Thomas Penz

Unity in Variety—The Pan-Genome of the Chlamydiae

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

In situ architecture, function, and evolution of a contractile injection system

Identification of a new injection system To interact with other cells, bacteria use contractile machines that function similarly to membrane-puncturing bacteriophages. The so-called type 6 secretion system (T6SS) functions from inside a bacterial cell. Böck et al. used modern electron microscopy methods and functional assays to resolve the structure and function of a T6SS in the cellular context. They identified three modules and showed large-scale structural changes upon firing. T6SSs are organized in multibarrel gun-like arrays and may contribute to the survival of bacteria inside their host. Science, this issue p. 713 A model type 6 secretion system reveals the structure, dynamics, and evolution of apparatus attachment to the target membrane. Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail–like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus. The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.

Bacteriocyte-associated gammaproteobacterial symbionts of the Adelges nordmannianae/piceae complex (Hemiptera: Adelgidae).

Adelgids (Insecta: Hemiptera: Adelgidae) are known as severe pests of various conifers in North America, Canada, Europe and Asia. Here, we present the first molecular identification of bacteriocyte-associated symbionts in these plant sap-sucking insects. Three geographically distant populations of members of the Adelges nordmannianae/piceae complex, identified based on coI and ef1alpha gene sequences, were investigated. Electron and light microscopy revealed two morphologically different endosymbionts, coccoid or polymorphic, which are located in distinct bacteriocytes. Phylogenetic analyses of their 16S and 23S rRNA gene sequences assigned both symbionts to novel lineages within the Gammaproteobacteria sharing <92% 16S rRNA sequence similarity with each other and showing no close relationship with known symbionts of insects. Their identity and intracellular location were confirmed by fluorescence in situ hybridization, and the names ‘Candidatus Steffania adelgidicola’ and ‘Candidatus Ecksteinia adelgidicola’ are proposed for tentative classification. Both symbionts were present in all individuals of all investigated populations and in different adelgid life stages including eggs, suggesting vertical transmission from mother to offspring. An 85 kb genome fragment of ‘Candidatus S. adelgidicola’ was reconstructed based on a metagenomic library created from purified symbionts. Genomic features including the frequency of pseudogenes, the average length of intergenic regions and the presence of several genes which are absent in other long-term obligate symbionts, suggested that ‘Candidatus S. adelgidicola’ is an evolutionarily young bacteriocyte-associated symbiont, which has been acquired after diversification of adelgids from their aphid sister group.

The Endosymbiont Amoebophilus asiaticus Encodes an S-Adenosylmethionine Carrier That Compensates for Its Missing Methylation Cycle

All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes).

Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont

Chlamydiae are known as major bacterial pathogens of humans, causing the ancient disease trachoma, but they are also frequently found in the environment where they infect ubiquitous protists such as amoebae. All known chlamydiae require a eukaryotic host cell to thrive. Using the environmental chlamydia Protochlamydia amoebophila within its natural host, Acanthamoeba castellanii, we investigated gene expression dynamics in vivo and throughout the complete chlamydial developmental cycle for the first time. This allowed us to infer how a major virulence mechanism, the type III secretion system, is regulated and employed, and we show that the physiology of chlamydiae undergoes a complete shift regarding carbon metabolism and energy generation. This study provides comprehensive insights into the infection strategy of chlamydiae and reveals a unique adaptation to life within a eukaryotic host cell. ABSTRACT Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as the carbon source during initial stages and a postreplicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes. IMPORTANCE Chlamydiae are known as major bacterial pathogens of humans, causing the ancient disease trachoma, but they are also frequently found in the environment where they infect ubiquitous protists such as amoebae. All known chlamydiae require a eukaryotic host cell to thrive. Using the environmental chlamydia Protochlamydia amoebophila within its natural host, Acanthamoeba castellanii, we investigated gene expression dynamics in vivo and throughout the complete chlamydial developmental cycle for the first time. This allowed us to infer how a major virulence mechanism, the type III secretion system, is regulated and employed, and we show that the physiology of chlamydiae undergoes a complete shift regarding carbon metabolism and energy generation. This study provides comprehensive insights into the infection strategy of chlamydiae and reveals a unique adaptation to life within a eukaryotic host cell.