Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and E. coli K-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHEC's but not to K-12's growth in vitro. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Over 200 mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote EHEC resistance to host-derived stresses. cvpA is also required for intestinal growth of several other enteric pathogens, and proved to be required for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt deoxycholate, highlighting the important role of this previously uncharacterized protein in pathogen survival. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine.

A Vibrio cholerae BolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity.

BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

A New Suite of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes.

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counterselection escape, which prevents isolation of cells with the desired mutation, occasionally limits their utility. To circumvent this, we engineered a series of "pTOX" allelic-exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome-binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the gene encoding blue AmilCP or magenta TsPurple nonfluorescent chromoprotein facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 paralogous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC ß-lactamase activity and the corresponding ß-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high-level ampC derepression and ß-lactam resistance. These data suggest why ß-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens, like E. cloacae Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria.IMPORTANCE Targeted modification of bacterial genomes is critical for genetic analysis of microorganisms. Allelic exchange is a technique that relies on homologous recombination to replace native loci with engineered sequences. However, current allelic-exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic-exchange vectors, pTOX, which were validated in several medically important proteobacteria. They encode visible nonfluorescent chromoproteins that enable easy identification of colonies bearing integrated vectors and permit stringent selection for the second step of homologous recombination. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on ß-lactam antibiotic resistance.

Unsupervised Learning Approach for Comparing Multiple Transposon Insertion Sequencing Studies.

Transposon insertion sequencing (TIS) is a widely used technique for conducting genome-scale forward genetic screens in bacteria. However, few methods enable comparison of TIS data across multiple replicates of a screen or across independent screens, including screens performed in different organisms. Here, we introduce a post hoc analytic framework, comparative TIS (CompTIS), which utilizes unsupervised learning to enable meta-analysis of multiple TIS data sets. CompTIS first implements screen-level principal-component analysis (PCA) and clustering to identify variation between the TIS screens. This initial screen-level analysis facilitates the selection of related screens for additional analyses, reveals the relatedness of complex environments based on growth phenotypes measured by TIS, and provides a useful quality control step. Subsequently, PCA is performed on genes to identify loci whose corresponding mutants lead to concordant/discordant phenotypes across all or in a subset of screens. We used CompTIS to analyze published intestinal colonization TIS data sets from two vibrio species. Gene-level analyses identified both pan-vibrio genes required for intestinal colonization and conserved genes that displayed species-specific requirements. CompTIS is applicable to virtually any combination of TIS screens and can be implemented without regard to either the number of screens or the methods used for upstream data analysis.IMPORTANCE Forward genetic screens are powerful tools for functional genomics. The comparison of similar forward genetic screens performed in different organisms enables the identification of genes with similar or different phenotypes across organisms. Transposon insertion sequencing is a widely used method for conducting genome-scale forward genetic screens in bacteria, yet few bioinformatic approaches have been developed to compare the results of screen replicates and different screens conducted across species or strains. Here, we used principal-component analysis (PCA) and hierarchical clustering, two unsupervised learning approaches, to analyze the relatedness of multiple in vivo screens of pathogenic vibrios. This analytic framework reveals both shared pan-vibrio requirements for intestinal colonization and strain-specific dependencies. Our findings suggest that PCA-based analytics will be a straightforward widely applicable approach for comparing diverse transposon insertion sequencing screens.

CRISPR Screen Reveals that EHEC's T3SS and Shiga Toxin Rely on Shared Host Factors for Infection.

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen's key virulence factors.

Time-Resolved Transposon Insertion Sequencing Reveals Genome-Wide Fitness Dynamics during Infection.

Transposon insertion sequencing (TIS) is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection). Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE). From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant's fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen) collected over a 2-week infection period from a natural host (the flatfish turbot). PACE uncovered more genes that affect E. piscicida's fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses.IMPORTANCE Transposon insertion sequencing (TIS) enables genome-wide mapping of the genetic determinants of fitness, typically based on observations at a single sampling point. Here, we move beyond analysis of endpoint TIS data to create a framework for analysis of time series TIS data, termed pattern analysis of conditional essentiality (PACE). We applied PACE to identify genes that contribute to colonization of a natural host by the fish pathogen Edwardsiella piscicida. PACE uncovered more genes that affect E. piscicida's fitness in vivo than were detected using a terminal sampling point, and its clustering of mutants with related fitness profiles informed design of new live vaccine candidates. PACE yields insights into patterns of fitness dynamics and circumvents major limitations of existing methodologies. Finally, the PACE method should be applicable to additional "omic" time series data, including screens based on clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR/Cas9).

Deciphering the landscape of host barriers to Listeria monocytogenes infection.

Listeria monocytogenes is a common food-borne pathogen that can disseminate from the intestine and infect multiple organs. Here, we used sequence tag-based analysis of microbial populations (STAMP) to investigate Lmonocytogenes population dynamics during infection. We created a genetically barcoded library of murinized Lmonocytogenes and then used deep sequencing to track the pathogen's dissemination routes and quantify its founding population (Nb) sizes in different organs. We found that the pathogen disseminates from the gastrointestinal tract to distal sites through multiple independent routes and that Nb sizes vary greatly among tissues, indicative of diverse host barriers to infection. Unexpectedly, comparative analyses of sequence tags revealed that fecally excreted organisms are largely derived from the very small number of L. monocytogenes cells that colonize the gallbladder. Immune depletion studies suggest that distinct innate immune cells restrict the pathogen's capacity to establish replicative niches in the spleen and liver. Finally, studies in germ-free mice suggest that the microbiota plays a critical role in the development of the splenic, but not the hepatic, barriers that prevent L. monocytogenes from seeding these organs. Collectively, these observations illustrate the potency of the STAMP approach to decipher the impact of host factors on population dynamics of pathogens during infection.

Interplay between microbial d-amino acids and host d-amino acid oxidase modifies murine mucosal defence and gut microbiota.

L-Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d-amino acids (d-aa) have important non-ribosome-based functions(1). Mammals synthesize d-Ser and d-Asp, primarily in the central nervous system, where d-Ser is critical for neurotransmission(2). Bacteria synthesize a largely distinct set of d-aa, which become integral components of the cell wall and are also released as free d-aa(3,4). However, the impact of free microbial d-aa on host physiology at the host-microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d-aa that are derived from the microbiota. Furthermore, the microbiota induces production of d-amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d-aa by DAO, which yields the antimicrobial product H2O2, protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d-aa and DAO as previously unrecognized mediators of microbe-host interplay and homeostasis on the epithelial surface of the small intestine.

The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes.

UNLABELLED: Transposon insertion sequencing (TIS; also known as TnSeq) is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS) protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt) and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences. IMPORTANCE: Transposon insertion sequencing (TIS) is often used to assess the relative frequency with which genetic loci can be disrupted, which is taken as an indicator of their importance for bacterial fitness. Here, we report that biological factors other than the relative levels of fitness of insertion mutants can influence TIS output. We found that the presence of the DNA binding protein H-NS, which preferentially recognizes AT-rich sequences, is linked to significant underrepresentation of mutations within AT-rich loci in transposon insertion libraries. Furthermore, there is a marked correspondence between loci bound by H-NS and loci with an increased frequency of disruption in a Δhns insertion library relative to a wt library. Our data suggest that factors other than genetic fitness (e.g., DNA binding proteins such as H-NS) can systematically modulate the frequency of transposon insertions in TIS studies and add a note of caution for interpretation of TIS data.

CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity.

Type III secretion systems (T3SSs) inject bacterial effector proteins into host cells and underlie the virulence of many gram-negative pathogens. Studies have illuminated bacterial factors required for T3SS function, but the required host processes remain largely undefined. We coupled CRISPR/Cas9 genome editing technology with the cytotoxicity of two Vibrio parahaemolyticus T3SSs (T3SS1 and T3SS2) to identify human genome disruptions conferring resistance to T3SS-dependent cytotoxicity. We identity non-overlapping genes required for T3SS1- and T3SS2-mediated cytotoxicity. Genetic ablation of cell surface sulfation reduces bacterial adhesion and thereby alters the kinetics of T3SS1-mediated cytotoxicity. Cell surface fucosylation is required for T3SS2-dependent killing, and genetic inhibition of fucosylation prevents membrane insertion of the T3SS2 translocon complex. These findings reveal the importance of ubiquitous surface modifications for T3SS function, potentially explaining the broad tropism of V. parahaemolyticus, and highlight the utility of genome-wide CRISPR/Cas9 screens to discover processes underlying host-pathogen interactions.

Genetic analysis of Vibrio parahaemolyticus intestinal colonization.

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.

A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics.

Gram-negative bacteria are notoriously resistant to a variety of high-molecular-weight antibiotics due to the limited permeability of their outer membrane (OM). The basis of OM barrier function and the genetic factors required for its maintenance remain incompletely understood. Here, we employed transposon insertion sequencing to identify genes required for Vibrio cholerae resistance to vancomycin and bacitracin, antibiotics that are thought to be too large to efficiently penetrate the OM. The screen yielded several genes whose protein products are predicted to participate in processes important for OM barrier functions and for biofilm formation. In addition, we identified a novel factor, designated vigA (for vancomycin inhibits growth), that has not previously been characterized or linked to outer membrane function. The vigA open reading frame (ORF) codes for an inner membrane protein, and in its absence, cells became highly sensitive to glycopeptide antibiotics (vancomycin and ramoplanin) and bacitracin but not to other large antibiotics or detergents. In contrast to wild-type (WT) cells, the vigA mutant was stained with fluorescent vancomycin. These observations suggest that VigA specifically prevents the periplasmic accumulation of certain large antibiotics without exerting a general role in the maintenance of OM integrity. We also observed marked interspecies variability in the susceptibilities of Gram-negative pathogens to glycopeptides and bacitracin. Collectively, our findings suggest that the OM barrier is not absolute but rather depends on specific OM-antibiotic interactions.

Chemoproteomic profiling of host and pathogen enzymes active in cholera.

Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human choleric stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, and genetic disruption of all four proteases increased the abundance of intelectin, an intestinal lectin, and its binding to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting that it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialog in an animal model of infection.

The design and analysis of transposon insertion sequencing experiments.

Transposon insertion sequencing (TIS) is a powerful approach that can be extensively applied to the genome-wide definition of loci that are required for bacterial growth under diverse conditions. However, experimental design choices and stochastic biological processes can heavily influence the results of TIS experiments and affect downstream statistical analysis. In this Opinion article, 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 the computational analysis of TIS data.

A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance.

The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall-acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall-acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

Correction: A Cytosine Methytransferase Modulates the Cell Envelope Stress Response in the Cholera Pathogen.

[This corrects the article DOI: 10.1371/journal.pgen.1005666.].

A Cytosine Methyltransferase Modulates the Cell Envelope Stress Response in the Cholera Pathogen [corrected].

DNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM's DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential σE cell envelope stress pathway is dispensable in ∆vchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some cell envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex cell circuits to control critical housekeeping processes.

Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome.

UNLABELLED: Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII. IMPORTANCE: The genome of the enteric pathogen Vibrio cholerae consists of two chromosomes. While the chromosome I replication origin and its cognate replication initiator protein resemble those of Escherichia coli, the factors responsible for chromosome II replication initiation display no similarity to any other known initiation systems. Here, to enhance our understanding of how this DNA sequence, oriCII, and its initiator protein, RctB, function, we used both targeted mutagenesis and a new random-mutagenesis approach (OriSeq) to finely map the oriCII structural features and sequences required for RctB-mediated DNA replication. Collectively, our findings reveal the extraordinary evolutionary honing of the architecture and motifs that constitute oriCII and reveal a new role for methylation in oriCII-based replication. Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis-acting sequences.