Open Access

Complete genome sequence of Beutenbergia cavernae type strain (HKI 0122T)

  • Miriam Land1, 2,
  • Rüdiger Pukall3,
  • Birte Abt3,
  • Markus Göker3,
  • Manfred Rohde4,
  • Tijana Glavina Del Rio1,
  • Hope Tice1,
  • Alex Copeland1,
  • Jan-Fang Cheng1,
  • Susan Lucas1,
  • Feng Chen1,
  • Matt Nolan1,
  • David Bruce1, 5,
  • Lynne Goodwin1, 5,
  • Sam Pitluck1,
  • Natalia Ivanova1,
  • Konstantinos Mavromatis1,
  • Galina Ovchinnikova1,
  • Amrita Pati1,
  • Amy Chen6,
  • Krishna Palaniappan6,
  • Loren Hauser1, 2,
  • Yun-Juan Chang1, 2,
  • Cynthia C. Jefferies1, 2,
  • Elizabeth Saunders5,
  • Thomas Brettin1, 5,
  • John C. Detter1, 5,
  • Cliff Han1, 5,
  • Patrick Chain1, 7,
  • James Bristow1,
  • Jonathan A. Eisen1, 8,
  • Victor Markowitz6,
  • Philip Hugenholtz1,
  • Nikos C. Kyrpides1,
  • Hans-Peter Klenk3 and
  • Alla Lapidus1
Standards in Genomic Sciences20091:1010021

https://doi.org/10.4056/sigs.1162

Published: 20 July 2009

Abstract

Beutenbergia cavernae (Groth et al. 1999) is the type species of the genus and is of phylogenetic interest because of its isolated location in the actinobacterial suborder Micrococcineae. B. cavernae HKI 0122T is a Gram-positive, non-motile, non-spore-forming bacterium isolated from a cave in Guangxi (China). B. cavernae grows best under aerobic conditions and shows a rod-coccus growth cycle. Its cell wall peptidoglycan contains the diagnostic L-lysine ← L-glutamate interpeptide bridge. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the poorly populated micrococcineal family Beutenbergiaceae, and this 4,669,183 bp long single replicon genome with its 4225 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords

mesophilenon-pathogenicaerobic and microaerophilicrod-coccus growth cycleMK-8(H4)actinomycete Micrococcineae

Introduction

Beutenbergia cavernae strain HKI 0122T (DSM 12333 = ATCC BAA-8 = JCM 11478) is the type strain of the species, which represents the type species of the genus Beutenbergia, the type genus of the family Beutenbergiaceae [1]. B. cavernae was described by Groth et al. 1999 as Gram-positive, non-motile and non-spore-forming [1]. The organism is of significant interest for its position in the tree of life within the small (2 type strains) family Beutenbergiaceae Zhi, et al, 2009 emend. Schumann et al. 2009 in the actinobacterial suborder Micrococcineae [2], which in addition to the genus Beutenbergia contains only the genus Salana [3,4] (Figure 1), also otherwise stated in a recent overview on the class Actinobacteria [2]. Here we present a summary classification and a set of features for B. cavernae strain HKI 0122T (Table 1), together with the description of the complete genome sequencing and annotation.
Figure 1.

Phylogenetic tree of B. cavernae HKI 0122T and all type strains of the genus Beutenbergia, inferred from 1411 aligned characters [5,6] of the 16S rRNA sequence under the maximum likelihood criterion [7]. The tree was rooted with species from the genera Isoptericola and Oerskovia, both also members of the actinobacterial suborder Micrococcineae. 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%. Strains with a genome-sequencing project registered in GOLD [8] are printed in blue; published genomes in bold.

Table 1.

Classification and general features of B. cavernae HKI 0122T based on the MIGS recommendations [9]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

 
 

Phylum Actinobacteria

 
 

Class Actinobacteria

TAS [10]

 

Order Actinomycetales

TAS [10]

 

Suborder Micrococcineae

TAS [2]

 

Family Beutenbergiaceae

TAS [2]

 

Genus Beutenbergia

TAS [1]

 

Species Beutenbergia cavernae

TAS [1]

 

Type strain HKI 0122

 
 

Gram stain

positive

TAS [1]

 

Cell shape

varies; rod-coccus growth cycle

TAS [1]

 

Motility

nonmotile

TAS [1]

 

Sporulation

non-sporulating

TAS [1]

 

Temperature range

mesophile

TAS [1]

 

Optimum temperature

28°C

TAS [1]

 

Salinity

tolerance of 2–4% (w/v) NaCl

TAS [1]

MIGS-22

Oxygen requirement

aerobic and microaerobic, no growth under anaerobic conditions

TAS [1]

 

Carbon source

glucose, maltose, mannose, cellobiose

TAS [1]

 

Energy source

unknown

 

MIGS-6

Habitat

cave (soil)

TAS [1]

MIGS-15

Biotic relationship

  

MIGS-14

Pathogenicity

none

NAS

 

Biosafety level

1

TAS [11]

 

Isolation

cave, soil between rocks

TAS [1]

MIGS-4

Geographic location

Guangxi, China

TAS [1]

MIGS-5

Sample collection time

about 1999

TAS [1]

MIGS-4.1

Longitude - Latitude

110.263306 - 25.307878

TAS [1]

MIGS-4.2

MIGS-4.3

Depth

not reported

 

MIGS-4.4

Altitude

not reported

 

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 the Gene Ontology project [12]. If the evidence code is IDA the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

In addition to strain HKI 0122T, only one additional strain (HKI 0132) was isolated from the soil sample collected in the Reed Flute Cave near Guilin, Guangxi, China. HKI 0132 was also classified in the species B. cavernae [1]. No closely related isolates and uncultivated clones with more than 97% 16S rRNA gene sequence identity are recorded in the microbiological literature, nor can any phylotype from environmental samples or genomic surveys be directly linked to B. cavernae.

B. cavernae cells vary in shape and colonies grown on rich medium vary in color from cream to bright yellow. In young cultures, cells are irregular rods arranged in palisades, clusters or in pairs at an angle to give V-formations (Figure 2) [1]. Cells in stationary cultures are predominantly coccoid, occurring singly, in pairs, irregular clusters and short chains. During growth in complex media a rod-coccus growth cycle was observed [1]. B. cavernae grow well under aerobic and microaerophilic conditions, but not under anaerobic conditions [1]. The optimal growth temperature is 28°C [1].
Figure 2.

Scanning electron micrograph of B. cavernae HKI 0122T

B. cavernae is able to degrade casein, esculin, gelatin and potato starch. Acids are produced from L-arabinose, D-cellobiose, dextrin, D-fructose, D-galactose, D-glucose, glycerol, inulin, maltose, D-mannose, D-raffinose, L-rhamnose, D-ribose, salicin, sucrose, starch, trehalose and D-xylose. There is no acid production from D-glucitol, lactose and D-mannitol. Nitrate is reduced to nitrite, H2S is produced [1].

Classification and features

Figure 1. shows the phylogenetic neighborhood of B. cavernae strain HKI 0122T in a 16S rRNA based tree. Analysis of the two identical 16S rRNA gene sequences in the genome of strain HKI differed by four nucleotides from the previously published 16S rRNA sequence generated from DSM 12333 (Y18378). The slight differences between the genome data and the reported 16S rRNA gene sequence is most likely due to sequencing errors in the previously reported sequence data.

Chemotaxonomy

The peptidoglycan of B. cavernae HKI 0122T contains D- and L-alanine, D- and L-glutamic acid and L-lysine, with the latter widely distributed among actinobacteria [1]. The strain possesses a type A4〈 peptidoglycan with a diagnostic LLys←L-Glu interpeptide bridge, type A11.54 according to http://www.dsmz.de/microorganisms/. Glucose, mannose and galactose are the cell wall sugars [1]. The fatty acid profile of strain B. cavernae HKI 0122T is dominated by 13-methyl tetradecanoic (iso-C15:0; 43.7%) and 12-methyl tetradecanoic (anteiso-C15:0; 34.6%) saturated, branched chain acids. Other predominantly saturated fatty acids play a minor role in the cellular fatty acid composition of the strain: iso-C14:0 (0.9%), C14:0 (1.9%); C15:0 (0.9%) isoC16:0 (2.3%), C16:0 (6.8%), isoC17:0 (3.1%), anteiso-C17:0 (4.9%), und C18:1 (0.9%) [1]. Mycolic acids are not present [1]. MK-8(H4) is the major menaquinone, complemented by minor amounts of MK-8(H2), MK-8 and MK-9(H4) [1]. The combination of the LLys←L-Glu interpeptide bridge and MK-8(H4) as the dominating menaquinone is shared with the organisms from the neighboring genera Bogoriella and Georgenia. The polar lipids of strain HKI 0122T consist of phosphatidylinositol and diphosphatidylglycerol together with three yet unidentified phospholipids [1].

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 Genomes OnLine Database [8] and the complete genome sequence in GenBank (CP001618). 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-31

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

MIGS-31.2

Sequencing coverage

8.56x Sanger; 10.86x pyrosequence

MIGS-30

Assemblers

Newbler version 1.1.02.15, phrap

MIGS-32

Gene calling method

Prodigal

 

INSDC / Genbank ID

CP001618

 

Genbank Date of Release

07-MAY-2009

 

GOLD ID

Gc01025

 

NCBI project ID

20827

 

Database: IMG-GEBA

2501416922

MIGS-13

Source material identifier

DSM 12333

 

Project relevance

Tree of Life, GEBA

Growth conditions and DNA isolation

B. cavernae HKI 0122T, DSM 12333, was grown in DSMZ medium 736 (Rich Medium) [13] at 28°C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modification of the standard protocol for cell lysis in first freezing for 20 min. (−70°C), then heating 5 min. (98°C), and cooling 15 min to 37°C; adding 1.5 ml lysozyme (standard: 0.3 ml, only), 1.0 ml achromopeptidase, 0.12 ml lysostaphine, 0.12 ml mutanolysine, 1.5 ml proteinase K (standard: 0.5 ml, only). Over night incubation at 35°C.

Genome sequencing and assembly

The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 5,256 overlapping fragments of 1000 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 parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [14]. Gaps between contigs were closed by editing in Consed, custom primer walking or PCR amplification. A total of 1627 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 19.42x coverage of the genome.

Genome annotation

Genes were identified using Prodigal [15] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGIGenePRIMP pipeline [16]. 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 (IMG-ER) platform [17].

Genome properties

The genome is 4,669,183 bp long and comprises one main circular chromosome with a 73.1% GC content. (Table 3 and Figure 3). Of the 4278 genes predicted, 4225 were protein coding genes, and 53 RNAs. Twenty eight pseudogenes were also identified. The majority of the genes (74.3%) 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.
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, rRNAs red, other RNAs black), GC content, GC skew

Table 3.

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

4,669,183

 

DNA Coding region (bp)

4,347,731

93.12%

DNA G+C content (bp)

3,413,947

73.12%

Number of replicons

1

 

Extrachromosomal elements

0

 

Total genes

4278

100.00%

RNA genes

53

1.24%

rRNA operons

2

 

Protein-coding genes

4225

98.76%

Pseudo genes

28

0.65%

Genes with function prediction

3183

74.40%

Genes in paralog clusters

689

16.11%

Genes assigned to COGs

3109

72.67%

Genes assigned Pfam domains

3246

75.88%

Genes with signal peptides

1034

24.17%

Genes with transmembrane helices

1135

26.53%

CRISPR repeats

1

 
Table 4.

Number of genes associated with the 21 general COG functional categories

Code

Value

%

Description

J

169

4

Translation, ribosomal structure and biogenesis

A

4

0.1

RNA processing and modification

K

384

9.1

Transcription

L

122

2.9

Replication, recombination and repair

B

1

0

Chromatin structure and dynamics

D

25

0.6

Cell cycle control, mitosis and meiosis

Y

0

0

Nuclear structure

V

95

2.3

Defense mechanisms

T

138

3.3

Signal transduction mechanisms

M

166

3.9

Cell wall/membrane biogenesis

N

1

0

Cell motility

Z

0

0

Cytoskeleton

W

0

0

Extracellular structures

U

27

0.6

Intracellular trafficking and secretion

O

89

2.1

Posttranslational modification, protein turnover, chaperones

J

169

4

Translation, ribosomal structure and biogenesis

G

546

12.9

Carbohydrate transport and metabolism

E

264

6.3

Amino acid transport and metabolism

F

92

2.2

Nucleotide transport and metabolism

H

129

3.1

Coenzyme transport and metabolism

I

101

2.4

Lipid transport and metabolism

P

183

4.3

Inorganic ion transport and metabolism

Q

62

1.5

Secondary metabolites biosynthesis, transport and catabolism

R

433

10.3

General function prediction only

S

249

5.9

Function unknown

-

1116

26.4

Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Katja Steenblock for growing B. cavernae 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.

Authors’ Affiliations

(1)
DOE Joint Genome Institute
(2)
Oak Ridge National Laboratory
(3)
DSMZ - German Collection of Microorganisms and Cell Cultures GmbH
(4)
HZI - Helmholtz Centre for Infection Research
(5)
Bioscience Division, Los Alamos National Laboratory
(6)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory
(7)
Lawrence Livermore National Laboratory
(8)
University of California Davis Genome Center

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Copyright

© The Author(s) 2009