Revealing Phylogenetic Relationships and CRISPR-Cas Functionality in Legionella pneumophila using Next Generation Sequencing

Legionella pneumophila is a Gram-negative intracellular pathogen of diverse protozoan hosts in the aquatic environment. The generalist evolutionary strategy enables the bacterium to also replicate within human macrophages and cause Legionnaires' disease. The environmental challenges that shape L. pneumophila evolution, however, had not been well characterized. In my first data chapter, I employed next generation sequencing approaches to reveal the evolutionary relationships of L. pneumophila isolates. Two laboratory lineages were domesticated from the clinical strain collected during the eponymous 1976 Philadelphia outbreak of Legionnaires' disease. Through whole genome sequencing, I reconstructed the phylogenies of these commonly used laboratory strains and showed that certain laboratory manipulations would render both desired and unexpected mutations. Next, I characterized the genomes of a 2005 Toronto outbreak strain and several local strains of a similar genotype. Using comparative genomics and plasmid transformation, I demonstrated for the first time an active genome defense role of CRISPR-Cas systems in L. pneumophila. CRISPR-Cas is known as an adaptive immune system in prokaryotes against mobile genetic elements. Using CRISPR-Cas as a window into the environmental challenges to the bacterium, I revealed that L. pneumophila employs multiple distinct CRISPR-Cas systems to protect against a recurrent invasive DNA termed LME-1. To combat the mutational escape of mobile genetic elements, CRISPR-Cas can rapidly renew its immunological arsenal by acquiring secondary spacers through a priming process. In my last data chapter, using deep sequencing approaches I characterized the molecular patterns of primed spacer acquisition in L. pneumophila type I-C CRISPR-Cas. My examination of spacer dynamics suggested a close interplay between CRISPR-Cas interference and adaptation machineries under priming. Taken together, my thesis provided insights into L. pneumophila adaptations in the laboratory and to the environmental life style that involves generalist parasitism and extensive horizontal gene transfers.Ph.D

Legionella pneumophila Philadelphia-1 clinical genome (GenBank format)

GenBank formatted file derived from editing NC_002942 (Legionella pneumophila subsp. pneumophila str. Philadelphia 1). Corrects several discrepancies between the 1976 Philadelphia isolate given to the Isberg laboratory by Barry Fields and the original sequence

Data from: Phylogenetic reconstruction of the Legionella pneumophila Philadelphia-1 laboratory strains through comparative genomics.

Over 20 years ago, two groups independently domesticated Legionella pneumophila from a clinical isolate of bacteria collected during the first recognized outbreak of Legionnaires’ disease (at the 1976 American Legion’s convention in Philadelphia). These two laboratory strains, JR32 and Lp01, along with their derivatives, have been disseminated to a number of laboratories around the world and form the cornerstone of much of the research conducted on this important pathogen to date. Nevertheless, no exhaustive examination of the genetic distance between these strains and their clinical progenitor has been performed thus far. Such information is of paramount importance for making sense of several phenotypic differences observed between these strains. As environmental replication of L. pneumophila is thought to exclusively occur within natural protozoan hosts, retrospective analysis of the domestication and axenic culture of the Philadelphia-1 progenitor strain by two independent groups also provides an excellent opportunity to uncover evidence of adaptation to the laboratory environment. To reconstruct the phylogenetic relationships between the common laboratory strains of L. pneumophila Philadelphia-1 and their clinical ancestor, we performed whole-genome Illumina resequencing of the two founders of each laboratory lineage: JR32 and Lp01. As expected from earlier, targeted studies, Lp01 and JR32 contain large deletions in the lvh and tra regions, respectively. By sequencing additional strains derived from Lp01 (Lp02 and Lp03), we retraced the phylogeny of these strains relative to their reported ancestor, thereby reconstructing the evolutionary dynamics of each laboratory lineage from genomic data

Lp03-AWE genome (GenBank format)

GenBank formatted file derived from editing NC_002942 (Legionella pneumophila subsp. pneumophila str. Philadelphia 1). Lp03 is a dotA mutant, derived from a thymidine auxotroph, originally thought to be Lp02. This sequence reflects the genome of the Lp03 isolate in Alexander Ensminger's strain collection

JR32-AWE (GenBank format)

GenBank formatted file derived from editing NC_002942 (Legionella pneumophila subsp. pneumophila str. Philadelphia 1). JR32 is a common laboratory strain, a streptomycin resistant, restriction-minus derivative of the Philadelphia-1 clinical strain. This sequence reflects the genome of the JR32 isolate in Alexander Ensminger's strain collection

Lp01 "core" genome (GenBank format)

GenBank formatted file derived from editing NC_002942 (Legionella pneumophila subsp. pneumophila str. Philadelphia 1). Lp01 is a common laboratory strain, a streptomycin resistant, restriction-minus derivative of the Philadelphia-1 clinical strain. This "core" sequence reflects the genome of the original Lp01 isolate, calculated based on the phylogeny between contemporary Lp01 strains and Lp02

Lp01-CR genome (GenBank format)

GenBank formatted file derived from editing NC_002942 (Legionella pneumophila subsp. pneumophila str. Philadelphia 1). Lp01 is a common laboratory strain. This Lp01-CR sequence reflects the genome of the Lp01 isolate in Chitong Rao's strain collection

Lp02-AWE genome (GenBank format)

GenBank formatted file derived from editing NC_002942 (Legionella pneumophila subsp. pneumophila str. Philadelphia 1). Lp02 is a thymidine auxotroph, derived from Lp01 by trimethoprim selection. This sequence reflects the genome of the original Lp02 isolate in Alexander Ensminger's strain collection

Ecological rules for the assembly of microbiome communities.

Humans and many other hosts establish a diverse community of beneficial microbes anew each generation. The order and identity of incoming symbionts is critical for health, but what determines the success of the assembly process remains poorly understood. Here we develop ecological theory to identify factors important for microbial community assembly. Our method maps out all feasible pathways for the assembly of a given microbiome-with analogies to the mutational maps underlying fitness landscapes in evolutionary biology. Building these "assembly maps" reveals a tradeoff at the heart of the assembly process. Ecological dependencies between members of the microbiota make assembly predictable-and can provide metabolic benefits to the host-but these dependencies may also create barriers to assembly. This effect occurs because interdependent species can fail to establish when each relies on the other to colonize first. We support our predictions with published data from the assembly of the preterm infant microbiota, where we find that ecological dependence is associated with a predictable order of arrival. Our models also suggest that hosts can overcome barriers to assembly via mechanisms that either promote the uptake of multiple symbiont species in one step or feed early colonizers. This predicted importance of host feeding is supported by published data on the impacts of breast milk in the assembly of the human microbiome. We conclude that both microbe to microbe and host to microbe interactions are important for the trajectory of microbiome assembly