of 3
Draft Genome Assembly of
Stutzerimonas
sp. Strain S1 and
Achromobacter spanius
Strain S4, Two Syringol-Metabolizing
Bacteria Isolated from Compost Soil
Daniel P. Brink
,
a
Elin M. Larsson
,
a,b
Javier García-Hidalgo
,
a
Helga Helgadóttir
,
a
Marie F. Gorwa-Grauslund
a
a
Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, Sweden
b
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA
ABSTRACT
Two bacterial strains able to use syringol as a sole carbon source were
isolated from compost. The isolates, named S1 and S4, were sequenced using the Illumina
platform. The
fi
nal assemblies contained 4.2 Mbp, 63% GC, and 3,912 genes for S1 and
6.2 Mbp, 64% GC, and 5,503 genes for S4.
S
yringol is a major depolymerization product from lignins rich in sinapyl alcohol units
(S-units), such as hardwood, cornstalk, and wheat straw lignin (1
3). Compared to other
types of lignin units, microbes able to metabolize S-unit-derived aromatics are underrepre-
sented in the literature (4), and among them only a single syringol-utilizing species has been
previously reported (5). Here, we present the isolation of two novel bacterial strains capable
of growth on syringol as a sole carbon source.
Coarse-grained compost was provided by the Sysav waste company (Malmö, Sweden).
Isolation was performed by adding 52.9 g compost to 200 mL NaCl (0.8%, wt/vol), and
vortexing vigorously. After settling, 1 mL supernatant was used to inoculate 250-mL
shake-
fl
asks with 25 mL M9 medium (6) supplemented with trace elements (7), 2 mM syringol,
and 100
m
g/mL cycloheximide to prevent fungal growth. After 2 days of incubation at 30°C
and 180 rpm, the culture was streaked on agar plates with the same medium. Several se-
quential restreaks on fresh plates were performed to verify the isolation of pure strains and
stability of the syringol-utilization phenotype. Both strains were isolated from the same shake-
fl
ask culture.
Genomes from cells cultivated overnight (5 mL M9 medium in conical tubes) were
extracted using the GeneJET genomic DNA puri
fi
cation kit (Thermo Fisher, MA, USA).
Sequencing was performed at Euro
fi
ns Genomics Europe (Germany) using Euro
fi
ns
NGSelect
DNA library preparation and the Illumina NovaSeq 6000 S2 PE150 XP platform. The total
number of reads was 10,282,622 for strain S1 and 11,937,525 for S4. Read quality and trimming
were performed with FastQC v0.11.9 (8) and Trimmomatic v0.39 (9), respectively. Assembly
was done with SPAdes v3.15.3 with options: -k 21,33,55,77 --only-assembler --careful (10). An
additional SPAdes run with the --plasmid
fl
ag (11) was also performed. Assembly metrics
were assessed with Quast v4.5.4 (12). Coverage was calculated with BBMap v38.18 (13). Gene
prediction and annotation were done with PGAP v6.3 (14, 15). The presence of aromatic fun-
neling pathways was analyzed based on homology with known bacterial proteins (16, 17)
using blastp v2.12.0
1
(18). Default parameters were used except where otherwise noted.
The assembly of strain S1 resulted in 4.19 Mbp split over 22 contigs, a 63.08% GC content,
an
N
50
of 428,008 bp, and 686

6
115

average coverage. The S4 assembly had 6.18 Mbp,
33 contigs, a 64.19% GC content, an
N
50
of 370,688 bp, and 541

6
90

average coverage.
No plasmids were successfully assembled. Based on an average nucleotide identity analysis
(Table 1), we propose to name the isolates
Stutzerimonas
sp. strain S1 and
Achromobacter
spanius
S4. Totals of 3,912 and 5,503 protein-encoding genes were predicted in the S1 and
S4 annotations, respectively. Putative genes from aromatic metabolic pathways were identi
fi
ed
Editor
Catherine Putonti, Loyola University
Chicago
Copyright
© 2023 Brink et al. This is an open-
access article distributed under the terms of
the
Creative Commons Attribution 4.0
International license
.
Address correspondence to Daniel P. Brink,
daniel.brink@tmb.lth.se.
The authors declare no con
fl
ict of interest.
Received
3 November 2022
Accepted
12 February 2023
Published
23 February 2023
March 2023 Volume 12 Issue 3
10.1128/mra.01150-22
1
GENOME SEQUENCES
in both isolates, including beta-ketoadipate, ferulate, and syringate pathways. Syringol metabo-
lism in bacteria is not well understood, but homologs to a recently described engineered
guaiacol-demethylating cytochrome P450 system that enabled conversion of syringol to py-
rogallol in
Pseudomonas putida
(19) were identi
fi
ed in both strains at 24 to 35% sequence
identity (locus tags in S1, N8H22_08560 and N8H22_17570; locus tags in S4, N8H69_18620
and N8H69_24105).
Data availability.
The annotated genome assemblies of strains S1 and S4 have been
deposited at DDBJ/ENA/GenBank under the accession numbers
JAOEIY000000000
and
JAOEJJ000000000
, respectively. The versions described in this paper are
JAOEIY010000000
and
JAOEJJ010000000
, respectively. The raw reads for S1 and S4 have been uploaded to the
NCBI Sequence Read Archive as
SRR21673156
and
SRX17674200
, respectively. The BioProject
numbers for the data sets are
PRJNA883164
and
PRJNA883223
.
ACKNOWLEDGMENTS
This work was
fi
nanced by the Swedish Foundation for Strategic Research through the
grant contract RBP14-0052. Elin Larsson also acknowledges funding from the Resnick
Sustainability Institute, California Institute of Technology, Pasadena, CA, USA. The computations
and data handling were enabled by resources provided by the Swedish National Infrastructure
for Computing (SNIC) at UPPMAX partially funded by the Swedish Research Council through
grant agreement no. 2018-05973. Computations were performed in projects SNIC 2020/15-218
and SNIC 2021/22-698.
TABLE 1
Results of the average nucleotide identity analysis used for taxonomic classi
fi
cation of strains S1 and S4
a
Reference assembly (NCBI URL)
ANIb
identity (%)
ANIb alignment
coverage (%)
ANIm
identity (%)
ANIm alignment
coverage (%)
Strain S1
Stutzerimonas degradans
FDAARGOS 876 (
GCF_016028635.1
)
85.64
77.29
87.76
68.59
Pseudomonas phenolilytica
RBPA9 (
GCF_021432765.1
)
85.61
79.97
87.60
70.78
Stutzerimonas frequens
TPA3 (
GCF_017577085.1
)
83.31
72.13
87.50
52.64
Stutzerimonas stutzeri
YWX-1 (
GCF_019203165.1
)
83.21
70.54
87.12
52.78
Stutzerimonas chloritidismutans
6L11 (
GCF_020783375.1
)
83.05
72.76
87.13
52.99
Stutzerimonas kunmingensis
DSM 25974 (
GCF_024397575.1
)
83.04
70.75
87.17
51.83
Stutzerimonas balearica
L1 (
GCF_021713615.1
)
81.07
67.88
86.06
42.84
[
Pseudomonas
]
nosocomialis
CCUG 58779 (
GCF_005876875.1
)
79.90
55.22
85.03
33.20
Stutzerimonas azoti
fi
gens
DSM 17556 (
GCF_000425625.1
)
79.76
51.18
84.97
31.95
Stutzerimonas kirkiae
P28C (
GCF_004327175.1
)
79.23
34.83
84.68
20.03
Pseudomonas putida
KT2440 (
GCF_000007565.2
)
76.65
35.06
83.63
12.63
Escherichia coli
K-12 substrain MG1655 (
GCF_000005845.2
)
70.52
6.00
85.33
0.13
Strain S4
Achromobacter spanius
UQ283 (
GCF_003994415.1
)
98.83
*
94.88
98.87
*
95.95
Achromobacter
sp. strain B7 (
GCF_003600685.1
)
87.35
76.79
88.69
75.19
Achromobacter
sp. strain ES-001 (
GCF_019448355.1
)
87.35
77.11
88.67
75.44
Achromobacter piechaudii
LMG 1861 (
GCF_902860155.1
)
86.91
75.12
88.34
71.79
Achromobacter deleyi
As-55 (
GCF_013116765.2
)
84.65
64.95
86.92
57.57
Achromobacter pestifer
FDAARGOS 790 (
GCF_013267355.1
)
84.12
60.38
86.74
51.99
Achromobacter denitri
fi
cans
FDAARGOS 787 (
GCF_013343095.1
)
83.87
60.04
86.59
51.40
Achromobacter xylosoxidans
A8 (
GCF_000165835.1
)
83.85
61.41
86.71
52.00
Bordetella petrii
DSM 12804 (
GCF_000067205.1
)
78.99
36.69
84.62
19.84
Burkholderia pyrrocinia
LWK2 (
GCF_024732225.1
)
73.11
12.85
82.20
1.74
Pseudomonas putida
KT2440 (
GCF_000007565.2
)
71.29
7.12
82.01
0.44
Escherichia coli
K-12 substrain MG1655 (
GCF_000005845.2
)
69.60
2.59
81.85
0.03
a
Ribosomal 16S sequences were extracted from the assemblies with Barrnap v0.9 (20) and used to select reference genomes from relevant genera to perfor
m average
nucleotide identity (ANI) analysis. FastANI v1.33 (21) was used to rapidly query the S1 assembly against the 348 genomes from the
Pseudomonadaceae
family available in
the representative RefSeq collection in the NCBI database; in a second stage, S1 was also queried against the 441 genomes from the recently delineated
Stutzerimonas
genus (22). S4 was queried against 734 representative RefSeq
Burkholderiales
genomes. The results from the FastANI screening and BLAST analysis of the 16S sequence
were used to decide which genomes to include for the
fi
nal ANI analysis, which used PyANI v0.2.12 (23) calling on the standard ANI algorithms MUMmer v4.0.0rc1 (24) and
BLAST v2.12.0
1
(18). To illustrate the taxonomic diversity, only one representative genome from each species was used in the
fi
nal analysis. Model organisms
P. putida
and
Escherichia coli
were included as controls. ANIb, BLAST-based ANI; ANIm, MUMmer-based ANI. A cutoff
.
95% ANI percent identity was used for species delineation as
previously recommended (25). Values that ful
fi
ll this criterion are marked with asterisks. The table is sorted by ANIb percent identity in descending order.
Announcement
Microbiology Resource Announcements
March 2023 Volume 12 Issue 3
10.1128/mra.01150-22
2
We also thank Sysav South Scania Waste Company (Malmö, Sweden) for kindly providing
the compost sample used for the isolation.
D.P.B. and E.M.L. performed the bioinformatics analysis. J.G.-H. and H.H. isolated the
strains. D.P.B. designed the bioinformatics work
fl
ow and wrote the manuscript. M.F.G.-G.
conceived the study and revised the manuscript. All authors read and approved the
fi
nal
manuscript.
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