Open Access

Complete genome sequence of Kribbella flavida type strain (IFO 14399T)

  • Rüdiger Pukall
  • , Alla Lapidus
  • , Tijana Glavina Del Rio
  • , Alex Copeland
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Susan Lucas
  • , Feng Chen
  • , Matt Nolan
  • , Kurt LaButti
  • , Amrita Pati
  • , 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,
  • , Manfred Rohde
  • , Markus Göker
  • , Jim Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Thomas Brettin,
Corresponding author

DOI: 10.4056/sigs.731321

Received: 30 March 2010

Published: 30 April 2010

Abstract

The genus Kribbella consists of 15 species, with Kribbella flavida (Park et al. 1999) as the type species. The name Kribbella was formed from the acronym of the Korea Research Institute of Bioscience and Biotechnology, KRIBB. Strains of the various Kribbella species were originally isolated from soil, potato, alum slate mine, patinas of catacombs or from horse racecourses. Here we describe the features of K. flavida together with the complete genome sequence and annotation. In addition to the 5.3 Mbp genome of Nocardioides sp. JS614, this is only the second completed genome sequence of the family Nocardioidaceae. The 7,579,488 bp long genome with its 7,086 protein-coding and 60 RNA genes and is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

ActinobacteriaaerobicsoilmyceliaLL-diaminopimelic acidPropionibacterineaeNocardioidaceaeGEBA

Introduction

Strain IFO 14399T (= DSM 17836 = KCTC 9580 = JCM 10339 = NBRC 14399) is the type strain the species Kribbella flavida, which is the type species of the genus Kribbella. Strain IFO 14399T was originally isolated from soil in China and first described as ‘Nocardioides fulvus’ by Ruan and Zhang, 1979 [1]. In 1999, the strain was reclassified into the novel genus Kribbella on the basis of comparative chemotaxonomic and 16S rRNA sequence analysis [2]. K. flavida exhibits mycelia on several media used for growing the strain. The mycelium consists of hyphae, which are extensively branched and penetrate into the agar medium. The hyphae often fragment into rod to coccus-like elements [2]. Here we present a summary classification and a set of features for K. flavida IFO 14399T, together with the description of the complete genomic sequencing and annotation.

Classification and features

The type strain IFO 14399T was isolated from soil in China. Genbank contains only one additional 16S rRNA gene sequence with at least 99% similarity, derived from a strain isolated from scabby potatoes (EU80972). No phylotypes from environmental samples or genomic surveys be directly linked to K. flavida, indicating rare occurrence of the species in so far screened habitats (October 2009). Figure 1 shows the phylogenetic neighborhood of K. flavida IFO 14399T in a 16S rRNA based tree. The sequence of the two 16S rRNA genes in the genome of strain 14399T differ by two nucleotides from each other and by up to two nucleotides from the previously published 16S rRNA sequence generated from KACC 20258 (AY253863).

Figure 1

Tree highlighting the position of K. flavida IFO 14399T relative to the other type strains of the genus Kribbella and the type strains of the other genera within the families Nocardioidaceae and Propionibacteriaceae. The tree was inferred from 1,343 aligned characters [3,4] of the 16S rRNA gene sequence under the maximum likelihood criterion [5] and rooted in accordance with current taxonomy. 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 [6] are shown in blue, published genomes in bold.

K. flavida is a Gram-positive, aerobic and non-acid-fast actinomycete (Table 1), characterized by primary mycelium (Figure 2), with branched hyphae that penetrate into the agar medium. Aerial mycelium is also developed and can break up into short to elongated rod-like elements. Growth occurs between pH 5 and 9 and between 20 and 37°C. The strain shows positive activity for catalase, oxidase and urease. It utilizes D-glucose, D-cellobiose, maltose, D-melibiose, sucrose, D-trehalose, melezitose, D-raffinose, adonitol, myo-inositol, D-mannitol, inulin, disodium succinate and disodium fumarate as sole carbon and energy source [2].

Table 1

Classification and general features of K. flavida IFO 14399T according to the MIGS recommendations [7]

MIGS ID

   Property

    Term

    Evidence code

   Current classification

    Domain Bacteria

    TAS [8]

    Phylum Actinobacteria

    TAS [9]

    Class Actinobacteria

    TAS [10]

    Order Actinomycetales

    TAS [11]

    Suborder Propionibacterineae

    TAS [10]

    Family Nocardioidaceae

    TAS [12]

    Genus Kribbella

    TAS [2]

    Species Kribbella flavida

    TAS [2]

    Type strain IFO 14399

    TAS [2]

   Gram stain

    positive

    TAS [2]

   Cell shape

    hyphae, fragmented into rod to    coccoid elements

    TAS [2]

   Motility

    nonmotile

    NAS

   Sporulation

    nonsporulating

    NAS

   Temperature range

    20°C-37°C

    TAS [2]

   Optimum temperature

    not reported

   Salinity

    not reported

MIGS-22

   Oxygen requirement

    strictly aerobic

    TAS [2]

   Carbon source

    saccharolytic

    TAS [2]

   Energy source

    carbohydrates

    TAS [2]

MIGS-6

   Habitat

    soil

    TAS [2]

MIGS-15

   Biotic relationship

    free living

    NAS

MIGS-14

   Pathogenicity

    none

    NAS

   Biosafety level

    1

    TAS [13]

   Isolation

    soil

    TAS [1,2]

MIGS-4

   Geographic location

    Beijing, China

    TAS [1]

MIGS-5

   Sample collection time

    NAS

MIGS-4.1MIGS-4.2

   Latitude    Longitude

    39.55    116.25

    NAS

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 of the Gene Ontology project [14]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Figure 2

Scanning electron micrograph of K. flavida IFO 14399T

Chemotaxonomy

One of the meaningful characteristics of the genus Kribbella is the presence of LL-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan [2]. The predominant menaquinone is a tetrahydrogenated menaquinone with nine isoprenoid units MK-9(H4) [2]. The major fatty acids detected in K. flavida are anteiso-C15:0 and iso-C16:0 [2]. Phosphatidylcholine is the main polar lipid [2]. The genus Kribbella differs from other LL-diaminopimelic acid and MK-9(H4) containing taxa, by having a typical hyphal morphology [2].

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 [15] 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.

Table 2

Genome sequencing project information

MIGS ID

    Property

    Term

MIGS-31

    Finishing quality

    Finished

MIGS-28

    Libraries used

    Two Sanger libraries: 8kb pMCL200 and fosmid pcc1Fos    One 454 pyrosequence standard library and    one Standard Illumina library

MIGS-29

    Sequencing platforms

    ABI3730, 454 GS FLX, Illumina GA

MIGS-31.2

    Sequencing coverage

    7.4× Sanger; 13.4× pyrosequence

MIGS-30

    Assemblers

    Newbler 1.1.01.20, phrap

MIGS-32

    Gene calling method

    Prodigal, GenePRIMP

    INSDC ID

    CP001736

    Genbank Date of Release

    January 13, 2010

    GOLD ID

    Gc01192

    NCBI project ID

    21089

    Database: IMG-GEBA

    2501939632

MIGS-13

    Source material identifier

    DSM 17836

    Project relevance

    Tree of Life, GEBA

Growth conditions and DNA isolation

K. flavida IFO 14399T, DSM 17836, was grown in DSM medium 830 [15] at 28°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. [16].

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 can be found on the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.01.20 (Roche). Large Newbler contigs were broken into 8,548 overlapping fragments of 1,000 bp and entered into 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 [17] or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 2,850 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher). The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 51.2× coverage of the genome. The final assembly contains 59,008 Sanger and 433,053 pyrosequence reads.

Genome annotation

Genes were identified using Prodigal [18] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [19]. 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 manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [20].

Genome properties

The genome is 7,579,488 bp long with a 70.6% GC content (Table 3 and Figure 3). Of the 7,146 genes predicted, 7,086 were protein-coding genes, and 60 RNAs; 143 pseudogenes were also identified. The majority of the protein-coding genes (70.7%) were assigned with a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is summarized in Table 4.

Table 3

Genome Statistics

Attribute

  Value

   % of Total

Genome size (bp)

  7,579,488

   100.00%

DNA coding region (bp)

  6,893,122

   90.94%

DNA G+C content (bp)

  5,348,686

   70.57%

Number of replicons

  1

Extrachromosomal elements

  0

Total genes

  7,146

   100.00%

RNA genes

  60

   0.84%

rRNA operons

  2

Protein-coding genes

  7,086

   99.16%

Pseudo genes

  143

   2.00%

Genes with function prediction

  5,049

   70.65%

Genes in paralog clusters

  1,595

   22.32%

Genes assigned to COGs

  4,877

   68.25%

Genes assigned Pfam domains

  5,174

   72.40%

Genes with signal peptides

  1,721

   24.08%

Genes with transmembrane helices

  1,675

   23.44%

CRISPR repeats

  0

   0

Figure 3

Graphical circular 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, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code

   Value

   %age

   Description

J

   225

   4.1

   Translation, ribosomal structure and biogenesis

A

   2

   0.0

   RNA processing and modification

K

   762

   13.8

   Transcription

L

   184

   3.3

   Replication, recombination and repair

B

   1

   0.0

   Chromatin structure and dynamics

D

   38

   0.7

   Cell cycle control, cell division, chromosome partitioning

Y

   0

   0.0

   Nuclear structure

V

   136

   2.5

   Defense mechanisms

T

   261

   4.7

   Signal transduction mechanisms

M

   239

   4.3

   Cell wall/membrane biogenesis

N

   2

   0.0

   Cell motility

Z

   2

   0.0

   Cytoskeleton

W

   0

   0.0

   Extracellular structures

U

   46

   0.8

   Intracellular trafficking and secretion

O

   143

   2.6

   Posttranslational modification, protein turnover, chaperones

C

   308

   5.6

   Energy production and conversion

G

   636

   11.5

   Carbohydrate transport and metabolism

E

   397

   7.2

   Amino acid transport and metabolism

F

   100

   1.9

   Nucleotide transport and metabolism

H

   264

   4.8

   Coenzyme transport and metabolism

I

   212

   3.8

   Lipid transport and metabolism

P

   218

   3.9

   Inorganic ion transport and metabolism

Q

   175

   3.2

   Secondary metabolites biosynthesis, transport and catabolism

R

   802

   14.5

   General function prediction only

S

   367

   6.7

   Function unknown

-

   2,269

   31.8

   Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Katja Steenblock for growing K. flavida 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, 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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