bioRxiv ScienceSearch

bioRxiv · 10.1101/028977

CRISPR system acquisition and evolution of an obligate intracellular Chlamydia-related bacterium

Abstract

Recently, a new Chlamydia-related organism, Protochlamydia naegleriophila KNic, was discovered within a Naegleria amoeba. To decipher the mechanisms at play in the modeling of genomes from the Protochlamydia genus, we sequenced de novo the full genome of Pr. naegleriophila combining the advantages of two second-generation sequencing technologies. The assembled complete genome comprises a 2,885,111 bp chromosome and a 145,285 bp megaplasmid. For the first time within the Chlamydiales order, a CRISPR system, the immune system of bacteria, was discovered on the chromosome. It is composed of a small CRISPR locus comprising eight repeats and the associated cas and cse genes of the subtype I-E. A CRISPR locus was also found within Chlamydia sp. Diamant, another Pr. naegleriophila strain whose genome was recently released, suggesting that the CRISPR system was acquired by a common ancestor of these two members of Pr. naegleriophila, after the divergence from Pr. amoebophila. The plasmid encodes an F-type conjugative system similar to that found in the Pam100G genomic island of Pr. amoebophila suggesting an acquisition of this conjugative system before the divergence of both Protochlamydia species and the integration of a putative Pr. amoebophila plasmid into its main chromosome giving rise to the Pam100G genomic island. Overall, this new Pr. naegleriophila genome sequence enables to investigate further the dynamic processes shaping the genomes of Chlamydia-related bacteria.

Source connections

Explore related subjects

Keep this discovery

BibTeXRIS

Claire Bertelli, Ousmane Cisse, Brigida Rusconi, Carole Kebbi-Beghdadi, Antony Croxatto, Alexander Goesmann, Francois Collyn, Gilbert Greub. 2015-10-13. CRISPR system acquisition and evolution of an obligate intracellular Chlamydia-related bacterium. https://doi.org/10.1101/028977

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

In vivo bioluminescent imaging reveals temporal and spatial dynamics of giardiasis

Giardia lamblia is the most prevalent protistan parasite, causing acute and chronic diarrheal disease in over one billion people worldwide. Vertebrate hosts ingest Giardia cysts from contaminated sources, and these cysts excyst in the gut to become motile trophozoites. Trophozoites colonize the small intestine by attaching to the intestinal villi and later differentiate into infectious cysts that are released into the environment, completing Giardias life cycle. Due to the limited accessibility of the gastrointestinal tract, our understanding of in in vivo temporal and spatial dynamics of giardiasis is largely inferred from parasite physiology in laboratory culture. Yet parasite growth under in vitro culture conditions may not mirror in vivo parasite physiology in the host. Here we develop bioluminescent imaging (BLI) methods to directly interrogate the temporal and spatial dynamics of giardiasis in mice, providing an improved animal model for the evaluation of anti-Giardia drugs. This non-invasive method of imaging giardiasis allows unprecedented and precise quantification of in vivo temporal and spatial patterns of infection. By infecting mice with parasites expressing constitutive or encystation-specific luciferase bioreporters, we show that parasite colonization of the gut is not uniform. Metabolically active parasites primarily colonize the proximal small intestine in \"hot spots\"--high density foci of infection that likely result in localized pathology to the gut epithelium. Using in vivo and ex vivo BLI of encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and parasites encyst throughout the entire duration of infection. We also find that encystation is initiated in high density foci in the proximal small intestine, rather than the colon as has been previously assumed, and show that the initiation of encystation is magnified in parasites incubated at high density in laboratory culture. Prior models have suggested that chemical cues cause parasites to encyst as they are dislodged from the upper gut and travel to more distal regions of the gastrointestinal tract. We suggest a model of encystation in which parasites reach a threshold density that results the induction of encystation due to local nutrient depletion. The in vivo imaging of giardiasis has redefined the dynamics of the Giardia life cycle in the host, paving the way for future mechanistic studies of density-dependent processes in this highly prevalent, yet understudied parasite.

Microbiology

A bacterial chaperone is required for plastid function in malaria parasites

Apicomplexan parasites such as Plasmodium falciparum, the causative agent of malaria, contain a non-photosynthetic plastid known as the apicoplast that functions to produce essential metabolic compounds. It was previously reported that several members of the Clp family of chaperones and proteases localize to the apicoplast. In bacteria and in chloroplasts these proteins form complexes that degrade proteins in a proteasome-like manner to regulate key cellular processes, but their function in the apicoplast is completely unknown. In this study, we generated a conditional mutant of the P. falciparum apicoplast-targeted pfclpc gene and found that under normal conditions it localizes to the apicoplast. Knockdown of PfClpC results in growth inhibition and morphological defects, indicating that PfClpC is essential for parasite viability. Upon inhibition, PfClpC loses its apicoplast localization and appears in vesicle-like structures. Other apicoplast-targeted proteins also localize to these structures, suggesting that organelle integrity is compromised. Addition of isopentynyl pyrophosphate completely rescued the growth inhibition, indicating that the only essential function of PfClpC is related to the apicoplast. Moreover, cellular assays suggest that PfClpC inhibition interferes with the ability of the schizont-stage parasites to properly sort functional apicoplast organelles into daughter-merozoites. These data show that PfClpC is an essential gene that functions to maintain apicoplast integrity.\n\nAuthor SummaryThe deadly human malaria parasite, Plasmodium falciparum, contains a unique organelle called the apicoplast, a non-photosynthetic plastid that produces vital metabolites. Members of the prokaryotic-derived Clp family were previously reported to localize to the apicoplast. In bacteria and plant chloroplasts, Clp homologs form a proteasome-like complex that degrade proteins but their function in parasite biology is unknown. Here we took a conditional knockdown approach to study an apicoplast localized Clp proteins, PfClpC, which we found to be essential for parasite viability. Inhibition of PfClpC results in a growth arrest phenotype that correlates with a reduced replication rate. We observed that PfClpC localizes to the apicoplast, however upon inhibition it is found dispersed in vesicle-like structures suggesting a complete breakdown of organelle integrity. Our ability to rescue the phenotype by adding an essential apicoplast-derived metabolite proved that the only essential function of PfClpC is linked to apicoplast function. Furthermore, we have found evidence supporting a role for PfClpC in apicoplast sorting into daughter cells. Therefore, we propose PfClpC as a potential drug target due to its essentiality, prokaryotic origin and absence from the human host.

Microbiology

Functional analysis of a biosynthetic cluster essential for production of 4-formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6

Rhizosphere-associated Pseudomonas fluorescens WH6 produces the germination-arrest factor, 4-formylaminooxyvinylglycine (FVG). FVG has previously been shown to both arrest the germination of weedy grasses and to inhibit the growth of the bacterial plant pathogen Erwinia amylovora. Very little is known about the mechanism by which FVG is produced. Although a previous study identified a region of the genome that may be involved in FVG biosynthesis, it has not yet been determined which genes within that region are sufficient and necessary for FVG production. In the current study, we explored the role of each of the putative genes encoded in that region by constructing deletion mutations. Mutant strains were assayed for their ability to produce FVG with a combination of biological assays and thin-layer chromatographic analyses. This work defined the core FVG biosynthetic gene cluster and revealed several interesting characteristics of FVG production. We determined that FVG biosynthesis requires two small open reading frames of less than 150 nucleotides and that multiple transporters have overlapping but distinct functionality. In addition, two genes in the center of the biosynthetic gene cluster are not required for FVG production, suggesting that additional products may be produced from the cluster. Transcriptional analysis indicated that at least three active promoters play a role in the expression of genes within this cluster. The results of this study enrich our knowledge regarding the diversity of mechanisms by which bacteria produce non-proteinogenic amino acids like vinylglycines.

Microbiology