Chlamydia trachomatis is a Gram-negative, obligate intracellular pathogen that is the causative agent of sexually transmitted infections and the ocular disease trachoma. Chlamydia trachomatis undergoes a biphasic developmental cycle differentiating between the infectious elementary body (EB) and the replicative reticulate body (RB). Under certain stress conditions, C. trachomatis can stall its developmental cycle and enter an aberrant state termed persistence. While in a persistent state, C. trachomatis is refractory toward antibiotics, can evade the host immune response, and becomes undetectable using standard clinical detection methods. Environmental and other pathogenic microbes are known to utilize partner switching mechanisms (PSM) to regulate sigma factors used to initiate a stress response. For this reason, this study focuses on the chlamydial PSM, its role in regulating the availability of the housekeeping sigma factor σ66, and its role in the developmental cycle and stress response of C. trachomatis. The chlamydial PSM is composed of five known proteins: the anti-sigma factor RsbW, two anti-anti-sigma factors RsbV1 and RsbV2, a regulatory phosphatase RsbU, and a second phosphatase-like protein CTL0852. In order to test the role of the PSM in the chlamydial stress response, a panel of C. trachomatis rsbV1 mutants were generated, persistence inducing iron starvation and tryptophan starvation cell culture conditions were optimized, and growth of the rsbV1 mutants under iron starvation conditions were assayed. No significant differences were seen between rsbV1 mutants under iron starvation nor recovery conditions as determined by progeny production and inclusion size analysis. Furthermore, this study generated PSM protein producing Escherichia coli strains for in vitro protein work and performed operon mapping of the PSM genes of C. trachomatis to help aid in future studies of the chlamydial PSM by facilitating the development of new chlamydial PSM mutants. This study gives phylogenetic support to the classification of ctl0852 as a chlamydial PSM gene by comparing relative mutations rates of PSM genes across chlamydial species.

Partner-switching regulatory systems (PSRSs) are utilized by many different bacteria to regulate a wide array of cellular responses, from stress response to expression of virulence factors. The filamentous cyanobacterium Nostoc punctiforme can transiently differentiate motile filaments, called hormogonia, in response to various changes in the environment. Hormogonia utilize a Type IV pilus (T4P) complex in conjunction with a secreted polysaccharide for gliding motility along solid surfaces. This study identified three genes, designated hmpU, hmpW, and hmpV, encoding the protein components of a PSRS involved in regulation of hormogonium motility in N. punctiforme. Although mutant strains with in-frame deletions in hmpU, hmpW, and hmpV differentiated morphologically distinct hormogonium-like filaments, further phenotypic analysis demonstrated significant distinctions among the strains. The ∆hmpW strain contained a higher percentage of motile filaments that moved faster than the wild-type strain, while the ∆hmpU and ∆hmpV strains consisted of fewer motile filaments that moved at a slower rate compared to wild type. Immunoblotting and immunofluorescence of PilA, the major component of the pilus in the T4P system, showed that although all mutant strains appeared to express similar levels of PilA protein, the ∆hmpU and ∆hmpV strains displayed reduced extracellular PilA. Lectin blotting and staining with fluorescently-labeled UEA lectin demonstrated a decrease in extracellular hormogonium polysaccharide in the ∆hmpU and ∆hmpV strains, consistent with the current understanding that the polysaccharide is secreted via the T4P system. Epistasis analysis demonstrated that the ∆hmpW, ∆hmpV double-deletion mutant strain displayed reduced spreading in plate motility assays, similar to the ∆hmpV single mutant. Together, these results support a model in which the HmpU phosphatase and HmpW serine kinase control the phosphorylation state of the HmpV protein, modulating its activity on a downstream target to ultimately promote activation of the T4P motor complex and enhance hormogonium motility.

The phylum of Chlamydiota is composed of gram negative obligate intracellular bacteria that live as symbionts of diverse eukaryotes, from protists to animals and humans. Members of the phylum can be split into two groups: the environmental Chlamydia, which includes symbionts of amoeba, and the pathogenic Chlamydia, which includes species infecting animals, birds, and humans and includes Chlamydia trachomatis the leading cause of reportable, bacterial sexually transmitted infections and the ocular infection, trachoma. The characterized phylum members undergo a biphasic developmental cycle alternating between the infectious elementary body (EB) and the replicative reticulate body (RB), with each form having distinct morphological and physiological properties. Differentiation between these forms occurs within a host cell membrane-derived vacuole termed the inclusion. The molecular mechanisms governing and executing bacterial development and RB growth remain unclear. The essentiality and uniqueness of development makes it a prime target for the development of novel, chlamydial-specific therapeutics. Reductive evolution has resulted in the loss of or fragmentation of numerous metabolic pathways, particularly in the pathogenic Chlamydia (~1 Mbp genome) as compared to the environmental Chlamydia (~2.5 Mbp). We hypothesize that the bacterium senses environmental changes (host cytoplasm) to ensure that development and growth coincide with host cell energy and metabolite levels. We predict that an encoded partner switching mechanism (PSM) plays a key role in: 1) regulation of growth by acting as a molecular throttle through regulation of the housekeeping sigma factor, and 2) differentiation by impacting the composition of the sigma factor pool allowing for transcriptional changes needed for developmental transitions. We also predict that PSM regulation occurs through sensing of nucleotide triphosphates, TCA-cycle intermediates, metal concentrations, and redox. Canonical PSMs have a PP2C-type sensor phosphatase (SP), an anti-sigma factor (ASF, serine kinase), an anti-anti-sigma factor (AASF, substrate of the SP and ASF) and a stress-response related alternative sigma factor. The PSM in pathogenic Chlamydia is atypical, and despite its reduced genome, is comprised of two SPs (RsbU which responds to α-ketoglutarate and CTL0852), two AASFs (RsbV1 and RsbV2), one ASF (RsbW), and, unusually, the ASF regulates the availability of the “housekeeping” sigma factor, σ66. To test our hypotheses, we first constructed and purified a variety of amino acid point mutants of the two AASFs, ASF, and the SP for in vitro analyses. Kinase and phosphatase activity towards RsbV1/V2 was measured in the presence of different metals, phosphate donors, and pH and redox conditions. Phos-tag acrylamide gels were used to assess protein phosphorylation status. We discovered that metalation impacts enzyme activity and the substrate specificity of RsbU, and that RsbW can use multiple phosphate donors. Prior work, and our data, found that RsbW and RsbU have higher enzymatic activity towards RsbV1 than RsbV2, leading us to explore the importance of RsbV2 in chlamydial biology. Genome gazing revealed that environmental Chlamydia possess a single AASF, and bioinformatic analyses support that it is more similar to RsbV2 than RsbV1 suggesting that the pathogenic Chlamydia gained RsbV1. Comparing the biochemical features of the two AASFs provides potential reasons for the different enzyme affinities. To flesh out the in vivo importance of each AASF, we characterized bacterial growth, infectious progeny production, and the levels of RsbV1/V2 in a cell culture infection model using a collection of C. trachomatis L2 rsbV1 null or rsbV2 knockdown strains. We also overexpressed the AASFs in strains grown with different glucose levels. Note that C. trachomatis is an auxotroph for glucose-6-phosphate. In normal chlamydial culture glucose medium levels, the rsbV1 null strain showed an ~1 log reduction in infectious progeny numbers while the rsbV2 knockdown or AASF overexpression strains had no defects. We also observed that the rsbV1 null strain has a developmental delay and exhibits growth differences in response to glucose levels, i.e. a functional PSM seems to set a “growth cap” in response to different glucose availability. Immunoblotting analysis of RsbV1/V2 demonstrated the presence of both proteins throughout development, and protein levels remained the same in low or high glucose levels and in the wild type or rsbV1 null strains (measuring RsbV2 only for the RsbV1 null strain). These results tell us that the AASF levels have minimal impact on chlamydial biology, suggesting that phosphorylation status is key to regulation. To assess phosphorylation, we used protein pulldown assays and Phos-tag gels to assess RsbV1 and RsbV2 phosphorylation during development. Both RsbV1 and RsbV2 were phosphorylated during the EB stage, which is similar to our prior results using Chlamydia caviae. In conjunction with the in vivo phosphorylation data, we hypothesize that stage-dependent inhibition of AASF/RsbW interactions frees RsbW to sequester σ66. Reduced pools of σ66 would promote RB-EB conversion through increased RNAP binding to the late gene sigma factors σ54 and σ28. Supporting this model, overexpression of a non-phosphorylatable RsbV2 S55A mutant (an RsbW “trap”), but not overexpression of RsbV1 S56A, resulted in a 3 log reduction in infectious progeny production without gross changes in inclusion morphology or bacterial numbers, while causing a reduction in σ54 and σ28 regulated EB-specific proteins and inhibition of RB-EB transition shown via transmission electron microscopy. As an alternative approach to assess the consequence of reduced “free” RsbW, we used a CRISPRi knockdown system targeting rsbW and observed a reduction in infectious progeny production under some conditions, which is consistent with the RsbV2 S55A expression strain results. The rsbW CRISPRi-associated phenotype was weaker than the RsbV2 S55A phenotype. As bacterial redox status changes throughout development (RBs are reduced and EBs are oxidized), we also assessed whether the cysteine-rich proteins RsbV2 and RsbW were redox responsive. In parallel to the unique AASF expansion in the pathogenic Chlamydia, RsbV2 in the pathogenic Chlamydia has a CXCC motif that is not found in the RsbV homolog in the environmental Chlamydia. Our in vitro studies found that, under oxidizing conditions, RsbV2 is dimerized, and the dimer form inhibits phosphorylation of RsbV2 by RsbW. We predict that retention of RsbV2 after RsbV1 acquisition has been selected for, in part, owing to a unique redox-sensing role compared to RsbV1 and that the presence of two AASFs enables more sensitive tuning of growth and development in response to metabolite levels. The different phenotypes when overexpressing non-phosphorylatable RsbV1 and RsbV2 also hints at a potential non-PSM or expanded PSM role for RsbV1. The in vitro redox findings need to be further explored in an in vivo model. Collectively, we think the expansion of the PSM, in addition to other gene gain events, facilitated infection of multi-cellular organisms. Additionally, our data support that the PSM regulates growth/cell differentiation in response to energy/nutrients, and that redox levels and biochemical features of RsbV1 and RsbV2 govern PSM-component interactions. As disruption of normal PSM function significantly reduces production of infectious progeny, compounds targeting the PSM components could serve as novel, narrow spectrum inhibitors.

Kooperatives Verhalten lässt sich in vielen Bereichen menschlichen Zusammenlebens sowie im gesamten Tierreich beobachten. In evolutionären Modellen wurde gezeigt, dass Netzwerkstrukturen die Kooperation erhöhen können. Empirische Studien versuchten vergeblich diesen Mechanismus auch bei Menschen nachzuweisen. Es scheint, als würden Netzwerke nur dann die Kooperation erhöhen, wenn die Strukturen nicht statisch sind, sondern dynamisch. Das heißt, dass die Individuen die Möglichkeit haben, ihre Partner zu wechseln. Eine wichtige – aber bislang unerforschte – Eigenschaft dynamischer Netzwerke ist jedoch, dass derartige Wechsel von Partnern in der Regel Kosten verursachen, ob in Form von Zeit oder Ressourcen. Kapitel I meiner Arbeit schließt diese Lücke, in dem es sich mit den Effekten von Kosten auf dynamischen Netzwerken befasst. Ich konnte nachweisen, dass Menschen seltener Interaktionen mit Partnern beendeten, wenn die Kontaktaufnahme mit einem neuen Partner mit Kosten verbunden war. Bei sehr hohen Kosten, wurden Partner so selten gewechselt, dass das Netzwerk fast statisch war. Interessanterweise blieb die Kooperation dennoch sehr hoch. Das bedeutet, dass für kooperatives Verhalten entscheidend ist, ob man die Möglichkeit hat, Partner zu wechseln. Im Gegensatz zu bisherigen Annahmen ist es daher nicht wichtig, wie oft tatsächlich Partner gewechselt werden, sondern lediglich ob es die Möglichkeit dazu gibt. In Kapitel II beschäftige ich mich mit optimalem Entscheidungsverhalten. Im sogenannten Judge-Advisor-System geht es darum, dass eine Person, der Judge, eine unbekannte numerische Größe schätzen will. Dazu erhält der Judge eine zweite unabhängige Schätzung als Rat von einer zweiten Person, des Advisor. Schließlich ist die Frage, wie der Judge optimal den Rat verwerten kann um seine Anfangsschätzung zu verbessern. Bisherige Forschung konzentrierte sich hauptsächlich auf zwei mögliche Methoden, (i) das Bilden des Mittelwerts und (ii) das Wählen der besseren Anfangsschätzung. Das Hauptargument für diese einfachen Methoden ist deren häufige Verwendung in bisherigen Experimenten. Allerdings wurden sehr wohl auch andere Gewichtungen beobachtet und daher ist eine gründliche Analyse der optimalen Gewichtung erforderlich. In der vorliegenden Arbeit leitete ich ein normatives Modell her, das beschreibt, unter welchen Bedingungen welche Methode das bestmögliche Ergebnis liefert. Es wurden drei Methoden verglichen: (i) das Bilden des Durchschnitts, (ii) das Wählen der besseren Anfangsschätzung, und (iii) das Bilden eines gewichtetet Mittelwerts, wobei das Gewicht vom Kompetenzunterschied abhängt. Welche Methode optimal ist, hängt davon ab, wie groß der Kompetenzunterschied ist und wie gut er vom Judge erkannt wird. Die Durchschnittbildung ist immer dann vorteilhaft, wenn der Kompetenzunterschied nicht groß ist, oder nur schwer richtig eingeschätzt werden kann. Wenig überraschend lohnt sich das Wählen der besseren Anfangsschätzung, wenn der Kompetenzunterschied hinreichend groß ist, vorausgesetzt es wird tatsächlich die bessere Anfangsschätzung gewählt. Wenn der Kompetenzunterschied vom Judge gut eingeschätzt werden kann, ist eine Entsprechende Gewichtung immer die beste Methode, unabhängig vom tatsächlichen Unterschied. In Übereinstimmung mit bisheriger Forschung wurde auch die Kombination von Durchschnittbildung und Wählen der besseren Anfangsschätzung untersucht. Diese Kombinationsmethode beruht darauf, bei als gering eingeschätztem Kompetenzunterschied den Durchschnitt zu bilden und ansonsten die bessere Anfangsschätzung zu wählen. Interessanterweise schneidet diese Kombinationsmethode sehr schlecht ab, was hauptsächlich daran liegt, dass zu oft die falsche Anfangsschätzung genommen würde. Insgesamt ist das gewichtete Mittel also eine geeignete Methode für einen großen Parameterbereich.