Restricted accessResearch articleFirst published online 2025-12
Novel Modification Sites of dPreQ 0 in Aminobacter niigataensis Phage Erebus Provide New Insights into the Role of 7-Deazaguanine Modifications in Bacteriophages
Bacteriophages protect themselves against host-encoded defense systems through DNA modifications. This study introduces Erebus, a newly identified phage infecting Aminobacter niigataensis, a bacterium capable of mineralizing 2,6-dichlorobenzamide- a common pesticide metabolite. The use of such bacterial degraders has been proposed for the bioremediation of contaminated groundwater. However, the presence of bacteriophages targeting these degraders poses a potential challenge to the success of such strategies.
Materials and Methods:
The Erebus phage was isolated and subjected to whole-genome sequencing. Phylogenetic analysis was performed to determine its taxonomic placement and genomic synteny. DNA modifications were identified using a combination of liquid chromatography–mass spectrometry (LC-MS) and Oxford Nanopore Technologies sequencing. Transmission electron microscopy was used to determine the phage morphology.
Results:
Phylogenetic analysis revealed that Erebus belongs to an unclassified genus, showing high synteny with Rhizobium phages of the Kleczkowskaviridae family. The phage possesses a double-stranded DNA genome of 52,229 base pairs, which includes a functional 7-deazaguanine DNA-modification system. Nanopore sequencing and LC-MS analysis confirmed the presence of PreQ0 modifications at novel GG and AG motifs, conferring resistance against multiple restriction endonucleases.
Conclusions:
This is the first report of a phage infecting the genus Aminobacter, highlighting the potential impact of bacteriophages on microbial biodegradation strategies. The findings underscore the importance of considering phage-host interactions when deploying bacterial degraders for environmental remediation.
OfirG, SorekR. Contemporary phage biology: From classic models to new insights. Cell, 2018; 172(6):1260–1270.
2.
WeigeleP, RaleighEA. Biosynthesis and function of modified bases in bacteria and their viruses. Chem Rev, 2016; 116(20):12655–12687.
3.
HutinetG, KotW, CuiL, et al.7-Deazaguanine modifications protect phage DNA from host restriction systems. Nat Commun, 2019; 10(1):5442.
4.
CuiL, BalamkunduS, LiuCF, et al.Four additional natural 7-deazaguanine derivatives in phages and how to make them. Nucleic Acids Res, 2023; 51(21):11980.
5.
KotW, OlsenNS, NielsenTK, et al.Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs. Nucleic Acids Res, 2020; 48(18):10383–10396.
6.
OlsenNS, NielsenTK, CuiL, et al.A novel Queuovirinae lineage of Pseudomonas aeruginosa phages encode dPreQ0 DNA modifications with a single GA motif that provide restriction and CRISPR Cas9 protection in vitro. Nucleic Acids Res, 2023; 51(16):8663–8676.
7.
SorensenSR, HoltzeMS, SimonsenA, et al.Degradation and mineralization of nanomolar concentrations of the herbicide dichlobenil and its persistent metabolite 2,6-dichlorobenzamide by Aminobacter spp. isolated from dichlobenil-treated soils. Appl Environ Microbiol, 2007; 73(2):399–406.
8.
JohnsenA. Grundvand Status og udvikling 1989–2015 (technical report).ThorlingL. (ed), GEUS, Denmark; 2016. pp. 81–105 (in Danish).
9.
PorazziE, Pardo MartinezM, FanelliR, et al.GC-MS analysis of dichlobenil and its metabolites in groundwater. Talanta, 2005; 68(1):146–154.
10.
PukkilaV, KontroMH. Dichlobenil and 2,6-dichlorobenzamide (BAM) dissipation in topsoil and deposits from groundwater environment within the boreal region in southern Finland. Environ Sci Pollut Res Int, 2014; 21(3):2289–2297.
11.
VandermaesenJ, HoremansB, DegryseJ, et al.Mineralization of the common groundwater pollutant 2,6-Dichlorobenzamide (BAM) and its metabolite 2,6-dichlorobenzoic acid (2,6-DCBA) in sand filter units of drinking water treatment plants. Environ Sci Technol, 2016; 50(18):10114–10122.
12.
98/83/EC C directive. European Council, Drinking Water Directive (COUNCIL DIRECTIVE 98/83/EC). Official Journal of the European Communities, Brussels, 1998.
13.
AlbersCN, FeldL, Ellegaard-JensenL, et al.Degradation of trace concentrations of the persistent groundwater pollutant 2,6-dichlorobenzamide (BAM) in bioaugmented rapid sand filters. Water Res, 2015; 83:61–70.
14.
HoremansB, RaesB, VandermaesenJ, et al.Biocarriers improve bioaugmentation efficiency of a rapid sand filter for the treatment of 2,6-dichlorobenzamide-contaminated drinking water. Environ Sci Technol, 2017a;51(3):1616–1625.
15.
SchostagMD, GobbiA, FiniMN, et al.Combining reverse osmosis and microbial degradation for remediation of drinking water contaminated with recalcitrant pesticide residue. Water Res, 2022; 216:118352.
16.
NielsenTK, HoremansB, LoodC, et al.The complete genome of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of plasmids. Sci Rep, 2021; 11(1):18943.
17.
Ellegaard-JensenL, HoremansB, RaesB, et al.Groundwater contamination with 2,6-dichlorobenzamide (BAM) and perspectives for its microbial removal. Appl Microbiol Biotechnol, 2017; 101(13):5235–5245.
18.
AlbersAN, JacobsenOS, AamandJ. Using 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. in flow through biofilters—initial adhesion and BAM degradation potentials. Appl Microbiol Biotechnol, 2014; 98(2):957–967.
19.
Ellegaard-JensenL, SchostagMD, Nikbakht FiniM, et al.Bioaugmented sand filter columns provide stable removal of pesticide residue from membrane retentate. Front Water, 2020; 2:603567.
20.
HoremansB, VandermaesenJ, SekharA, et al.Aminobacter sp. MSH1 invades sand filter community biofilms while retaining 2,6-dichlorobenzamide degradation functionality under C- and N-limiting conditions. FEMS Microbiol Ecol, 2017b;93(6).
21.
HyllingO, Nikbakht FiniM, Ellegaard-JensenL, et al.A novel hybrid concept for implementation in drinking water treatment targets micropollutant removal by combining membrane filtration with biodegradation. Sci Total Environ, 2019; 694:133710.
22.
T’SyenJ, RaesB, HoremansB, et al.Catabolism of the groundwater micropollutant 2,6-dichlorobenzamide beyond 2,6-dichlorobenzoate is plasmid encoded in Aminobacter sp. MSH1. Appl Microbiol Biotechnol, 2018; 102(18):7963–7979.
23.
ThurmanEM, MalcolmRL. Preparative isolation of aquatic humic substances. Environ Sci Technol, 1981; 15(4):463–466.
24.
EgliT. How to live at very low substrate concentration. Water Res, 2010; 44(17):4826–4837.
25.
ClokieMRJ, KropinskiAM. Bacteriophages: Methods and Protocols: Isolation, Characterization, and Interactions, Vol. 1. Humana Press: New York, NY; 2009.
26.
KropinskiAM, MazzoccoA, WaddelT, et al.Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay. Bacteriophages. (ClokieMRJ, KropinskiAM, eds). Vol. 501, Methods in Molecular Biology: Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay. Humana Press; 2009; 69–76.
27.
ClokieMRJ, KropinskiAM. Introduction: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects. Methods in Molecular Biology, vol 502. In: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects Methods in Molecular Biology, vol 502. Humana Press Inc; 2009; pp. 13–22.
28.
HyllingO, CarstensAB, KotW, et al.Two novel bacteriophage genera from a groundwater reservoir highlight subsurface environments as underexplored biotopes in bacteriophage ecology. Sci Rep, 2020; 10(1):11879.
29.
KotW, VogensenFK, SorensenSJ, et al.DPS – a rapid method for genome sequencing of DNA-containing bacteriophages directly from a single plaque. J Virol Methods, 2014; 196:152–156.
30.
LavigneR, NobenJP, HertveldtK, et al.The structural proteome of Pseudomonas aeruginosa bacteriophage ϕKMV. Microbiology (Reading), 2006; 152(Pt 2):529–534.
31.
ThiavilleJJ, KellnerSM, YuanY, et al.Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc Natl Acad Sci U S A, 2016; 113(11):E1452–E1459.
32.
StoiberMH, QuickJ, EganR, et al.De novo identification of DNA modifications enabled by genome-guided nanopore signal processing. bioRxiv, 2017; 094672.
33.
AzizRK, BartelsD, BestAA, et al.The RAST Server: Rapid annotations using subsystems technology. BMC Genomics, 2008; 9(1):75.
34.
AltschulSF, MaddenTL, SchäfferAA, et al.Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res, 1997; 25(17):3389–3402.
35.
El-GebaliS, MistryJ, BatemanA, et al.The Pfam protein families database in 2019. Nucleic Acids Res, 2019; 47(D1):D427–D432.
36.
SödingJ, BiegertA, LupasAN. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res, 2005; 33(Web Server issue):W244–W8.
37.
KelleyLA, MezulisS, YatesCM, et al.The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc, 2015; 10(6):845–858.
38.
MoraruC, VarsaniA, KropinskiAM. VIRIDIC—A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses, 2020; 12(11):1268.
LangmeadB, SalzbergSL. Fast gapped-read alignment with Bowtie 2. Nat Methods, 2012; 9(4):357–359.
41.
LiH, HandsakerB, WysokerA, et al.; 1000 Genome Project Data Processing Subgroup. The sequence alignment/map format and SAMtools. Bioinformatics, 2009; 25(16):2078–2079.
42.
QuinlanAR, HallIM. BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics, 2010; 26(6):841–842.
43.
AltschulSF, GishW, MillerW, et al.Basic local alignment search tool. J Mol Biol, 1990; 215(3):403–410.
EdgarRC. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 2004; 32(5):1792–1797.
46.
KumarS, StecherG, TamuraK. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016; 33(7):1870–1874.
47.
AdriaenssensE, BristerJR. How to name and classify your phage: An informal guide. Viruses, 2017; 9(4):70.
48.
SmithRJ, JeffriesTC, RoudnewB, et al.Confined aquifers as viral reservoirs. Environ Microbiol Rep, 2013; 5(5):725–730.
49.
EydalHSC, JägevallS, HermanssonM, et al.Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep groundwater. Isme J, 2009; 3(10):1139–1147.
50.
KyleJE, EydalHSC, FerrisFG, et al.Viruses in granitic groundwater from 69 to 450 m depth of the spö hard rock laboratory, Sweden. Isme J, 2008; 2(5):571–574.
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