI: 10.4056/sigs.601105

Received: 28 January 2010

Published: 28 February 2010

Abstract

Sphaerobacter thermophilus Demharter et al. 1989 is the sole and type species of the genus Sphaerobacter, which is the type genus of the family Sphaerobacteraceae, the order Sphaerobacterales and the subclass Sphaerobacteridae. Phylogenetically, it belongs to the genomically little studied class of the Thermomicrobia in the bacterial phylum Chloroflexi. Here, the genome of strain S 6022T is described which is an obligate aerobe that was originally isolated from an aerated laboratory-scale fermentor that was pulse fed with municipal sewage sludge. We describe the features of this organism, together with the complete genome and annotation. This is the first complete genome sequence of the thermomicrobial subclass Sphaerobacteridae, and the second sequence from the chloroflexal class Thermomicrobia. The 3,993,764 bp genome with its 3,525 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

SphaerobacteridaeThermomicrobiathermophileobligate aerobicsewage sludge isolatepleomorphicnon-motilenon-sporeformingGEBA

Introduction

Strain S 6022T (DSM 20745 = ATCC 49802 = NCIMB 13125) is the type strain of the species Sphaerobacter thermophilus, representing the type species of the genus Sphaerobacter. S. thermophilus was described by Demharter et al. in 1989 [1]. It is Gram-positive, non-motile and non-sporeforming. It was originally isolated from thermal treated municipal sewage sludge from München-Grosslappen, Germany [2]. Cells of S. thermophilus were also identified in three other municipal sludge stabilization plants spread across Germany (Isenbüttel, Nettetal, and Gemmingen) using an immunolabelling procedure. From the operating parameters of these plants a minimum temperature growth range of 40-65°C can be predicted [2]. Here we present a summary classification and a set of features for S. thermophilus strain S 6022T, together with the description of the complete genomic sequencing and annotation.

Classification and features

The closest related cultivated organism with a 16S rRNA sequence recorded in Genbank is Thermomicrobium roseum (DSM 5159) [3,4], which shares a mere 87% sequence similarity with strain S 6022T, indicating that S. thermophilus is phylogenetically one of the most isolated bacterial species. Only some uncultivated bacterial clones show a slightly closer relationship, e.g. clone Amb_16S_1237 (EF018775) isolated from Populus tremula (trembling aspen, 92%), EU035785 and EF643378 from soil in a radish-rich area in Jaunpur (India), clone AKYG1722 from farm soil adjacent to a silage storage bunker in Minnesota (89%), and AM935838 from a pilot-scale bioremediation process of hydrocarbon-contaminated soil in France (88%). None of the phylotypes sequenced during environmental screenings or genomic surveys surpassed 82% sequence similarity with strain S 6022T, expressly underlining the phylogenetically isolated and rare occurrence of S. thermophilus (status May 2009).

Figure 1 shows the phylogenetic neighborhood of S. thermophilus strain S 6022T in a 16S rRNA based tree. The sequence of the sole 16S rRNA gene in the genome of strain S 6022T differs by six nucleotides (0.4%) from the previously published 16S rRNA sequence generated from DSM 20745 (AJ420142). The difference between the genome data and the previously reported 16S rRNA in GenBank gene sequence is most likely due to sequencing errors in the latter.

Figure 1

Phylogenetic tree of S. thermophilus strain S 6022T and all type strains of the phylum Chloroflexi, inferred from 1,304 aligned characters [5,6] of the 16S rRNA gene sequence under the maximum likelihood criterion [7]. The tree was rooted with the members of Anaerolineae and Caldilineae within the Chloroflexi. 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 [8] are shown in blue, published genomes in bold.

S. thermophilus S 6022T cells are coccoid (Figure 2), but are also described as coccoid rods, 1-1.5 by 1.5-3 µm, in older cultures or in glucose-free medium irregular club- or dumb-bell shaped forms [1]. Branched cells are not observed. Colonies on Ottow Medium (DSMZ Medium No. 467) [9] are opaque, circular with entire margin and reach a diameter of 1-2 mm after 3 days of incubation at 60°C. The strain grows strictly aerobically with optimal growth at 55°C and pH 8.5 (Table 1). There is no acid production from glucose. Strain S 6022T possesses catalase and oxidase and hydrolyzes starch but not gelatin, casein or cellulose [1]. Strain S 6022T shares many features such as thermophilia, optimal pH for growth, and lack of motility with its closest relative, T. roseum (DSM 5159) [3,4]. The genome sequence as presented here might contribute to the solution of the question if S. thermophilus, like T. roseum encodes a complete flagellar system [4], although neither strain is motile. Interestingly, none of the other species in the Chloroflexi for which a genome sequence currently exists encode for any flagellar structural components [4].

Figure 2

Scanning electron micrograph of S. thermophilus S strain 6022T

Table 1

Classification and general features of S. thermophilus S 6022T in accordance to the MIGS recommendations [10]

MIGS ID

Property

Term

Evidence code

Current classification

Domain Bacteria

TAS [11]

Phylum Chloroflexi

TAS [12]

Class Thermomicrobia

TAS [11-14]

Order Sphaerobacterales

TAS [15]

Suborder Sphaerobacterineae

TAS [16]

Family Sphaerobacteraceae

TAS [15]

Genus Sphaerobacter

TAS [1]

Species Sphaerobacter thermophilus

TAS [1]

Type strain S 6022

Gram stain

positive

TAS [1]

Cell shape

coccoid rods, irregular club- or dump-bell shaped

TAS [1]

Motility

nonmotile

TAS [1]

Sporulation

nonsporulating

TAS [1]

Temperature range

thermophile, minimum 40-65°C

TAS [1,2]

Optimum temperature

55°C, pH 8.5

TAS [1]

Salinity

not reported

MIGS-22

Oxygen requirement

obligate aerobic

TAS [1]

Carbon source

starch

NAS

Energy source

unknown

MIGS-6

Habitat

thermal treated sewage sludge

TAS [2]

MIGS-15

Biotic relationship

free living

NAS

MIGS-14

Pathogenicity

none

NAS

Biosafety level

1

TAS [17]

Isolation

thermal treated sewage sludge

TAS [2]

MIGS-4

Geographic location

Munich, Germany

TAS [2]

MIGS-5

Sample collection time

between 1973 and 1988, probably 1984

TAS [1]

MIGS-4.1,MIGS-4.2

Latitude, Longitude

48.139, 11.58

NAS

MIGS-4.3

Depth

0 m

NAS

MIGS-4.4

Altitude

518 m

NAS

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 from of 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.

Chemotaxonomy

Acid hydrolysates of the cell wall of strain S 6022T yielded a ratio of glutamic acid to ornithine to alanine to β-alanine to muramic acid to glucosamine = 1:1.1:1.2:1.6:0.9:1.1. The murein structure type belongs to the murein variation A3β [19] with cross-linking via β-alanine [1]. The cell wall is unusually rich in protein content [1]. The principal isoprenoid quinone is an unsaturated menaquinone of type MK-8/0. MK-6/0, MK-7/0, MK-10/0 appear as minor constituents (4.8%, 7.7%, 12.8%) MK-6/0 [1]. Nothing is known about the spectrum of cellular fatty acids in the organism.

Genome sequencing information

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 Genomes OnLine Database [8] and the complete genome sequence 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.

Table 2

Genome sequencing project information

MIGS ID

  Property

   Term

MIGS-13

  Finishing quality

   Finished

MIGS-28

  Libraries used

   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

   7.4× Sanger; 28.5× Pyrosequence

MIGS-30

  Assemblers

   Newbler version 1.1.02.15, Arachne

MIGS-32

  Gene calling method

   Prodigal, GenePRIMP

  INSDC ID

   CP001823 (chromosome), CP001824 (plasmid)

  GenBank date of release

   November 23, 2009

  GOLD ID

   Gc01151

  NCBI project ID

   21087

  Database: IMG-GEBA

   2502082099

MIGS-13

  Source material identifier

   DSM 20745

  Project relevance

   Tree of Life, GEBA

Growth conditions and DNA isolation

S. thermophilus S 6022T, DSM 20745, was grown in DSMZ medium 467 [20] at 55°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions with modification st/FT for cell lysis according to Wu et al. [21].

Genome sequencing and assembly

The genome was sequenced using a combination of Sanger, 454 and Illumina sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at Web Site. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,435 overlapping fragments of 1,000 bp and entered into the assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the Arachne assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by custom primer walks from sub-clones or PCR products. A total of 109 Sanger finishing reads were produced. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher – publication in preparation). The final assembly consists of 35,091 Sanger and 516,954 Roche/454 reads. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 35.9× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [22] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline (Web Site) [23]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (Web Site) platform [24].

Genome properties

The two replicons containing genome is 3,993,764 bp long with a 68.1% GC content (Table 3 and Figure 3). Of the 3,582 genes predicted, 3525 were protein coding genes, and 57 RNAs; 40 pseudogenes were also identified. The majority of the protein-coding genes (72.3%) were assigned a putative function while those remaining were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 4.

Table 3

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

3,993,764

100.00%

DNA coding region (bp)

3,461,586

86.67%

DNA G+C content (bp)

2,720,128

68.11%

Number of replicons

2

Extrachromosomal elements

0

Total genes

3,582

100.00%

RNA genes

57

1.59%

rRNA operons

2

Protein-coding genes

3,525

98.41%

Pseudo genes

40

1.12%

Genes with function prediction

2,591

72.33%

Genes in paralog clusters

677

18.90%

Genes assigned to COGs

2,619

73.12%

Genes assigned Pfam domains

2,679

74.79%

Genes with signal peptides

709

19.79%

Genes with transmembrane helices

908

25.35%

CRISPR repeats

1

Figure 3

Graphical circular map of the genome. Chromosome (left), plasmid (right), drown not in scale. 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, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code

value

%age

  Description

J

162

4.6

  Translation, ribosomal structure and biogenesis

A

0

0.0

  RNA processing and modification

K

162

4.6

  Transcription

L

121

3.4

  Replication, recombination and repair

B

2

0.1

  Chromatin structure and dynamics

D

26

0.7

  Cell cycle control, mitosis and meiosis

Y

0

0.0

  Nuclear structure

V

61

1.7

  Defense mechanisms

T

109

3.1

  Signal transduction mechanisms

M

173

4.9

  Cell wall/membrane biogenesis

N

36

1.0

  Cell motility

Z

0

0.0

  Cytoskeleton

W

0

0.0

  Extracellular structures

U

47

1.3

  Intracellular trafficking and secretion

O

108

3.1

  Posttranslational modification, protein turnover, chaperones

C

226

6.4

  Energy production and conversion

G

157

4.5

  Carbohydrate transport and metabolism

E

404

11.5

  Amino acid transport and metabolism

F

60

1.7

  Nucleotide transport and metabolism

H

145

4.1

  Coenzyme transport and metabolism

I

111

3.1

  Lipid transport and metabolism

P

163

4.6

  Inorganic ion transport and metabolism

Q

79

2.2

  Secondary metabolites biosynthesis, transport and catabolism

R

373

10.6

  General function prediction only

S

207

5.9

  Function unknown

-

963

27.3

  Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Gabriele Gehrich-Schröter for growing S. thermophilus cultures and Susanne Schneider for DNA extraction and quality analysis (both at DSMZ). 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, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1.


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