Exploring horizontal gene transfer and phage infections in a South African deep subsurface bacterial population
Mlandu, Cumisa Manzikazi
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Viruses (more specifically bacteriophages which infect both bacteria and archaea) are the most abundant microorganisms in the ecosystem. In extreme environments such as the deep subsurface, bacteriophages play an important role in the altering of biogeochemical cycles and in the evolvability and survival of their hosts. Unfortunately even in their abundance, little is known about phages and their interactions with their hosts and this is due to the fact that 99% of the microorganisms in the environment (especially in the deep subsurface) cannot be cultured. Instead the use of culture-independent techniques such as microscopy and next generation sequencing based techniques. Recent research done on microbial diversities in deep subsurface environments prefers the use of metagenome platforms that have 100% sequence coverage as it allows for comprehensive sequencing as such one is able to identify and assemble genomes of species that are present at low frequencies in environmental samples. These techniques also allow for the detection of phage genes and HGT events within the genomes of the host, thereby giving insight into the host-phage interactions in these extreme environments. Access to the microbial matter in deep terrestrial subsurface is through mines and several in South African deep mines have identified phage genes and HGT events within genomes of indigenous bacteria. Microscopic techniques such as EFM and TEM have been used to detect and morphologically characterize phages (respectively). The characterization of phages in extreme environments using TEM has only been published for deep marine environments, hot springs and solfataric fields and not for the deep mine fissure water. Therefore, phages in the deep terrestrial environment have only been studied through the use of metagenomic sequencing techniques. In this study the main objectives were to identify phage genes and the presence of horizontally acquired genes and their effect on the deep subsurface bacterial communities in terms of evolution and survival. Sampling and concentration (using tangential flow filtration) of fracture water from Star Diamonds mine, Fronteirs mining resulted in samples eligible for microscopic analysis (using SEM, EFM and TEM) and sequencing using whole metagenome sequencing to identify the phage related genes. SEM analysis revealed most of the bacterial communities are in biofilm structures and this is expected due to the unfavourable conditions of the environment. The free phages in the viral fraction fracture water were detected using EFM and the TEM analysis identified phages belonging to the Myoviridae and Podoviridae families which have been previously studied and found in the marine environments. This study is the first to identify free viral particles in the fissure water of deep subsurface South African mines. It is also the first study to detect free viral particles in the South African diamond mine. Whole metagenome sequencing of fracture water from Star Diamonds mine, Fronteir mining was done in order to achieve the above stated main objectives. All three domains of life (Bacteria, Archaea and Eukarya) were present in the fracture water and dominated by bacteria from the phylum Proteobacteria. The phages detected in the metagenome belonged to the order Caudovirales with the Siphoviridae family being the most abundant. The presence of the Myoviridae and Podoviridae families further confirmed the results from the phage characterization using TEM. Previous studies in deep subsurface samples from South Africa have discovered viral infections mostly in Firmicutes, which dominate older fracture water at deeper depths. In this study the majority of the phage sequences identified using VirSorter were from phages that infect hosts from the phylum Proteobacteria which is the most abundant phyla in the fracture water. This study therefore provides valuable insight into other host microorganisms such as the Proteobacteria which generally dominate in younger fracture water at shallower depths. Partially complete prophages were detected and annotated and the presence of prophages in South African deep mines has been previously identified by two researchers. The presence of prophages suggests that phages in this environment can be both lytic (as observed with the detection of free phages using TEM) and lysogenic, but the low abundance of phages detected using TEM and EFM suggests that they prefer lysogenic infections. The CRISPRs, mobile/transposable elements, transposase and retrons detected within the binned metgenome data are suggested to be markers for possible phage mediated HGT events by previous researchers. This study further identified genes suggested to be products of HGT events that were part of the nitrogen fixation, cobalamin synthesis and sulfide reduction pathways and motility and sporulation. These genes conferred novel capabilities to the host (that they were transduced into via HGTs) for survivability and evolution in the extreme deep subsurface environment. This is the first study to specifically look at the bacteria-phage interactions within the subsurface mines of South Africa and it is also the first to find viral infections within the Star Diamonds mine. As future research, phylogenetic analyses would need to be done in order to further confirm the identified HGT events.