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3. Sunlight Photolysis of Extracellular and Intracellular Antibiotic Resistance Genes tetA and sul2 in Photosensitizer-Free Water

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4. The life cycle-dependent transcriptional profile of the obligate intracellular amoeba symbiont Amoebophilus asiaticus

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   Abstract Free-living amoebae often harbor obligate intracellular bacterial symbionts. Amoebophilus (A.) asiaticus is a representative of a lineage of amoeba symbionts in the phylum Bacteroidota. Here, we analyse the transcriptome of A. asiaticus strain 5a2 at four time points during its infection cycle and replication within the Acanthamoeba host using RNA sequencing. Our results reveal a dynamic transcriptional landscape throughout different A. asiaticus life cycle stages. Many intracellular bacteria and pathogens utilize eukaryotic-like proteins (ELPs) for host cell interaction and the A. asiaticus 5a2 genome shows a particularly high abundance of ELPs. We show the expression of all genes encoding ELPs and found many ELPs to be differentially expressed. At the replicative stage of A. asiaticus, ankyrin repeat proteins and tetratricopeptide/Sel1-like repeat proteins were upregulated. At the later time points, high expression levels of a type 6 secretion system that likely prepares for a new infection cycle after lysing its host, were found. This study reveals comprehensive insights into the intracellular lifestyle of A. asiaticus and highlights candidate genes for host cell interaction. The results from this study have implications for other intracellular bacteria such as other amoeba-associated bacteria and the arthropod symbionts Cardinium forming the sister lineage of A. asiaticus. 

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5. Comparative transcriptomics and genomics from continuous axenic media growth identifies Coxiella burnetii intracellular survival strategies

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   Abstract Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a ‘reverse evolution’ approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (Sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic, and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media. 

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