Phage-mediated lysogenisation and transduction dynamics in Salmonella Typhimurium

Salmonella enterica is an important foodborne pathogen whose characteristics have been shaped by bacteriophages and horizontal gene transfer. Nevertheless, many of its interactions with phages remain poorly understood. In this context, this dissertation aimed to shine a light on the detailed lysogenisation and transduction dynamics stemming from the interactions between Salmonella enterica serovar Typhimurium and its temperate phage P22.

Earlier research on P22 lysogenisation in our group revealed a novel interaction instigated by the P22 ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon that is involved in D-galactonate metabolism. Moreover, the pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. In the current dissertation, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5′ untranslated region that is instructive for a secondary pid mRNA species, and has a 3′ Rho-independent termination loop that is responsible for stability of the pid transcript. Subsequently, a broad interactomics approach to reveal the molecular components underlying the Pid/dgoRKAT interaction led to the hypothesis that Pid might function as a secondary channel binding factor (SCBF) that affects RNA polymerase processivity via the support of another SCBF DksA. Further evidence in support of this hypothesis came from the observations that Pid and DksA share structural similarity, and that a deletion of dksA or mutations in the RNA polymerase could attenuate the Pid/dgoRKAT interaction. However, so far, the putative Pid/DksA/RNA polymerase interaction could not yet be confirmed in a more direct fashion.

Since phage-mediated transduction is an important driver of horizontal gene transfer, the transducing capacity of P22 was inferred by sequence-mapping P22 capsid content and compared to two other Salmonella phages (temperate phage ES18, and obligate lytic phage Det7). As such, we found evidence for the presence of genetic watermarks within the S. Typhimurium chromosome that resemble the packaging sequence of P22, and that tremendously increase the packaging and transduction frequencies of nearby regions via P22. More specifically, we have identified a ca. 561 kb host region flanked by such watermarks that is highly packaged and transduced during both P22 prophage induction and lytic infection. We propose that such watermarks in the host chromosome can become selected as sites next to which high frequency of transduction occurs.

In summary, this dissertation has provided novel molecular-genetic insights into how Salmonella Typhimurium undergoes P22 lysogenisation and transduction dynamics, and underscores that (even in well-studied model systems) such particular phage-host interactions deserve further exploration.