Transposon Insertion Research Articles

Transposable Elements Contribute to the Regulation of Long Noncoding RNAs in Drosophila melanogaster

Background: Transposable elements (TEs) and noncoding sequences are major components of the genome, yet their functional contributions to long noncoding RNAs (lncRNAs) are not well understood. Although many lncRNAs originating from TEs (TE-lncRNAs) have been identified across various organisms, their characteristics and regulatory roles, particularly in insects, remain largely unexplored. This study integrated multi-omics data to investigate TE-lncRNAs in D. melanogaster, focusing on the influence of transposons across different omics levels. Results: We identified 16,118 transposons overlapping with lncRNA sequences that constitute 2119 TE-lncRNAs (40.4% of all lncRNAs) using 256 public RNA-seq samples and 15 lncRNA-seq samples of Drosophila S2 cells treated with heavy metals. Of these, 67.2% of TE-lncRNAs contain more than one TE. The LTR/Gypsy family was the most common transposon insertion. Transposons preferred to insert into promoters, transcription starting sites, and intronic regions, especially in chromosome ends. Compared with lncRNAs, TE-lncRNAs showed longer lengths, a lower conservation, and lower levels but a higher specificity of expression. Multi-omics data analysis revealed positive correlations between transposon insertions and chromatin openness at the pre-transcriptional level. Notably, a total of 516 TE-lncRNAs provided transcriptional factor binding sites through transposon insertions. The regulatory network of a key transcription factor was rewired by transposons, potentially recruiting other transcription factors to exert regulatory functions under heavy metal stress. Additionally, 99 TE-lncRNAs were associated with m6A methylation modification sites, and 115 TE-lncRNAs potentially provided candidate small open reading frames through transposon insertions. Conclusions: Our data analysis demonstrated that TEs contribute to the regulation of lncRNAs. TEs not only promote the transcriptional regulation of lncRNAs, but also facilitate their post-transcriptional and epigenetic regulation.

Genome-wide identification of root colonization fitness genes in plant growth promoting Pseudomonas asiatica employing transposon-insertion sequencing

BackgroundPseudomonas spp. are well-studied plant growth promoters, particularly in the context of root colonization. However, the specific genetic factors that determine its fitness in the rhizosphere remain largely unexplored. This study breaks new ground by employing transposon insertion sequencing (Tn-Seq) to identify the genetic factors in Pseudomonas asiatica JR11 that are crucial for colonizing corn roots.ResultsWe created a transposon mutant library of P. asiatica JR11 with 91,884 insertion sites and subjected it to three consecutive enrichment cycles within the corn root system. A total of 79 genes were identified as essential for root colonization (negatively-selected), while 22 genes were found to counteract root colonization efficiency (positively-selected), with both sets being commonly present across all three cycles. These genes involve amino acid metabolism, cell wall biosynthesis, and protein functions. Additionally, we found four negatively-selected and four positively-selected hypothetical proteins that consistently influenced root colonization fitness.ConclusionsThe identification of these molecular determinants opens up exciting possibilities for further research. Understanding these pathways could lead to the development of novel strategies for enhancing the fitness of P. asiatica JR11 during corn root colonization, with potential implications for plant growth promotion and agricultural practices.

Menaquinone production in genetically engineered E. coli.

Menaquinone (MK) is an important electron transporter in Escherichia coli. This isoprenoid quinone can transfer electrons to many terminal acceptors, such as fumarate and nitrate, which helps this organism survive under diverse and challenging conditions. As the isoprenoid quinones with various length of isoprenyl tail are widely distributed in nature, the heterologous expression of polyprenyl diphosphate synthases (PDSs) has been investigated using its counterpart, ubiquinone (UQ). In this study, we investigated the MK production by the expression of various heterologous PDS genes from prokaryotic and eukaryotic species, including Saccharomyces cerevisiae COQ1 (hexa-PDS), Haemophilus influenzae hi0881 (hepta-PDS), Synechocystis sp. strain PCC6803 slr0611 (nona-PDS) and Glunocobacter suboxydans ddsA (deca-PDS) in E. coli. We detected specific isoforms of MK, including MK7, MK9, and MK10, via the expression of HI0881, Slr0611 and DdsA respectively, but rarely detect MK6 via the expression of Coq1. As UQ6 was detected in E. coli harboring COQ1, the acceptance of the side chain lengths by MenA (prenyl transferase for MK) was narrower than UbiA (prenyl transferase for UQ). We also identified a mutation in menA in the E. coli AN386 strain and a transposon insertion of IS186 in menC in E. coli KO229 (∆ispB) and its parental strain FS1576. Taken together, these results elucidate the different nature of MenA from UbiA.

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

Pseudomonas aeruginosa faces a fitness trade-off between mucosal colonization and antibiotic tolerance during airway infection.

Pseudomonas aeruginosa frequently causes antibiotic-recalcitrant pneumonia, but the mechanisms driving its adaptation during human infections remain unclear. To reveal the selective pressures and adaptation strategies at the mucosal surface, here we investigated P. aeruginosa growth and antibiotic tolerance in tissue-engineered airways by transposon insertion sequencing (Tn-seq). Metabolic modelling based on Tn-seq data revealed the nutritional requirements for P. aeruginosa growth, highlighting reliance on glucose and lactate and varying requirements for amino acid biosynthesis. Tn-seq also revealed selection against biofilm formation during mucosal growth in the absence of antibiotics. Live imaging in engineered organoids showed that biofilm-dwelling cells remained sessile while colonizing the mucosal surface, limiting nutrient foraging and reduced growth. Conversely, biofilm formation increased antibiotic tolerance at the mucosal surface. Moreover, mutants with exacerbated biofilm phenotypes protected less tolerant but more cytotoxic strains, contributing to phenotypic heterogeneity. P. aeruginosa must therefore navigate conflicting physical and biological selective pressures to establish chronic infections.

The evolutionary tale of lilies: Giant genomes derived from transposon insertions and polyploidization

The evolutionary tale of lilies: Giant genomes derived from transposon insertions and polyploidization

Phenotypic characterization and genetic mapping of the semi-dwarf mutant sdw9 in maize.

In summary, we characterized a maize semi-dwarf mutant, sdw9, and successfully isolated the responsible gene, which encodes a GRAS protein, through a combination of map-based cloning and Re-sequencing (Re-seq). Our findings demonstrate that the candidate gene ZmGRAS42 regulates BR signaling genes, thereby influencing internode development. This regulatory function likely involves processes such as cell division, cell cycle regulation and cell wall synthesis. Favorable variations of ZmGRAS42 identified in this study may hold promise for the development of lodging-resistant maize cultivars suitable for high-density planting, contributing to the improvement of maize breeding programs. Plant height and lateral root angle are crucial determinants of plant architecture in maize (Zea mays) which are closely related to lodging resistance at high planting density. These traits are intricately regulated by various phytohormones. Mutations affecting hormone biosynthesis and signaling often lead to reduced yield alongside diminished plant height, posing challenges in breeding dwarf maize varieties. In this study, the maize mutant sdw9 was characterized, which displays shorter stature and altered lateral root angle compared to WT, while showing potential to increase planting density and improve overall yield despite a slight reduction in single-ear yield. Employing positional cloning coupled with Re-seq techniques, we pinpointed a transposon insertion in the candidate gene ZmGRAS42, which encodes a GRAS transcription factor involved in BR signaling in maize. Transcriptome analysis revealed that ZmGRAS42 orchestrates the expression of several known dwarfing genes such as D8, Br2, and Na2, along with genes associated with cell wall organization, cell division, and cell cycle regulation, notably Cesa4, Cesa7, and Cyc11. Furthermore, identification of favorable ZmGRAS42 haplotypes linked to reduced plant height offers novel avenues for maize breeding strategies. These findings not only hold the potential for enhancing maize lodging resistance but also for optimizing land utilization through high-density planting practices.

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.

Transposable Element (TE) insertion predictions from RNAseq inputs and TE impact on RNA splicing and gene expression in Drosophila brain transcriptomes

Recent studies have suggested that Transposable Elements (TEs) residing in introns frequently splice into and alter primary gene-coding transcripts. To re-examine the exonization frequency of TEs into protein-coding gene transcripts, we re-analyzed a Drosophila neuron circadian rhythm RNAseq dataset and a deep long RNA fly midbrain RNAseq dataset using our Transposon Insertion and Depletion Analyzer (TIDAL) program. Our TIDAL results were able to predict several TE insertions from RNAseq data that were consistent with previous published studies. However, we also uncovered many discrepancies in TE-exonization calls, such as reads that mainly support intron retention of the TE and little support for chimeric mRNA spliced to the TE. We then deployed rigorous genomic DNA-PCR (gDNA-PCR) and RT-PCR procedures on TE-mRNA fusion candidates to see how many of bioinformatics predictions could be validated. By testing a w1118 strain from which the deeper long RNAseq data was derived and comparing to an OreR strain, only 9 of 23 TIDAL candidates (< 40%) could be validated as a novel TE insertion by gDNA-PCR, indicating that deeper study is needed when using RNAseq data as inputs into current TE-insertion prediction programs. Of these validated calls, our RT-PCR results only supported TE-intron retention. Lastly, in the Dscam2 and Bx genes of the w1118 strain that contained intronic TEs, gene expression was 23 times higher than the OreR genes lacking the TEs. This study's validation approach indicates that chimeric TE-mRNAs are infrequent and cautions that more optimization is required in bioinformatics programs to call TE insertions using RNAseq datasets.

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.

Role of R5 Pyocin in the Predominance of High-Risk Pseudomonas aeruginosa Isolates.

Infections with antimicrobial resistant pathogens, such as Pseudomonas aeruginosa, are a frequent occurrence in healthcare settings. Human P. aeruginosa infections are predominantly caused by a small number of sequence types (ST), such as ST235, ST111, and ST175. Although ST111 is recognized as one of the most prevalent high-risk P. aeruginosa clones worldwide and frequently exhibits multidrug-resistant or extensively drug-resistant phenotypes, the basis for this dominance remains unclear. In this study, we used a genome-wide transposon insertion library screen to discover that the competitive advantage of ST111 strains over certain non-ST111 strains is through production of R pyocins. We confirmed this finding by showing that competitive dominance was lost by ST111 mutants with R pyocin gene deletions. Further investigation showed that sensitivity to ST111 R pyocin (specifically R5 pyocin) is caused by deficiency in the O-antigen ligase waaL, which leaves lipopolysaccharide (LPS) bereft of O antigen, enabling pyocins to bind the LPS core. In contrast, sensitivity of waaL mutants to R1 or R2 pyocins depended on additional genomic changes. In addition, we found the PA14 mutants in lipopolysaccharide biosynthesis (waaL, wbpL, wbpM) that cause high susceptibility to R pyocins also exhibit poor swimming motility. Analysis of 5,135 typed P. aeruginosa strains revealed that several international, high-risk sequence types (including ST235, ST111, and ST175) are enriched for R5 pyocin production, indicating a correlation between these phenotypes and suggesting a novel approach for evaluating risk from emerging prevalent P. aeruginosa strains. Overall, our study sheds light on the mechanisms underlying the dominance of ST111 strains and highlighting the role of waaL in extending spectrum of R pyocin susceptibility.

Genomic and phenotypic imprints of microbial domestication on cheese starter cultures

Domestication – the artificial selection of wild species to obtain variants with traits of human interest – was integral to the rise of complex societies. The oversupply of food was probably associated with the formalization of food preservation strategies through microbial fermentation. While considerable literature exists on the antiquity of fermented food, only few eukaryotic microbes have been studied so far for signs of domestication, less is known for bacteria. Here, we tested if cheese starter cultures harbour typical hallmarks of domestication by characterising over 100 community samples and over 100 individual strains isolated from historical and modern traditional Swiss cheese starter cultures. We find that cheese starter cultures have low genetic diversity both at the species and strain-level and maintained stable phenotypic traits. Molecular clock dating further suggests that the evolutionary origin of the bacteria approximately coincided with the first archaeological records of cheese making. Finally, we find evidence for ongoing genome decay and pseudogenization via transposon insertion related to a reduction of their niche breadth. Future work documenting the prevalence of these hallmarks across diverse fermented food systems and geographic regions will be key to unveiling the joint history of humanity with fermented food microbes.

Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus

Staphylococcus aureus (S. aureus), particularly Methicillin-resistant S. aureus (MRSA), poses a significant global public health threat, necessitating advanced methodologies to enhance our understanding of this organism at the omics levels. This study introduces a refined protocol for constructing and curing high-density transposon mutant (tn-mutant) libraries in S. aureus, addressing the challenges associated with low transductant yields, and the complex genetic manipulation mechanism in Gram-positive bacteria. Our methodology employs a Himar1 transposon based on a two-plasmid system, leveraging Himar1’s high insertional efficiency in AT-rich organisms. Enhanced transduction efficiency was achieved through chloramphenicol pre-treatment and the use of modified enriched media. Complementing this, an optimized plasmid curing procedure ensured a representative and stable tn-mutant library. The protocol was successfully applied to multiple S. aureus strains, demonstrating an increase in mutant recovery and reduced post-curing impact. The method offers a robust approach for Transposon Insertion Sequencing (TIS) applications in S. aureus, enabling deeper insights into survival, resistance, and pathogenicity mechanisms. This protocol holds a significant potential for accelerating the construction of tn-mutant libraries in various S. aureus strains.

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

&lt;i&gt;Erwinia pyrifoliae&lt;/i&gt; 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 &lt;i&gt;E. amylovora&lt;/i&gt;, there is no specific explanation of its pathogenic genes and mechanisms. In this study, our investigation into &lt;i&gt;E. pyrifoliae&lt;/i&gt; 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 (&lt;i&gt;pdeF&lt;/i&gt;) gene, a key player in bacterial signaling, governing microbial behavior and adaptation to environmental changes. Our findings provide insights into the genetic regulation of &lt;i&gt;E. pyrifoliae&lt;/i&gt; pathogenicity, suggesting potential avenues for further research aimed at understanding and controlling this bacterial pathogen by targeting &lt;i&gt;pdeF&lt;/i&gt; to mitigate apple black shoot blight disease.

Dynamics of drug delivery determines course of evolution of antibiotic responses in bacteria.

To adjust to sudden shifts in conditions, microbes possess regulated genetic mechanisms that sense environmental challenges and induce the appropriate responses. The initial evolution of microbes in new environments is thought to be driven by regulatory mutations, but it is not clear how this evolution is affected by how quickly conditions change (i.e. dynamics). Here, we perform experimental evolution on continuous cultures of tetracycline resistant E. coli in different dynamical regimens of drug administration. We find that cultures evolved under gradually increasing drug concentrations acquire fine- tuning mutations adapting an alternative efflux pump to tetracycline. However, cultures that are instead periodically exposed to large drug doses evolve transposon insertions resulting in loss of regulation of the main mechanism of tetracycline resistance. A mathematical model shows that sudden drug exposures overwhelm regulated responses, which cannot induce resistance fast enough. These results help explain the frequent loss of regulation of resistance in clinical pathogens.

Pneumococcal transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of serotype 19F in Indonesia

Genetic evolution of resistance due to mutations and transposon insertions is the primary cause of antimicrobial resistance in Streptococcus pneumoniae. Resistance to macrolide, tetracycline, and chloramphenicol is caused by the insertion of specific genes that carried by transposon (Tn). This study aims to analyze transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of S. pneumoniae serotype 19F in Indonesia. S. pneumoniae serotype 19F isolates were collected from nasopharyngeal swab specimens from different regions in Indonesia. Genomic DNA was extracted from sixteen isolates and whole genome sequencing was performed on Illumina platform. Raw sequence data were analyzed using de novo assembly by ASA3P and Microscope server. The presence of transposons was identified with detection of int and xis genes and visualized by pyGenomeViz. The genome size of S. pneumoniae ranges from 2,040,117 bp to 2,437,939 bp, with a GC content of around 39 %. ST1464 (4/16) and ST271 (3/16) were found as the predominant sequence type among isolates. Tn2010 was the most common transposon among S. pneumoniae serotype 19F isolates (7/16) followed by Tn2009 (4/16), and Tn5253 (3/16). We identified two deletion sites within the tetM gene (2 bp and 58 bp) that confer tetracycline susceptibility from one isolate. This study suggests that genomic analysis can be employed for the detection and surveillance of antimicrobial resistance genes among S. pneumoniae strains isolated from various regions in Indonesia.

Transposon-mediated genic rearrangements underlie variation in small RNA pathways

Transposable elements (TEs) can alter host gene structure and expression, whereas host organisms develop mechanisms to repress TE activities. In the nematode Caenorhabditis elegans , a small interfering RNA pathway dependent on the helicase ERI-6/7 primarily silences retrotransposons and recent genes of likely viral origin. By studying gene expression variation among wild C. elegans strains, we found that structural variants and transposon remnants likely underlie expression variation in eri-6/7 and the pathway targets. We further found that multiple insertions of the DNA transposons, Polintons, reshuffled the eri-6/7 locus and induced inversion of eri-6 in some wild strains. In the inverted configuration, gene function was previously shown to be repaired by unusual trans-splicing mediated by direct repeats. We identified that these direct repeats originated from terminal inverted repeats of Polintons . Our findings highlight the role of host-transposon interactions in driving rapid host genome diversification among natural populations and shed light on evolutionary novelty in genes and splicing mechanisms.

Isolation, characterization, and genetic manipulation of cold-tolerant, manganese-oxidizing Pseudomonas sp. strains.

Manganese-oxidizing bacteria (MnOB) produce Mn oxide minerals that can be used by humans for bioremediation, but the purpose for the bacterium is less clear. This study describes the isolation and characterization of cold-tolerant MnOB strains isolated from a compost pile in Morris, Minnesota, USA: Pseudomonas sp. MS-1 and DSV-1. The strains were preliminarily identified as members of species Pseudomonas psychrophila by 16S rRNA analysis and a multi-locus phylogenetic study using a database of 88 genomes from the Pseudomonas genus. However, the average nucleotide identity between these strains and the P. psychrophila sp. CF149 type strain was less than 93%. Thus, the two strains are members of a novel species that diverged from P. psychrophila. DSV-1 and MS-1 are cold tolerant; both grow at 4°C but faster at 24°C. Unlike the mesophilic MnOB P. putida GB-1, both strains are capable of robustly oxidizing Mn at low temperatures. Both DSV-1 and MS-1 genomes contain homologs of several Mn oxidation genes found in P. putida GB-1 (mnxG, mcoA, mnxS1, mnxS2, and mnxR). Random mutagenesis by transposon insertion was successfully performed in both strains and identified genes involved in Mn oxidation that were similar to those found in P. putida GB-1. Our results show that MnOB can be isolated from compost, supporting a role for Mn oxidation in plant waste degradation. The novel isolates Pseudomonas spp. DSV-1 and MS-1 both can oxidize Mn at low temperature and likely employ similar mechanisms and regulation as P. putida GB-1.IMPORTANCEBiogenic Mn oxides have high sorptive capacity and are strong oxidants. These two characteristics make these oxides and the microbes that make them attractive tools for the bioremediation of wastewater and contaminated environments. Identifying MnOB that can be used for bioremediation is an active area of research. As cold-tolerant MnOB, Pseudomonas sp. DSV-1 and MS-1 have the potential to expand the environmental conditions in which biogenic Mn oxide bioremediation can be performed. The similarity of these organisms to the well-characterized MnOB P. putida GB-1 and the ability to manipulate their genomes raise the possibility of modifying them to improve their bioremediation ability.

Essential genes for Haemophilus parainfluenzae survival and biofilm growth.

Haemophilus parainfluenzae (Hp) is a Gram-negative, highly prevalent, and abundant commensal in the human oral cavity, and an infrequent extraoral opportunistic pathogen. Hp occupies multiple niches in the oral cavity, including the supragingival plaque biofilm. Little is known about how Hp interacts with its neighbors in healthy biofilms nor its mechanisms of pathogenesis as an opportunistic pathogen. To address this, we identified the essential genome and conditionally essential genes in in vitro biofilms aerobically and anaerobically. Using transposon insertion sequencing (TnSeq) with a highly saturated mariner transposon library in two strains, the ATCC33392 type-strain (Hp 392) and oral isolate EL1 (Hp EL1), we show that the essential genomes of Hp 392 and Hp EL1 are composed of 395 (20%) and 384 (19%) genes, respectively. The core essential genome, consisting of 341 (17%) essential genes conserved between both strains, was composed of genes associated with genetic information processing, carbohydrate, protein, and energy metabolism. We also identified conditionally essential genes for aerobic and anaerobic biofilm growth, which were associated with carbohydrate and energy metabolism in both strains. RNAseq analysis determined that most genes upregulated during anaerobic growth are not essential for Hp 392 anaerobic survival. The completion of this library and analysis under these conditions gives us a foundational insight into the basic biology of H. parainfluenzae in differing oxygen conditions, similar to its in vivo habitat. This library presents a valuable tool for investigation into conditionally essential genes for an organism that lives in close contact with many microbial species in the human oral habitat.IMPORTANCEHaemophilus parainfluenzae is a highly abundant human commensal microbe, present in most healthy individuals where it colonizes the mouth. H. parainfluenzae correlates with good oral health and may play a role in preservation of healthy host status. Also, H. parainfluenzae can cause opportunistic infections outside of the oral cavity. To date, little is known about how H. parainfluenzae colonizes the human host, despite being such a frequent and abundant part of our human microbiome. Here, we demonstrate the creation and use of a powerful tool, a TnSeq library, used to identify genes necessary for both the outright growth of this organism and also genes conditionally essential for growth in varying oxygen status which it can encounter in the human host. This tool and these data serve as a foundation for further study of this relatively unknown organism that may play a role in preserving human health.

Genome-wide fitness analysis of Salmonella enterica reveals aroA mutants are attenuated due to iron restriction in vitro.

Salmonella enterica is a globally disseminated pathogen that is the cause of over 100 million infections per year. The resulting diseases are dependent upon host susceptibility and the infecting serovar. As S. enterica serovar Typhimurium induces a typhoid-like disease in mice, this model has been used extensively to illuminate various aspects of Salmonella infection and host responses. Due to the severity of infection in this model, researchers often use strains of mice resistant to infection or attenuated Salmonella. Despite decades of research, many aspects of Salmonella infection and fundamental biology remain poorly understood. Here, we use a transposon insertion sequencing technique to interrogate the essential genomes of widely used isogenic wild-type and attenuated S. Typhimurium strains. We reveal differential essential pathways between strains in vitro and provide a direct link between iron starvation, DNA synthesis, and bacterial membrane integrity.IMPORTANCESalmonella enterica is an important clinical pathogen that causes a high number of deaths and is increasingly resistant to antibiotics. Importantly, S. enterica is used widely as a model to understand host responses to infection. Understanding how Salmonella survives in vivo is important for the design of new vaccines to combat this pathogen. Live attenuated vaccines have been used clinically for decades. A widely used mutation, aroA, is thought to attenuate Salmonella by restricting the ability of the bacterium to access particular amino acids. Here we show that this mutation limits the ability of Salmonella to acquire iron. These observations have implications for the interpretation of many previous studies and for the use of aroA in vaccine development.