A decade of advances in transposon-insertion sequencing.

It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.

Portrait of a killer: Uncovering resistance mechanisms and global spread of Acinetobacter baumannii.

Antibiotic resistance is a growing global concern in the field of medicine as it renders bacterial infections difficult to treat and often more severe. Acinetobacter baumannii is a gram-negative bacterial pathogen causing a wide range of infections, including pneumonia, sepsis, urinary tract infections, and wound infections. A. baumannii has emerged as a significant healthcare-associated pathogen due to its high level of antibiotic resistance. The global spread of antibiotic-resistant strains of A. baumannii has resulted in limited treatment options, leading to increased morbidity and mortality rates, especially in vulnerable populations such as the elderly and immunocompromised individuals, as well as longer hospital stays and higher healthcare costs. Further complicating the situation, multi- and pan-drug-resistant strains of A. baumannii are becoming increasingly common, and these deadly strains are resistant to all or almost all available antibiotics. A. baumannii employs various clever strategies to develop antibiotic resistance, including horizontal transfer of resistance genes, overexpression of inherent efflux pumps that remove drugs from the cell, intrinsic mutations, combined with natural selection under antibiotic selective pressure leading to emergence of successful resistance clones. The typical multidrug resistance phenotype of A. baumannii is, therefore, an orchestrated collimation of all these mechanisms combined with the worldwide spread of "global clones," rendering infections caused by this pathogen challenging to control and treat. To address the escalating problem of antibiotic resistance in A. baumannii, there is a need for increased surveillance, strict infection control measures, and the development of new treatment strategies, requiring a concerted effort by healthcare professionals, researchers, and policymakers.

DksA is a conserved master regulator of stress response in Acinetobacter baumannii.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

High-precision screening and sorting of double emulsion droplets.

Mounting evidence suggests that cell populations are extremely heterogeneous, with individual cells fulfilling different roles within the population. Flow cytometry (FC) is a high-throughput tool for single-cell analysis that works at high optical resolution. Sub-populations with unique properties can be screened, isolated and sorted through fluorescence-activated cell sorting (FACS), using intracellular fluorescent products or surface-tagged fluorescent products of interest. However, traditional FC and FACS methods cannot identify or isolate cells that secrete extracellular products of interest. Double emulsion (DE) droplets are an innovative approach to retaining these extracellular products so cells producing them can be identified and isolated with FC and FACS. The water-in-oil-in-water structure makes DE droplets compatible with the sheath flow of flow cytometry. Single cells can be encapsulated with other reagents into DEs, which act as pico-reactors. These droplets allow biological activities to take place while allowing for cell cultivation monitoring, rare mutant identification, and cellular events characterization. However, using DEs in FACS presents technical challenges, including rupture of DEs, poor accuracy and low sorting efficiency. This study presents high-performance sorting using fluorescent beads (as simulants for cells). This study aims to guide researchers in the use of DE-based flow cytometry, offering insights into how to resolve the technical difficulties associated with DE-based screening and sorting using FC.

Microfluidic Separation and Enrichment of Escherichia coli by Size Using Viscoelastic Flows.

Here, we achieve the separation and enrichment of Escherichia coli clusters from its singlets in a viscoelastic microfluidic device. E. coli, an important prokaryotic model organism and a widely used microbial factory, can aggregate in clusters, leading to biofilm development that can be detrimental to human health and industrial processes. The ability to obtain high-purity populations of E. coli clusters is of significance for biological, biomedical, and industrial applications. In this study, polystyrene particles of two different sizes, 1 and 4.8 µm, are used to mimic E. coli singlets and clusters, respectively. Experimental results show that particles migrate toward the channel center in a size-dependent manner, due to the combined effects of inertial and elastic forces; 4.8 and 1 µm particles are found to have lateral equilibrium positions closer to the channel centerline and sidewalls, respectively. The size-dependent separation performance of the microdevice is demonstrated to be affected by three main factors: channel length, the ratio of sheath to sample flow rate, and poly(ethylene oxide) (PEO) concentration. Further, the separation of E. coli singlets and clusters is achieved at the outlets, and the separation efficiency is evaluated in terms of purity and enrichment factor.

The Antimicrobial Properties of PdII - and RuII -pyta Complexes.

Infections associated with antimicrobial resistance (AMR) are poised to become the leading cause of death in the next few decades, a scenario that can be ascribed to two phenomena: antibiotic over-prescription and a lack of antibiotic drug development. The crowd-sourced initiative Community for Open Antimicrobial Drug Discovery (CO-ADD) has been testing research compounds contributed by researchers around the world to find new antimicrobials to combat AMR, and during this campaign has found that metallodrugs might be a promising, yet untapped source. To this end, we submitted 18 PdII - and RuII -pyridyl-1,2,3-triazolyl complexes that were developed as catalysts to assess their antimicrobial properties. It was found that the Pd complexes, especially Pd1, possessed potent antifungal activity with MICs between 0.06 and 0.125 µg mL-1 against Candida glabrata. The in-vitro studies were extended to in-vivo studies in Galleria mellonella larvae, where it was established that the compounds were nontoxic. Here, we effectively demonstrate the potential of PdII -pyta complexes as antifungal agents.

Inhibitors of bacterial RNA polymerase transcription complex.

A series of hybrid compounds that incorporated anthranilic acid with activated 1H-indoles through a glyoxylamide linker were designed to target bacterial RNA polymerase holoenzyme formation using computational docking. Synthesis, in vitro transcription inhibition assays, and biological testing of the hybrids identified a range of potent anti-transcription inhibitors with activity against a range of pathogenic bacteria with MICs as low as 3.1 µM. A structure activity relationship study identified the key structural components necessary for inhibition of both bacterial growth and transcription. Correlation of in vitro transcription inhibition activity with in vivo mechanism of action was established using fluorescence microscopy and resistance passaging using Gram-positive bacteria showed no resistance development over 30 days. Furthermore, no toxicity was observed from the compounds in a wax moth larvae model, establishing a platform for the development of a series of new antibacterial drugs with an established mode of action.

Elucidating Essential Genes in Plant-Associated Pseudomonas protegens Pf-5 Using Transposon Insertion Sequencing.

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacterium that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used for this species. In this study, we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses, and transport were underrepresented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant-associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed that the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico-predicted and in vitro-determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth.IMPORTANCE Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analyses of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of three other pseudomonads highlights how gene essentiality varies between closely related species.

The Molecular Basis of Acinetobacter baumannii Cadmium Toxicity and Resistance.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii, which is notorious for persisting in harsh environments. Here, we show that A. baumannii utilizes a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis.

AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

Nontoxic Cobalt(III) Schiff Base Complexes with Broad-Spectrum Antifungal Activity.

Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug-resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it is reasonable to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad-spectrum antifungal activity. Some of these complexes show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report the first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg kg-1 are tolerated without adverse effects, paving the way for further in vivo studies of these complexes.

O-Antigen-Dependent Colicin Insensitivity of Uropathogenic Escherichia coli.

The outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted by Escherichia coli, can target other E. coli cells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of various E. coli strains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why do E. coli strains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenic E. coli sequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen into E. coli K-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizing E. coli toward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to "last-resort" antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).

Genomic analysis of Klebsiella pneumoniae isolates from Malawi reveals acquisition of multiple ESBL determinants across diverse lineages.

OBJECTIVES: ESBL-producing Klebsiella pneumoniae (KPN) pose a major threat to human health globally. We carried out a WGS study to understand the genetic background of ESBL-producing KPN in Malawi and place them in the context of other global isolates. METHODS: We sequenced genomes of 72 invasive and carriage KPN isolates collected from patients admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi. We performed phylogenetic and population structure analyses on these and previously published genomes from Kenya (n = 66) and from outside sub-Saharan Africa (n = 67). We screened for presence of antimicrobial resistance (AMR) genetic determinants and carried out association analyses by genomic sequence cluster, AMR phenotype and time. RESULTS: Malawian isolates fit within the global population structure of KPN, clustering into the major lineages of KpI, KpII and KpIII. KpI isolates from Malawi were more related to those from Kenya, with both collections exhibiting more clonality than isolates from the rest of the world. We identified multiple ESBL genes, including blaCTX-M-15, several blaSHV, blaTEM-63 and blaOXA-10, and other AMR genes, across diverse lineages of the KPN isolates from Malawi. No carbapenem resistance genes were detected; however, we detected IncFII and IncFIB plasmids that were similar to the carbapenem resistance-associated plasmid pNDM-mar. CONCLUSIONS: There are multiple ESBL genes across diverse KPN lineages in Malawi and plasmids in circulation that are capable of carrying carbapenem resistance. Unless appropriate interventions are rapidly put in place, these may lead to a high burden of locally untreatable infection in vulnerable populations.

Defining the ABC of gene essentiality in streptococci.

BACKGROUND: Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. RESULTS: Six barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species. CONCLUSIONS: The use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

Genomic landscape of extended-spectrum ß-lactamase resistance in Escherichia coli from an urban African setting.

Objectives: Efforts to treat Escherichia coli infections are increasingly being compromised by the rapid, global spread of antimicrobial resistance (AMR). Whilst AMR in E. coli has been extensively investigated in resource-rich settings, in sub-Saharan Africa molecular patterns of AMR are not well described. In this study, we have begun to explore the population structure and molecular determinants of AMR amongst E. coli isolates from Malawi. Methods: Ninety-four E. coli isolates from patients admitted to Queen's Hospital, Malawi, were whole-genome sequenced. The isolates were selected on the basis of diversity of phenotypic resistance profiles and clinical source of isolation (blood, CSF and rectal swab). Sequence data were analysed using comparative genomics and phylogenetics. Results: Our results revealed the presence of five clades, which were strongly associated with E. coli phylogroups A, B1, B2, D and F. We identified 43 multilocus STs, of which ST131 (14.9%) and ST12 (9.6%) were the most common. We identified 25 AMR genes. The most common ESBL gene was bla CTX-M-15 and it was present in all five phylogroups and 11 STs, and most commonly detected in ST391 (4/4 isolates), ST648 (3/3 isolates) and ST131 [3/14 (21.4%) isolates]. Conclusions: This study has revealed a high diversity of lineages associated with AMR, including ESBL and fluoroquinolone resistance, in Malawi. The data highlight the value of longitudinal bacteraemia surveillance coupled with detailed molecular epidemiology in all settings, including low-income settings, in describing the global epidemiology of ESBL resistance.

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

UNLABELLED: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology. AVAILABILITY AND IMPLEMENTATION: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-Tradis CONTACT: parkhill@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aspartate-Based CXCR4 Chemokine Receptor Binding of Cross-Bridged Tetraazamacrocyclic Copper(II) and Zinc(II) Complexes.

The CXCR4 chemokine receptor is implicated in a number of diseases including HIV infection and cancer development and metastasis. Previous studies have demonstrated that configurationally restricted bis-tetraazamacrocyclic metal complexes are high-affinity CXCR4 antagonists. Here, we present the synthesis of Cu(2+) and Zn(2+) acetate complexes of six cross-bridged tetraazamacrocycles to mimic their coordination interaction with the aspartate side chains known to bind them to CXCR4. X-ray crystal structures for three new Cu(2+) acetate complexes and two new Zn(2+) acetate complexes demonstrate metal-ion-dependent differences in the mode of binding the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Concurrent density functional theory molecular modelling studies produced an energetic rationale for the unexpected [Zn(OAc)(H2 O)](+) coordination motif present in all of the Zn(2+) cross-bridged tetraazamacrocycle crystal structures, which differs from the chelating acetate [Zn(OAc)](+) structures of known unbridged and side-bridged tetraazamacrocyclic Zn(2+) -containing CXCR4 antagonists.

Synthesis and antimalarial activity of metal complexes of cross-bridged tetraazamacrocyclic ligands.

Using transition metals such as manganese(II), iron(II), cobalt(II), nickel(II), copper(II), and zinc(II), several new metal complexes of cross-bridged tetraazamacrocyclic chelators namely, cyclen- and cyclam-analogs with benzyl groups, were synthesized and screened for in vitro antimalarial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum. The metal-free chelators tested showed little or no antimalarial activity. All the metal complexes of the dibenzyl cross-bridged cyclam ligand exhibited potent antimalarial activity. The Mn(2+) complex of this ligand was the most potent with IC50s of 0.127 and 0.157µM against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) P. falciparum strains, respectively. In general, the dibenzyl hydrophobic ligands showed better anti-malarial activity compared to the activity of monobenzyl ligands, potentially because of their higher lipophilicity and thus better cell penetration ability. The higher antimalarial activity displayed by the manganese complex for the cyclam ligand in comparison to that of the cyclen, correlates with the larger pocket of cyclam compared to that of cyclen which produces a more stable complex with the Mn(2+). Few of the Cu(2+) and Fe(2+) complexes also showed improvement in activity but Ni(2+), Co(2+) and Zn(2+) complexes did not show any improvement in activity upon the metal-free ligands for anti-malarial development.

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria.

Functional genomics techniques, such as transposon insertion sequencing and RNA-sequencing, are key to studying relative differences in bacterial mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy numbers along the chromosome. This can lead to artifacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control. Further, relative differences in gene-wise read counts may result from alterations in chromosomal replication dynamics, rather than selection or direct gene regulation. We term this artifact "chromosomal location bias" and implement a principled statistical approach to correct it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated into statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analyzing a ciprofloxacin-treated transposon insertion sequencing data set in Escherichia coli as a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress.IMPORTANCEAltered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.

Transposons Carrying the aacC2e Aminoglycoside and blaTEM Beta-Lactam Resistance Genes in Acinetobacter.

This study examines the genetic contexts and evolutionary steps responsible for the formation of the widely spread transposon Tn6925 carrying blaTEM and aacC2e, which confers resistance to beta-lactam and aminoglycoside antibiotics in Gram-negative bacteria. The blaTEM-1 and aacC2e genes were found in several transposons. They were first observed within an IS26 bounded 3.7 kb transposon (Tn6925) on several Acinetobacter baumannii plasmids located within a 4.7 kb dif module. Truncated and expanded variations of Tn6925 were found across other A. baumannii plasmids, as well as in other Gram-negative bacteria (including Vibrio cholerae). Moreover, blaTEM-1 and aacC2e were in much larger resistance-heavy transposons including the ISAba1-bounded 24.6 kb (here called Tn6927), found in an A. baumannii chromosome. A novel ISKpn12-bounded transposon was also observed to contain blaTEM and aacC2e which was found interrupting Tn5393 along with an IS26 pseudo-compound transposon to form a 24.9 kb resistance island in an Acinetobacter pittii plasmid. Multiple mobile genetic elements are involved in the formation of transposon structures that circulate blaTEM and aacC2e. Among these, IS26 and ISAba1 appear to have played a major role in the formation and spread of these elements in the Acinetobacter species.