Transposon-insertion sequencing (TIS) is a powerful approach for deciphering genetic requirements for bacterial growth in different conditions, as it enables simultaneous genome-wide analysis of the fitness of thousands of mutants. However, current methods for comparative analysis of TIS data do not adjust for stochastic experimental variation between datasets and are limited to interrogation of annotated genomic elements. Here, we present ARTIST, an accessible TIS analysis pipeline for identifying essential regions that are required for growth under optimal conditions as well as conditionally essential loci that participate in survival only under specific conditions. ARTIST uses simulation-based normalization to model and compensate for experimental noise, and thereby enhances the statistical power in conditional TIS analyses. ARTIST also employs a novel adaptation of the hidden Markov model to generate statistically robust, high-resolution, annotation-independent maps of fitness-linked loci across the entire genome. Using ARTIST, we sensitively and comprehensively define Mycobacterium tuberculosis and Vibrio cholerae loci required for host infection while limiting inclusion of false positive loci. ARTIST is applicable to a broad range of organisms and will facilitate TIS-based dissection of pathways required for microbial growth and survival under a multitude of conditions.

Preface Transposon-insertion sequencing (TIS) is a powerful approach that can be widely applied to genome-wide definition of loci that are required for growth in diverse conditions. However, experimental design choices and stochastic biological processes can heavily influence the results of TIS experiments and affect downstream statistical analysis. Here, we discuss TIS experimental parameters and how these factors relate to the benefits and limitations of the various statistical frameworks that can be applied to computational analysis of TIS data.

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Vibrio parahaemolyticus is the most common cause of seafood-borne gastroenteritis worldwide and a blight on global aquaculture. This organism requires a horizontally acquired type III secretion system (T3SS2) to infect the small intestine, but knowledge of additional factors that underlie V. parahaemolyticus pathogenicity is limited. We used transposon-insertion sequencing to screen for genes that contribute to viability of V. parahaemolyticus in vitro and in the mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic contributions to in vivo fitness. We identified genes that contribute to V. parahaemolyticus colonization of the intestine independent of known virulence mechanisms in addition to uncharacterized components of T3SS2. Our study revealed that toxR, an ancestral locus in Vibrio species, is required for V. parahaemolyticus fitness in vivo and for induction of T3SS2 gene expression. The regulatory mechanism by which V. parahaemolyticus ToxR activates expression of T3SS2 resembles Vibrio cholerae ToxR regulation of distinct virulence elements acquired via lateral gene transfer. Thus, disparate horizontally acquired virulence systems have been placed under the control of this ancestral transcription factor across independently evolved human pathogens.Vibrio parahaemolyticus | transposon-insertion sequencing | type III secretion | bacterial pathogenesis | pathogen evolution T he gram-negative γ-proteobacterium Vibrio parahaemolyticus thrives in either pathogenic or symbiotic association with marine organisms and as a planktonic bacterium (1). This facultative human pathogen, abundant in aquatic environments, was first isolated following a food poisoning outbreak in 1952 and has emerged as the leading cause of seafood-associated gastroenteritis worldwide and a blight on global aquaculture (2, 3). Sequencing of the V. parahaemolyticus genome and the development of animal models of infection have demonstrated a critical role for type III secretion in V. parahaemolyticus virulence (4, 5).Pathogenic isolates of V. parahaemolyticus encode two type III secretion systems (T3SSs), which are multiprotein structures that mediate the translocation of bacterial effector proteins directly into eukaryotic cells (4, 6). All V. parahaemolyticus strains encode a T3SS on the large chromosome (T3SS1), and the vast majority of clinical isolates, but few environmental isolates possess a horizontally acquired pathogenicity island (VPaI-7) encoding a second T3SS (T3SS2) and one or more pore-forming toxins (TDH) (7). Studies using the infant rabbit model of V. parahaemolyticus infection, which recapitulates manifestations of human gastrointestinal disease (e.g., profuse diarrhea, enteritis, epithelial disruption), revealed that although T3SS1 and TDH are dispensable for intestinal colonization and pathogenesis, colonization and pathology are dependent on T3SS2, consistent with the epidemiological association between T3SS2 and pathogenicity (5). Furthermor...