Standards in Genomic Sciences

, 12:23

Complete genome sequence of Pseudomonas stutzeri strain RCH2 isolated from a Hexavalent Chromium [Cr(VI)] contaminated site

  • Romy Chakraborty
  • Hannah Woo
  • Paramvir Dehal
  • Robert Walker
  • Marcin Zemla
  • Manfred Auer
  • Lynne A. Goodwin
  • Alexey Kazakov
  • Pavel Novichkov
  • Adam P. Arkin
  • Terry C. Hazen
Open Access
Short genome report

DOI: 10.1186/s40793-017-0233-7

Cite this article as:
Chakraborty, R., Woo, H., Dehal, P. et al. Stand in Genomic Sci (2017) 12: 23. doi:10.1186/s40793-017-0233-7

Abstract

Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water in several DOE sites, including Hanford 100 H area. In order to stimulate microbially mediated reduction of Cr(VI) at this site, a poly-lactate hydrogen release compound was injected into the chromium contaminated aquifer. Targeted enrichment of dominant nitrate-reducing bacteria post injection resulted in the isolation of Pseudomonas stutzeri strain RCH2. P. stutzeri strain RCH2 was isolated using acetate as the electron donor and is a complete denitrifier. Experiments with anaerobic washed cell suspension of strain RCH2 revealed it could reduce Cr(VI) and Fe(III). The genome of strain RCH2 was sequenced using a combination of Illumina and 454 sequencing technologies and contained a circular chromosome of 4.6 Mb and three plasmids. Global genome comparisons of strain RCH2 with six other fully sequenced P. stutzeri strains revealed most genomic regions are conserved, however strain RCH2 has an additional 244 genes, some of which are involved in chemotaxis, Flp pilus biogenesis and pyruvate/2-oxogluturate complex formation.

Keywords

Pseudomonas Nitrate reduction Chromium Hanford 100H 

Abbreviations

DOE

Department of Energy

gDNA

Genomic DNA

JGI

Joint Genome Institute

NCBI

National Center for Biotechnology Information (Bethesda, MD, USA)

RDP

Ribosomal Database Project (East Lansing, MI, USA)

Introduction

Hexavalent Cr(VI) is a highly toxic and mobile contaminant in the environment. At the DOE site in Hanford, WA, Cr(VI) concentrations reached as high as 50 ppm as a result of nuclear weapon production waste released into the groundwater and soil. In order to reduce Cr(VI) to non-toxic immobilized Cr(III), the bioremediative strategy at the site has been to stimulate indigenous microorganisms [1] by injecting environmentally safe, food quality polylactate ester Hydrogen Release Compound. The slow release electron donor induced biologically mediated reduction of Cr(VI) to Cr(III) by indigenous microorganisms, and as a result, Cr(IV) concentrations were reduced to below 50 ppb in all parts of the Hanford 100 H site [1]. Some group of organisms including Pseudomonadaceae were enriched concomitant to decrease in Cr(VI) concentrations after HRC injection, and continued to remain high [1]. Pseudomonas stutzeri strain RCH2, was isolated from a monitoring well post injection.

Pseudomonas spp. are well-characterized heterotrophs known to degrade several hydrocarbons [2, 3, 4, 5], and reduce metals such as Cr(VI) [6, 7, 8, 9]. They have commonly been detected in several DOE contaminated sites [10, 11, 12, 13] including Uranium contaminated Oakridge Field Research Center [14, 15]. Prolific cultivation of Pseudomonas spp. from such unique contaminated environments is imperative in elucidating the metabolic potential, biochemical and physiological characteristics and the genetic determinants of key pathways of this ubiquitous group of bacteria in the environment. The genome sequence of RCH2 allows for detailed examination of this and closely related microbes in response to environmental perturbations at the genetic level, and provides a basis for investigating response, adaptation and evolution in presence of metal contaminants [16].

Organism features

Classification and features

Enrichments were initiated in Minimal Fresh Water medium [17] with 10 mM acetate as the sole electron donor and 10 mM nitrate as the electron acceptor. All enrichments were incubated in the dark at 30 °C. Periodic transfers of positive enrichments as identified by microscopy or visual turbidity, were made into fresh media. After 5 such transfers, a pure culture of strain RCH2 was obtained by the agar shake tube method [18, 19]. For routine culturing, strain RCH2 was grown in MFW medium under anaerobic conditions, using either lactate or acetate as electron donor and nitrate as electron acceptor. All culturing was done in sealed serum vials with N2:CO2 gas (80:20) in the headspace, as the medium contained 30 mM bicarbonate buffer.

For initial genotyping, gDNA was extracted using the MoBio UltraClean Microbial DNA Isolation Kit (MoBio Inc, Carlsbad, CA). PCR amplification was carried out using universal bacterial 16S ribosomal RNA gene (16S rRNA) primers 1492R and 27 F in 50 μl reactions. The small subunit ribosomal RNA gene was sequenced by Sanger sequencing using universal primers 8 F and 1492R [25] at University of California, Berkeley sequencing facility. 16S rRNA sequence analysis places strain RCH2 in the family Pseudomonadaceae.

Cells in exponential phase of strain RCH2 are rod shaped, approximately 2 μm long and 0.25-0.3 μm wide (Figs. 1 and 2). Anaerobically, P. stutzeri strain RCH2 grew optimally in MFW medium at 37 °C. While best growth was observed at pH 7.2, strain RCH2 could grow at pH between 6.5–8.0. Growth was observed to decrease with increasing salinity of the medium. Strain RCH2 was tested with and can utilize 10 mM acetate, lactate, fumarate, succinate, pyruvate, glucose and sucrose as electron donors and carbon source while grown under nitrate-reducing conditions. Strain RCH2 can also grow under aerobic conditions as is typical of Pseudomonas spp. Strain RCH2 could also grow in complex media such as LB and R2A broth under aerobic conditions. Strain RCH2 reduced Cr(VI) when tested with washed cell suspension. Briefly, strain RCH2 was grown in MFW medium to mid-log phase (optical density of 0.2–0.3 at 600 nm), with lactate as electron donor and nitrate as electron acceptor. Cells were collected by centrifugation, and the cell pellet washed with 30 mM phosphate buffer. Centrifugation and washing were repeated to minimize potential carryover of nitrate in the Cr(VI) reduction experiments. The cell pellet was then resuspended in phosphate buffer and sealed in anaerobic serum vials. To all the vials, 200 μM potassium dichromate was added as electron acceptor, and 10 mM lactate was added as the electron donor. Electron donor addition was left out of the control treatments. The vials were incubated in the dark at 32 °C. Samples were withdrawn periodically for analysis of Cr(VI). Changes in Cr(VI) concentration was determined colorimetrically at 540 nm using the diphenyl carbazide (DPC) assay [24]. The cell suspension experiment demonstrated that after 5 hours, almost 135 μM Cr(VI) was readily reduced by the active cells of strain RCH2 (Fig. 3). In the absence of lactate as the electron donor in the controls, almost no Cr(VI) reduction occurred after 30 min. The reduction of Cr(VI) in the initial period of time could be attributed to abiotic Cr(VI) reduction or carry over lactate from the growth culture despite the washing of the cell pellet.
Fig. 1

A scanning electron micrograph of P. stutzeri strain RCH2 in exponential phase. Scale bar, 1 μm

Fig. 2

Sequence data were aligned using the Clustal W program [20] with Pseudomonas spp. downloaded with strain and accession numbers from the RDP [21] database were sequence identity was 97–100% to P. stutzeri strain RCH2. SeaView v4.0 [22] was used to reconstruct the phylogenetic position of P. stutzeri strain RCH2 within the genus Psuedomonas based on 16S rRNA gene sequence by maximum likelihood following a Tamura-Nei, 93 model and the phylogeny was tested using Approximate Likelihood-Ratio Test (aLRT) (given as a percentage) [23], only values greater than 60% are shown. Azotobacter species were included for comparison and Cellvibrio species were used for the out-group

Fig. 3

Chromium(VI) reduction by cell suspension of P. stutzeri strain RCH2

Genome sequencing information

Genome project history

The genome was selected based on the isolate’s ability to thrive in a chromium contaminated aquifer at Hanford 100 H and its ability to reduce toxic Cr(VI). The genome sequence was submitted to NCBI and released on September 6, 2011. Finishing was completed at Los Alamos National Laboratory. A summary of the project information is shown in Tables 1 and 2, which also presents the project information and its association with MIGS version 2.0 compliance.
Table 1

Classification and general features of Pseudomonas stutzeri strain RCH2 according to the MIGS recommendations [25]

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain Bacteria

Phylum Proteobacteria

Class Gammaproteobacteria

Order Pseudomonadales

Family Pseudomonadaceae

Genus Pseudomonas

Species stutzeri

Strain RCH2

TAS [26]

TAS [27]

TAS [28]

TAS [29, 30]

TAS [29, 31]

TAS [29, 32, 33]

 

Gram stain

Negative

NAS

 

Cell shape

Rod-shaped

IDA

 

Motility

Motile

IDA

 

Sporulation

Non-sporulating

IDA

 

Temperature range

Mesophile

IDA

 

Optimum temperature

37 °C

IDA

 

Carbon source

pH range

Optimum pH

Lactate, Pyruvate,

6.5–8.0

7.2

IDA

 

Terminal electron receptor

Nitrate, Oxygen,

IDA

MIGS-6

Habitat

Cr(VI) contaminated aquifer

IDA

MIGS-6.3

Salinity

Optimal growth at 0.35% salinity

IDA

MIGS-22

Oxygen

Facultative anaerobe

IDA

MIGS-15

Biotic relationship

Free-living

IDA

MIGS-14

Pathogenicity

Unknown

IDA

MIGS-4

Geographic location

Benton County, Washington

IDA

MIGS-5

Sample collection time

2005

IDA

MIGS-4.1

Latitude

Centered on 46°38′51″N

IDA

MIGS-4.2

Longitude

119°35′55″W/46.6475°N 119.59861°W

IDA

MIGS-4.3

Depth

 

Not reported

MIGS-4.4

Altitude

115.8 m

IDA

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 [34]

Table 2

Genome sequencing project information for Pseudomonas stutzeri strain RCH2

MIGS ID

Property

Term

MIGS 31

Finishing quality

Finished

MIGS-28

Libraries used

454 titanium standard library, 454 paired end library, Illumina GAii shotgun library

MIGS 29

Sequencing platforms

454-GS-FLX, Illumina GAii

MIGS 31.2

Fold coverage

454: 32.2x

Illumina GAii: 127.1x

MIGS 30

Assemblers

Newbler, Velvet

MIGS 32

Gene calling method

GenePrimp, Prodigal 1.4

 

Locus Tag

PSEST

 

Genbank ID

CP003071.1- CP003074.1

 

Genbank Date of Release

September 6, 2011

 

GOLD ID

Gp0005131

 

BIOPROJECT

PRJNA60029

MIGS 13

Source Material Identifier

 
 

Project relevance

Chromium (VI) reduction, nitrate reduction

Growth conditions and genomic DNA preparation

P. stutzeri strain RCH2 was grown under anaerobic conditions at 37 °C in basal medium containing 20 mM lactate as the sole electron donor and carbon source and 10 mM nitrate as the terminal electron acceptor. Cells were harvested for DNA extraction when they reached mid-log phase of growth.

Genomic DNA was extracted from a 50 ml culture using the CTAB extraction method recommended by JGI, USA [35]. JGI DNA mass standards were used to ascertain the quantity and quality of the extracted gDNA. JGI protocol for running the gel electrophoresis was followed.

Genome sequencing and assembly

The genome of P. stutzeri strain RCH2 was generated at the DOE JGI using a combination of Illumina [36] and 454 technologies [37]. For this genome we constructed and sequenced an Illumina GAii shotgun library which generated 16,378,443 reads totaling 589.6 Mb, a 454 Titanium standard library which generated 255,080 reads and 2 paired end 454 libraries with an average insert size of 9 kb, and 19 kb which generated 582,773 reads totaling 216.3 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI [35]. The initial draft assembly contained 32 contigs in 1 scaffold. The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data were assembled with VELVET, version 1.0.13 [38], and the consensus sequence were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS −4.24 (High Performance Software, LLC). The software Consed [39, 40, 41] was used in the following finishing process. Illumina data were used to correct potential base errors and increase consensus quality using the software Polisher developed at Joint Genome Institute (JGI) (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gap Resolution (Cliff Han, unpublished), Dupfinisher [42], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 68 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The total size of the genome is 4,600,489 bp and the final assembly is based on 148 Mb of 454 draft data which provides an average 32.2x coverage of the genome and 584.6 Mb of Illumina draft data which provides an average 127.1x coverage of the genome.

Genome annotation

Genes were identified using Prodigal [43] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [44]. 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. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [45], RNAMMer [46], Rfam [47], TMHMM [48], and signalP [49].

Genome properties

The genome consists of one circular chromosome of 4,575,057 bp (62.49% GC content) and includes 3 circular plasmids of 12,763 bp, 9,865 bp and 2,804 bp for a total genome size of 4,600,489 bp. There are 4322 protein-coding genes of which 3593 genes were assigned to a putative function and the 729 remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Tables 3, 4 and 5 and Fig. 4.
Table 3

Summary of genome: 1 chromosome and 3 plasmids

Label

Size (Mb)

Topology

INSDC identifier

RefSeq ID

Chromosome

4.575

circular

CP003071.1

NC_019936.1

Plasmid pPSEST01

0.013

circular

CP003072.1

NC_019937.1

Plasmid pPSEST02

0.010

circular

CP003073.1

NC_019938.1

Plasmid pPSEST03

0.003

circular

CP003074.1

NC_019939.1

Table 4

Genome statistics for Pseudomonas stutzeri strain RCH2

Attribute

Value

% of Total

Genome size (bp)

4,600,489

100.00

DNA coding (bp)

4,159,553

90.42

DNA G + C (bp)

2,874,963

62.49a

DNA scaffolds

4

100.00

Total genes

4,412

100.00

Protein coding genes

4,322

97.96

RNA genes

90

2.04

Pseudo genes

57

1.29b

Genes in internal clusters

NA

 

Genes with function prediction

3,593

81.44

Genes assigned to COGs

3,195

72.42

Genes with Pfam domains

3,786

85.81

Genes with signal peptides

477

10.81

Genes with transmembrane helices

1,118

25.34

CRISPR repeats

NA

 

aGC percentage shown as count of G's and C's divided by the total number of bases. The total number of bases is not necessarily synonymous with a total number of G's, C's, A's, and T's

bPseudogenes may also be counted as protein coding or RNA genes, so is not additive under total gene count

Table 5

Number of genes associated with the general COG functional categories

Code

Value

% of totala

Description

J

227

6.27

Translation, ribosomal structure and biogenesis

A

1

0.03

RNA processing and modification

K

237

6.55

Transcription

L

129

3.56

Replication, recombination and repair

B

1

0.03

Chromatin structure and dynamics

D

39

1.08

Cell cycle control, Cell division, chromosome partitioning

V

83

2.29

Defense mechanisms

T

243

6.71

Signal transduction mechanisms

M

216

5.97

Cell wall/membrane biogenesis

N

158

4.37

Cell motility

U

78

2.16

Intracellular trafficking and secretion

O

155

4.28

Posttranslational modification, protein turnover, chaperones

C

251

6.94

Energy production and conversion

G

169

4.67

Carbohydrate transport and metabolism

E

285

7.88

Amino acid transport and metabolism

F

85

2.35

Nucleotide transport and metabolism

H

171

4.73

Coenzyme transport and metabolism

I

172

4.75

Lipid transport and metabolism

P

241

6.66

Inorganic ion transport and metabolism

Q

91

2.51

Secondary metabolites biosynthesis, transport and catabolism

R

302

8.34

General function prediction only

S

230

6.36

Function unknown

-

1217

27.58

Not in COGs

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

Fig. 4

Graphical map of the chromosome and plasmids of P. stuzeri strain RCH2. From outside to center of each map: Genes on forward strand (color by COG categories as denoted by the JGI Integrated Microbial Genomes (IMG) platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew

Insights from the genome sequence

Global genomic comparison of six fully sequenced P. stutzeri strains (RCH2, A1501, ATCC 17588, CCUG 29243, DSM 4166, DSM 10701) demonstrated that most of the genomic regions are conserved but there are some differences between genome of RCH2 and other genomes (Fig. 5). We identified genes that are differentially present in RCH2 and other fully sequenced P. stutzeri strains by using “Compare two proteomes” tool of DOE Systems Biology Knowledgebase [www.kbase.us]. For 4231 proteins encoded by chromosomal genes of RCH2 strain, there are 3696, 3677, 3534, 3526 and 3199 orthologous genes in CCUG 29243, DSM 4166, A1501, ATCC 17588 and DSM 10701 strains, respectively. No orthologs for plasmid genes of RCH2 were found in five other P. stutzeri strains. We identified 244 P. stutzeri genes that are present in RCH2 chromosome but absent in all other fully sequenced strains. Approximately 48% of those genes encode hypothetical proteins. Particularly noticeable are RCH2-specific gene clusters encoding chemotaxis (Psest_0653-Psest_0662), pyruvate/2-oxoglutarate complex (Psest_2217-Psest_2220) and Flp pilus biogenesis (Psest_2616-Psest_2630) proteins (Fig. 5). We identified 18 strain-specific genes encoding transcriptional regulators, thus the regulatory network of P. stutzeri RCH2 may differ significantly from closely related bacteria.
Fig. 5

Global comparison of six P. stutzeri strains with reference to the RCH2 strain chromosome sequence. We aligned each of the individual genome sequences against the RCH2 chromosome sequence using Basic Local Alignment Search Tool BLASTN [51]. The innermost ring indicates the genomic position. The next ring is a plot of G + C content. Next five rings indicate the presence or absence of BLAST hits in that position, with each ring corresponding to one of P. stutzeri strains. The outermost ring indicates positions of RCH2-specific genes, with clusters of chemotaxis, pyruvate dehydrogenase and Flp pili genes marked orange, purple and green, respectively. The graphical view of the alignments was rendered using BLAST Ring Image Generator [52]

Extended insights

We searched for regulatory interactions in P. stutzeri strain RCH2 using an automated conservative propagation procedure described earlier [50]. By comparison with the RegPrecise database, this procedure identified 27 regulons in P. stutzeri RCH2 genome. Of those regulons, 11 contain genes for central carbon metabolism and utilization of various carbon sources. Other regulatory systems control metabolism of amino acids (MetR, PhhR), nitrogen (NtrC) and phosphonate (PhnF), biosynthesis of biotin (BirA), lipopolysaccharide (GlmR) and nucleotides (NrdR, RutR), metal homeostasis (CadR, CueR, Zur), DNA repair (LexA) and biogenesis of iron-sulfur clusters (IscR). At the same time, P. stutzeri strain RCH2 lacks several transcription factors conserved in various Gammaproteobacteria, like PdxR (regulator of pyridoxine biosynthesis), FabR (regulator of fatty acid biosynthesis) and SoxR (regulator of superoxide stress response).

Conclusion

Pseudomonas stutzeri strain RCH2 isolated from chromium-contaminated aquifer, is a complete denitrifier that can couple nitrate reduction to oxidation of several organic carbon. When supplemented with lactate, robust culture of strain RCH2 reduces Cr(VI) rapidly and this feature contributes to the versatility of this organism to survive in such chromium(VI) contaminated areas. The genome of strain RCH2 reveals differences when compared to closely related strains, and contains an additional 244 genes, mostly of unknown function. Clusters that are specific to strain RCH2 include chemotaxis and Flp pilus biogenesis and these clusters are absent from the five closely related strains examined. The genome sequence of strain RCH2 will assist in further research into the underlying mechanisms of adaption and persistence in metal and/or nitrate contaminated sites.

Acknowledgements

The work conducted at Lawrence Berkeley National Lab by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program) and at the Joint Genome Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH1123. The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Authors’ contributions

RC isolated the organism and performed laboratory experiments. RC, HW and RW drafted the manuscript. RC, HW, PD, LG, AK, PN, RW, APA, TCH sequenced, assembled and annotated the genome. MZ and MA performed the SEM studies. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Funding information

Funder NameGrant NumberFunding Note
Biological and Environmental Research
  • DE-AC02-05CH1123

Copyright information

© The Author(s). 2017

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.

Authors and Affiliations

  • Romy Chakraborty
    • 1
  • Hannah Woo
    • 2
  • Paramvir Dehal
    • 1
  • Robert Walker
    • 1
  • Marcin Zemla
    • 1
  • Manfred Auer
    • 1
  • Lynne A. Goodwin
    • 3
  • Alexey Kazakov
    • 1
  • Pavel Novichkov
    • 1
  • Adam P. Arkin
    • 1
  • Terry C. Hazen
    • 1
    • 2
  1. 1.Lawrence Berkeley National LaboratoryBerkeleyUSA
  2. 2.University Of TennesseeKnoxvilleUSA
  3. 3.Department of Energy Joint Genome InstituteWalnut CreekUSA

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