Two novel bacteriophage genera from a groundwater reservoir highlight subsurface environments as underexplored biotopes in bacteriophage ecology

Abstract

Although bacteriophages are central entities in bacterial ecology and population dynamics, there is currently no literature on the genomes of bacteriophages isolated from groundwater. Using a collection of bacterial isolates from an aquifer as hosts, this study isolated, sequenced and characterised two bacteriophages native to the groundwater reservoir. Host phylogenetic analyses revealed that the phages targeted B. mycoides and a novel Pseudomonas species. these results suggest that both bacteriophages represent new genera, highlighting that groundwater reservoirs, and probably other subsurface environments as well, are underexplored biotopes in terms of the presence and ecology of bacteriophages. Despite metagenomics revealing that groundwater reservoirs harbour complex bacterial communities that are closely associated with biogeochemical cycling, much remains uncharted about their microbial ecology 1. In this context, it could be argued that bacteriophages (phages) have been studied to an even lesser extent, but they are generally considered to play a fundamental role in shaping bacterial communities and consequently influencing biogeochemical cycling 2, 3. While viruses (including phages) only constitute 0.04% of the earth's biomass 4 , they are widely regarded to constitute the largest and most diverse family of biological entities 5. Thus, mapping out their taxonomy, distribution and ecological role is a daunting task. Currently, there are no genome sequences of phages, isolated from groundwater systems, despite descriptions of phage abundances and viromes in these systems 6,7. This study isolated, sequenced and characterised two novel phages from a groundwater reservoir. To the authors' knowledge, this is the first report on groundwater phages that includes their sequenced genome and phylogenomic affiliation. The aim of the present study was to describe the first isolated groundwater bacteriophages that target actual bacterial isolates from the groundwater reservoir through isolation, genome sequencing, bioinformatics and protein characterisation. Predator-prey pairs are of relevance not only in the study of environmental microbiology, including the food webs of groundwater aquifers, but also in the context of bioaugmentation for purification of contaminated groundwater, where knowledge of indigenous enemies is crucial to the survival of introduced degrader bacteria. Results and discussion Phage isolation and phage host identification. Using a collection of natural bacterial groundwater isolates as hosts, two phages-Anath (Genbank accession MG983742.1) and Lana (Genbank accession MK473373.1)-were successfully obtained and their hosts identified as Bacillus mycoides and Pseudomonas sp., respectively, by means of the publicly available online tool for strain identification Type (Strain) Genome Server (TYGS) (https ://www.tygs.dsmz.de) 8. Besides being the first sequenced groundwater phages, this is also the

Lytic archaeal viruses infect abundant primary producers in Earth’s crust

“…In addition, pronounced morphological diversity of bacteriophages with presumably lytic representatives has been found in granitic groundwater of up to 450 m depth 12 , and might be the result of recombination events, horizontal gene transfer, and lysogeny known to shape microbial communities of the subsurface 13 . The recent recovery of two novel bacteriophage genera with lytic genes from groundwater highlights the potential of subsurface environments for being huge reservoirs of previously unknown viruses 14 . Furthermore, a study on predominant Halanaerobium spp.…”

Characterization of the first Pseudomonas grimontii bacteriophage, PMBT3

“…HMWF031 (GBAN PTU03211; coverage, 99%; identity, 60.4%) and Pseudomonas sp. B1(2018) (GBAN WP_116583222; coverage, 99%; identity, 61.7%), and less similarity to TerB of the first described member of the genus Lanavirus , i.e., the Pseudomonas phage Lana [ 29 ] (GBAN YP_009820378.1; coverage, 99%; identity, 50.3%). An amino acid sequence alignment with selected TerB proteins deduced from bacterial and phage genomes (i.e., e-values from 1e-53 to 3e-43) showed a distribution of homologous amino acids over the entire length of the protein, as demonstrated by the consensus sequence and identical amino acids in the individual sequences shown in Supplementary Fig.…”

“…A search of the databases using megablast [ 24 ] indicated that the phage PMBT3 genome showed only low similarity at the nucleic acid level (i.e., coverage, 35%; identity, 81.7%) to Pseudomonas phage Lana [ 29 ] (GBAN NC_048166 [ 38 ]), which has a similar genome size of 88,342 bp. The resulting amino-acid-based phylogenetic GBDP tree generated with VICTOR [ 28 ] for the phages PMBT3, Lana, PMBT14, and 30 other Pseudomonas phages is shown in Fig.…”

“…Furthermore, the Virus Classification and Tree Building Online Resource (VICTOR) [ 28 ] (freely available at https://victor.dsmz.de ) was used for whole-amino-acid-sequence-based phylogeny and classification. For this purpose, the phage PMBT3 proteome was compared to that of its closest relative, Pseudomonas phage Lana [ 29 ], and 30 Pseudomonas phages in the databases, with a focus on P. fluorescens phages, including the recently described P. fluorescens phage PMBT14 [ 30 ] (GBANs and their characteristics are listed in Supplementary Table S7 in Online Resource 4). All pairwise amino acid sequence comparisons were conducted using the Genome-BLAST Distance Phylogeny (GBDP) method with optimal settings as described by Meier-Kolthoff et al [ 28 ].…”

Genome-informed microscopy reveals infections of uncultivated carbon-fixing archaea by lytic viruses in Earth’s crust

“…In addition, pronounced morphological diversity of bacteriophages with presumably lytic representatives has been found in granitic groundwater of up to 450 m depth [11], and might be the result of recombination events, horizontal gene transfer and lysogeny known to shape microbial communities of the subsurface [12]. The recent recovery of two novel bacteriophage genera with lytic genes from groundwater highlights the potential of subsurface environments for being huge reservoirs of previously unknown viruses [13]. Furthermore, a study on predominant Halanaerobium spp.…”