Dispensable Regions Research Articles

Comprehensive deletion scan of anti-CRISPR AcrIIA4 reveals essential and dispensable domains for Cas9 inhibition.

Delineating a protein's essential and dispensable domains provides critical insight into how it carries out its function. Here, we developed a high-throughput method to synthesize and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Our approach generates precise deletions using a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. We applied our method to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9. Extensive structural characterization has shown that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains; nonetheless, the importance of each AcrIIA4 interaction for SpCas9 inhibition is unknown. We used our approach to determine the essential and dispensable regions of AcrIIA4. Surprisingly, not all contacts with SpCas9 were required, and in particular, we found that the AcrIIA4 loop that inserts into SpCas9's RuvC catalytic domain can be deleted. Our results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding, and that its interaction with the SpCas9 catalytic domain is inessential.

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships.IMPORTANCEGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

Attenuation hotspots in neurotropic human astroviruses.

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3' end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.

Greedy reduction of Bacillus subtilis genome yields emergent phenotypes of high resistance to a DNA damaging agent and low evolvability.

Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.

Expanding repertoire of SARS-CoV-2 deletion mutations contributes to evolution of highly transmissible variants

The emergence of highly transmissible SARS-CoV-2 variants and vaccine breakthrough infections globally mandated the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 2.13 million SARS-CoV-2 genomes from 188 countries/territories (up to June 2021) and performed whole-genome viral sequencing from 102 COVID-19 patients, including 43 vaccine breakthrough infections. We identified 92 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a 3-month window. Deletions in the Spike protein N-terminal domain were highly enriched for these ‘surge-associated mutations’ (Odds Ratio = 14.19, 95% CI 6.15–32.75, p value = 3.41 × 10–10). Based on a longitudinal analysis of mutational prevalence globally, we found an expanding repertoire of Spike protein deletions proximal to an antigenic supersite in the N-terminal domain that may be one of the key contributors to the evolution of highly transmissible variants. Finally, we generated clinically annotated SARS-CoV-2 whole genome sequences from 102 patients and identified 107 unique mutations, including 78 substitutions and 29 deletions. In five patients, we identified distinct deletions between residues 85–90, which reside within a linear B cell epitope. Deletions in this region arose contemporaneously on a diverse background of variants across the globe since December 2020. Overall, our findings based on genomic-epidemiology and clinical surveillance suggest that the genomic deletion of dispensable antigenic regions in SARS-CoV-2 may contribute to the evasion of immune responses and the evolution of highly transmissible variants.

DELEAT: gene essentiality prediction and deletion design for bacterial genome reduction

BackgroundThe study of gene essentiality is fundamental to understand the basic principles of life, as well as for applications in many fields. In recent decades, dozens of sets of essential genes have been determined using different experimental and bioinformatics approaches, and this information has been useful for genome reduction of model organisms. Multiple in silico strategies have been developed to predict gene essentiality, but no optimal algorithm or set of gene features has been found yet, especially for non-model organisms with incomplete functional annotation.ResultsWe have developed DELEAT v0.1 (DELetion design by Essentiality Analysis Tool), an easy-to-use bioinformatic tool which integrates an in silico gene essentiality classifier in a pipeline allowing automatic design of large-scale deletions in any bacterial genome. The essentiality classifier consists of a novel logistic regression model based on only six gene features which are not dependent on experimental data or functional annotation. As a proof of concept, we have applied this pipeline to the determination of dispensable regions in the genome of Bartonella quintana str. Toulouse. In this already reduced genome, 35 possible deletions have been delimited, spanning 29% of the genome.ConclusionsBuilt on in silico gene essentiality predictions, we have developed an analysis pipeline which assists researchers throughout multiple stages of bacterial genome reduction projects, and created a novel classifier which is simple, fast, and universally applicable to any bacterial organism with a GenBank annotation file.

Comprehensive dissection of dispensable genomic regions in Streptomyces based on comparative analysis approach

BackgroundLarge-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches.ResultsPhylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis.ConclusionsThe integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.

Accessories Make the Outfit: Accessory Chromosomes and Other Dispensable DNA Regions in Plant-Pathogenic Fungi

Fungal pathogen genomes can often be divided into core and accessory regions. Accessory regions ARs) may be comprised of either ARs (within core chromosomes (CCs) or wholly dispensable (accessory) chromosomes (ACs). Fungal ACs and ARs typically accumulate mutations and structural rearrangements more rapidly over time than CCs and many harbor genes relevant to host-pathogen interactions. These regions are of particular interest in plant pathology and include host-specific virulence factors and secondary metabolite synthesis gene clusters. This review outlines known ACs and ARs in fungal genomes, methods used for their detection, their common properties that differentiate them from the core genome, and what is currently known of their various roles in pathogenicity. Reports on the evolutionary processes generating and shaping AC and AR compartments are discussed, including repeat induced point mutation and breakage fusion bridge cycles. Previously ACs have been studied extensively within key genera, including Fusarium, Zymoseptoria, and Alternaria, but are growing in frequency of observation and perceived importance across a wider range of fungal species. Recent advances in sequencing technologies permit affordable genome assembly and resequencing of populations that will facilitate further discovery and routine screening of ACs.

Highly parallel genome variant engineering with CRISPR-Cas9.

Understanding the functional effects of DNA sequence variants is of critical importance for studies of basic biology, evolution, and medical genetics; however, measuring these effects in a high-throughput manner is a major challenge. One promising avenue is precise editing with the CRISPR-Cas9 system, which allows for generation of DNA double-strand breaks (DSBs) at genomic sites matching the targeting sequence of a guide RNA (gRNA). Recent studies have used CRISPR libraries to generate many frameshift mutations genome wide through faulty repair of CRISPR-directed breaks by nonhomologous end joining (NHEJ) 1 . Here, we developed a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering. We used our method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes in yeast. We found that most PTCs were highly deleterious unless they occurred close to the 3' end of the gene and did not affect an annotated protein domain. Unexpectedly, we discovered that some putatively essential genes are dispensable, whereas others have large dispensable regions. This approach can be used to profile the effects of large classes of variants in a high-throughput manner.

MinGenome: An In Silico Top-Down Approach for the Synthesis of Minimized Genomes.

Genome minimized strains offer advantages as production chassis by reducing transcriptional cost, eliminating competing functions and limiting unwanted regulatory interactions. Existing approaches for identifying stretches of DNA to remove are largely ad hoc based on information on presumably dispensable regions through experimentally determined nonessential genes and comparative genomics. Here we introduce a versatile genome reduction algorithm MinGenome that implements a mixed-integer linear programming (MILP) algorithm to identify in size descending order all dispensable contiguous sequences without affecting the organism's growth or other desirable traits. Known essential genes or genes that cause significant fitness or performance loss can be flagged and their deletion can be prohibited. MinGenome also preserves needed transcription factors and promoter regions ensuring that retained genes will be properly transcribed while also avoiding the simultaneous deletion of synthetic lethal pairs. The potential benefit of removing even larger contiguous stretches of DNA if only one or two essential genes (to be reinserted elsewhere) are within the deleted sequence is explored. We applied the algorithm to design a minimized E.coli strain and found that we were able to recapitulate the long deletions identified in previous experimental studies and discover alternative combinations of deletions that have not yet been explored in vivo.

Focus on Effector-Triggered Susceptibility.

Effector biology exhibits diversity at every level. Effector proteins play key roles in the molecular interplay between plants and plant-associated organisms, and effector biology remains one of the most active areas in the research field of molecular plant-microbe interactions. Using effectors as probes, much has been learned about pathogen virulence and host immunity, which has broad implications in developing disease-resistant crops that are essential for global food security. Thus, the MPMI Editorial Board is publishing this Focus Issue to showcase recent progress in this area.

Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

BackgroundSoil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world’s second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula.ResultsFocusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp.ConclusionsWe demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn’t share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2486-8) contains supplementary material, which is available to authorized users.

Non-manifesting AHI1 truncations indicate localized loss-of-function tolerance in a severe Mendelian disease gene.

Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening ('Kingsmore panel') do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screening.

Highly Acidic C-Terminal Region of Cytomegalovirus pUL96 Determines Its Functions during Virus Maturation Independently of a Direct pp150 Interaction

Tegument proteins pp150 and pUL96 function at a late step in cytomegalovirus (CMV) maturation. Here, we show that pp150 interacts directly with pUL96; however, the N-terminal region of pp150 and the C-terminal region of pUL96, which are critical for these proteins to function, are not required for this interaction. Moreover, the largely dispensable C-terminal region of pp150 is critical for pp150-pUL96 interaction. To further study the role of pUL96, several point and clustered mutations were engineered into the CMV Towne bacterial artificial chromosome (Towne-BAC) genome, replacing the conserved negatively charged C-terminal residues of pUL96. Although individual point mutations (E122A, D124A, and D125A) reduced virus growth slightly, the clustered mutations of 122EVDDAV127 significantly reduced virus growth, produced small syncytial plaque phenotypes, and impacted a late stage of virus maturation. When the UL96 C-terminal alanine conversion mutant (B6-BAC) virus was serially passaged in cell culture, it gained a plaque size comparable to that of Towne-BAC, displayed an altered restriction fragment length pattern, and replicated with increased growth kinetics. Whole-genome sequencing of this passaged virus (UL96P10) and the similarly passaged Towne-BAC virus revealed major differences only in the RNA4.9 and UL96 regions. When one of the mutations in the UL96 coding region was engineered into the B6-BAC virus, it significantly increased the plaque size and rescued the virus growth rate. Thus, accumulation of compensatory mutations only in UL96 in this revertant and the specific involvement of functionally dispensable regions of pp150 in the pUL96-pp150 interaction point toward a role for pUL96 in virus maturation that does not depend upon pp150. Human cytomegalovirus causes significant medical problems in newborns, as well as in people with low immunity. In this study, we investigated the functions of two essential virus proteins, pp150 and pUL96, and determined the impact of their mutual interaction on virus replication. These studies provide valuable information that is critical for the development of targeted antiviral therapies.

Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate large-scale metabolic model

The nonessential regions in bacterial chromosomes are ill-defined due to incomplete functional information. Here, we establish a comprehensive repertoire of the genome regions that are dispensable for growth of Bacillus subtilis in a variety of media conditions. In complex medium, we attempted deletion of 157 individual regions ranging in size from 2 to 159 kb. A total of 146 deletions were successful in complex medium, whereas the remaining regions were subdivided to identify new essential genes (4) and coessential gene sets (7). Overall, our repertoire covers ∼76% of the genome. We screened for viability of mutant strains in rich defined medium and glucose minimal media. Experimental observations were compared with predictions by the iBsu1103 model, revealing discrepancies that led to numerous model changes, including the large-scale application of model reconciliation techniques. We ultimately produced the iBsu1103V2 model and generated predictions of metabolites that could restore the growth of unviable strains. These predictions were experimentally tested and demonstrated to be correct for 27 strains, validating the refinements made to the model. The iBsu1103V2 model has improved considerably at predicting loss of viability, and many insights gained from the model revisions have been integrated into the Model SEED to improve reconstruction of other microbial models.

Identification of non-essential regions in nucleocapsid protein of porcine reproductive and respiratory syndrome virus for replication in cell culture

Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) plays a central role in virus replication. In this study, serial N- and C-terminal truncations of N protein were performed in the context of type 2 PRRSV infectious cDNA clone, and our results revealed that a stretch of inter-genotypic variable N terminal residues aa 5–13 ( 5NGKQQKKK 13K) and the last four inter-genotypic variable aa residues ( 120SPS 123A) at the C terminus of N protein were dispensable for type 2 PRRSV infectivity. All the recovered deletion mutant viruses had spontaneous mutations in the N coding region, including substitution, deletion and insertion. We re-engineered the additional internal deletion with or without the original C-terminal deletion back into wild-type APRRS and found that the internal domain spanning the inter-genotypic variable residues 39–42 ( 39KGP 42G) and conserved residues 48–52 ( 48KNPE 52K), respectively, were dispensable for type 2 PRRSV viability. These results demonstrated that N protein contains non-essential regions for virus viability in cell culture. Such dispensable regions could be utilized as insertion site for foreign tag expression and the rescued viruses could be the candidates for marker vaccine.

Superpositioning of Deletions Promotes Growth of Escherichia coli with a Reduced Genome

Escherichia coli has dispensable genome regions and eliminating them may improve cell use by reducing unnecessary metabolic pathways and complex regulatory networks. Although several strains with reduced genomes have already been constructed, there have been no reports of strains constructed with deletions assayed for influence on growth. To retain robust growth and fundamental metabolic pathways, the growth of each deletion strain and combination effects of deletions were checked using M9 minimal medium. Then a new strain, MGF-01, with a 1 Mb reduced genome was constructed by integrating deletions that did not affect growth. MGF-01 grew as well as the wild type in the exponential phase and continued growing after the wild type had entered the stationary phase. The final cell density of MGF-01 was 1.5 times higher than that of the wild-type strain. Using MGF-01 as a production host, a 2.4-fold increase in l-threonine production was achieved.

The inherent property of polyhydroxyalkanoate synthase to form spherical PHA granules at the cell poles: The core region is required for polar localization

Only the PHA synthase is required for formation of spherical intracellular PHA granules emerging at cell poles. This study aims to assign the polar targeting signal in the PHA synthase and to provide insight into molecular mechanisms of granule formation. Random in-frame insertion mutagenesis indicated dispensable and essential regions suggesting that only the N terminus (<100 aa) is dispensable and forms a random coil structure. The inactive PHA synthase (C319A) is still localized to cell poles, indicating that the nascent PHA chain does not serve as an anchor or signal for subcellular localization and granule formation. Deletion of the N terminus did neither affect subcellular localization nor PHA granule formation. The deletion of the hydrophobic C terminus (68 aa) did not impact on subcellular localization of the PHA synthase, but abolished PHA synthase activity. The structural protein PhaP1 was found to be not required for subcellular localization and initiation of granule formation. PhaP1 only localizes to the cell poles, when PHA granules are formed. These data suggested that the PHA synthase itself localizes to the cell poles via its core region (93–521 aa), which is structurally constraint and comprises the polar positional information for self-assembly of PHA granules at the cell poles.

Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis.

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

Phenotypic Screening of Escherichia coli K-12 Tn 5 Insertion Libraries, Using Whole-Genome Oligonucleotide Microarrays

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.