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Legionella pneumophila is a gram‐negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase‐like metabolic changes via alternative sigma factors, and two‐component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity. [ABSTRACT FROM AUTHOR]

Legionella pneumophila possesses a unique intracellular lifecycle featuring distinct morphological stages that include replicative forms and transmissive cyst forms. Expression of genes associated with virulence traits and cyst morphogenesis is concomitant, and governed by a complex stringent response based‐regulatory network and the stationary phase sigma factor RpoS. In Pseudomonas spp., rpoS expression is controlled by the autorepressor PsrA, and orthologs of PsrA and RpoS are required for cyst formation in Azotobacter. Here we report that the L. pneumophila psrA ortholog, expressed as a leaderless monocistronic transcript, is also an autorepressor, but is not a regulator of rpoS expression. Further, the binding site sequence recognized by L. pneumophila PsrA is different from that of Pseudomonas PsrA, suggesting a repertoire of target genes unique to L. pneumophila. While PsrA was dispensable for growth in human U937‐derived macrophages, lack of PsrA affected bacterial intracellular growth in Acanthamoeba castellanii protozoa, but also increased the quantity of poly‐3‐hydroxybutyrate (PHB) inclusions in matured transmissive cysts. Interestingly, overexpression of PsrA increased the size and bacterial load of the replicative vacuole in both host cell types. Taken together, we report that PsrA is a host‐specific requirement for optimal temporal progression of L. pneumophila intracellular lifecycle in A. castellanii. [ABSTRACT FROM AUTHOR]

Comprehensive approaches to detect protein protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p Mms1p and Sae2p Mre11p. The Rtt101p Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies. [ABSTRACT FROM AUTHOR]