Viruses of Bacteria (bacteriophages) and Archaea have the ability to significantly alter the structure and function of microbial communities. Thus, it is critical to obtain a greater understanding of the dynamic interaction between microbial hosts and their associated viral populations. The CRISPR-Cas system in Bacteria and Archaea serves as a method to connect viruses to their hosts and provides insight into virus-host interactions. A clustered regularly interspaced short palindromic repeat (CRISPR) locus and CRISPR-associated (Cas) proteins function together in the CRISPR-Cas adaptive immune system. Transcripts of the spacers that separate the repeats in the CRISPR locus confer immunity through sequence identity with targeted viral, plasmid, or other foreign DNA. The CRISPR locus can be interpreted as a historical timeline of virus exposure as spacers are incorporated in a unidirectional manner at the leader end of the CRISPR locus.
Metagenomic approaches were employed to simultaneously analyze CRISPR loci in microbial hosts and sequences of their associated viruses to examine: 1) the viral response to CRISPR spacer diversification in a closed model system, 2) the retention of older spacers without co-existing targets through time, 3) the information about population histories revealed from the CRISPR locus, and 4) the factors impacting phage and viral diversity in a model natural system. The host-phage system of Streptococcus thermophilus DGCC7710 and phage 2972 was used as a closed model system whereas the low diversity microbial communities growing as biofilms atop acid mine drainage (AMD) within the Richmond Mine at Iron Mountain, CA, USA was used as a model natural system.
S. thermophilus DGCC7710 was challenged with phage 2972 and the resulting host and phage populations were examined after one week of co-culturing in order to explore how co-existing, co-evolving hosts and phage populations establish. Additions of new spacers converted the clonal CRISPR locus into a diversified locus, with multiple sub-dominant CRISPR strain lineages present in the final S. thermophilus population. All phage mutations that circumvented three early-acquired spacers were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutate in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages.
The CRISPR loci of Bacteria and Archaea and their associated viral and plasmid populations from AMD biofilm microbial communities were examined from samples collected over eight years. Notably, CRISPR loci were present in most populations. It was shown that CRISPR loci in some AMD microorganisms retain older CRISPR spacers over long time periods, despite not targeting any abundant coexisting viruses or plasmids. In order to investigate this phenomenon, the two CRISPR loci from a dominant archaeal G-plasma population were reconstructed and viral targets were identified via spacer matches. A polyclonal bloom of viruses was detected, and G-plasma population with highly diverse CRISPR loci emerged. In collaboration, a mathematical model was developed to link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. The subset of hosts that retain old spacers seemingly lacking any matches to current viruses may indicate tuning of CRISPR-mediated immunity against low abundance viruses that may re-emerge.
Through retention of older spacers as well as the acquisition of new spacer sequences, CRISPR-Cas systems can provide insight into recent population history. The link between spacers and their viral targets and the relative position and order of spacers in the loci are key characteristics that can be uncovered via population metagenomic analysis. New bioinformatics methods were developed and used to analyze the CRISPR loci of Leptospirillum group II bacterial population and its associated bacteriophage AMDV1 in biofilms sampled over five years in the AMD site. Spacers throughout the locus target the same phage population (AMDV1), but there are blocks of consecutive spacers without AMDV1 targets and only newer spacers target plasmid populations. This suggests the consistent co-existence of the bacteria with the AMDV1 phage population, with periods when this phage was prominent, and a fluctuating plasmid pool. The approach of examining the pattern of CRISPR spacers with targets may have direct application to tracking the potential sources of medically- and defense-relevant microbial strains. Spacer matches can identify phage or plasmids previously existing in the host's environment and indicate phage and plasmid diversity levels over time, thus constraining the past history of the population.
CRISPR spacers can be used to identify sequences derived from viruses or plasmids within a metagenomic dataset. In fact, with metagenomic datasets sequenced from AMD biofilms, CRISPR spacer targeting enabled detection of a very wide variety of previously unknown viruses and plasmids. Fine scale examination of the AMDV1 phage population diversity over time revealed meter-scale spatial variation, but somewhat reproducible seasonal genotypic abundance patterns. There is evidence in CRISPR loci of Leptospirillum group II that excision events removed large blocks of spacers that only match phage or plasmid sequences present at earlier times. These CRISPR locus and phage diversity patterns suggest that there is viral-imposed selection for host strains as well as host-related selection for virus types.
The function of the CRISPR-Cas system was only recognized a little over five years ago. In the intervening period, the majority of research has focused on unraveling the biochemical and mechanistic underpinnings of Cas and spacer function. In this thesis, the emphasis has been on understanding the importance of population diversification for long-term CRISPR-Cas system-based immunity, the significance of spacer retention, and the utility of locus analysis for ecological and evolutionary studies. Hosts and viruses were examined in two different systems. The challenge experiment between S. thermophilus and phage 2972 isolate cultures demonstrated the rapid evolution of host and phage populations as the direct result of the addition of CRISPR spacers and the fixation of phage mutations. Examination of CRISPR loci and associated viruses and plasmids populations in the AMD system suggests CRISPR loci reconstruction enables investigation of past viral and plasmid exposure. It was shown that older spacers may be retained in order to target reappearing or low abundance viruses. Spacers with co-existing matches provide host immunity and offer a method for detection and genomic analysis of new viruses. Evaluating results from both systems has provided a more complete understanding of the CRISPR-mediated host-virus dynamics in microbial communities.