Open Access

Complete genome sequence of the halophilic bacterium Spirochaeta africana type strain (Z-7692T) from the alkaline Lake Magadi in the East African Rift

  • Konstantinos Liolos
  • , Birte Abt
  • , Carmen Scheuner
  • , Hazuki Teshima
  • , Brittany Held
  • , Alla Lapidus
  • , Matt Nolan
  • , Susan Lucas
  • , Shweta Deshpande
  • , Jan-Fang Cheng
  • , Roxanne Tapia,
  • , Lynne A. Goodwin,
  • , Sam Pitluck
  • , Ioanna Pagani
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Marcel Huntemann
  • , Amrita Pati
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Manfred Rohde
  • , Brian J. Tindall
  • , John C. Detter
  • , Markus Göker
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz,
  • , Tanja Woyke
  • , Hans-Peter Klenk
  • and Nikos C. Kyrpides
Corresponding author

DOI: 10.4056/sigs.3607108

Received: 20 May 2013

Published: 15 June 2013

Abstract

Spirochaeta africana Zhilina et al. 1996 is an anaerobic, aerotolerant, spiral-shaped bacterium that is motile via periplasmic flagella. The type strain of the species, Z-7692T, was isolated in 1993 or earlier from a bacterial bloom in the brine under the trona layer in a shallow lagoon of the alkaline equatorial Lake Magadi in Kenya. Here we describe the features of this organism, together with the complete genome sequence, and annotation. Considering the pending reclassification of S. caldaria to the genus Treponema, S. africana is only the second 'true' member of the genus Spirochaeta with a genome-sequenced type strain to be published. The 3,285,855 bp long genome of strain Z-7692T with its 2,817 protein-coding and 57 RNA genes is a part of the G enomic E ncyclopedia of B acteria and A rchaea project.

Keywords:

anaerobicaerotolerantmesophilichalophilicspiral-shapedmotileperiplasmic flagellaGram-negativechemoorganotrophicSpirochaetaceaeGEBA

Introduction

Strain Z-7692T (= DSM 8902 = ATCC 700263) is the type strain of the species Spirochaeta africana [1]. The genus Spirochaeta currently consists of 18 validly named species [2]. The genus name was derived from the latinized Greek words 'speira' meaning 'a coil' and 'chaitê' meaning 'hair', yielding the Neo-Latin word 'Spirochaeta', a 'coiled hair' [2]. The species epithet was derived from the Latin word 'africana', of African continent, found in the African alkaline Lake Magadi [1]. Here we present a summary classification and a set of features for S. africana strain Z-7692T, together with the description of the complete genome sequencing and annotation.

Classification and features

16S rRNA analysis

A representative genomic 16S rRNA sequence of strain Z-7692T was compared using NCBI BLAST [3,4] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [5] and the relative frequencies of taxa and keywords (reduced to their stem [6]) were determined, weighted by BLAST scores. The most frequently occurring genera were Spirochaeta (91.1%), Treponema (5.8%) and Cytophaga (3.1%) (29 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 99.6%, whereas the average coverage by HSPs was 99.0%. Regarding the 19 hits to sequences from other members of the genus, the average identity within HSPs was 89.1%, whereas the average coverage by HSPs was 78.9%. Among all other species, the one yielding the highest score was Spirochaeta asiatica (NR_026300), which corresponded to an identity of 96.6% and an HSP coverage of 98.8%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was AF454308 (Greengenes short name 'spirochete clone ML320J-13'), which showed an identity of 90.6% and an HSP coverage of 99.3%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'microbi' (10.5%), 'mat' (8.8%), 'hypersalin' (6.3%), 'new' (4.2%) and 'world' (4.1%) (221 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found, indicating that this species is rarely found in environmental sequencing.

Figure 1 shows the phylogenetic neighborhood of S. africana in a 16S rRNA based tree. The sequences of the three identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (X93928).

Figure 1

Phylogenetic tree highlighting the position of S. africana relative to the type strains of the other species within the phylum 'Spirochaetes'. The tree was inferred from 1,332 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [9]. Rooting was done initially using the midpoint method [10] and then checked for its agreement with the current classification (Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 350 ML bootstrap replicates [11] (left) and from 1,000 maximum-parsimony bootstrap replicates [12] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [13] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks (see [14-20] and CP003155 for Sphaerochaeta pleomorpha, CP002903 for Spirochaeta thermophila, CP002696 for Treponema brennaborense, CP001841 for T. azotonutricium and CP001843 for T. primitia. Note: Spirochaeta caldaria, S. stenostrepta and S. zuelzerae were effectively renamed to T. caldaria, T. stenostrepta and T. zuelzerae in [15], however, the names have not yet been validily published.

Morphology and physiology

Cells of strain Z-7692T are 0.25 to 0.3 µm in diameter and 15 to 30 µm (occasionally 7 to 40 µm) in length and form regular, stable primary coils [1] (Figure 2); spherical bodies were seen in stationary-phase cultures (not visible in Figure 2). The cells are motile by periplasmic flagella [1] (not visible in Figure 2). The cell mass is orange [1]. S. africana is a Gram-negative, anaerobic, aerotolerant, mesophilic microorganism (Table 1) with an optimal growth temperature between 30°C and 37°C, and no growth observed above 47°C [1]. The optimum pH is 8.8-9.8, no growth is observed at pH 8 or pH 10.8 [1]. S. africana is halophilic and does not grows at NaCl concentrations below 3% or above 10% (wt/vol) [1].

Figure 2

Scanning electron micrograph of S. africana strain Z-7692T

Table 1

Classification and general features of S. africana Z-7692T according to the MIGS recommendations [21] and the NamesforLife database [22].

MIGS ID

      Property

      Term

     Evidence code

      Current classification

      Domain Bacteria

     TAS [23]

      Phylum Spirochaetae

     TAS [24,25]

      Class Spirochaetes

     TAS [25,26]

      Order Spirochaetales

     TAS [27,28]

      Family Spirochaetaceae

     TAS [27,29]

      Genus Spirochaeta

     TAS [27,30-32]

      Species Spirochaeta africana

     TAS [1]

      Type strain Z-7692

     TAS [1]

      Gram stain

      negative

     TAS [1]

      Cell shape

      spiral shaped

     TAS [1]

      Motility

      motile

     TAS [1]

      Sporulation

      none

     TAS [1]

      Temperature range

      mesophile

     TAS [1]

      Optimum temperature

      30 - 37°C

     TAS [1]

      Salinity

      halophile

     TAS [1]

MIGS-22

      Oxygen requirement

      anaerobic, aerotolerant

     TAS [1]

      Carbon source

      saccharolytic, utilize carbohydrates

     TAS [1]

      Energy metabolism

      chemoorganotroph

     TAS [1]

MIGS-6

      Habitat

      alkaline salt lakes, fresh water

     TAS [1]

MIGS-15

      Biotic relationship

      free living

     TAS [1]

MIGS-14

      Pathogenicity

      none

     TAS [1]

      Biosafety level

      1

     TAS [33]

      Isolation

      bacterial bloom in the brine under trona from alkaline lake

     TAS [1]

MIGS-4

      Geographic location

      Lake Magadi (Kenya)

     TAS [1]

MIGS-5

      Sample collection time

      1993 or before

     NAS

MIGS-4.1

      Latitude

      -1.945

     NAS

MIGS-4.2

      Longitude

      36.253

     NAS

MIGS-4.3

      Depth

      not reported

MIGS-4.4

      Altitude

      not reported

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 [34].

S. africana utilizes mainly mono- and disaccharides as carbon and energy sources. Amino acids cannot be fermented. Glucose is fermented to acetate, ethanol and H2 as the main fermentation products, with a minor amount of lactate produced in stationary phase [1]. Strain Z-7692T is able to ferment fructose, maltose, trehalose, saccharose, cellobiose, glucose, glycogen, starch. Poor growth was observed with mannose and or xylose, no growth with galactose, N-acetylglucosamin or ribose. A supplement of vitamins is required [1].

Chemotaxonomy

Major components detected in the fatty acid analysis are the fatty acids C14:0 (6.6%), C16:1cis9 (6.3%), C16:0 (19.0%), C18:1cis-9 (1.4%), summed feature 10 (C18:1cis11/trans9/trans6 and/or an unknown fatty acid with an equivalent chain length of 17.834) (34.9%), C18:0 (1.8%), C20:1cis13/trans11 (2.4%), as well as dimethyl acetals (DMA)/aldehydes (ALDE) probably derived from plasmalogens, C14:0 DMA (5.0%), C16:0 ALDE (3.8%), C16:1cis-9 DMA (1.1%), C16:0 DMA (15.3%), C18:1cis11 DMA (0.8%) [35]. No data are available on polar lipid, quinone or other cell wall/envelope components that may be taxonomically significant

Taxonomic perspective

The data presented in Figure 1, based on an evaluation of the 16S rRNA gene sequence data provide an interesting insight into the nomenclature and classification of members of the genus Spirochaeta. In determining which species currently placed in this genus should remain members of this genus it is important to note that the primary criterion is which species group with the type strain of the type species of the genus Spirochaeta. It should be noted that the type species of this genus is Spirochaeta plicatilis and only a description serves as the type since no type strain appears to be available. This makes it difficult to determine which species represented by living type strains belong within the genus Spirochaeta. This is important because the monophyletic group delineated by the majority of the members of the genus Spirochaeta and members of the genus Borrelia does not split into two monophyletic groups corresponding with the members of the genus Spirochaeta and Borrelia, but causes the members of the genus Spirochaeta to appear to be paraphyletic. If one of the goals of modern taxonomy is to classify species in a single genus only if the members of the genus constitute a monophyletic group, then there are three possible solutions. The first is that all members of the genus Borrelia should be transferred to the genus Spirochaeta, although this is also complicated by the fact that a type strain for the type species of the genus Borrelia, Borrelia anserine has never been designated. The second alternative would be to create a number of genera based on monophyletic groups to be found within the current analysis of members of the genus Spirochaeta. The third alternative would be to accept the status quo whereby members of the genus Spirochaeta appear to constitute a paraphyetic group. However, a key factor in attempting to undertake such a reclassification would be the absence of type strains of the type species of the genera Spirochaeta and Borrelia. There are already indications that the evolutionary group constituting members of the genera Spirochaeta and Borrelia show an interesting degree of diversity at the level of morphology, physiology and the genome.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [36,37], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [38]. The genome project is deposited in the Genomes On Line Database [13] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology [39]. A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information

MIGS ID

      Property

     Term

MIGS-31

      Finishing quality

     Finished

MIGS-28

      Libraries used

     Four genomic libraries: one 454 pyrosequence standard library,     two 454 PE libraries (4 kb and 6 kb insert size), one Illumina library

MIGS-29

      Sequencing platforms

     Illumina GAii, 454 GS FLX Titanium

MIGS-31.2

      Sequencing coverage

     123.6 × Illumina; 23.4 × pyrosequence

MIGS-30

      Assemblers

     Newbler version 2.3-PreRelease-6/30/2009, Velvet 1.0.13,     phrap version SPS - 4.244

MIGS-32

      Gene calling method

     Prodigal 1.4, GenePRIMP

      INSDC ID

     CP003282

      GenBank Date of Release

     April 2, 2012

      GOLD ID

     Gc02193

      NCBI project ID

     52939

      Database: IMG

     2509276057

MIGS-13

      Source material identifier

     DSM 8902

      Project relevance

     Tree of Life, GEBA

Growth conditions and DNA isolation

S. africana strain Z-7692T, DSM 8902, was grown anaerobically in DSMZ medium 700 (Alkaliphilic Spirochaea medium) [40] at 37°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/LALM for cell lysis as described in Wu et al. 2009 [41]. DNA is available through the DNA Bank Network [42].

Genome sequencing and assembly

The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [43]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 511 contigs in one scaffold was converted into a phrap [44] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (459.3 Mb) was assembled with Velvet [45] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 234.5 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 21. The Phred/Phrap/Consed software package [44] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [43], Dupfinisher [46], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 132 additional reactions were necessary to close some gaps and to raise the quality of the final contigs. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [47]. The error rate of the final genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 480.9 x coverage of the genome. The final assembly contained 509,107 pyrosequence and 12,708,968 Illumina reads.

Genome annotation

Genes were identified using Prodigal [48] as part of the DOE-JGI genome annotation pipeline [20], followed by a round of manual curation using the JGI GenePRIMP pipeline [49]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [50].

Genome properties

The genome consists of a 3,285,855 bp long chromosome with a G+C content of 57.8% (Table 3 and Figure 3). Of the 2,874 genes predicted, 2,817 were protein-coding genes, and 57 RNAs; 35 pseudogenes were also identified. The majority of the protein-coding genes (74.2%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics

Attribute

   Value

   % of Total

Genome size (bp)

   3,285,855

   100.00%

DNA coding region (bp)

   3,080,373

   93.75%

DNA G+C content (bp)

   1,898,112

   57.77%

Number of replicons

   1

Extrachromosomal elements

   0

Total genes

   2,874

   100.00%

RNA genes

   57

   1.98%

rRNA operons

   3

Protein-coding genes

   2,817

   98.02%

Pseudo genes

   35

   1.22%

Genes with function prediction

   2,133

   74.22%

Genes in paralog clusters

   1,205

   41.93%

Genes assigned to COGs

   2,153

   74.91%

Genes assigned Pfam domains

2,235

77.77

Genes with signal peptides

247

8.59

Genes with transmembrane helices

847

29.47%

CRISPR repeats

1

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).

Table 4

Number of genes associated with the general COG functional categories

Code

   Value

   %age

   Description

J

   151

   6.4

   Translation, ribosomal structure and biogenesis

A

   0

   0.0

   RNA processing and modification

K

   137

   5.8

   Transcription

L

   138

   5.8

   Replication, recombination and repair

B

   0

   0.0

   Chromatin structure and dynamics

D

   24

   1.0

   Cell cycle control, cell division, chromosome partitioning

Y

   0

   0.0

   Nuclear structure

V

   49

   2.1

   Defense mechanisms

T

   239

   10.1

   Signal transduction mechanisms

M

   149

   6.3

   Cell wall/membrane/envelope biogenesis

N

   93

   3.9

   Cell motility

Z

   0

   0.0

   Cytoskeleton

W

   1

   0.0

   Extracellular structures

U

   60

   2.5

   Intracellular trafficking, secretion, and vesicular transport

O

   100

   4.2

   Posttranslational modification, protein turnover, chaperones

C

   109

   4.6

   Energy production and conversion

G

   185

   7.8

   Carbohydrate transport and metabolism

E

   179

   7.5

   Amino acid transport and metabolism

F

   62

   2.6

   Nucleotide transport and metabolism

H

   68

   2,9

   Coenzyme transport and metabolism

I

   57

   2.4

   Lipid transport and metabolism

P

   103

   4.3

   Inorganic ion transport and metabolism

Q

   23

   1.0

   Secondary metabolites biosynthesis, transport and catabolism

R

   267

   11.3

   General function prediction only

S

   181

   7.6

   Function unknown

-

   721

   25.1

   Not in COGs

Insights from the genome sequence

Phylogenomic analyses

According to the results from 16S rRNA gene analysis (Figure 1), for a comparative analysis the genome sequences of S. africana (GenBank ID CP003282), S. alkalica (GenBank ID PRJNA169743), S. caldaria (CP002868) and S. smaragdinae (CP002116) were used. The genomes of S. caldaria (3.2 Mb, 2,928 protein-coding genes), S. africana (3.3 Mb, 2,874 protein-coding genes) and S. alkalica (3.4 Mb, 2,938 protein-coding genes) have a similar size, whereas the genome of S. smaragdinae (4.7 Mb, 4,363 protein-coding gene) is significantly larger in size. S. caldaria and S. smaragdinae have similar G+C contents, 46% and 49%, respectively. The G+C contents of S. alkalica and S. africana are significantly higher, 61% and 58%, respectively.

An estimate of the overall similarity between the genomes of S. africana, and those of the other Spirochaeta species was generated with the GGDC-Genome-to-Genome Distance Calculator [51,52]. This system calculates the distances by comparing the genomes to obtain HSPs (high-scoring segment pairs) and interfering distances from the set of formulas (1, HSP length / total length; 2, identities / HSP length; 3, identities / total length). Table 5 shows the results of the pairwise comparison.

Table 5

Pairwise comparison of S. africana with S. alkalica, S. caldaria, and S. smaragdinae, using the GGDC-Genome-to-Genome Distance Calculator.

HSP length /total length [%]

identities /HSP length [%]

identities /total length [%]

S. africana

S. alkalica

5.21

86.44

4.51

S. africana

S. caldaria

1.62

84.50

1.37

S. africana

S. smaragdinae

1.64

83.52

1.37

S. smaragdinaeS. smaragdinaeS. caldaria

S. alkalicaS. caldariaS. alkalica

2.511.522.08

87.7183.9188.57

2.201.281.85

The comparison of S. africana with S. alkalica reached the highest scores using the GGDC, 5.2% of the average of genome length are covered with HSPs. The identity within the HSPs was 86.4%, whereas the identity over the whole genome was 4.5%. Lower similarity scores were observed in the comparison of S. africana with S. caldaria and with S. smaragdinae only 1.62% and 1.64%, respectively, of the average of both genome lengths are covered with HSPs. The identity within these HSPs was 84.5% and 83.5%, respectively, whereas the identity over the whole genome was only 1.4% in both comparisons. S. alkalica shows the highest GGDC scores with S. smaragdinae: 2.5% of the average of genome length are covered with HSPs and the identity within the HSPs was 87.7%, whereas the identity over the whole genome was 2.2% [51].

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Helga Pomrenke for growing S. africana cultures and Evelyne-Marie Brambilla for DNA extraction and quality control (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, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.

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