scholarly journals Codon Usage Heterogeneity in the Multipartite Prokaryote Genome: Selection-Based Coding Bias Associated with Gene Location, Expression Level, and Ancestry

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
J. L. López ◽  
M. J. Lozano ◽  
A. Lagares ◽  
M. L. Fabre ◽  
W. O. Draghi ◽  
...  
Keyword(s):  
Codon Usage ◽  
Sinorhizobium Meliloti ◽  
Bacterial Species ◽  
Gene Location ◽  
Translation Efficiency ◽  
Content Type ◽  
Translational Machinery ◽  
New Genes ◽  
A Genome ◽  
Core Genes

ABSTRACT Prokaryotes represent an ancestral lineage in the tree of life and constitute optimal resources for investigating the evolution of genomes in unicellular organisms. Many bacterial species possess multipartite genomes offering opportunities to study functional variations among replicons, how and where new genes integrate into a genome, and how genetic information within a lineage becomes encoded and evolves. To analyze these issues, we focused on the model soil bacterium Sinorhizobium meliloti, which harbors a chromosome, a chromid (pSymB), a megaplasmid (pSymA), and, in many strains, one or more accessory plasmids. The analysis of several genomes, together with 1.4 Mb of accessory plasmid DNA that we purified and sequenced, revealed clearly different functional profiles associated with each genomic entity. pSymA, in particular, exhibited remarkable interstrain variation and a high density of singletons (unique, exclusive genes) featuring functionalities and modal codon usages that were very similar to those of the plasmidome. All this evidence reinforces the idea of a close relationship between pSymA and the plasmidome. Correspondence analyses revealed that adaptation of codon usages to the translational machinery increased from plasmidome to pSymA to pSymB to chromosome, corresponding as such to the ancestry of each replicon in the lineage. We demonstrated that chromosomal core genes gradually adapted to the translational machinery, reminiscent of observations in several bacterial taxa for genes with high expression levels. Such findings indicate a previously undiscovered codon usage adaptation associated with the chromosomal core information that likely operates to improve bacterial fitness. We present a comprehensive model illustrating the central findings described here, discussed in the context of the changes occurring during the evolution of a multipartite prokaryote genome. IMPORTANCE Bacterial genomes usually include many thousands of genes which are expressed with diverse spatial-temporal patterns and intensities. A well-known evidence is that highly expressed genes, such as the ribosomal and other translation-related proteins (RTRPs), have accommodated their codon usage to optimize translation efficiency and accuracy. Using a bioinformatic approach, we identify core-genes sets with different ancestries, and demonstrate that selection processes that optimize codon usage are not restricted to RTRPs but extended at a genome-wide scale. Such findings highlight, for the first time, a previously undiscovered adaptation strategy associated with the chromosomal-core information. Contrasted with the translationally more adapted genes, singletons (i.e., exclusive genes, including those of the plasmidome) appear as the gene pool with the less-ameliorated codon usage in the lineage. A comprehensive summary describing the inter- and intra-replicon heterogeneity of codon usages in a complex prokaryote genome is presented.

scholarly journals Evolutionary responses to codon usage of horizontally transferred genes in Pseudomonas aeruginosa: gene retention, amelioration and compensatory evolution

2021 ◽  
Vol 7 (6) ◽  
Author(s):  
Martijn Callens ◽  
Celine Scornavacca ◽  
Stéphanie Bedhomme
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Codon Usage ◽  
Bacterial Species ◽  
Trna Genes ◽  
Content Type ◽  
Compensatory Evolution ◽  
Synonymous Mutations ◽  
The Core ◽  
Selective Retention ◽  
Core Genes

Prokaryote genome evolution is characterized by the frequent gain of genes through horizontal gene transfer (HGT). For a gene, being horizontally transferred can represent a strong change in its genomic and physiological context. If the codon usage of a transferred gene deviates from that of the receiving organism, the fitness benefits it provides can be reduced due to a mismatch with the expression machinery. Consequently, transferred genes with a deviating codon usage can be selected against or elicit evolutionary responses that enhance their integration, such as gene amelioration and compensatory evolution. Within bacterial species, the extent and relative importance of these different mechanisms has never been considered altogether. In this study, a phylogeny-based method was used to investigate the occurrence of these different evolutionary responses in Pseudomonas aeruginosa . Selection on codon usage of genes acquired through HGT was observed over evolutionary time, with the overall codon usage converging towards that of the core genome. Gene amelioration, through the accumulation of synonymous mutations after HGT, did not seem to systematically affect transferred genes. This pattern therefore seemed to be mainly driven by selective retention of transferred genes with an initial codon usage similar to that of the core genes. Additionally, variation in the copy number of tRNA genes was often associated with the acquisition of genes for which the observed variation could enhance their expression. This provides evidence that compensatory evolution might be an important mechanism for the integration of horizontally transferred genes.

Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis

2021 ◽  
Vol 71 (11) ◽  
Author(s):  
Héléna Cuny ◽  
Clément Offret ◽  
Amine M. Boukerb ◽  
Leila Parizadeh ◽  
Olivier Lesouhaitier ◽  
...  
Keyword(s):  
Type Species ◽  
Bacterial Species ◽  
Acid Methyl Ester ◽  
Phenotypic Traits ◽  
Rrna Gene ◽  
Ostrea Edulis ◽  
Content Type ◽  
Link Type ◽  
A Genome ◽  
Flat Oyster

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA–DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1–2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7–8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas . In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

scholarly journals PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
George C. diCenzo ◽  
Harsh Sharthiya ◽  
Anish Nanda ◽  
Maryam Zamani ◽  
Turlough M. Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Bacterial Species ◽  
Transport Rate ◽  
Phosphate Limitation ◽  
Rapid Adaptation ◽  
Two Component System ◽  
Phosphate Homeostasis ◽  
Component System ◽  
Content Type ◽  
Two Component

ABSTRACT Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating phoU, including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of Sinorhizobium meliloti that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that phoU cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, phoU deletions were readily recovered in phosphate-free media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Site-directed mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import. IMPORTANCE Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the Sinorhizobium meliloti PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.

scholarly journals Relaxed Selection Drives a Noisy Noncoding Transcriptome in Members of the Mycobacterium tuberculosis Complex

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Adam M. Dinan ◽  
Pin Tong ◽  
Amanda J. Lohan ◽  
Kevin M. Conlon ◽  
Aleksandra A. Miranda-CasoLuengo ◽  
...  
Keyword(s):  
Related Species ◽  
Noncoding Rna ◽  
Bacterial Species ◽  
Population History ◽  
Broad Host Range ◽  
Potential Contribution ◽  
Relaxed Selection ◽  
Content Type ◽  
A Genome ◽  
The Impact

ABSTRACT Related species are often used to understand the molecular underpinning of virulence through examination of a shared set of biological features attributable to a core genome of orthologous genes. An important but insufficiently studied issue, however, is the extent to which the regulatory architectures are similarly conserved. A small number of studies have compared the primary transcriptomes of different bacterial species, but few have compared closely related species with clearly divergent evolutionary histories. We addressed the impact of differing modes of evolution within the genus Mycobacterium through comparison of the primary transcriptome of M. marinum with that of a closely related lineage, M. bovis. Both are thought to have evolved from an ancestral generalist species, with M. bovis and other members of the M. tuberculosis complex having subsequently undergone downsizing of their genomes during the transition to obligate pathogenicity. M. marinum, in contrast, has retained a large genome, appropriate for an environmental organism, and is a broad-host-range pathogen. We also examined changes over a shorter evolutionary time period through comparison of the primary transcriptome of M. bovis with that of another member of the M. tuberculosis complex (M. tuberculosis) which possesses an almost identical genome but maintains a distinct host preference. IMPORTANCE Our comparison of the transcriptional start site (TSS) maps of M. marinum and M. bovis uncovers a pillar of conserved promoters, noncoding RNA (NCRNA), and a genome-wide signal in the −35 promoter regions of both species. We identify evolutionarily conserved transcriptional attenuation and highlight its potential contribution to multidrug resistance mediated through the transcriptional regulator whiB7. We show that a species population history is reflected in its transcriptome and posit relaxed selection as the main driver of an abundance of canonical −10 promoter sites in M. bovis relative to M. marinum. It appears that transcriptome composition in mycobacteria is driven primarily by the availability of such sites and that their frequencies diverge significantly across the mycobacterial clade. Finally, through comparison of M. bovis and M. tuberculosis, we illustrate that single nucleotide polymorphism (SNP)-driven promoter differences likely underpin many of the transcriptional differences between M. tuberculosis complex lineages.

scholarly journals Sinorhizobium meliloti, a Slow-Growing Bacterium, Exhibits Growth Rate Dependence of Cell Size under Nutrient Limitation

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Xiongfeng Dai ◽  
Zichu Shen ◽  
Yiheng Wang ◽  
Manlu Zhu
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Cell Size ◽  
Sinorhizobium Meliloti ◽  
Cell Cycle Progression ◽  
Bacterial Species ◽  
Progression Rate ◽  
Content Type ◽  
E Coli ◽  
Slow Growing

ABSTRACTBacterial cells need to coordinate the cell cycle with biomass growth to maintain cell size homeostasis. For fast-growing bacterial species likeEscherichia coliandBacillus subtilis, it is well-known that cell size exhibits a strong dependence on the growth rate under different nutrient conditions (known as the nutrient growth law). However, cell size changes little with slow growth (doubling time of >90 min) forE. coli, posing the interesting question of whether slow-growing bacteria species also observe the nutrient growth law. Here, we quantitatively characterize the cell size and cell cycle parameter of a slow-growing bacterium,Sinorhizobium meliloti, at different nutrient conditions. We find thatS. melilotiexhibits a threefold change in its cell size when its doubling time varies from 2 h to 6 h. Moreover, the progression rate of its cell cycle is much longer than that ofE. coli, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate. Our study shows that the nutrient growth law holds robustly regardless of the growth capacity of the bacterial species, generalizing its applicability among the bacterial kingdom.IMPORTANCEThe dependence of cell size on growth rate is a fundamental principle in the field of bacterial cell size regulation. Previous studies of cell size regulation mainly focus on fast-growing bacterial species such asEscherichia coliandBacillussubtilis. We find here thatSinorhizobium meliloti, a slow-growing bacterium, exhibits a remarkable growth rate-dependent cell size pattern under nutrient limitation, generalizing the applicability of the empirical nutrient growth law of cell size. Moreover,S. melilotiexhibits a much slower speed of cell cycle progression thanE. colidoes, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate.

scholarly journals Species Designations Belie Phenotypic and Genotypic Heterogeneity in Oral Streptococci

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Irina M. Velsko ◽  
Brinta Chakraborty ◽  
Marcelle M. Nascimento ◽  
Robert A. Burne ◽  
Vincent P. Richards
Keyword(s):  
Genetic Basis ◽  
Genome Wide Association Study ◽  
Bacterial Species ◽  
Arginine Deiminase ◽  
Poor Correlation ◽  
Oral Streptococci ◽  
Single Strain ◽  
Content Type ◽  
A Genome ◽  
Probiotic Strains

ABSTRACTHealth-associated oralStreptococcusspecies are promising probiotic candidates to protect against dental caries. Ammonia production through the arginine deiminase system (ADS), which can increase the pH of oral biofilms, and direct antagonism of caries-associated bacterial species are desirable properties for oral probiotic strains. ADS and antagonistic activities can vary dramatically among individuals, but the genetic basis for these differences is unknown. We sequenced whole genomes of a diverse set of clinical oralStreptococcusisolates and examined the genetic basis of variability in ADS and antagonistic activities. A total of 113 isolates were included and represented 10 species:Streptococcus australis, A12-like,S. cristatus,S. gordonii,S. intermedius,S. mitis,S. oralisincludingS. oralissubsp.dentisani,S. parasanguinis,S. salivarius, andS. sanguinis. Mean ADS activity and antagonism onStreptococcus mutansUA159 were measured for each isolate, and each isolate was whole genome shotgun sequenced on an Illumina MiSeq. Phylogenies were built of genes known to be involved in ADS activity and antagonism. Several approaches to correlate the pan-genome with phenotypes were performed. Phylogenies of genes previously identified in ADS activity and antagonism grouped isolates by species, but not by phenotype. A genome-wide association study (GWAS) identified additional genes potentially involved in ADS activity or antagonism across all the isolates we sequenced as well as within several species. Phenotypic heterogeneity in oral streptococci is not necessarily reflected by genotype and is not species specific. Probiotic strains must be carefully selected based on characterization of each strain and not based on inclusion within a certain species.IMPORTANCERepresentative type strains are commonly used to characterize bacterial species, yet species are phenotypically and genotypically heterogeneous. Conclusions about strain physiology and activity based on a single strain therefore may be inappropriate and misleading. When selecting strains for probiotic use, the assumption that all strains within a species share the same desired probiotic characteristics may result in selection of a strain that lacks the desired traits, and therefore makes a minimally effective or ineffective probiotic. Health-associated oral streptococci are promising candidates for anticaries probiotics, but strains need to be carefully selected based on observed phenotypes. We characterized the genotypes and anticaries phenotypes of strains from 10 species of oral streptococci and demonstrate poor correlation between genotype and phenotype across all species.

scholarly journals Identification of a Membrane-Bound Transcriptional Regulator That Links Chitin and Natural Competence in Vibrio cholerae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Ankur B. Dalia ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Vibrio Cholerae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Active Form ◽  
Transcriptional Regulator ◽  
Natural Competence ◽  
Exogenous Dna ◽  
Content Type ◽  
New Genes ◽  
Membrane Bound

ABSTRACTVibrio choleraeis naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhancestfoXtranslation. We show that this regulation of thetfoRpromoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system inEscherichia coli. This transcriptional regulator was recently identified independently and was named “TfoS” (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence inV. choleraein the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous hostE. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at thetfoRpromoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence inV. cholerae.IMPORTANCENaturally competent bacteria can take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. This ability to take up exogenous DNA is shared by diverse bacterial species and serves as a mechanism to acquire new genes to enhance the fitness of the organism. Several members of the familyVibrionaceaebecome naturally competent when grown on chitin; however, a molecular understanding of how chitin activates competence is lacking. Here, we identify a novel membrane-bound transcriptional regulator that is required for natural transformation in the human pathogenVibrio cholerae. We demonstrate that this regulator senses chitin oligosaccharides to activate the competence cascade, thus, uncovering the molecular link between chitin and natural competence in thisVibriospecies.

scholarly journals The non-neutral, multi-level, phenotypic impact of synonymous mutations revealed through heterologous gene expression in human cells.

2022 ◽  
Author(s):  
Marion A. L. Picard ◽  
Fiona Leblay ◽  
Cecile Cassan ◽  
Mathilde Decourcelle ◽  
Anouk Willemsen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antibiotic Resistance ◽  
Codon Usage ◽  
Heterologous Gene Expression ◽  
Human Cells ◽  
Heterologous Gene ◽  
Hek293 Cells ◽  
Translation Efficiency ◽  
Cellular Phenotype ◽  
A Genome

Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between mRNA molecules carrying synonymous polymorphisms, with potential phenotypic impact at the molecular and at the organismal level. A combination of neutral and selective processes determines the global genome codon usage preferences, as well as local differences between genes within a genome and between positions along a single gene. The relative contribution of evolutionary forces at shaping codon usage bias in eukaryotes is a matter of debate, especially in mammals. The main riddle remains understanding the sharp contrast between the strong molecular impact of gene expression differences arising from codon usage preferences and the thin evidence for codon usage selection at the organismal level. Here we report a multiscale analysis of the consequences of alternative codon usage on heterologous gene expression in human cells. We generated synonymous versions of the shble antibiotic resistance gene, fused to a fluorescent reporter, and expressed independently them in human HEK293 cells. We analysed: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single cell-level construct expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cellular fitness. Our results show that differences in codon usage preferences in our focal gene strongly impacted the molecular and the cellular phenotype: i) they elicited large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduced splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity as different multimodal distributions of cellular fluorescence EGFP; iv) they resulted in a trade-off between burden of heterologous expression and antibiotic resistance. While certain codon usage-related variables monotonically correlated with protein expression, other variables (e.g. CpG content or mRNA folding energy) displayed a bell-like behaviour. We interpret that codon usage preferences strongly shape the molecular and cellular phenotype in human cells through a direct impact on gene expression.

scholarly journals ADAR1 A-to-I RNA editing alters codon usage

2018 ◽  
Author(s):  
Pavla Brachova ◽  
Nehemiah S. Alvarez ◽  
Xiaoman Hong ◽  
Kailey A. Vincent ◽  
Keith E. Latham ◽  
...  
Keyword(s):  
Codon Usage ◽  
Rna Editing ◽  
Mammalian Cells ◽  
Germinal Vesicle ◽  
Translation Efficiency ◽  
Nucleotide Substitutions ◽  
Cellular Processes ◽  
Wobble Position ◽  
A Genome ◽  
Transcriptional Gene Regulation

AbstractBackgroundFully grown mammalian oocytes and eggs are transcriptionally quiescent, and therefore have a unique RNA environment in which cellular processes depend on post-transcriptional regulation. RNA editing of adenosines into inosines (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a common post-transcriptional gene regulatory mechanism, yet it has not been systematically studied in oocytes.ResultsA genome-wide RNA editing analysis of transcriptionally active growing oocytes from postnatal day 12 (PND12) mice, fully grown germinal vesicle (GV) oocytes, and transcriptionally quiescent metaphase II (MII) eggs indicates an abundant amount of A-to-I editing of mRNA transcripts. Editing of mRNA was greatest in GV oocyte and MII eggs compared to the PND12 immature oocytes, this was consistent with ADAR1 levels within these cells. Compared to somatic tissues, oocytes exhibited a different pattern of RNA editing, with a high proportion of RNA edits occurring in the coding regions. These edits resulted in nucleotide substitutions that were enriched at the third nucleotide of the codon (wobble position). Codon usage can affect mRNA stability and translation efficiency.ConclusionsRNA editing in mouse oocytes is distinct from RNA editing in somatic cells due to increased frequencies of coding sequence RNA edits. We provide evidence in support of a previously unreported phenomenon of selective ADAR1 editing of the codon wobble position. Editing of the wobble position has the potential to fine tune post-transcriptional gene regulation through altering codon usage. This important observation advances our current understanding of RNA editing in mammalian cells.

scholarly journals Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium

2012 ◽  
Vol 78 (9) ◽  
pp. 3098-3107 ◽  
Author(s):  
Anita Khatiwara ◽  
Tieshan Jiang ◽  
Sam-Sun Sung ◽  
Turki Dawoud ◽  
Jeong Nam Kim ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Bacterial Species ◽  
Growth Conditions ◽  
Luria Bertani ◽  
Mutant Library ◽  
Content Type ◽  
A Genome ◽  
Gene Functions ◽  
New Gene

ABSTRACTAs more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturatingSalmonella entericaserotype Typhimurium mutant library recovered from selection under 3 differentin vitrogrowth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes inS. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD,glnL,recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes inSalmonellaand the other approximately 70 bacterial species for which EZ:Tn5mutagenesis has been established.

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