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Complete Genome Sequence of Sulfurospirillum Strain ACSTCE, a Tetrachloroethene-Respiring Anaerobe Isolated from Contaminated Soil

  • Institute for Applied Ecology Chinese Academy of Sciences

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Here, we report the complete genome sequence of the tetrachloroethene-to-trichloroethene dechlorinator Sulfurospirillum sp. strain ACSTCE The genome consists of a 38.05-kb circular plasmid and a 2.69-Mb circular chromosome, which encodes 3 identical reductive dehalogenases with 91.47% amino acid identity to the PceA of Sulfurospirillum multivorans strain DSM 12446.
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Complete Genome Sequence of Sulfurospirillum Strain ACS
a Tetrachloroethene-Respiring Anaerobe Isolated from
Contaminated Soil
Leitao Huo,
Yi Yang,
Yan Lv,
Xiuying Li,
Frank E. Löffler,
Jun Yan
Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, China
University of Chinese Academy of Sciences, Beijing, China
Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA
Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USA
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Joint Institute for Biological Sciences (JIBS), Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee, USA
Department of Biosystems Engineering & Soil Science, University of Tennessee, Knoxville, Tennessee, USA
ABSTRACT Here, we report the complete genome sequence of the tetrachloroethene-
to-trichloroethene dechlorinator Sulfurospirillum sp. strain ACS
. The genome con-
sists of a 38.05-kb circular plasmid and a 2.69-Mb circular chromosome, which en-
codes 3 identical reductive dehalogenases with 91.47% amino acid identity to the
PceA of Sulfurospirillum multivorans strain DSM 12446.
Some members of the genus Sulfurospirillum have versatile energy metabolisms,
including organohalide respiration, a vital process for the turnover of chlorinated
compounds in environmental systems (1–4). Sulfurospirillum sp. strain ACS
, which
reductively dechlorinates tetrachloroethene (PCE) to trichloroethene, was isolated
from a PCE-dechlorinating consortium derived from contaminated soil collected at
the Axton Cross Superfund site near Holliston, MA (5). Sanger sequencing revealed
that strain ACS
shares 98.6% 16S rRNA gene sequence identity with the PCE-to-
cis-1,2-dichloroethene dechlorinator Sulfurospirillum multivorans strain DSM 12446
(4, 5).
Strain ACS
was grown in anoxic, bicarbonate-buffered mineral salt medium under
PCE-dechlorinating conditions (5). Cells were harvested by centrifugation at 21,000 g
for 30 min, and genomic DNA was extracted using the cetyltrimethylammonium bro-
mide method (6). The genome was sequenced using a dual-platform approach. For
Illumina sequencing, DNA was sonicated to generate 500-bp fragments, followed by
T-A ligation to add adaptors and library preparation using the VAHTS universal DNA
library prep kit (Vazyme Biotech Co., Nanjing, China) following the manufacturer’s
instructions. Paired-end sequencing (2 150 bp) of the DNA library with an average
insert size of 350 bp was performed on a HiSeq 2000 instrument (Illumina, San Diego,
CA, USA). For PacBio sequencing, DNA was sheared using g-TUBEs (Covaris, Inc.,
Woburn, WA, USA) to generate 10-kb fragments, which were subsequently end repaired
and ligated with universal hairpin adapters using the SMRTbell template prep kit 1.0
(Pacific Biosciences, Menlo Park, CA, USA) following the manufacturer’s instructions. The
library was sequenced in a single single-molecule real-time (SMRT) cell using a PacBio
RS II/Sequel SMRT instrument (7). The PacBio raw read N
value is 2,794 bp. The
genome sequence was assembled with 840,203 PacBio raw long reads (coverage,
712) using WGS-Assembler version 8.2 and polished with 12,672,060 Illumina short
reads (coverage, 701) using Pilon version 1.22 (8, 9). Circlator version 1.5.1 was used
Citation Huo L, Yang Y, Lv Y, Li X, Löffler FE,
Yan J. 2020. Complete genome sequence of
Sulfurospirillum strain ACS
tetrachloroethene-respiring anaerobe isolated
from contaminated soil. Microbiol Resour
Announc 9:e00941-20.
Editor J. Cameron Thrash, University of
Southern California
Copyright © 2020 Huo et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Jun Yan,
Received 9 August 2020
Accepted 14 September 2020
Published 1 October 2020
Volume 9 Issue 40 e00941-20 1
to evaluate genome completeness, remove overlapping ends, and circulate the ge-
nome (10). Default parameters were used for all software unless otherwise specified.
Functional annotation was performed using the NCBI Prokaryotic Genome Annotation
Pipeline (PGAP) (11).
The final assembly resulted in a 2,685,870-bp circular chromosome and a 38,046-bp
circular plasmid (Table 1). Using the genome of S. multivorans strain DSM 12446
(GenBank accession number CP007201.1) as a reference, the replication origin in the
chromosome of strain ACS
was rotated to the 159-bp position (noncoding region)
upstream of the dnaA gene, which encodes the replication initiator protein DnaA. The
strain ACS
chromosome harbors two rRNA operons organized in the order 16S-23S-
5S. Four reductive dehalogenase (RDase) A genes, of which three are identical and
adjacent to a downstream RDase B gene encoding a membrane-bound anchor protein,
are present on the chromosome. The protein encoded by the three identical RDase A
genes shares 91.47% amino acid identity with the PceA RDase (GenBank accession
number AHJ12791)ofS. multivorans strain DSM 12446 (12, 13). The chromosome also
harbors complete nap and nrf clusters and a fumarate reductase gene (frdA), suggesting
other electron acceptors support growth. On the strain ACS
plasmid, genes encod-
ing the plasmid replication initiator protein RepB (FA592_13925) and subunits
(FA592_13805 and FA592_13810) of a type II toxin-antitoxin system were functionally
annotated by PGAP. The majority of plasmid-associated genes encode proteins with
unknown function. The genome and novel RDase genes reported in this study can be
used to better understand the physiology and evolution in organohalide-respiring
Sulfurospirillum members.
Data availability. The genome sequences and assembly projects reported in this
article have been deposited in DDBJ/ENA/GenBank under the accession numbers listed
in Table 1. Raw reads have been deposited in the Sequence Read Archive (SRA) under
accession numbers SRR12303098 (Illumina) and SRR12303097 (PacBio).
This work was funded by the National Natural Science Foundation of China (grant
numbers 41673126, 41977295, and 41907287), Key Research Program of Frontier
Science of Chinese Academy of Sciences (ZDBS-LY-DQC038), and Liaoning Revitaliza-
tion Talents Program (XLYC1807139). F.E.L. acknowledges support from the Strategic
and Environmental Research and Development Program (SERDP).
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Dehalospirillum multivorans to the genus Sulfurospirillum as Sulfurospiril-
TABLE 1 Genome features of strain ACS
Data for:
Chromosome Plasmid
Assembly length (bp) 2,685,870 38,046
GC content (%) 38.84 35.71
No. of assembled contigs 1 1
No. of coding sequences 2,704 62
No. of tRNAs 45 0
No. of rRNAs (5S, 16S, 23S) 6 (2, 2, 2) 0
GenBank accession no. CP045453 CP045454
BioSample accession no. SAMN11478962 SAMN11478962
BioProject accession no. PRJNA534163 PRJNA534163
Huo et al.
Volume 9 Issue 40 e00941-20 2
lum multivorans comb. nov. Int J Syst Evol Microbiol 53:787–793. https://
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Microbiology Resource Announcement
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... A number of the taxa identified here are known to degrade toxic compounds. For example, Sulfurospirillum is often the dominant species in oil-contaminated samples (21) and some can respire organohalides (22). Desulfobacterales, Sulfurimonas, and Colwellia have been shown to degrade hydrocarbons in marine systems (23)(24)(25). ...
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Methane hydrates represent vast reserves of natural gas with roles in global carbon cycling and climate change. This study provided the first analysis of metagenomes associated with Sirsoe methanicola , the only polychaete species known to colonize methane hydrates.
... There are also several strains within the Sulfurospirillum sp. (strains JPD-1, ACS TCE , and ACE DCE ) and S. carboxydovorans (strain MV) with optimal growth temperatures within the mesophilic growth range (∼21-30 • C; Table S1, Supporting Information; Laanbroek et al. 1977, Campbell et al. 2001, Pietari 2002, Jensen and Finster 2005, Yang et al. 2017a, Huo et al. 2020. ...
Temperature is a key factor affecting microbial activity and ecology. An increase in temperature generally increases rates of microbial processes up to a certain threshold, above which rates decline rapidly. In the subsurface, temperature of groundwater is usually stable and related to the annual average temperature at the surface. However, anthropogenic activities related to the use of the subsurface, e.g. for thermal heat management, foremost heat storage, will affect the temperature of groundwater locally. This mini-review intends to summarize the current knowledge on reductive dehalogenation activities of the chlorinated ethenes, common urban groundwater contaminants, at different temperatures. This includes an overview of activity and dehalogenation extent at different temperatures in laboratory isolates and enrichment cultures, the effect of shifts in temperature in micro- and mesocosm studies as well as observed biotransformation at different natural and induced temperatures at contaminated field sites. Furthermore, we address indirect effects on biotransformation, e.g. changes in fermentation, methanogenesis and sulfate reduction as competing or synergetic microbial processes. Finally, we address the current gaps in knowledge regarding bioremediation of chlorinated ethenes, microbial community shifts and bottlenecks for active combination with thermal energy storage, and necessities for bioaugmentation and/or natural re-populations after exposure to high temperature.
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Two anaerobic, tetrachloroethene- (PCE-) respiring bacterial isolates, designated strain ACS DCE T and strain ACS TCE , were characterized using a polyphasic approach. Cells were Gram-stain-negative, motile, non-spore-forming and shared a vibrioid- to spirillum-shaped morphology. Optimum growth occurred at 30 °C and 0.1–0.4 % salinity. The pH range for growth was pH 5.5–7.5, with an optimum at pH 7.2. Hydrogen, formate, pyruvate and lactate as electron donors supported respiratory reductive dechlorination of PCE to cis -1,2-dichloroethene ( c DCE) in strain ACS DCE T and of PCE to trichloroethene (TCE) in strain ACS TCE . Both strains were able to grow with pyruvate under microaerobic conditions. Nitrate, elemental sulphur, and thiosulphate were alternative electron acceptors. Autotrophic growth was not observed and acetate served as carbon source for both strains. The major cellular fatty acids were C 16 : 1 ω 7 c , C 16 : 0 , C 14 : 0 and C 18 : 1 ω 7 c . Both genomes feature a circular plasmid. Strains ACS DCE T and ACS TCE were previously assigned to the candidate species 'Sulfurospirillum acididehalogenans'. Here, based on key genomic features and pairwise comparisons of whole-genome sequences, including average nucleotide identity, digital DNA–DNA hybridization and average amino acid identity, strains ACS DCE T and ACS TCE , ' Ca . Sulfurospirillum diekertiae' strains SL2-1 and SL2-2, and the unclassified Sulfurospirillum sp. strain SPD-1 are grouped into one distinct species separate from previously described Sulfurospirillum species. Compared to Sulfurospirillum multivorans and Sulfurospirillum halorespirans , which dechlorinate PCE to c DCE without substantial TCE accumulation, these five strains produce TCE or c DCE as the end product. In addition, some cellular fatty acids (e.g., C 16 : 0 3OH, C 17 : 0 iso 3OH, C 17 : 0 2OH) were detected in strains ACS DCE T and ACS TCE but not in other Sulfurospirillum species. On the basis of phylogenetic, physiological and phenotypic characteristics, 'Ca . Sulfurospirillum acididehalogenans' and 'Ca . Sulfurospirillum diekertiae' are proposed to be merged into one novel species within the genus Sulfurospirillum , for which the name Sulfurospirillum diekertiae sp. nov. is proposed. The type strain is ACS DCE T (=JCM 33349 T = KCTC 15819 T =CGMCC 1.5292 T ).
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Organohalide-respiring bacteria (OHRB) play critical roles in the detoxification of chlorinated pollutants and bioremediation of subsurface environments (e.g., groundwater and sediment) impacted by anthropogenic chlorinated solvents. The majority of known OHRB cannot perform reductive dechlorination below neutral pH, hampering the applications of OHRB for remediating acidified groundwater due to fermentation and reductive dechlorination ( 1 , 2 ).
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Sulfurospirillum sp. strain ACS DCE couples growth with reductive dechlorination of tetrachloroethene to cis -1,2-dichloroethene at pH values as low as 5.5. The genome sequence of strain ACS DCE consists of a circular 2,737,849-bp chromosome and a 39,868-bp plasmid and carries 2,737 protein-coding sequences, including two reductive dehalogenase genes.
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A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H2 as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H2, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H2. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160mgrM), a dechlorination rate of about 50 nmol min-1 (mg cell protein)-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.
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Recent technological advances have opened unprecedented opportunities for large-scale sequencing and analysis of populations of pathogenic species in disease outbreaks, as well as for large-scale diversity studies aimed at expanding our knowledge across the whole domain of prokaryotes. To meet the challenge of timely interpretation of structure, function and meaning of this vast genetic information, a comprehensive approach to automatic genome annotation is critically needed. In collaboration with Georgia Tech, NCBI has developed a new approach to genome annotation that combines alignment based methods with methods of predicting protein-coding and RNA genes and other functional elements directly from sequence. A new gene finding tool, GeneMarkS+, uses the combined evidence of protein and RNA placement by homology as an initial map of annotation to generate and modify ab initio gene predictions across the whole genome. Thus, the new NCBI's Prokaryotic Genome Annotation Pipeline (PGAP) relies more on sequence similarity when confident comparative data are available, while it relies more on statistical predictions in the absence of external evidence. The pipeline provides a framework for generation and analysis of annotation on the full breadth of prokaryotic taxonomy. For additional information on PGAP see and the NCBI Handbook,
Long-read, single-molecule real-time (SMRT) sequencing is routinely used to finish microbial genomes, but available assembly methods have not scaled well to larger genomes. We introduce the MinHash Alignment Process (MHAP) for overlapping noisy, long reads using probabilistic, locality-sensitive hashing. Integrating MHAP with the Celera Assembler enabled reference-grade de novo assemblies of Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and a human hydatidiform mole cell line (CHM1) from SMRT sequencing. The resulting assemblies are highly continuous, include fully resolved chromosome arms and close persistent gaps in these reference genomes. Our assembly of D. melanogaster revealed previously unknown heterochromatic and telomeric transition sequences, and we assembled low-complexity sequences from CHM1 that fill gaps in the human GRCh38 reference. Using MHAP and the Celera Assembler, single-molecule sequencing can produce de novo near-complete eukaryotic assemblies that are 99.99% accurate when compared with available reference genomes.
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Physiological tests, redetermination of G+C values with HPLC and DNA-DNA hybridization were used to determine the taxonomic affiliation of Spirillum 5175. This facultatively sulfur-reducing bacterium was compared to the type strains of the phenotypically most similar species Wolinella succinogenes and Campylobacter sputorum biovar bubulus. In addition to morphology, the following physiological properties were in common: use of elemental sulfur, nitrate, nitrite, aspartate, fumarate or malate as electron acceptor for growth with hydrogen or formate under anoxic conditions; microaerobic growth with 2% (v/v) oxygen. The G+C content of Wolinella succinogenes (51.8 mol%) and Campylobacter sputorum biovar bubulus (30.4 mol%) differs about 10 mol% from the G+C content of Spirillum 5175 (40.6 mol%). No significant DNA homology could be detected between the three strains. These differences excluded affiliation of Spirillum 5175 with the genera Wolinella or Campylobacter despite phenotypic similarities. On the basis of our results and DNA-rRNA hybridization studies by other authors, we established the new genus Sulfurospirillum for the freeliving Campylobacter-like bacteria Spirillum 5175 and Campylobacter spec. DSM 806. Strain Spirillum 5175 is described as the type strain of the new genus and species Sulfurospirillum deleyianum.
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