Open Access

Insights into Cedecea neteri strain M006 through complete genome sequence, a rare bacterium from aquatic environment

Standards in Genomic Sciences201712:40

https://doi.org/10.1186/s40793-017-0255-1

Received: 10 April 2017

Accepted: 12 July 2017

Published: 21 July 2017

Abstract

Cedecea neteri M006 is a rare bacterium typically found as an environmental isolate from the tropical rainforest Sungai Tua waterfall (Gombak, Selangor, Malaysia). It is a Gram-reaction-negative, facultative anaerobic, bacillus. Here, we explore the features of Cedecea neteri M006, together with its genome sequence and annotation. The genome comprised 4,965,436 bp with 4447 protein-coding genes and 103 RNA genes.

Keywords

Cedecea Gram-negative Facultative anaerobic Genome

Introduction

The Cedecea genus is an extremely rare member of the Enterobacteriaceae family [1]. The name Cedecea was proposed in 1980 for a new genus formerly designated as CDC Enteric Group 15 [1, 2]. Cedecea is characterized by positive lipase activity, resistance to colistin and cephalothin, and the inability to hydrolyze gelatin or DNA [35]. Discovery was from human sources where its natural environmental habitat remains unknown, Cedecea constitutes a rare pathogen of rising importance [6]. To date, only a few species of Cedecea have been identified: C. davisae , C. lapagei and C. neteri . All three species exhibit different behaviors in the human body. C. davisae has been reported to be associated with scrotal abscess [7] and, most recently, to cause bacteraemia in patients with sigmoid colon cancer [8]. On the other hand, C. lapagei has mostly been reported to be involved in pneumonia cases [5, 9]. C. neteri is associated with bacteremia in heart disease patients [4] and patients with systemic lupus erythematosus [10].

Strain M006 is a strain of Cedecea neteri and is an aquatic isolate from the Sungai Tua Waterfall, a Malaysian tropical rainforest waterfall (N 03 19.91′ E 101 42.15′). In this study, we present an overview of the classification and features of C. neteri M006 as well as its genome sequence and annotation. There are a few C. neteri aquatic isolates deposited in GenBank and C. neteri strain M006 was one of the few isolates discovered from a waterfall which its genome feature has not been reported. Hence, here we firstly reported the genome information of C. neteri M006 isolated from a waterfall environment.

Organisms Information

Classification and features

Strain M006 was categorized as a member of the genus Cedecea by 16S rRNA phylogeny and phenotypic characteristics (Table 1). The EzTaxon database [11] was used as the preliminary 16S rRNA gene sequence-based identification. Strain M006 was most closely related to C. neteri GTC 1717T (GenBank accession = AB086230) with a sequence similarity of 99.78%. Subsequent phylogenetic analysis was performed comparing the 16S rRNA gene sequences of strain M006 and related species (Fig. 1). The sequences were aligned and phylogenic trees were built using neighbor-joining (NJ) and maximum-likelihood (ML) methods implemented in MEGA version 5 [12].
Table 1

Classification and general features of Cedecea neteri M006 according to MIGS recommendations [14]

MIGS ID

Property

Term

Evidence code

 

Classification

Domain Bacteria

TAS [22]

Phylum Proteobacteria

TAS [23, 24]

Class Gammaproteobacteria

TAS [2527]

Order unknown

TAS [23]

Family Enterobacteriaceae

TAS [2830]

Genus Cedecea

TAS [4]

Species Cedecea neteri

IDA

Strain: M006

Gram stain

negative

TAS [4, 10]

Cell shape

bacillus

TAS [4, 10]

Motility

motile

TAS [4]

Sporulation

Non-spore forming

NAS

Temperature range

4-28 °C

IDA

Optimum temperature

28 °C

IDA

pH range; Optimum

e.g., 5.0-8.0; 7

IDA

Carbon source

D-sorbitol, Sucrose, D-xylose, malonate

TAS [4]

MIGS-6

Habitat

waterfall

IDA

MIGS-6.3

Salinity

unknown

IDA

MIGS-22

Oxygen requirement

Facultative anaerobic

TAS [4, 10]

MIGS-15

Biotic relationship

Free-living

TAS [4]

MIGS-14

Pathogenicity

Non-pathogen

IDA

MIGS-4

Geographic location

Sungai Tua Waterfall, Malaysia

IDA

MIGS-5

Sample collection

2013

IDA

MIGS-4.1

Latitude

N 03 19.91′

IDA

MIGS-4.2

Longitude

E 101 42.15′

IDA

MIGS-4.4

Altitude

586 m

IDA

Evidence 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 [31]

Fig. 1

Phylogenetic tree highlighting the position of Cedecea neteri M006 relative to the type strains of other species within the genus of Cedecea. The strains and their corresponding GenBank accession numbers of 16S rRNA genes are indicated in parentheses. The sequences were aligned and the phylogenetic inferences were obtained using the maximum-likelihood method with MEGA version 5 [12]. The numbers at the nodes are the percentage of bootstrap values obtained by 500 replicates. Bar, 0.01 substitutions per nucleotide positions

C. neteri M006 cells are Gram-negative, bacillus in shape (0.6-0.7 × 1.3-1.9 μm), are facultatively anaerobic and are motile with 5-9 peritrichous flagella. Colonies formed on nutrient agar are 1.5 mm in diameter and non-pigmented. Scanning electron micrograph pictures of nutrient broth grown cultures showed free-floating cells and clotted cells (Fig. 2). The carbon sources utilized by C. neteri are D-sorbitol, sucrose, D-xylose and malonate. C. neteri is reported to be unable to utilize dulcitol, adoitol, L-rhamnose, erythritol, glycerol and mucate. The optimal temperature for strain M006 is 28 °C.
Fig. 2

Scanning electron micrograph of Cedecea neteri M006. Scale bar 3.0 μm

C. neteri M006 cells are Gram-negative, bacillus in shape, survive facultative anaerobically and are motile. The colonies formed on nutrient agar are 1.5 mm in diameter and are non-pigmented. The colony is whitish in color and the appearance is round with a smooth edge. Signaling molecules, known as N-acylhomoserine lactone, are produced for communication purposes in order to regulate physiological properties. The preliminary screening of strain M006 using the bacterial biosensor Chromobacterium violaceum (CV026) showed the purple pigmentation indicative the presence of signaling molecules (Fig. 3).
Fig. 3

Preliminary screening for AHL. AHL screening of strain M006 with CV026. E. carotovora PNP22 and E. carotovora GS101 served as negative and positive controls respectively

Genome sequencing information

Genome project history

Strain M006 was selected for the sequencing based on its phylogenetic position and the similarity of its 16S rRNA to other members of the genus Cedecea , The genome project was deposited in the Genomes On-Line Database [13] and the genome sequence was deposited in GenBank (CP009458.1). A summary of the project and the Minimum Information about a Genome Sequence (MIGS) [14] are shown in Table 2.
Table 2

Genome sequencing project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Complete

MIGS-28

Libraries used

PacBio

MIGS 29

Sequencing platforms

PacBio

MIGS 31.2

Fold coverage

74.34×

MIGS 30

Assemblers

HGAP V 2.1.1

MIGS 32

Gene calling method

IMG-ER

 

Locus Tag

LH23

 

Genbank ID

CP009458

 

Genbank Date of Release

2014/10/22

 

GOLD ID

Gp0109502

 

BIOPROJECT

PRJNA260769

MIGS 13

Source List Identifier

M006

 

Project relevance

Environmental

Growth conditions and genomic DNA preparation

Cedecea neteri M006 was cultured aerobically on Luria-Bertani (LB) agar medium at 28 °C overnight (16-18 h). Genomic DNA was extracted using the MasterPure™ DNA Purification Kit (Epicentre Inc., Madison, WI, USA). The extracted genomic DNA was examined via a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) for its quality.

Genome sequencing and assembly

The genome of strain M006 was sequenced at the microbiome lab, High Impact Research, University Malaya, using a Pacific Biosciences single-molecule real-time (PacBio SMRT) sequencer. The sequencing was carried out using P5 chemistry on two SMRT cells with a 20-kb prepared SMRTbell library [15]. De novo assembly of 41,094 reads using the hierarchical genome assembly process in the SMRT version 2.1.1 portal resulted with one contig of 3.96 Mb in size. The sequencing average coverage is 74.34 × and this genome has a GC content of 54.41%.

Genome annotation

After genome assembly, it was analyzed using Rapid Annotation using Subsystem Technology server databases (version 2.0) [16], which identified 4423 predicted coding sequences with a total of 103 RNA genes. The predicted open reading frames were annotated by searching clusters of orthologous groups [17] using the Integrated Microbial Genomes Expert Review [18]. The different groups of RNAs (rRNA and tRNA) were identified by using RNAmmer 1.2 [19] and tRNAscan-SE 1.23 [20] respectively. The additional gene prediction analysis and functional annotation were performed within IMG-ER platform.

Genome properties

The genome comprised a circular chromosome with a length of 4,965,436 bp and 54.41% G + C content (Fig. 4 and Table 3). It is composed of one contig and of the 4550 predicted genes, 4447 were protein-coding genes. The properties of and the statistics for the genome are summarized in Table 3. The distribution of genes into COG functional categories is presented in Table 4.
Fig. 4

Graphical circular map of the genome. Starting from the outermost circle and moving inwards, each ring of the circle contains information on a genome: tRNA/rRNA, genes on the reverse and forward strands, GC skew and GC ratio

Table 3

Genome statistics

Attribute

Value

% of total

Genome size (bp)

4,965,436

100

DNA coding (bp)

4,350,834

87.62

DNA G + C (bp)

2,701,616

54.41

DNA scaffolds

1

100

Total genes

4550

100

Protein coding genes

4447

97.74

RNA genes

103

2.26

rRNA genes

22

0.48

tRNA

80

1.76

Pseudo genes

24

0.53

Genes in paralog clusters

3462

76.09

Genes with function prediction

4091

89.91

Genes assignmed to COGs

3611

79.36

Genes with Pfam peptides

4095

90.00

Genes with signal peptides

466

10.24

Genes with transmembrane helices

1079

23.71

CRISPR repeats

0

0.00

Table 4

Number of genes associated with general COG functional categories

Code

Value

% agea

Description

J

189

4.70

Translation, ribosomal structure and biogenesis

A

1

0.02

RNA processing and modification

K

395

9.82

Transcription

L

133

3.31

Replication, recombination and repair

B

0

0.00

Chromatin structure and dynamics

D

32

0.80

Cell cycle control, Cell division, chromosome partitioning

V

47

1.17

Defense mechanisms

T

181

4.50

Signal transduction mechanisms

M

224

5.57

Cell wall/membrane biogenesis

N

117

2.91

Cell motility

U

105

2.61

Intracellular trafficking and secretion

O

145

3.60

Posttranslational modification, protein turnover, chaperones

C

231

5.74

Energy production and conversion

G

362

9.00

Carbohydrate transport and metabolism

E

412

10.24

Amino acid transport and metabolism

F

96

2.39

Nucleotide transport and metabolism

H

158

3.93

Coenzyme transport and metabolism

I

109

2.71

Lipid transport and metabolism

P

266

6.61

Inorganic ion transport and metabolism

Q

75

1.86

Secondary metabolites biosynthesis, transport and catabolism

R

409

10.16

General function prediction only

S

337

8.37

Function unknown

-

939

20.64

Not in COGs

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

Insights from the genome sequence

RAST annotation allowed the insight of subsystem category distribution of C. neteri strain M006. This category enabled the understanding of various functional roles such as protein classes, amino acid biosynthesis and metabolic pathways. There are 552 subsystems. The most abundant subsystem feature belonged to carbohydrate metabolism (n = 576; out of a total of 3760 subsystem feature counts), followed by amino acid and derivatives (n = 495) and protein metabolism (n = 299) (Fig. 5). One of the subsystem features grouped as regulation and cell signaling was focused to allow functional genes related to quorum sensing (QS) activity to be searched. The in-silico study identified the novel LuxIR homologue of C. neteri , which was later designated as CneIR. The complete open reading frame of C. neteri strain M006 cneI and cneR homologues were found and are 462 bp and 723 bp, respectively. The complete genome sequencing allows deeper understanding of the genetic makeup that may help in identifying the linkage of pathogenicity and virulence factors with its QS properties [15].
Fig. 5

RAST annotation of C. neteri strain M006. This annotation pipeline allows a view of the subsystem category distribution of C. neteri strain M006. Genes responsible for QS activity in this strain can be found in regulation and cell signaling subsystem (red arrow)

Currently, the availability of genomes of this genus is low. Only 5 complete genomes of C. neteri strains including strain M006 and a draft genome of type strain NBRC 105707 are deposited in NCBI. A matrix and dendrogram were generated based on AAI calculation that provide estimation of the average amino acid identity using best hits (one-way AAI) and reciprocal best hits (two-way AAI) between several genomic datasets of proteins [21], C. davisae type strain DSM 4568 was included in the analyses. From the analyses, we can see closer protein clustering between strain M004 and strain ND14a (Fig. 6). Some of the basic comparisons of the genomes are listed in Table 5.
Fig. 6

AAI calculation for 6 C. neteri strains and 1 C. davisae strain. Analyses of conserved genes in the core genome computed based on AAI calculator provided (a) an AAI matrix; and (b) AAI-based phylogenetic distance tree, clustered according to distance pattern. The AAI-distance tree was clustered based on BIONJ method

Table 5

Comparison of several strains of C. neteri

Organism/Name

Strain

Size (Mb)

GC%

Gene

Protein

C. neteri

M006

4.97

54.40

4703

4531

ND02

4.31

53.90

4053

3884

ND14b

5.05

56.90

4491

4295

ND14a

4.66

54.80

4426

4215

SSMD04

4.88

55.10

4622

4416

NBRC 105707

5.20

54.10

4944

4739

Conclusion

This study provides phenotypic and genomic insights into Cedecea neteri strain M006. It reports the isolation of C. neteri from an aquatic environment for the first time. This study also revealed of the QS ability of C. neteri .

Abbreviations

AHL: 

N-acylhomoserine lactone

HGAP: 

hierarchical genome assembly process

LB: 

Luria-Bertani

ML: 

Maximum-likelihood

NJ: 

Neighbor-joining

QS: 

Quorum sensing

RAST: 

Rapid Annotation using System Technology

Declarations

Acknowledgements

This research was supported by the University of Malaya via High Impact Research Grants (UM.C/625/1/HIR/MOHE/CHAN/01, Grant No. A-000001-50001, UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027 and GA001-2016) and University of Malaya IPPP grant No. PG090-2015B awarded to Kok-Gan Chan. The authors thank Dr. Andrew Sanderson (PhD, Nottingham) for proofreading this manuscript.

Authors’ contributions

WST carried out the experiment, KGC conceived the idea and supervised the whole project, all authors wrote and proofread the paper. Both authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya
(2)
Vice Chancellor Office, Jiangsu University

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© The Author(s). 2017