Transposon Mutagenesis Research Articles

High-throughput transposon mutagenesis in the family Enterobacteriaceae reveals core essential genes and rapid turnover of essentiality.

The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the model organism Escherichia coli, as well as major human pathogens including Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with the Keio E. coli single-gene deletion library often regarded as a gold standard. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of 13 sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first assess several gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations, we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes. Despite the presence of a large cohort of variably essential genes, we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently acquired essentiality multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival and identifies several instances of essential stress responses masked by redundancy in free-living bacteria.IMPORTANCEThe essential genome, that is the set of genes absolutely required to sustain life, is a core concept in genetics. Essential genes in bacteria serve as drug targets, put constraints on the engineering of biological chassis for technological or industrial purposes, and are key to constructing synthetic life. Despite decades of study, relatively little is known about how gene essentiality varies across related bacteria. In this study, we have collected gene essentiality data for 13 bacteria related to the model organism Escherichia coli, including several human pathogens, and investigated the conservation of essentiality. We find that approximately a third of the genes essential in any particular strain are non-essential in another related strain. Surprisingly, we do not find evidence for essential genes unique to specific genera; rather it appears a substantial fraction of the essential genome rapidly gains or loses essentiality during evolution. This suggests that essentiality is not an immutable characteristic but depends crucially on the genomic context. We illustrate this through a comparison of our essential genes in free-living bacteria to genes conserved in 34 insect endosymbionts with naturally reduced genomes, finding several cases where genes generally regarded as being important for specific stress responses appear to have become essential in endosymbionts due to a loss of functional redundancy in the genome.

Metagenomic analysis during capecitabine therapy reveals microbial chemoprotective mechanisms and predicts drug toxicity in colorectal cancer patients.

Unpredictable chemotherapy side effects are a major barrier to successful treatment. Cell culture and mouse experiments indicate that the gut microbiota is influenced by and influences anti-cancer drugs. However, metagenomic data from patients paired to careful side effect monitoring remains limited. Herein, we focus on the oral fluoropyrimidine capecitabine (CAP). We investigate CAP-microbiome interactions through metagenomic sequencing of longitudinal stool sampling from a cohort of advanced colorectal cancer (CRC) patients. We established a prospective cohort study including 56 patients with advanced CRC treated with CAP monotherapy across 4 centers in the Netherlands. Stool samples and clinical questionnaires were collected at baseline, during cycle 3, and post-treatment. Metagenomic sequencing to assess microbial community structure and gene abundance was paired with transposon mutagenesis, targeted gene deletion, and media supplementation experiments. An independent US cohort was used for model validation. CAP treatment significantly altered gut microbial composition and pathway abundance, enriching for menaquinol (vitamin K2) biosynthesis genes. Transposon library screens, targeted gene deletions, and media supplementation confirmed that menaquinol biosynthesis protects Escherichia coli from drug toxicity. Microbial menaquinol biosynthesis genes were associated with decreased peripheral sensory neuropathy. Machine learning models trained in this cohort predicted hand-foot syndrome and dose reductions in an independent cohort. These results suggest treatment-associated increases in microbial vitamin biosynthesis serve a chemoprotective role for bacterial and host cells, with implications for toxicities outside the gastrointestinal tract. We provide a proof-of-concept for the use of microbiome profiling and machine learning to predict drug toxicities across independent cohorts. These observations provide a foundation for future human intervention studies, more in-depth mechanistic dissection in preclinical models, and extension to other cancer treatments.

Burkholderia pseudomallei produces 2-alkylquinolone derivatives important for host virulence and competition with bacteria that employ naphthoquinones for aerobic respiration.

Melioidosis is caused by Burkholderia pseudomallei, an opportunistic Gram-negative pathogen that inhabits soil and water in tropical and subtropical regions. B. pseudomallei infections often occur following contact with contaminated water or soil or by inhalation of contaminated dust and water droplets. There is limited knowledge about how B. pseudomallei is able to survive in harsh environmental conditions and compete with the microbes that inhabit these niches. Previous research demonstrated that 3-methyl-2-alkylquinolones (MAQs), and their corresponding N-oxides (MAQNOs), are produced by B. pseudomallei and provide a competitive advantage when grown in the presence of Gram-positive bacteria. In this study, 39 Gram-negative environmental bacteria in the Pseudomonadota and Bacteroidota phyla were isolated and characterized. Intriguingly, B. pseudomallei inhibited 71% of bacteria in the phylum Bacteroidota in zone of inhibition and coculture competition assays, but no Pseudomonadota isolates were similarly inhibited. Transposon mutagenesis was utilized to identify B. pseudomallei genes required for the inhibition of Sphingobacterium sp. ST4, a representative member of the Bacteroidota. Three mutations mapped to hmqA-G, the locus encoding 2-alkylquinolone derivatives, and two mutations were identified in scmR, a gene encoding a quorum-sensing controlled LysR-type transcriptional regulator. B. pseudomallei strains with deletion mutations in hmqD and scmR were unable to produce 2-alkylquinolone derivatives or inhibit Bacteroidota isolates in competition assays. RAW264.7 murine macrophage cells were infected with B. pseudomallei 1026b and 1026b ΔhmqD and there was a 94-fold reduction in the number of intracellular 1026b ΔhmqD bacteria relative to 1026b. The 50% lethal dose (LD50) of 1026b and 1026b ΔhmqD in BALB/c mice was determined to be 3 x 105 colony forming units (CFU) and > 1 x 106 CFU, respectively. Taken together, the results indicate that the products of the B. pseudomallei hmqA-G locus are important for intracellular replication in murine macrophages, virulence in a mouse model of melioidosis, and competition with bacteria that utilize naphthoquinones for aerobic respiration.

A highly conserved SusCD transporter determines the import and species-specific antagonism of Bacteroides ubiquitin homologues

Efficient interbacterial competitions and diverse defensive strategies employed by various bacteria play a crucial role in acquiring a hold within a dense microbial community. The gut symbiont Bacteroides fragilis secretes an antimicrobial ubiquitin homologue (BfUbb) that targets an essential periplasmic PPIase to drive intraspecies bacterial competition. However, the mechanisms by which BfUbb enters the periplasm and its potential for interspecies antagonism remain poorly understood. Here, we employ transposon mutagenesis and identify a highly conserved TonB-dependent transporter SusCD (designated as ButCD) in B. fragilis as the BfUbb transporter. As a putative protein-related nutrient utilization system, ButCD is widely distributed across diverse Bacteroides species with varying sequence similarity, resulting in distinct import efficiency of Bacteroides ubiquitin homologues (BUbb) and thereby determining the species-specific toxicity of BUbb. Cryo-EM structural and functional investigations of the BfUbb–ButCD complex uncover distinctive structural features of ButC that are crucial for its targeting by BfUbb. Animal studies further demonstrate the specific and efficient elimination of enterotoxigenic B. fragilis (ETBF) in the murine gut by BfUbb, suggesting its potential as a therapeutic against ETBF-associated inflammatory bowel disease and colorectal cancer. Our findings provide a comprehensive elucidation of the species-specific toxicity exhibited by BUbb and explore its potential applications.

L-asparaginase-driven antibiosis in Pseudomonas fluorescens EK007: A promising biocontrol strategy against fire blight

Fire blight, caused by Erwinia amylovora, is a destructive bacterial disease affecting pear and apple trees. The biocontrol ability of Pseudomonas fluorescens EK007 suppresses E. amylovora through competitive exclusion. In this study, EK007 was isolated from the pear phylloplane and characterized as an effective biological agent through antibacterial compounds. To identify the mechanisms underlying EK007's biocontrol activity, physiological tests, transposon insertion mutant libraries, allelic exchange, and whole-genome sequencing were performed. A transposon mutation in the massC homolog gene, part of the massetolide A lipopeptide biosynthesis cluster, reduced the biocontrol efficiency. Allelic exchange confirmed cyclic lipopeptide (CLP) as part of the mechanism. Additionally, a gacA mutant isolated by transposon mutagenesis showed deficient inhibition activity. Culture conditions and nutritional sources clearly influenced EK007's antimicrobial activity against E. amylovora. Growth yield generally correlated with antibiotic production, with amino acids and iron affecting production. Asparagine and aspartate shut down biocontrol activity. This study presents preliminary findings on a novel CLP that may contribute to EK007's antibacterial activity against E. amylovora. While EK007 shows promise as a biocontrol candidate compared to related strains, these results are based solely on in vitro studies, highlighting the need for further investigations to evaluate its efficacy in natural environments.

TML1 AND TML2 SYNERGISTICALLY REGULATE NODULATION AND AFFECT ARBUSCULAR MYCORRHIZA IN MEDICAGO TRUNCATULA.

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant's need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of Nodulation (AON) and Autoregulation of Mycorrhization (AOM) both negatively regulate their respective processes and share multiple components - plants that make too many nodules usually have higher AM fungal root colonization. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON in Lotus japonicus . M. truncatula has two sequence homologs: Mt TML1 and Mt TML2. We report the generation of stable single and double mutants harboring multiple allelic variations in MtTML1 and MtTML2 using CRISPR-Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations in Mt TML1 or Mt TML2 produced 2-3 times the nodules of wild-type plants whereas plants containing mutations in both genes displayed a synergistic effect, forming 20x more nodules compared to wild type plants. Examination of expression and heterozygote effects suggest genetic compensation may play a role in the observed synergy. Plants with mutations in both TMLs only showed mild increases in AM fungal root colonization at later timepoints in our experiments, suggesting these genes may also play a minor role in AM symbiosis regulation. The mutants created will be useful tools to dissect the mechanism of synergistic action of Mt TML1 and Mt TML2 in M. truncatula symbiosis with beneficial microbes.

Uncovering Candidate Pathogenicity Genes in Erwinia pyrifoliae YKB12327 via Tn5-insertion Mutagenesis

<i>Erwinia pyrifoliae</i> is a gram-negative bacterial pathogen that commonly causes black shoot blight in pear and apple tree. Although the pathogenicity of this bacterial species is very similar to <i>E. amylovora</i>, there is no specific explanation of its pathogenic genes and mechanisms. In this study, our investigation into <i>E. pyrifoliae</i> pathogenicity involved generating seven YKB12327 mutant strains using Tn5 transposon mutagenesis. Observations revealed weakened growth rate and loss of pathogenicity in these mutants. Whole-genome sequencing and alignment analysis identified transposon insertions within the coding sequences of five strains and in the intergenic region of two strains. Annotation analysis elucidated genes directly or indirectly associated with pathogenicity. Notably, mutant strain MT16 displayed a transposon insertion mutation in the cyclic-di-GMP phosphodiesterase (<i>pdeF</i>) gene, a key player in bacterial signaling, governing microbial behavior and adaptation to environmental changes. Our findings provide insights into the genetic regulation of <i>E. pyrifoliae</i> pathogenicity, suggesting potential avenues for further research aimed at understanding and controlling this bacterial pathogen by targeting <i>pdeF</i> to mitigate apple black shoot blight disease.

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an invivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with α-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

The role of cydB gene in the biofilm formation by Campylobacter jejuni.

Campylobacter jejuni is a major cause of food- and water-borne bacterial infections in humans. A key factor helping bacteria to survive adverse environmental conditions is biofilm formation ability. Nonetheless, the molecular basis underlying biofilm formation by C. jejuni remains poorly understood. Around thirty genes involved in the regulation and dynamics of C. jejuni biofilm formation have been described so far. We applied random transposon mutagenesis to identify new biofilm-associated genes in C. jejuni strain 81-176. Of 1350 mutants, twenty-four had a decreased ability to produce biofilm compared to the wild-type strain. Some mutants contained insertions in genes previously reported to affect the biofilm formation process. The majority of identified genes encoded hypothetical proteins. In the library of EZ-Tn5 insertion mutants, we found the cydB gene associated with respiration that was not previously linked with biofilm formation in Campylobacter. To study the involvement of the cydB gene in biofilm formation, we constructed a non-marked deletion cydB mutant together with a complemented mutant. We found that the cydB deletion-mutant formed a weaker biofilm of loosely organized structure and lower volume than the parent strain. In the present study, we demonstrated the role of the cydB gene in biofilm formation by C. jejuni.

The mntH gene of Burkholderia cenocepacia influences motility and quorum sensing to control virulence.

Quorum sensing (QS) signals widely exist in bacteria to control biological functions in response to populations of cells. Burkholderia cenocepacia, an important opportunistic pathogen in patients with cystic fibrosis (CF), is commonly found in the environment and mostly utilizes the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) QS systems to control biological functions. Our previous study illuminated the detailed mechanism by which B.cenocepacia integrates BDSF and cyclic diguanosine monophosphate (c-di-GMP) signals to control virulence. Here, we employed Tn5 transposon mutagenesis to identify genes related to the BDSF QS system. One of the most significantly attenuated mutants had an insertion in the mntH gene. Here, we showed that MntH (Bcam0836), a manganese transport protein, controls QS-regulated phenotypes, including motility, biofilm formation and virulence. We also found that. BDSF production was attenuated at both the gene and signaling levels in the Bcam0836 mutant, and that Bcam0836 influenced the expression of some genes regulated by the BDSF receptor RpfR and the downstream regulator GtrR. These results show that the manganese transport protein. MntH modulates a subset of genes and functions regulated by the QS system in B. cenocepacia.

Double-Wing Motif Protein is a Novel Biofilm Regulatory Factor of the Plant Disease Biocontrol Agent, Bacillus subtilis.

Transposon mutagenesis screening of Bacillus subtilis YB-1471, a novel rhizosphere biocontrol agent of Fusarium crown rot (FCR) of wheat, resulted in the identification of orf04391, linked to reduced biofilm formation. The gene encodes a protein possessing a putative tertiary structure of a "double-wing" DNA-binding domain. Expression of orf04391 increased during biofilm development in stationary cultures and during rapid growth in shaking cultures. An orf04391 deletion strain showed reduced biofilm production related to lower levels of the extracellular matrix, and the mutant also had reduced sporulation, adhesion, root colonization, and FCR biocontrol efficiency. Transcriptome analysis of YB-1471 and Δorf04391 in stationary culture showed that the loss of orf04391 resulted in altered expression of numerous genes, including sinI, an initiator of biofilm formation. DNA binding was shown with his-tagged Orf04391 binding to the sinIR operon in vivo and in vitro. Orf04391 appears to be a transcriptional regulator of biofilm formation in B. subtilis through the Spo0A-SinI/SinR pathway.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus.

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

The Generation of an ocular anti-inflammatory biotherapeutic to enhance wound healing.

Microbes exist at and colonize mucosal surfaces striking a balance with the host immune system, so that these microbes can thrive on host tissues without causing pathology. Because of this, mucosal barrier-colonizing bacteria can be leveraged to act as long-term delivery vehicles for naturally derived therapeutics. Here, we use a mouse model of corneal wound healing to show that the eye-colonizing bacterium, Corynebacterium mastitidis (C. mast) can be engineered to produce and secrete bioactive murine anti-inflammatory interleukin (mIL)-10. Specifically, we used transposon mutagenesis to identify a native C. mast-specific secretion signal that was used to direct C. mast to secrete mIL-10. Mini-transposons were generated to deliver secretion capable mIL-10 to the bacterial genome. After screening, two isolates were identified that can: 1) colonize the eye, 2) produce and secrete mIL-10, and 3) enhance wound healing in an IL-10-dependent manner. This proof of concept illustrates that eye-colonizing bacteria can be engineered to deliver therapeutics to the ocular surface for the alleviation of ocular surface disease(s).

Lactate promotes the biofilm-to-invasive-planktonic transition in Salmonella enterica serovar Typhimurium via the de novo purine pathway.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection triggers an inflammatory response that changes the concentration of metabolites in the gut impacting the luminal environment. Some of these environmental adjustments are conducive to S. Typhimurium growth, such as the increased concentrations of nitrate and tetrathionate or the reduced levels of Clostridia-produced butyrate. We recently demonstrated that S. Typhimurium can form biofilms within the host environment and respond to nitrate as a signaling molecule, enabling it to transition between sessile and planktonic states. To investigate whether S. Typhimurium utilizes additional metabolites to regulate its behavior, our study delved into the impact of inflammatory metabolites on biofilm formation. The results revealed that lactate, the most prevalent metabolite in the inflammatory environment, impedes biofilm development by reducing intracellular c-di-GMP levels, suppressing the expression of curli and cellulose, and increasing the expression of flagellar genes. A transcriptomic analysis determined that the expression of the de novo purine pathway increases during high lactate conditions, and a transposon mutagenesis genetic screen identified that PurA and PurG, in particular, play a significant role in the inhibition of curli expression and biofilm formation. Lactate also increases the transcription of the type III secretion system genes involved in tissue invasion. Finally, we show that the pyruvate-modulated two-component system BtsSR is activated in the presence of high lactate, which suggests that lactate-derived pyruvate activates BtsSR system after being exported from the cytosol. All these findings propose that lactate is an important inflammatory metabolite used by S. Typhimurium to transition from a biofilm to a motile state and fine-tune its virulence.IMPORTANCEWhen colonizing the gut, Salmonella enterica serovar Typhimurium (S. Typhimurium) adopts a dynamic lifestyle that alternates between a virulent planktonic state and a multicellular biofilm state. The coexistence of biofilm formers and planktonic S. Typhimurium in the gut suggests the presence of regulatory mechanisms that control planktonic-to-sessile transition. The signals triggering the transition of S. Typhimurium between these two lifestyles are not fully explored. In this work, we demonstrated that in the presence of lactate, the most dominant host-derived metabolite in the inflamed gut, there is a reduction of c-di-GMP in S. Typhimurium, which subsequently inhibits biofilm formation and induces the expression of its invasion machinery, motility genes, and de novo purine metabolic pathway genes. Furthermore, high levels of lactate activate the BtsSR two-component system. Collectively, this work presents new insights toward the comprehension of host metabolism and gut microenvironment roles in the regulation of S. Typhimurium biology during infection.

Saturation transposon mutagenesis enables genome-wide identification of genes required for growth and fluconazole resistance in the human fungal pathogen Cryptococcus neoformans.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the Cryptococcus neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that 5' insertions of transposons can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

EfpA is required for regrowth of Mycobacterium tuberculosis following isoniazid exposure.

Efflux of antibiotics is an important survival strategy in bacteria. Mycobacterium tuberculosis has approximately sixty efflux pumps, but little is known about the role of each pump or the substrates they efflux. The putative efflux pump, EfpA, is a member of the major facilitator superfamily and has been shown to be essential by saturation transposon mutagenesis studies. It has been implicated in the efflux of isoniazid (INH), which is a first-line drug used to treat tuberculosis (TB). This is supported by evidence from transcriptional profiling showing that efpA is induced in response to INH exposure. However, its roles in the physiology and adaptation of M. tuberculosis to antibiotics have yet to be determined. In this study, we describe the repression of efpA in M. tuberculosis, using CRISPR interference (CRISPRi) to knockdown the expression of this essential gene and the direct effect of this on the ability of M. tuberculosis to survive exposure to INH over a 45-day time course. We determined that wild-type levels of efpA were required for recovery of M. tuberculosis following INH exposure and that, after 45 days of INH exposure, only a few viable colonies were recoverable from efpA-repressed M. tuberculosis. We conclude that EfpA is required for recovery of M. tuberculosis following INH exposure, which could reduce the efficacy of INH in vivo, and that EfpA may have a role in the development of resistance during drug therapy.

Interruption of mycothiol synthesis and intracellular redox status impact iron-regulated reporter activation in Mycobacterium smegmatis.

Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The Mycobacterium tuberculosis iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The ideR gene is essential in M. tuberculosis and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and loxP mediated recombination in Mycobacterium smegmatis. Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene mshA results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mshA mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCEMycobacterium smegmatis is a tractable organism to study mycobacterial gene regulation. We used M. smegmatis to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene mshA is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by Mycobacterium tuberculosis to overcome temporary or local changes in iron availability during infection.

Multiple regulatory inputs including cell envelope stress orchestrate expression of the Escherichia coli rpoN operon.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ54, hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σE also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality.