Abstract

The phosphite assimilating bacterium, P. glucosidilyticus DD6b, was isolated from the gut of the zooplankton Daphnia magna. Its 3,872,381 bp high-quality draft genome is arranged into 93 contigs containing 3311 predicted protein-coding and 41 RNA-encoding genes. This genome report presents the specific properties and common features of P. glucosidilyticus DD6b genome in comparison with the genomes of P. glucosidilyticus type strain DSM 23,534, and another five Pedobacter type strains with publicly available completely sequenced genomes. Here, we present the first journal report on P. glucosidilyticus genome sequence and provide information on a new specific physiological determinant of P. glucosidilyticus species.

Keywords

Pedobacter glucosidilyticusPhosphite assimilationSphingobacteriia

Introduction

Pedobacter glucosidilyticus strain DD6b was isolated from the crustacean Daphnia magna gut microbial community. During a study on nutritional needs of D. magna, the gut microbiota was investigated over time, under starvation stress and after host death [1, 2].

Daphnia spp. are small members of the zooplankton and key organisms in freshwater food webs. Heterotrophic bacteria contribute significantly to the nutrition of Daphnia species [3, 4] and are often characterized by high P:C values [5], indicating that they are a rich source of phosphorus for zooplankton [6].

Furthermore, some bacteria can assimilate reduced inorganic and organic P compounds (phosphite [+III] and organophosphonates, [7–15]) under phosphate starvation. Organophosphonates have been found in a variety of biologically produced molecules including antibiotics, phosphonolipids, phosphonoproteins, phosphonoglycans [7]. The most common naturally occurring phosphonate 2-AEP or ciliatine serves as a precursor in the biosynthesis of phosphonolipids in marine single celled organisms, sea anemones or ciliated protozoan. Recently, a sphingophosphonolipid was found in Bacteriovorax stolpii, a facultative predator that parasitizes larger Gram-negative bacteria [16].

Tests with newly isolated P. glucosidilyticus DD6b revealed growth with phosphite as a single P-source. This ability of the strain DD6b, together with the lack of information in the literature concerning phosphite or organophosphonate assimilation abilities of the other members of genus Pedobacter was the reason to investigate the genome of P. glucosidilyticus DD6b.

The cells of P. glucosidilyticus strain DD6b are non-flagellated, non-spore-forming, flexible gliding rods with slightly rounded or tapered ends. They have protruded surfaces, and vary in size ranging from 1.0–1.2 μm in length and 0.2–0.3 μm in width (Fig. 1). Colonies (0.8–1 mm in diameter) appear after 6–7 days. They have orange-pink color on nutrient agar at 25 °C (Fig. 1, Right). Strain DD6b exhibits moderate growth, with a doubling time of 15–20 h, when grown on complex media such as nutrient broth. On chemically defined minimal medium MDS3 the strain had a doubling time of a) 7.5 h with phosphate and b) 20–23 h with phosphite as single P-source. Growth occurred at 15–28 °C, pH 7.0–7.2, and 0.2–0.5 % NaCl in the medium. Strain DD6b is motile via gliding.

The ability of P. glucosidilyticus strain DD6b to grow on phosphite was proved by cultivating it successfully after 3 successive transfers on MDS3 medium, supplemented with 1 mM phosphite as sole P-source. The concentration of phosphite in the medium was monitored during the growth experiments as described previously [10].

Current taxonomic classification and general features of P. glucosidilyticus DD6b according to the minimum information about the genome sequence requirements are provided in Table 1. Additional information about the genome is available in the associated MIGS record table (Additional file 1: Table S1).

Table 1

Classification and general features of P. glucosidilyticus strain DD6b [22]

aEvidence codes - IDA Inferred from Direct Assay, 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 [45]

Phylogenetic tree based on 16S rRNA gene sequences of all types trains of Sphingobacteriaceae: The dendrogram was calculated with MEGA5 [18] using the Maximum Likelihood method based on the Jukes-Cantor model [19]. The analyzed sequences were aligned by CLUSTALW [21]. The clustering of the sequences was tested by the bootstrap approach with 1000 resamplings. The length of the tree branches was scaled according to the number of substitutions per site (see size bar). P. glucosidilyticus DD6b is marked in red, Pedobacter species with publicly available genome sequences are marked in blue. All other genome-sequenced species are marked with an asterisk

The phylogenetic tree was produced with MEGA5 [18] using the Maximum Likelihood method based on the Jukes Cantor model [19]. Sequences of all Sphingobacteriaceae type strains were downloaded from RDP [20], aligned by CLUSTALW [21] and tested by the bootstrap approach with 1000 resamplings. The length of the tree branches was scaled according the number of substitutions per site. Strain DD6b clustered clearly within the Pedobacter group and next to P. glucosidilyticus 1-2T.

Genome sequencing information

Genome project history

This organism was selected for sequencing on the basis of its environmental relevance to issues in global P cycle and the source of isolation. The genome project is deposited in GenBank database (JMTN00000000) and an improved high-quality-draft genome sequence in IMG (2590828803). Genome sequencing and annotation were done at Göttingen Genomics Laboratory (Georg-August-University Göttingen), while cultivation and analysis were performed at the University of Konstanz. A summary of the project information and its association with MIGS version 2.0 compliance [22] is shown in Table 2.

Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Improved-high-quality draft

MIGS-28

Libraries used

Illumina paired-end library; Nextera XT

MIGS 29

Sequencing platforms

Illumina XT

MIGS 31.2

Fold coverage

120 × Illumina

MIGS 30

Assemblers

SPAdes

MIGS 32

Gene calling method

Prodigal

Locus Tag

PBAC

Genbank ID

JMTN00000000

GenBank Date of Release

December 8th, 2014

GOLD ID

Gp0043583

BIOPROJECT

PRJNA246159

MIGS 13

Source Material Identifier

DD6b

Project relevance

Ecology

Growth conditions and genomic DNA preparation

P. glucosidilyticus DD6b was grown in nutrient broth. A newly developed chemically defined medium MDS3 was used to study carbohydrate, phosphite and phosphate assimilation. Phosphite and phosphate assimilation tests were performed in MDS3 medium supplemented with 0.1 to 1 mM phosphite or phosphate as single P- source. Glucose 10 mM final concentration was used as C- source. The chemical composition and preparation of MDS3 medium are given in Additional file 2: Data S2. The DNA extraction procedure was performed in the following way: 4 ml of a well grown fresh culture reaching its maximum optical density (OD600nm = 0.291 ± 0.040) were spun down at 13 000 × g in a benchtop micro centrifuge for 5 min. The supernatant was discarded and the cell pellet was suspended in the cell lysis solution from the Purgene Core Kit B (Qiagen, Hilden, Germany). Further, the extraction was performed as per manufacturer’s instructions, following the protocol for Gram-negative bacteria. The genomic DNA yield was 47.7 ng/μl. The purity of the preparation was estimated with NanoDrop ND-1000 (Thermo Fisher Scientific, Germany), with an UV absorbance ratio at 260/280 nm of 2.33, and an UV absorbance ratio at 260/230 of 0.84.

Genome sequencing and assembly

The extracted genomic DNA was used for whole genome sequencing employing a Genome Analyzer II (Illumina, San Diego, CA, USA). Shotgun libraries were prepared according to the protocol of the manufacturer. Sequencing resulted in 12,380,618 paired-end Illumina reads of 112 bp. Reads were trimmed using Trimmomatic 0.32 [23] to get rid of sequences with quality scores lower than 20 (Illumina 1.9 encoding) and remaining adaptor sequences. SPAdes 2.5 software [24] was employed for the initial de novo assembly and 4,150,000 reads. The final assembly resulted in 93 contigs larger than 0.5 kb from which 84 were larger than 1 kb including 68 contigs larger than 3 kb. This assembly had an average coverage of 120, N50 value of 97,360 bp and N90 value of 24,905 bp, respectively.

Genome annotation

The software tool Prodigal [25] was used for automatic gene prediction. rRNA and tRNA genes were identified with RNAmmer and tRNAscan, respectively [26, 27]. Automatic annotation was carried out with the IMG-ER system [28, 29] and afterwards manually curated by employing BLASTP, Swiss-Prot, TrEMBL, and InterPro databases [30].

Genome properties

The statistics of the genome are given in Table 3. The high quality draft genome was assembled into 93 contigs with a total size of 3876 Mb and an overall GC content of 34.74 mol%. A total of 3352 genes were predicted of which 3311 were protein-encoding and 41 RNAs genes (3 rRNA and 38 tRNA). Of the protein-encoding genes 2610 (77.86 %) were assigned to a putative function and the remaining 701 (20.91 %) were annotated as hypothetical proteins. The distribution of the genes into COG functional categories [31] is shown in Table 4. One CRISPR array of 46 repeats with a direct repeat length of 46 nt adjacent to an incomplete cas cluster comprising cas1, cas2 and cas9 was identified in the genome of P. glucosidilyticus DD6b. However, potential prophage regions were not present in the genome sequence.

Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

3,872,381

100.00

DNA coding (bp)

3,510,386

90.65

DNA G + C (bp)

1,344,522

34.72

DNA scaffolds

93

100.00

Total genes

3352

100.00

Protein coding genes

3311

98.78

RNA genes

41

1.22

Pseudo genes

0

0

Genes in internal clusters

2465

73.54

Genes with function prediction

2610

77.86

Genes assigned to COGs

1910

73.54

Genes with Pfam domains

2646

56.98

Genes with signal peptides

482

14.38

Genes with transmembrane helices

749

22.34

CRISPR repeats

1

0

Table 4

Number of genes associated with general COG functional categories

Code

Value

% age

Description

J

149

7.17

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

129

6.20

Transcription

L

102

4.91

Replication, recombination and repair

B

1

0.04

Chromatin structure and dynamics

D

20

0.96

Cell cycle control, Cell division, chromosome partitioning

V

35

1.68

Defense mechanisms

T

69

3.32

Signal transduction mechanisms

M

185

8.90

Cell wall/membrane biogenesis

N

4

0.19

Cell motility

U

27

1.30

Intracellular trafficking and secretion

O

76

3.66

Posttranslational modification, protein turnover, chaperones

C

123

5.92

Energy production and conversion

G

173

8.32

Carbohydrate transport and metabolism

E

154

7.41

Amino acid transport and metabolism

F

64

3.08

Nucleotide transport and metabolism

H

117

5.63

Coenzyme transport and metabolism

I

77

3.70

Lipid transport and metabolism

P

119

7.72

Inorganic ion transport and metabolism

Q

36

1.73

Secondary metabolites biosynthesis, transport and catabolism

R

255

12.27

General function prediction only

S

164

7.89

Function unknown

-

1442

43.02

Not in COGs

The total is based on the total number of protein coding genes in the genome

Another specific property of P. glucosidilyticus DD6b in comparison with the P. glucosidilyticus type strain, is the presence of a complete DNRA pathway allowing the reduction of nitrate to ammonium, where the reduction of nitrate to nitrite proceeds through an assimilatory ferredoxin-nitrate reductase narB (PBAC_22000) and the reduction of nitrite to ammonia by a dissimilatory nitrite reductase nirBD (PBAC_21900; PBAC_21910). The positions of the gene clusters for DNRA pathway and phoPR are shown in Fig. 3 (circle 5, clusters 1 and 2, clockwise).

Strain DD6b assimilates phosphite and most probably can assimilate phosphonoacetate under phosphate starvation to support its growth. Phosphite oxidation in strain DD6b proceeds via a sec-dependent periplasmic alkaline phosphatase (PBAC_31300), analogously to E.coli [15]. The genome sequence of strain DD6b suggests that this bacterium should be able to assimilate phosphonoacetate under phosphate starvation, as the gene encoding phosphonoacetate hydrolase phnA (PBAC_28850) catalyzing the hydrolysis of phosphonoacetate to acetate and phosphate is present. Strain DD6b does not harbor a complete 2-aminoethylphosphonate degradation pathway, despite the presence of a phosphonoacetate hydrolase encoding gene. Also, neither genes encoding carbon-phosphorus lyase complex, nor genes coding for phosphonopyruvate hydrolase or phosphonoacetaldehyde hydrolase were detected in the genome. The regulation of the phosphorus homeostasis in Gram negative bacteria is under the control of the two-component signal transduction pathway of the Pho regulon, encoded by phoPR. Also, 8 copies of the gene encoding alkaline phosphatase synthesis sensor protein PhoR were identified in the genome of P. glucosidilyticus DD6b one of which (PBAC_27380) was specific for this genome, without present orthologs in the rest of the Pedobacter type species whole genome sequences. The second component PhoP is present with two orthologous genes in the genome of DD6b.

Also, P. glucosidilyticus DD6b has 447 singletons, the majority of which encode proteins of unknown function. Amongst the unique genes with assigned functions were found those encoding the synthesis of a green-light absorbing proteorhodopsin (PBAC_30230) playing a role in the generation of phototrophic energy (Fig. 3, position 3), proteins involved in the synthesis of EPS and capsule formation. Specific for the genome of P. glucosidilyticus DD6b are a rhamnogalacturonate lyase (PBAC_06010) and a second specific pectate lyase coding genes (PBAC_05900) participating in the degradation of rhamnogalacturonan I and pectin [38]. A comprehensive genome properties comparison of the 5 publicly available Pedobacter type strains genomes and the P. glucosidilyticus DD6b used in this study is presented in Additional file 3: Table S3.

Specific for P. glucosidilyticus DD6b genome is the presence of genes related to phytopathogenicity and pectine degradation, as well as for assimilative nitrate reduction.

Based on physiological experiments, we proved that P. glucosidilyticus DD6b assimilates phosphite as single phosphorus source, in agreement with the presence of a periplasmic alkaline phosphatase-encoding gene in the genome of the strain. Furthermore, the presence of an orthologous alkaline phosphatase gene in the genome of P. glucosidilyticus DSM 23,534 strongly suggests that the type strain might possess this ability too. Common and specific only for both P. glucosidilyticus genomes was the presence of a phosphonoacetate hydrolase (PhnA) encoding gene, suggesting phosphonoacetate utilization ability for both strains. However, their genomes do not encode a complete 2-aminoethylphosphonate degradation pathway. Finally, none of the 6 analyzed Pedobacter genomes encoded any of the rest known organophosphonate degradation pathways. Overall members of genus Pedobacter species are characterized by low diversity and distribution of inorganic and organophosphonate degradation pathways. However, in future studies the phosphite assimilation property of P. glucosidilyticus species can be regarded as specific physiological determinant within genus Pedobacter (Additional file 4).

Abbreviations

2-AEP:

2-aminoethylphosphonate

Declarations

Acknowledgements

We thank Heike M. Freese for supplying the strain, Bernhard Schink for support, Joachim Hentschel for the scanning electron micrograph of the strain, Bernd Gahr, Sylke Wiechmann, Frauke-Dorothee Meyer and Kathleen Gollnow for technical assistance. This work was supported by the University of Konstanz. D.D.S. was partially supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn – Bad Godesberg, Germany (SI 1300/4-1).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.