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Complete genome sequence of Thauera aminoaromatica strain MZ1T


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Thauera aminoaromatica strain MZ1T, an isolate belonging to genus Thauera, of the family Rhodocyclaceae and the class the Betaproteobacteria, has been characterized for its ability to produce abundant exopolysaccharide and degrade various aromatic compounds with nitrate as an electron acceptor. These properties, if fully understood at the genome-sequence level, can aid in environmental processing of organic matter in anaerobic cycles by short-circuiting a central anaerobic metabolite, acetate, from microbiological conversion to methane, a critical greenhouse gas. Strain MZ1T is the first strain from the genus Thauera with a completely sequenced genome. The 4,496,212 bp chromosome and 78,374 bp plasmid contain 4,071 protein-coding and 71 RNA genes, and were sequenced as part of the DOE Community Sequencing Program CSP_776774.
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Standards in Genomic Sciences (2012) 6:325-335 DOI:10.4056/sigs.2696029
The Genomic Standards Consortium
Complete genome sequence of Thauera aminoaromatica
strain MZ1T
Ke Jiang1, John Sanseverino1, Archana Chauhan1, Susan Lucas2, Alex Copeland2, Alla
Lapidus2, Tijana Glavina Del Rio2, Eileen Dalin2, Hope Tice2, David Bruce2, Lynne
Goodwin2, Sam Pitluck2, David Sims3, Thomas Brettin2, John C. Detter 2, Cliff Han3, Y.J.
Chang4, Frank Larimer4, Miriam Land4, Loren Hauser4, Nikos C. Kyrpides2, Natalia
Mikhailova2, Scott Moser1, Patricia Jegier1, Dan Close1, Jennifer M. DeBruyn5, Ying Wang1,
Alice C. Layton1, Michael S. Allen6 and Gary S. Sayler1*
1Center for Environmental Biotechnology, The University of Tennessee, Knoxville,
Tennessee, USA
2DOE Joint Genome Institute, Walnut Creek, California, USA
3Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA
4Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
5Department of Biosystems Engineering and Soil Science, The University of Tennessee,
Knoxville, Tennessee, USA
6Department of Biological Sciences, University of North Texas, Denton, Texas, USA
*Corresponding Author: Gary S. Sayler (
Keywords: Thauera aminoaromatica, MZ1T, genome.
Thauera aminoaromatica strain MZ1T, an isolate belonging to genus Thauera, of the family
Rhodocyclaceae and the class the Betaproteobacteria, has been characterized for its ability to
produce abundant exopolysaccharide and degrade various aromatic compounds with nitrate
as an electron acceptor. These properties, if fully understood at the genome-sequence level,
can aid in environmental processing of organic matter in anaerobic cycles by short-circuiting
a central anaerobic metabolite, acetate, from microbiological conversion to methane, a criti-
cal greenhouse gas. Strain MZ1T is the first strain from the genus Thauera with a completely
sequenced genome. The 4,496,212 bp chromosome and 78,374 bp plasmid contain 4,071
protein-coding and 71 RNA genes, and were sequenced as part of the DOE Community Se-
quencing Program CSP_776774.
Strain MZ1T (=DSM 25461 =MTCC 11151=LMG
26735), a Gram-negative bacterium, was isolated
from activated sludge samples from the industrial
wastewater treatment facility of Eastman Chemi-
cal Company, Kingsport, Tennessee [1]. It is relat-
ed to the genera Azoarcus and another prominent
community member of activated sludge, Zoogloea.
Strain MZ1T was identified as a significant com-
ponent of microbial clusters formed during vis-
cous bulking that resulted in poor sludge
dewaterability and increased costs for dewatering,
incineration and disposal [2]. Subsequently, MZ1T
was found to produce a novel exopolysaccharide
which contributed to the viscous bulking phe-
nomenon. The genus Thauera is named after the
German microbiologist Rudolf Thauer and was
described by Macy et al. [3]. Currently, this genus
consists of nine species with validly published
names. These species have been isolated from a
wide range of environments including wastewater
activated sludge, water and soil, and typically de-
grade aromatic compounds such as benzoic acid
or toluene under anaerobic conditions [3-8]. Here
we present a summary classification and a set of
features for T. aminoaromatica MZ1T, along with
the description of the complete genomic sequenc-
ing and annotation.
Thauera aminoaromatica strain MZ1T
326 Standards in Genomic Sciences
Classification and features
Strain MZ1T originally was identified as belonging
to Thauera genus based on the 16S rRNA phyloge-
netic analysis [1].The sequences of the four 16S
rRNA gene copies in the genome do not differ from
each other. However, they differ from the previous-
ly published 16S rRNA sequence (AF110005),
which contains one gap and eleven ambiguous base
calls. Figure 1 shows the phylogenetic relationship
of T. aminoaromatica MZ1T in a 16S rRNA based
tree to other Thauera species. Based on this tree,
strain MZ1T is closely grouped with T.
aminoaromatica S2, T. phenylacetica B4P and T.
selenatis and the cluster of these four strains is
well-separated from strains of T. aromatica, T.
chlorobenzoica, T. mechernichensis, T. terpenica, T.
butanivorans and T. linaloolentis.
DNA-DNA hybridization was performed between
strain MZ1T and T. selenatis ATCC 55363, T.
phenylacetica B4P DSM 14743 and T.
aminoaromatica S2 DSM 14742 by Deutsche
Sammlung von Mikroorganismen und Zellkulturen
GmbH (DSMZ) (Braunschweig, Germany). DNA-
DNA hybridization studies showed that MZ1T was
100% similar to strain S2, 78.9% to strain B4P and
59.6% to T. selenatis ATCC 55363, respectively.
When the recommended threshold value of 70%
DNA-DNA similarity is used for the definition of
bacterial species [4], MZ1T does not belong to the
same species as T. selenatis ATCC 55363 but does
belong to the same species as strain S2. Based on
these results we recommend MZ1T be classified as
Thauera aminoaromatica strain MZ1T.
Morphologically, cells of strain MZ1T are Gram
negative, short rods (0.5 x 1.1-1.8 µm) and motile
due to the presence of a polar flagellum (Figure 2).
Colonies are slimy, creamy white in color at the op-
timal growth temperature of 30 ºC and pH 7.2, re-
spectively. Strain MZ1T grows aerobically in
Stoke’s medium at 30 ºC shaking at 150 rpm and
produces copious quantities of extracellular poly-
saccharide from relatively simple short chain fatty
acids at early stationery stage [2]. However, when
grown on agar plates, no obvious
exopolysaccharide is observed. Under aerobic con-
ditions, benzoate, succinate, aspartate, glutamate,
proline, leucine, serine and alanine are utilized. Un-
der anaerobic conditions MZ1T is capable of
growth on benzoate with nitrate as the terminal
electron acceptor. The characteristic features of the
organism are listed in Table 1.
Figure 1. 16S rDNA based phylogenetic tree depicting the relationship between Thauera aminoaromatica MZ1T
and other members of the genus Thauera. The tree was constructed by using the Neighbor-Joining method and
Jukes & Cantor evolutionary distance matrix from aligned 16S rDNA gene sequences and rooted using Azoarcus
indigens as the outgroup. Bootstrap values (expressed as percentage of 500 replications) greater than 50 % are
shown at the branch points. The branches are scaled as the number of base substitutions per site.
Jiang et al. 327
Table 1. Classification and general features of T. aminoaromatica MZ1T according to the MIGS
recommendations [5].
MIGS ID Property Term Evidence code
Domain Bacteria TAS [6]
Phylum ‘Proteobacteria TAS [7]]
Class Betaproteobacteria TAS [8,9]
Order Rhodocyclales TAS [8,10]
Current classification Family Rhodocyclaceae TAS [8,11]
Genus Thauera TAS [3,12]
Species Thauera aminoaromatica IDA [3,13,14]
Strain MZ1T TAS [1]
Gram stain negative TAS [1]
Cell shape rod TAS [1]
Motility motile TAS [1]
Sporulation not reported
Temperature range 28-37 oC TAS [1]
Optimum temperature 30 oC TAS [1]
Salinity not reported
MIGS-22 Oxygen requirement aerobic, facultative TAS [1]
Carbon source numerous 1- and multi-C compounds TAS [1]
Energy metabolism chemolithoautotroph TAS [1]
MIGS-6 Habitat fresh water, waste water TAS [1]
MIGS-15 Biotic relationship free living NAS
MIGS-14 Pathogenicity none NAS
Biosafety level 1 TAS [1]
Isolation wastewater treatment plant TAS [1]
MIGS-4 Geographic location Kingsport, Tennessee, USA TAS [1]
MIGS-5 Sample collection time 1997 TAS [1]
MIGS-4.1 Latitude 36.548 NAS
MIGS-4.2 Longitude -82.561 NAS
MIGS-4.3 Depth NA
MIGS-4.4 Altitude 369.11 m NAS
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 the Gene Ontology project
[15]. If the evidence code is IDA, the property was directly observed by one of the authors or an
expert mentioned in the acknowledgements.
Thauera aminoaromatica strain MZ1T
328 Standards in Genomic Sciences
Figure 2. Scanning and transmission electronic microscopic images of T. aminoaromatica
MZ1T (A and B), S2 (C) and B4P (D).
The predominant fatty acids found in strain MZ1T
are C16:1 ω7c (50.65%), C16:0 (25.81%), C18:1 ω7c
(9.37%), C12:0 (6.3%), C10:0 3-OH (3.87%) and C12:0 3-
OH (3.16%). The fatty acid C12:0 3-OH is generally not
found in the Thauera genus but has been found in
T. selenatis [12,13]. Therefore, MZ1T is similar to
T. selenatis based on membrane fatty acid compo-
Genome sequencing and Annotation
Genome project history
This organism was selected for sequencing under
the DOE Joint Genome Institute (JGI) Community
Sequencing Program (CSP). The genome project is
deposited in the Genome On Line Database
(GOLD) [16] and the complete genome sequence is
deposited in GenBank (CP001281). Sequencing,
finishing and annotation were performed by the
DOE JGI. A summary of the project information is
shown in Table 2.
Growth conditions and DNA isolation
Strain MZ1T was grown aerobically in Stoke’s me-
dium at 30 ºC shaking at 150 rpm [2]. Genomic
DNA was extracted using a modified Cetyl
Trimethyl Ammonium Bromide (CTAB) DNA ex-
traction protocol [17]. Briefly, 100 ml of overnight
culture was used for DNA isolation. After incuba-
tion with CTAB extraction buffer at 60 oC for 1 hr,
cells were lysed and proteins precipitated using
an equal volume of chloroform-isoamyl alcohol
(24:1), and the aqueous phase was separated, to
which one half volume of 5 M NaCl was added fol-
lowed by two volumes of cold ~ 95% ethanol to
precipitate DNA. DNA was dissolved in Tris-EDTA
(TE) overnight at (4 to 6 oC). After RNase treat-
ment followed by phenol/chloroform extraction,
1/10 volume of 2 M sodium acetate and 2 volumes
absolute ethanol were added to re-precipitate
DNA. Finally, DNA was dissolved in TE. The purity,
quality and size of the bulk gDNA preparation
were assessed by JGI according to DOE-JGI guide-
Jiang et al. 329
Table 2. Genome sequencing project information T. aminoaromatica MZ1T.
MIGS ID Property Term
MIGS-31 Finishing quality Finished
MIGS-28 Libraries used FOSX random whole genome shotgun library
MIGS-29 Sequencing platforms ABI3730, 454-GS-FLX-Titanium
MIGS-31.2 Sequencing coverage 9.3 × with Sanger, 20 × with 454
Phrap, Newbler version 2.3
MIGS-34 Gene calling method Prodigal 1.4, GenePRIMP
INSDC ID CP001281 (chromosome)
CP001282 (plasmid)
Genbank Date of Release
August 1, 2009
GOLD ID Gc00901
NCBI project ID 20091
MIGS-13 Source material identifier MTCC 11151, DSM 25461, LMG 26735
Project relevance Bioenergy, Biotechnological, Ecological, Environmental, CSP_776774
Genome sequencing and assembly
The genome of T. aminoaromatica strain MZ1T
was sequenced at the JGI using a combination of 8
kb and 40 kb fosmid DNA libraries. In addition to
Sanger sequencing, 454 pyrosequencing was done
to a depth of 20 × coverage. All general aspects of
library construction and sequencing performed by
JGI can be found at the JGI website [18]. Draft as-
semblies were based on 47,422 total reads. The
combined libraries provided 9.0 × coverage. The
Phred/Phrap/Consed software package [19] was
used for sequence assembly and quality assess-
ment [20-22]. After the shotgun stage, reads were
assembled with parallel phrap (High Performance
Software, LLC). Possible misassemblies were cor-
rected with Dupfinisher [23] or transposon bomb-
ing of bridging clones (Epicentre Biotechnologies,
Madison, WI). Gaps between contigs were closed
by editing in Consed, custom primer walk or PCR
amplification (Roche Applied Science, Indianapo-
lis, IN). A total of 2,230 additional reactions were
necessary to close gaps and to raise the quality of
the finished sequence. The completed genome se-
quences of T. aminoaromatica strain MZ1T con-
tains 49,771 reads in the chromosome and 2,819
reads in the plasmid, achieving an average of 9.3 ×
coverage in the chromosome and 29.8 × in the
plasmid per base with an error rate 0 in 100,000.
Genome annotation
The genes were annotated through the Oak Ridge
National Laboratory genome annotation pipeline
using Prodigal [24] followed by a round of manual
curation using the JGI GenePRIMP pipeline [25].
Predicted CDSs were translated and used to
search the National Center for Biotechnology In-
formation (NCBI) nonredundant database,
UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and
InterPro databases. Data sources were then com-
bined to assert a product description for each
predicted protein. Non-coding genes and miscel-
laneous features were predicted using tRNAscan-
SE [26], RNAMMer [27], Rfam [28], TMHMM [29]
and signalP [30].
Genome properties
The genome contains one chromosome and one
plasmid for a total genome size of 4.5 Mb. (Table 3,
Figure 3A and Figure 3B). The circular chromo-
some is 4,496,212 bp in length with a coding den-
sity of 89%, a GC content of 68%, 4,071 protein
coding genes, 71 structural RNA genes, 93 pseudo
genes and 4 copies each of 5S, 16S and 23S rRNA
genes. About 62% of predicted genes begin with
ATG, 30% begin with TTG, and 7% begin with
GTG. Table 4 shows the distribution of genes in
COG categories. The plasmid (pTha01) is 78,374
bp in size and has a GC content of 62%, 77% cod-
ing density, 75 protein coding genes, 4 pseudo
genes and nonstructural RNA genes.
Thauera aminoaromatica strain MZ1T
330 Standards in Genomic Sciences
Figure 3A. Graphical circular map of the T. aminoaromatica MZ1T genome. The outermost two circles (circles 1
and 2) show the genes in the forward and reverse strands, respectively; different colors indicate different function
categories. The next circle (circle 3) shows RNA genes (tRNAs green, rRNAs red, other RNAs black); circle 4
shows the GC content, and circle 5 shows the GC skew.
Insights from the genome
Annotation of the genome indicated that strain
MZ1T has complete glycolytic and citric acid cycle
pathways along with two complete acetate assimi-
lation pathways with the key enzymes being ace-
tate-CoA ligase and acetate kinase-phosphate ace-
tyl transferase, respectively, thereby allowing
MZ1T to utilize acetate as a carbon source [31].
Three putative gene clusters responsible for
exopolysaccharide biosynthesis, polymerization
and export were found. The discovery of the wzy
gene in one of the cluster implicates a Wzy-
dependent pathway of polysaccharide synthesis
and export in MZ1T [32-34]. Unlike other related
Thauera spp [35-37], MZ1T does not appear to
have genes for anaerobic toluene or phenol deg-
radation; however, genes for both anaerobic and
Jiang et al. 331
aerobic benzoate degradation are present. The
genome of MZ1T contains a total of six sigma fac-
tors controlling global gene regulation. These in-
clude the housekeeping sigma factor σ70, the ni-
trogen regulator σ54, the heat shock sigma factor
σ32, as well as three copies of extracytoplasmic
function (ECF) sigma factor [38]. MZ1T has a large
number of genes encoding diverse transporter
proteins and those involved in chemotaxis. More
than ten copies of two component regulatory sys-
tems, genes known to be related to toxin-antitoxin
plasmid addiction systems, replication- partition
systems and stabilization factors such as Par-like
systems were found distributed in both the plas-
mid and chromosome. Additionally, genes encod-
ing efflux pumps for heavy metal resistance to ar-
senic, cadmium, lead, silver, zinc but not for sele-
nium have been found on the plasmid. Further-
more, both the plasmid and chromosome contain
numerous transposases, integrases and
recombinases which demonstrate that genetic re-
arrangement is widely occurring in this strain.
Figure 3B. Graphical circular map of the T. aminoaromatica MZ1T plasmid pTha01. The outermost two cir-
cles (circles 1 and 2) show the genes in the forward and reverse strands, respectively; different colors indicate
different function categories. The next circle (circle 3) shows RNA genes (tRNAs green, rRNAs red, other RNAs
black); circle 4 shows the GC content, and circle 5 shows the GC skew.
Thauera aminoaromatica strain MZ1T
332 Standards in Genomic Sciences
Table 3. Genome Statistics for T. aminoaromatica strain MZ1T.
% of Totala
Genome size (bp) 4,574,586 100.00%
DNA coding region (bp) 4,088,809 89.38%
DNA G+C content (bp) 3,124,403 68.30%
Number of replicons 2
Extrachromosomal elements 1
Total genes 4,142 100.00%
RNA genes
rRNA operons 4
Protein-coding genes 4,071 98.29%
Pseudo genes
Genes with function prediction 2,980 71.95%
Genes in paralog clusters 2177 52.56%
Genes assigned to COGs 3,163 76.36%
Genes assigned Pfam domains 3330 80.40%
Genes with signal peptides 919 22.19%
Genes with transmembrane helices 976 23.56%
CRISPR repeats
Table 4. Number of genes associated with the general COG functional categories
% age
J 175 5.01 Translation, ribosomal structure and biogenesis
A 1 0.03 RNA processing and modification
K 215 6.16 Transcription
L 215 6.16 Replication, recombination and repair
B 2 0.06 Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Y 0 0.0 Nuclear structure
V 68 1.98 Defense mechanisms
Signal transduction mechanisms
M 214 6.13 Cell wall/membrane/envelope biogenesis
N 94 2.69 Cell motility
Z 0 0.0 Cytoskeleton
W 0 0.0 Extracellular structures
U 105 3.01 Intracellular trafficking, secretion, and vesicular transport
O 155 4.44 Posttranslational modification, protein turnover, chaperones
Energy production and conversion
G 114 3.26 Carbohydrate transport and metabolism
E 276 7.90 Amino acid transport and metabolism
Nucleotide transport and metabolism
H 152 4.35 Coenzyme transport and metabolism
I 135 3.87 Lipid transport and metabolism
P 188 5.38 Inorganic ion transport and metabolism
Q 79 2.26 Secondary metabolites biosynthesis, transport and catabolism
R 378 10.82 General function prediction only
S 294 8.42 Function unknown
Not in COGs
Jiang et al. 333
In liquid culture, MZ1T grows as planktonic cells
until late log phase, during which it forms charac-
teristic flocs or cell clusters and then settles out. It
was hypothesized that this phenotype may be re-
lated to a quorum sensing mechanism. Genes with
possible roles in quorum sensing were identified
including an acyl-acyl-carrier protein synthase
and luxR response regulator (12 copies). However,
N-acyl-homoserine lactone synthetase or its hom-
ologue were not found, which does not support
the hypothesis of quorum sensing being one of the
mechanisms involved in floc formation. The ge-
nome also encodes adhesion related proteins
which could be linked to exopolysaccharide pro-
duction, quorum sensing or “clumping”. Therefore,
we speculate that the response of MZ1T to chang-
ing environmental conditions involves a complex
system involving exopolysaccharide production
and flocculation when the cells reach adequate
density. Thus, the complete genome sequence of
strain MZ1T provides an opportunity to study the
biology of important adaptive factors.
This work was supported by the Center for Environmen-
tal Biotechnology and the University of Tennessee Waste
Management Research and Education Institute and by
the Director, Office of Science, Office of Biological and
Environmental Research, Life Sciences Division, U.S. De-
partment of Energy under Contract No. DE-AC02-
05CH11231. We would like to thank the Community
Sequencing Program and the Joint Genome Institute for
sequencing and annotation of the MZ1T genome. We
would like to thank Dr. Georg Fuchs at University of
Freiburg for generously providing strain S2 and B4P.
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... Together with the published plasmids pEbN1_2 in Ar. aromaticum EbN1 T (Rabus et al., 2005) and pTX1 in T. sinica K11 T (GenBank accession number CP023440), we found a total of 11 plasmids in the 40 sequenced genomes of Thauera and Aromatoleum strains ( Figure 2; Table 1). Three of the 26 sequenced Thauera strains carry an IncP-1 type plasmid, two strains harbour an IncP-11 plasmid, and T. aminoaromatica MZ1T carries an unclassified plasmid that is not considered further in this study (Jiang et al., 2012). Among the 14 Aromatoleum genomes, four contain an IncP-1 type plasmid and one a chimeric plasmid with an IncP-1 β fragment. ...
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Self‐transferable plasmids of the incompatibility group P‐1 (IncP‐1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T, a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP‐1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP‐type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP‐1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k‐mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP‐1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria, and provide novel insights into the in situ host profile of these plasmids. This article is protected by copyright. All rights reserved.
... Burkholderia also has strong abilities to produce indole acetic acid and siderophores, helping plants decrease the oxidative damage of Cd (Dourado et al., 2013;Yang et al., 2016). Thauera, positively correlated to TCd (r = 0.67, p < 0.05) and ACd (r = 0.62, p < 0.05), was found to play more crucial roles in denitrification under higher Cd stress, and carried genes encoding efflux pumps for heavy metal tolerance to Cd (Jiang et al., 2012;Miao et al., 2018). Bradyrhizobium can accumulate a high amount of Cd within their cells by increasing the intracellular glutathione levels under high Cd stress (Bianucci et al., 2011;Guo and Chi, 2014). ...
The response of soil bacterial communities from farmland ecosystems to cadmium (Cd) pollution, in which a steep concentration gradient of more than 100 mg/kg has naturally formed, has not previously been fully reported. In this study, a field investigation was conducted in a typical severe Cd-polluted farmland ecosystem, and the bacterial community response to the steep Cd gradient was analyzed. The results showed that Cd concentration sharply decreased from 159.2 mg/kg to 4.18 mg/kg among four sampling sites alongside an irrigation canal over a distance of 150 m. Bacterial diversity and richness were significantly lower in highly polluted sites, and random forest analysis indicated that Cd gradient played a decisive role in reducing alpha diversity. Redundancy analysis (RDA) and co-occurrence network indicated that the synergistic effects of pH, Cd, and phosphorus were the main drivers shaping community structure. The functional results predicted by BugBase suggested that the bacterial community may adapt to the harsh environment by recruiting Cd-resistant microbes and improving oxidative stress tolerance of the whole community. Cd-resistant microorganisms such as Burkholderia, Bradyrhizobium, and Sulfurifustis, which directly or indirectly participate in diminishing oxidative damage of Cd, may play essential roles in maintaining community stability and might be potential bacterial resources for the bioremediation of Cd pollution.
... 28,29 However, it is unclear why in the study presented here, an increase in this bacterial group was observed when the production of EPSs and flocculation activity were the lowest. Further evaluations to investigate these observations may include further sequence analyses to determine if the identified Thauera are from the MZ1T strain, reported to be a significant cluster-forming organism in wastewater treatment, 30 and if the specific exopolysaccharides produced by this group are present in water samples. 28,29 Additionally, given the complexity of in situ evaluations, laboratory-based experimentation may be necessary to continue to explore these results, potentially through the establishment of environmentally controlled micro-and/or mesocosm approaches in which the most abundant taxa identified in our initial assessments are evaluated for their ability to produce EPSs and support the formation of flocs. ...
... Moreover, knowledge of the full genome sequence makes taxonomic identification easier and the databases built from the genomes serve as templates in the metagenomic databases, analysis. Many genomes of anaerobic hydrocarbon biodegraders have been published (Rabus et al., 2005;Mattes et al., 2008;Aklujkar et al., 2009;Selesi et al., 2010;Jiang et al., 2012;Martín-Moldes et al., 2015;Yin et al., 2017). Comparative analysis of the genome of the iron-reducing bacterium Geosporobacter ferrireducens IRF9 identified multiple anaerobic hydrocarbon activating genes (alkylsuccinate synthase) harbored by the strain (Jung et al., 2018). ...
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The accumulation of petroleum hydrocarbons in the environment substantially endangers terrestrial and aquatic ecosystems. Many microbial strains have been recognized to utilize aliphatic and aromatic hydrocarbons under aerobic conditions. Nevertheless, most of these pollutants are transferred by natural processes, including rain, into the underground anaerobic zones where their degradation is much more problematic. In oxic zones, anaerobic microenvironments can be formed as a consequence of the intensive respiratory activities of (facultative) aerobic microbes. Even though aerobic bioremediation has been well-characterized over the past few decades, ample research is yet to be done in the field of anaerobic hydrocarbon biodegradation. With the emergence of high-throughput techniques, known as omics (e.g., genomics and metagenomics), the individual biodegraders, hydrocarbon-degrading microbial communities and metabolic pathways, interactions can be described at a contaminated site. Omics approaches provide the opportunity to examine single microorganisms or microbial communities at the system level and elucidate the metabolic networks, interspecies interactions during hydrocarbon mineralization. Metatranscriptomics and metaproteomics, for example, can shed light on the active genes and proteins and functional importance of the less abundant species. Moreover, novel unculturable hydrocarbon-degrading strains and enzymes can be discovered and fit into the metabolic networks of the community. Our objective is to review the anaerobic hydrocarbon biodegradation processes, the most important hydrocarbon degraders and their diverse metabolic pathways, including the use of various terminal electron acceptors and various electron transfer processes. The review primarily focuses on the achievements obtained by the current high-throughput (multi-omics) techniques which opened new perspectives in understanding the processes at the system level including the metabolic routes of individual strains, metabolic/electric interaction of the members of microbial communities. Based on the multi-omics techniques, novel metabolic blocks can be designed and used for the construction of microbial strains/consortia for efficient removal of hydrocarbons in anaerobic zones.
... In wastewater treatment systems, members of the genera Thauera, Paracoccus, Hyphomicrobium, Comamonas, Azoarcus, Denitratisoma, Dechloromonas, and family Comamonadaceae are the major denitrifiers contributing into the nitrogen removal (Jiang et al., 2012;Baumann et al., 1996;Carvalho et al., 2007;Cowan et al., 2005;Neef et al., 1996;Martineau et al., 2013;Gumaelius et al., 2001;Khan et al., 2002). Interestingly, microbial species composition in wastewater affects nitrite accumulation in denitrification process. ...
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Water contamination is a growing environmental issue. Several harmful effects on human health and the environment are attributed to nitrogen contamination of water sources. Consequently, many countries have strict regulations on nitrogen compound concentrations in wastewater effluents. Wastewater treatment is carried out using energy- and cost-intensive biological processes, which convert nitrogen compounds into innocuous dinitrogen gas. On the other hand, nitrogen is also an essential nutrient. Artificial fertilizers are produced by fixing dinitrogen gas from the atmosphere, in an energy-intensive chemical process. Ideally, we should be able to spend less energy and chemicals to remove nitrogen from wastewater and instead recover a fraction of it for use in fertilizers and similar applications. In this review, we present an overview of various technologies of biological nitrogen removal including nitrification, denitrification, anaerobic ammonium oxidation (anammox), as well as bioelectrochemical systems and microalgal growth for nitrogen recovery. We highlighted the nitrogen removal efficiency of these systems at different temperatures and operating conditions. The advantages, practical challenges, and potential for nitrogen recovery of different treatment methods are discussed.
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Water contamination due to various nitrogenous pollutants generated from wastewater treatment plants is a crucial and ubiquitous environmental problem now-a-days. Nitrogen contaminated water has manifold detrimental effects on human health as well as aquatic life. Consequently, various biological treatment processes are employed to transform the undesirable forms of nitrogen in wastewater to safer ones for subsequent discharge. In this review, an overview of various conventional biological treatment processes (viz. nitrification, denitrification, and anammox) have been presented along with recent novel bioelectrochemical methods (viz. microbial fuel cells and microbial electrolysis cells). Additionally, nitrogen is an indispensable nutrient necessary to produce artificial fertilizers by fixing dinitrogen gas from the atmosphere. Thus, this study also explored the potential capability of various nitrogen recovery processes from wastewater (like microalgae, cyanobacteria, struvite precipitation, stripping, and zeolites) that are used in industries. Further, the trade-offs, challenges posed by these processes have been dwelt on along with other biological processes like CANON, SHARON, OLAND, and others.
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Dissolved oxygen (DO) is an imperative parameter of the activated sludge process (ASP) for wastewater bioremediation. The effect of DO on microbial communities and corresponding metabolic functions in wastewater bioremediation was investigated using next-generation analysis techniques in this study. Illumina-based whole genome sequencing was applied to analyze the composition of the microbial community along with their functional diversity in activated sludge systems operating at three different DO levels. Activated biomass was collected from lab-scale reactors maintained at 1, 2, and 4 mg/L DO levels. Metagenomes were sequenced on an Illumina platform and analyzed using various tools. Results revealed that Proteobacteria phylum and Pseudomonas, Nitrobacter, Thauera, and Alicyclipilus genera were abundant in all reactor samples. Despite distinct DO levels, the microbial communities were conserved and consisted of a common population forming the core group governing the metabolic functions. However, higher diversity was observed at functional level indicating that microbes evolve and adapt to serve their role in a typical ASP. Metabolic pathway related to benzoate dominated at 1 mg/L DO level, while pathways for degradation of aromatic compounds like phenol, toluene, and biphenyl via central metabolic pathway were found dominating at 4 mg/L DO level. Pathways corresponding to homogentisate, naphthalene, cresol, and salicylate degradation enriched at 2 mg/L DO level.
Involvements of quorum sensing (QS) in the formation of aerobic granules for wastewater treatment have been well recognized. In previous studies the evolution of the QS-related activities and communities during bioreactor start-up period has been extensively studied, while the variation of QS in long-term reactor operation remains unrevealed. Furthermore, information about the roles of quorum quenching (QQ) in bioreactors is very limited. In this work, both QS and QQ during the start-up and successive long-term operation period of an aerobic granule bioreactor were explored. The QS activity and communities increased in the start-up but gradually decreased in the long-term operation, while the QQ activity and communities remained stable. These results indicate the longer persistence of QQ than QS in the granules and the minor contribution of QS in the long-term operation. This work provides a new insight into the roles of QQ and QS in wastewater treatment bioreactors.
Anaerobic/anoxic denitrifying reactors, such as an upflow sludge blanket (USB) reactor, can retain high concentration of biomass inside of the reactor as granular sludge, which allows high nitrogen removal performance from wastewaters. However, granular sludge is critical to high nitrogen removal performance. In this study, simple granulation method was developed in a denitrifying USB reactor fed with municipal sewage and 50 mg-N⋅L−1 of sodium nitrate as an easily available nitrate source, and granular formation, nitrogen removal performance, and microbial community structure were investigated. A 11.4-L USB reactor was operated at an upflow velocity of 50 cm⋅h−1, and hydraulic retention time of 2.9 h under ambient temperature. Granular sludge with up to 3.8 mm of diameter was formed within 15 days, and the nitrate removal rate increased 9.8 mg-N⋅gMLVSS−1 ⋅h−1to 22.5 mg-N⋅ gMLVSS−1 ⋅h−1 at batch exam. 16S rRNA amplicon sequencing indicated that Cloacibacterium sp. was the most abundant in the granular sludge with detection rate of 6.11% to 12.95%. Also, propionate-producing bacterium Paludibacter sp., acetate-utilizing denitrifying bacterium Acidovorax sp. and Dechloromonas sp. were also abundant in the granular sludge. Denitrifying granular sludge was successfully formed in the USB reactor treating real municipal sewage with sodium nitrate feeding.
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Introduction DNA–DNA Reassociation and Gene Sequencing: Two Complementary Approaches The AD HOC Committee on Reconciliation of Approaches to Bacterial Systematics Relationship between DNA–DNA Similarities and rRNA Gene Similarities The AD HOC Committee for the Reevaluation of the Species Definition in Bacteriology Outlook References
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An ad hoc committee for the re-evaluation of the species definition in bacteriology met in Gent, Belgium, in February 2002. The committee made various recommendations regarding the species definition in the light of developments in methodologies available to systematists.
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Microscopic observations at an industrial wastewater treatment facility were conducted over a period of approximately 4 years to determine the relationship between the abundance of zoogleal clusters and sludge dewatering potential. Dewatering potential, measured as centrifuged solids content, was determined in sludge samples from an aeration basin. The abundance of zoogleal clusters and centrifuged solids content were negatively correlated, as determined by linear regression (r2 0.62). Curve-fitting using an exponentially weighted moving average and a polynomial equation yielded r2 0.82. Probing with small ribosomal subunit RNA (16S rRNA) oligonucleotide probes (ZRA and ZBE) indicated that the microorganisms forming these zoogleal clusters were not the same as previously classified Zoogloea ramigera strains (members of the beta subclass of Proteobacteria) isolated from other wastewater treatment systems. The microorganisms responsible for zoogleal cluster formation were identified using micromanipulator separation, cultivation, and 16S rRNA analysis. Distance matrix tree analysis of isolated strains revealed the presence of two types of microorganisms, referred to as mzt and mzl strains. The mzt isolates grouped most closely with members of the genus Thauera, whereas the mzl isolates grouped more closely with members of the genus Hydrogenophaga. Using 16S rRNA methods, a probe complementary to members of the genus Thauera (MZ1; S-G-Thau-0646-a-A-19) was designed to target mzt strains. Whole-cell hybridization with a fluorescein-labeled probe, coupled with epifluorescence microscopy, was used to verify the identity of the zoogleal cluster-forming organisms. Results indicated that the MZ1 probe hybridized specifically with zoogleal clusters present in sludge.
We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.
Lipopolysaccharides (LPSs) are complex glycolipids found in the outer membrane of Gram-negative bacteria. The lipid A–core component of the LPS molecule provides a versatile anchor to which a surface polymer:lipid A–core ligase enzyme can attach one or more structurally distinct surface polymers in a single bacterial strain. In some cases the same polymer can be found on the cell surface in both lipid A–core-linked and -unlinked forms. Analysis by SDS–PAGE of populations of LPS molecules extracted from bacterial cells indicates that there is extensive heterogeneity in their size distribution. Much of the heterogeneity results from complex modal distributions in the chain length of the polymers which are attached to lipid A–core. This is the result of preferential ligation of polymers with specific degrees of polymerization during the assembly of the LPS molecule. The surface architecture of the Gram-negative bacterial cell is therefore profoundly affected by the activities of the surface polymer:lipid A–core ligase and by molecular determinants of polymer chain length. Because of the involvement of cell-surface polymers in interactions between pathogenic bacteria and their hosts, these enzymatic activities also have an important impact on virulence. In this review, the organization of LPSs and related surface polymers will be described and the current understanding of the molecular mechanisms involved in surface diversity will be discussed. Emphasis is placed on the Enterobacteriaceae, but similarities to other bacteria suggest that aspects of the enterobacterial system will have broader significance.
Aromatic compounds are important growth substrates for microorganisms. They form a large group of diverse compounds including lignin monomers, amino acids, quinones, and flavonoids. Aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen which is essential for the hydroxylation and cleavage of aromatic ring structures. The anaerobic metabolism of low molecular mass soluble aromatic compounds requires, of necessity, a quite different strategy. In most known cases, aromaticity is broken by reduction and the ring is subsequently opened hydrolytically. A small number of different central aromatic intermediates can be reduced, the most common of which is benzoyl-CoA, a compound that is formed as a central intermediate in the degradation of a large number of aromatic growth substrates. This review concentrates on the anaerobic aromatic metabolism via the benzoyl-CoA pathway. The peripheral pathways that transform growth substrates to benzoyl-CoA include various types of novel reactions, for example carboxylation of phenolic compounds, reductive elimination of ring substituents like hydroxyl or amino groups, oxidation of methyl substituents, O-demethylation reactions and shortening of aliphatic side chains. The central benzoyl-CoA pathway differs in several aspects in the denitrifying, phototrophic and fermenting bacteria studied. In denitrifying and phototrophic bacteria it starts with the two-electron reduction of benzoyl-CoA to a cyclic dienoyl-CoA driven by the hydrolysis of two molecules of ATP to ADP+Pi. This ring reduction is catalyzed by benzoyl-CoA reductase and requires a low-potential ferredoxin as an electron donor. In Rhodopseudomonas palustris the cyclic diene is further reduced to cyclohex-1-ene-1-carboxyl-CoA. In the denitrifying species Thauera aromatica, the cyclic diene is hydrated to give 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Subsequent β-oxidation results in the formation of a cyclic β-oxo compound, followed by hydrolytic carbon ring opening yielding 3-hydroxypimelyl-CoA in the case of T. aromatica and pimelyl-CoA in the case of R. palustris. These intermediates are further β-oxidized via glutaryl-CoA; final products are 3 acetyl-CoA and 1 CO2. In fermenting bacteria benzoyl-CoA may possibly be reduced to the level of cyclohex-1-ene-1-carboxyl-CoA in an ATP-independent reaction. The genes coding for the enzymes of the central benzoyl-CoA pathway have been cloned and sequenced from R. palustris, T. aromatica, and Azoarcus evansii. Sequence analyses of the genes support the concept that phototrophic and denitrifying bacteria use two slightly different pathways to metabolize benzoyl-CoA. The gene sequences have in some cases been very helpful for the identification of possible catalytic mechanisms that were not obvious from initial characterizations of purified enzymes.
A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.
Conference Paper
Abstract Currently, the genome sequencing community is producing shotgun sequence data at a very high rate, but genome finishing is not keeping pace, even with the help from several automated finishing tools, such asautoFinish. One reason for the slow progress in finishing is that repetitive regions longer than the length of a ,sequencing ,read cannot be assembled correctly,with ,many ,current ,assembly ,tools. Therefore, most repeat regions have to be checked manually. If finishing rates are to increase further, most repetitive regions must be assembled ,correctly and,be finished ,in an ,automated ,fashion. The Dupfinisher, computer ,program ,is designed ,to finish repeats with minimal human,interaction. It can automatically detect repetitive regions, assemble each repeat individually using paired draft reads and primer walk reads, check the quality of these subassemblies, create artificial joins for finished and properly assembled ,repeats and run automated ,gap closure,scripts ,on unfinished ,subassemblies. Dupfinisher, is ,able to solve ,the majority ,of repeats in a microbial genome automatically, thus greatly reducing ,the amount ,of human ,attention