Screening for putative essential genes in Brucella abortus strains A19 and S2308 using transposon mutagenesis

Abstract

Brucella is a zoonotic pathogen that causes substantial economic losses in the livestock industry. However, the categorization of functional genes in Brucella requires further refinement. This knowledge gap was addressed by classifying gene essentiality through constructing a transposon mutant library. Mariner transposon mutant libraries were independently constructed for Brucella abortus A19 and S2308. Transposon sequencing was used to visualize the insertion sites within the mutant library, which revealed a uniform transposon distribution in both genomes and high library saturation. Furthermore, 451 and 393 putative essential genes were identified for B. abortus A19 and S2308, respectively. The functional enrichment analysis of these genes revealed that cofactor biosynthesis was the most highly represented, followed by ribosomal biogenesis, carbon metabolism, amino acid biosynthesis, and oxidative phosphorylation. Many of these genes encode known antimicrobial drug targets. This study provides a foundational resource for Brucella research, and the mutant library serves as a tool for screening functional Brucella genes.

Graphical abstract

Keywords

Brucella / Transposon mutant library / Transposon sequencing (Tn-seq) / Essential genes

1 INTRODUCTION

Brucella belongs to the phylum Proteobacteria and is a facultative intracellular parasite with a Gram-negative coccobacillus morphology (Corbel 1997). Brucella lacks endospores, capsules, and flagella. Brucellosis is caused by Brucella and is thus one of the most important zoonoses worldwide (Hasanjani Roushan and Ebrahimpour 2015). Brucellosis causes abortion and fever, posing serious threats to human and animal health. Brucella is classified into six species and 19 biovars based on pathogenicity and biochemical characteristics, among others (Abdallah et al. 2003). Brucella melitensis, Brucella abortus, and Brucella suis are the most prevalent and pathogenic (Alakavuklar et al. 2023). B. abortus A19 (vaccine strain) and S2308 (virulent wild-type strain) were selected for comparative analysis.

The persistent global burden of brucellosis underscores critical gaps in our understanding of Brucella biology. Decades of research have yet to fully elucidate the genetic requirements underlying bacterial survival and pathogenesis — knowledge essential for developing novel therapeutic interventions. Essential genes, defined as those indispensables for growth and survival under specific conditions, represent particularly attractive drug targets as their disruption proves lethal to the pathogen. Systematic identification of these genes therefore provides dual benefits: advancing fundamental understanding of bacterial physiology while revealing vulnerabilities for therapeutic exploitation.

Essential genes are genes that are crucial for the growth and survival of organisms under specific environmental conditions. The methods of identifying essential genes include single-gene knockout, RNA interference, CRISPR/Cas9, and transposon mutagenesis (Hayes 2003). In this study, we constructed a transposon mutant library for screening in B. abortus strains A19 and S2308.

A transposon is a discrete DNA segment capable of expressing its transposase. A transposon enzyme recognizes the terminal inverted repeats (TIRs) flanking the transposon, mediates transposon excision, and facilitates transposon transposition within the genome (Kleckner et al. 1975). Transposons can be transferred from a donor to a recipient genome via insertion between specific base pairs and are heritable genetic elements (Craig 1997). Transposons have been progressively used for screening the genes of prokaryotic and eukaryotic organisms since the initial discovery of transposons in the maize genome in the 1940s and the construction of the synthetic Tc1/Mariner-type transposon Sleeping Beauty in the late 20th century (van Opijnen and Camilli 2013).

Different transposon types exhibit distinct insertion site preferences: the Mariner transposon randomly inserts into thymine (T)–adenine (A) dinucleotide sites (Cain et al. 2020). Mu and Tn5 transposons exhibit a preference for inserting into DNA sequences containing guanine (G) and cytosine (C). The Tn7 insertion pattern is more random, showing a slight preference for A- and T-rich sequences (Green et al. 2012). The PiggyBac transposon is active in mammalian cells and randomly inserts at TTAA tetranucleotide junctions (Ding et al. 2005).

Highly random transposons are typically selected in molecular biology experiments for constructing highly saturated mutant libraries, ensuring the reliability of the subsequent analyses (Gray et al. 2015). Tn5 or Mariner transposons are commonly used for constructing bacterial transposon mutant libraries (Guo et al. 2013). Antibiotics are selected, and each bacterium within the mutant library typically harbors only a single transposon insertion site, enabling tens of thousands of mutant clones to be generated. We used the Mariner transposon to construct a saturated mutant library of B. abortus A19 and S2308.

Transposon insertion sites must be precisely mapped after constructing a random transposon mutant library. Transposon insertion sequencing (TIS) was developed for this task. TIS is a methodology that combines all transposon insertion mutations with high-throughput sequencing to assess the role and essentiality of each gene within the bacterial genome (Barquist et al. 2013). TIS enables the direct sequencing of transposon-flanking regions, simultaneously allowing individual insertion sites to be precisely identified and genomic regions devoid of insertions to be identified. Most or all nonessential genes are expected to harbor transposon insertions in a saturated mutant library; genes lacking insertions are highly likely to be essential. The library can be subjected to selective growth conditions, such as nutrient limitation; the library can be sequenced before and after selective pressure to determine the relative abundance of each mutant. This process facilitates the analysis of gene–phenotype associations under specific conditions (Chao et al. 2016).

Transposon sequencing (Tn-seq) is a specific implementation of TIS (van Opijnen and Camilli 2013). Tn-seq is commonly used for sequencing bacterial mutant libraries constructed using Mariner or Tn5 transposons. The type IIS restriction enzyme Mmel was originally used in Tn-seq to digest extracted genomic DNA from the mutant library (Morgan et al. 2009). However, this approach generated heterogeneous fragment sizes, some of which could be lost during the subsequent purification or PCR steps, biasing the sequencing results. The current commercial Tn-seq protocols typically involve the use of random fragmentation to shear genomic DNA into 200–500 bp fragments, mitigating the impact of size heterogeneity (Friedrich et al. 2017). The Tn-seq leverages massively parallel sequencing, providing highly reproducible and sensitive results. Tn-seq approach allows the contributions of gene fitness contributions in specific environments to be identified without relying on preconstructed knockout arrays (van Opijnen et al. 2009).

We employed a Mariner transposon system delivered via bacterial conjugation to construct mutant libraries of B. abortus strains A19 and S2308. The subsequent Tn-seq analysis enabled the genome-wide classification of gene essentiality and the preliminary characterization of essential genes.

2 RESULTS

2.1 Construction and sequencing analysis of mutant library

The results of three independent conjugation experiments showed that comprehensive transposon insertion libraries were successfully constructed, yielding 84,000 and 64,000 random mutant colonies of B. abortus strains A19 and S2308, respectively. Representative aliquots from each library were subjected to high-throughput sequencing analysis following standard quality control procedures. Sequencing generated 17,801,501 and 16,098,379 high-quality reads for the B. abortus A19 and S2308 libraries, respectively, with Q30 scores of 85.2% and 85.0%, respectively. This result confirmed that both libraries met the established quality standards for downstream analysis.

The B. abortus A19 genome contains 59,925 TA dinucleotide sites within the annotated open reading frames that are potential transposon insertion targets. The results of comprehensive sequencing analysis identified successful transposon insertions at 41,975 TA sites, covering 70.04% of the insertions across the genome. This high insertion frequency demonstrated that the mutant library was nearly saturated, providing robust coverage for determining essentiality (Xiao et al. 2023). Similarly, the B. abortus S2308 genome harbored 59,789 TA dinucleotide sites within open reading frames, with 37,444 confirmed insertion events detected through sequencing analysis, confirming that this library was also adequately saturated for reliably assessing gene essentiality.

The results of the statistical analysis of the TA site insertion frequencies revealed distinct bimodal distribution characteristics in the saturated transposon libraries. Genes with low and high insertion rates in the B. abortus A19 library formed well-separated, nonoverlapping populations at opposite extremes of the distribution spectrum. The saturation characteristics of the B. abortus S2308 library were broadly similar to those of the A19 library, albeit at a somewhat lower level. This difference is likely attributable to the smaller size of the S2308 library (Fig. 1). The genes with consistently low insertion frequencies (<20%) were predominantly essential for bacterial viability, whereas genes tolerating high insertion rates (>80%) were classified as nonessential for growth under standard laboratory conditions (Yang et al. 2017).

The insertion site data were comprehensively visualized using TRANSIT software (version 3.0.1), generating circular genome maps that simultaneously displayed read counts per insertion site, essential gene annotations, nonessential gene classifications, and genes of undetermined essentiality (Fig. 2). These results demonstrated the absence of insertion bias across nonessential genomic regions and indicated the markedly reduced or absent insertion densities within essential gene loci, confirming the reliability of the gene essentiality classifications.

The genes were quantitatively categorized using Gumbel statistical analysis, identifying 451, 2428, 82, and 211 essential genes, nonessential genes, genes of ambiguous essentiality, and genes with insufficient sequences for reliable analysis, respectively, in B. abortus A19. This analysis of B. abortus S2308 revealed 393 essential genes, 2466 nonessential genes, 98 ambiguous genes, and 212 genes with inadequate sequence lengths for classification (Fig. 3). The distribution of the essential genes across the bipartite Brucella genome showed pronounced chromosomal bias, with 398 of the 451 essential genes (88.2%) localized on chromosome I (Chr1) and 53 genes (11.8%) on chromosome II (Chr2) in B. abortus A19. B. abortus S2308 similarly displayed comparable distribution patterns, with 347 of the 393 essential genes (88.3%) residing on Chr1 and 46 genes (11.7%) residing on Chr2.

A detailed examination of the transposon insertion patterns within the putative essential genes revealed varying degrees of insertion tolerance. A total of 287 of the 451 (63.6%) candidate essential genes in B. abortus A19 contained zero transposon insertions, 150 (33.3%) harbored fewer than five insertions, and 14 (3.1%) contained more than five insertion events. Comparable patterns were observed in B. abortus S2308, where insertions were completely absent in 253 genes (64.4%), 131 genes (33.3%) contained fewer than five insertions, and 9 genes (2.3%) harbored more than five insertion events. The persistence of transposon insertions within certain essential genes likely reflects insertions occurring proximal to stop codons that do not disrupt critical gene function, which is consistent with the established principles of transposon mutagenesis.

2.2 Functions of essential genes

The functional enrichment of the putative essential genes identified in B. abortus strains A19 and S2308 through Gumbel analysis was comprehensively analyzed using the clusterProfiler package (version 4.14.6). The results revealed that these genes are predominantly associated with five core biological processes: cofactor biosynthesis, ribosomal biogenesis, carbon metabolism, amino acid biosynthesis, and oxidative phosphorylation (Tables 1–5). The enrichment profiles of B. abortus A19 and S2308 were similar, indicating conserved essential gene functions between these strains (Fig. 4).

2.3 Cofactor biosynthesis and metabolic enzyme functions

Cofactor biosynthesis was the most enriched functional category, reflecting its fundamental role in bacterial viability. Cofactors are essential structural components that bind inactive apoptoenzymes to form catalytically active holoenzymes, enabling critical physiological processes. These molecules directly regulate bacterial proliferation through modulating enzymatic activity, metabolic flux, and redox homeostasis (Sun et al. 2023). Cofactors can be functionally categorized into three distinct types based on their chemical structures and catalytic roles: catalysts, carriers, and substrates (Wang et al. 2019).

Dephospho-coenzyme A (CoA) kinase is an example of catalytic cofactor biosynthesis among the essential genes identified. Dephospho-CoA kinase enzyme catalyzes the ATP-dependent phosphorylation of dephospho-CoA to generate CoA, which represents the final step in CoA biosynthesis. This biosynthetic enzyme is indispensable for bacterial growth, given the extensive participation of CoA in diverse metabolic pathways, being highly conserved across prokaryotic and eukaryotic organisms (Shimosaka et al. 2019).

The essential gene library revealed numerous additional enzymes involved in cofactor metabolism and energy transduction, such as NAD+ kinase, NAD+ synthase, dihydropteroate synthase, 5-aminolevulinate synthase, nicotinate phosphoribosyltransferase, and serine hydroxymethyltransferase. These enzymes have various implications for bioengineering applications and antimicrobial target development. The overexpression of NAD+ synthase and nicotinate phosphoribosyltransferase in industrial strains in the bioengineering context enhances intracellular NAD(H) and NADP(H) concentrations, increasing the productivity of bacterial transformation systems (Jaroensuk et al. 2024). Dihydropteroate synthase is the primary molecular target of sulfonamide antibiotics. Dihydropteroate synthase catalyzes the condensation of p-aminobenzoic acid and dihydropteridine pyrophosphate to form dihydropteroate, the immediate precursor in tetrahydrofolate synthesis (Bermingham and Derrick 2002). Sulfonamides exert antimicrobial effects through competitively inhibiting dihydropteroate synthase, effectively disrupting folate biosynthesis in pathogenic bacteria (Duysak et al. 2023). However, the specific function of dihydropteroate synthase in B. abortus has not been validated.

2.4 Core cellular processes and carbon metabolism

Ribosomal biogenesis represents another major functional category that was enriched among the essential genes, reflecting the fundamental need to synthesize protein for bacterial viability. The identified essential proteins were exclusively involved in the assembly and maturation of 30S and 50S ribosomal subunits, underscoring the critical necessity of the translational machinery for cellular function.

Carbon metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway, provide the metabolic foundation required for bacterial energy production and biosynthetic precursor generation. The key enzymes identified in these interconnected pathways included transketolase from the pentose phosphate pathway; glucose-6-phosphate isomerase and triosephosphate isomerase from glycolysis; and citrate synthase, malate dehydrogenase, and NADP+-dependent isocitrate dehydrogenase from the tricarboxylic acid cycle. Additionally, essential genes involved in purine nucleotide biosynthesis, including ribose-phosphate pyrophosphokinase, and various enzymes involved in one-carbon metabolism were identified as critical for maintaining cellular nucleotide pools.

2.5 Amino acid biosynthesis and oxidative phosphorylation

The essential gene repertoire comprises numerous enzymes that are indispensable for amino acid biosynthesis, highlighting the need for de novo amino acid production under standard growth conditions. An example is chorismate mutase, a pivotal enzyme involved in aromatic amino acid biosynthesis that catalyzes the Claisen rearrangement of chorismate to prephenate, the immediate precursor of phenylalanine and tyrosine synthesis (van Neer et al. 2025). This enzyme participates in the shikimate pathway and is a potential target for the development of antibacterial inhibitors against pathogenic mycobacteria (Russ et al. 2020).

Most of the identified essential genes within the oxidative phosphorylation pathway encode the structural and catalytic subunits of the NADH dehydrogenase complex and ATP synthase. This reflects the fundamental need for aerobic energy production in Brucella species. These findings emphasize the critical dependence of B. abortus on oxidative metabolism for maintaining cellular energy homeostasis.

These five functional categories collectively encompassed most of the enrichment signals observed in the analysis. The representation of the other metabolic and biosynthetic pathways was substantially lower than that of the essential gene sets. This distribution pattern indicates that B. abortus essentiality is primarily driven by the core metabolic processes, protein synthesis machinery, and fundamental biosynthetic capabilities required for bacterial survival under laboratory culture conditions.

3 DISCUSSION

Tn-seq technology has been applied for screening virulence genes, growth factors, and drug resistance targets. We constructed a whole-genome mutant library for B. abortus strains A19 and S2308. The insertion site saturation exceeded 60% in both mutant libraries, indicating the high quality of the mutant library and the reliability of the subsequent results. The incubation time on the plates during library construction was strictly limited to 72 h to prevent sufficient growth of insertion mutants with growth defects at detrimental sites.

Although ribosomal protein genes are evolutionarily conserved, insertions were observed in rplQ, rpsH, rplT, and rplS within the B. abortus A19 library, which probably did not abolish normal function. Insertions were similarly observed in rpsA, rplS, and rplQ in the B. abortus S2308 library. A study on a B. melitensis NI mutant library also documented transposon insertions in the ribosomal protein genes rpsB and rpsI without affecting gene function, suggesting that certain ribosomal protein genes possess a degree of tolerance to transposon insertion (Cloeckaert et al. 2002; De et al. 2017).

Several of the potentially essential genes among those identified in the two libraries represented important drug targets, whereas others were associated with drug resistance or virulence in other pathogens. For example, dihydropteroate synthase is a key target for sulfonamide drugs (Venkatesan et al. 2023). MurC is a potential target for curcumin derivatives in Salmonella typhi (Debroy and Ramaiah 2022). FabF is a target for acyl sulfonamides in chlamydia (Mojica et al. 2017).

Several genes involved in lipopolysaccharide biosynthesis were identified in the two libraries. For instance, LptE in Pseudomonas aeruginosa promotes LptD maturation and stability (Lo Sciuto et al. 2018). LptE depletion not only reduces the virulence of P. aeruginosa but also causes severe membrane permeability defects, leading to a marked increase in susceptibility to Rifampin. Moreover, the susceptibility to Tetracycline and β-lactam antibiotics increased after LptE depletion (Shen et al. 2019). LptE is thus an essential gene in P. aeruginosa (Bolla et al. 2011). Lipopolysaccharide is a crucial antigen in Brucella that plays a key role in its pathogenicity. LptE may play a similar role in Brucella based on sequence homology analysis (Conde-Álvarez et al. 2013).

We constructed random mutant libraries of the B. abortus strains A19 and S2308. We used Tn-seq analysis to identify 451 and 393 putative essential genes required for survival on TSA plates in B. abortus strains A19 and S2308, respectively. Under different stress conditions or during host-pathogen interactions, the essentiality of certain genes may change, transitioning from non-essential to essential. These resources provide a foundation for further studying the essential genes governing Brucella survival in vitro and in vivo, as well as for developing novel vaccine candidates and drug targets against brucellosis.

4 MATERIALS AND METHODS

4.1 Bacterial strains, culture conditions, and plasmids

B. abortus strains A19 and S2308 were obtained from cryogenic laboratory storage and maintained in tryptic soy broth (TSB) or tryptic soy agar (Difco, BD, Franklin Lakes, NJ, USA) at 37°C under orbital shaking at 180 r/min. Transformants were selected using kanamycin sulfate (50 μg/mL). Escherichia coli strains were cultivated in Luria-Bertani medium supplemented with strain-specific antibiotics. Auxotrophic strain WM3064 required additional supplementation with 2,6-diaminopimelic acid (50 μg/mL; Sigma-Aldrich). Kanamycin (25 μg/mL) was used for selection. All bacterial cultures were preserved at −80°C in culture medium containing 35% (v/v) glycerol.

4.2 Constructing B. abortus A19 and S2308 mutant library

Comprehensive transposon insertion libraries were separately constructed for B. abortus strains A19 and S2308 using the Mariner himar1C9 transposase system delivered via the suicide vector pSam_Cam. Conjugative transfer was performed using E. coli WM3064 as the donor organism.

The donor strain harboring pSam_Mariner was cultured for 8 h in Luria-Bertani medium supplemented with kanamycin (25 μg/mL) and 2,6-diaminopimelic acid (50 μg/mL). The recipient B. abortus strain (A19 or S2308) was independently cultured in TSB medium for 48 h at 37°C to construct each library. Both bacterial cultures were harvested via centrifugation and washed with TSB. The resulting cell suspensions were collected into separate 50 mL centrifuge tubes.

Donor E. coli cells were separately mixed for conjugative mating with recipient B. abortus A19 or S2308 cells in a 3:1 volume ratio in 1.5 mL microcentrifuge tubes and transferred onto TSA plates, which were overlaid with nitrocellulose membranes. The mating plates were incubated at 37°C for 12 h. The bacteria were washed from the nitrocellulose membranes following conjugation using TSB, adjusted to a final volume of 20 mL, and induced with isopropyl β-D-1-thiogalactopyranoside at a final concentration of 1 mmol/L from a 1 mol/L stock solution under shaking conditions (180 r/min, 37°C) for 3 h.

Postinduction cells were pelleted via centrifugation (5000 r/min, 5 min), resuspended in fresh TSB to terminate isopropyl β-D-1-thiogalactopyranoside activity, and plated onto TSA containing kanamycin sulfate (50 μg/mL) using sterile glass beads to ensure uniform distribution. The transposon-positive colonies from each strain after 72 h of incubation at 37°C were separately processed to preserve the libraries. All colonies were harvested into 50 mL centrifuge tubes using TSB. A 15-mL aliquot was heat-inactivated (90°C, 20 min) for each library for the subsequent genomic sequencing analysis (Jiang et al. 2025). The remaining cell suspension was mixed with an equal volume of sterile 50% (v/v) glycerol, aliquoted into 2 mL cryovials, and archived at −80°C for long-term storage (Fig. 5).

4.3 Mutant library screening and characterization

The genomic DNA was extracted from the inactivated samples using a TIANamp Bacterial DNA Kit, and its quality was assessed. The DNA was randomly fragmented into 200–500 bp segments, which was followed by end repair and A-tailing. Adaptors were ligated to the repaired ends, and target fragments were enriched through primary PCR amplification using the transposon-specific primer R1 and adapter universal primer Tn-F. The amplified products were purified using 1X magnetic beads. Secondary PCR amplification was then performed using the P5 universal primer and the P7 index primer, followed by another 1X magnetic bead purification to obtain the final sequencing library. The quality of the resulting library was verified. The libraries were pooled after quality control according to the effective concentration and sequencing depth requirements for Illumina PE150 sequencing (Hangzhou Watson Biotechnologies). Sequence reads were filtered using the TPP tool within the TRANSIT software package based on primer-specific criteria to retain qualified sequences (Shen et al. 2016). The filtered reads were aligned against the reference genome sequence to map the precise insertion sites. The gene annotation files were converted to a TRANSIT-compatible format. Gene essentiality was determined using Gumbel analysis in TRANSIT, yielding separate essential gene calls based on the Gumbel (E) and binomial (EB) statistical models (DeJesus et al. 2015).

4.4 Putative essential gene enrichment analysis

After the transit Gumbel analysis, genes classified as “E” or “EB” were considered essential genes in the sample. KEGG enrichment analysis was performed on the identified essential genes using the clusterProfiler package (v4.14.6). The results from the enrichment analysis were visualized using the “dotplot” function.

References

[1] Abdallah AI, Commander NJ, Woodward MJ, Spencer S, Hart CA, Winstanley C (2003) Type III secretion homologs are present in Brucella melitensis, B. ovis, and B. suis biovars 1, 2, and 3. Curr Microbiol 46(4): 241–245

[2] Alakavuklar MA , Fiebig A , Crosson S . The Brucella cell envelope. Annu Rev Microbiol, 2023, 77: 233–253

[3] Barquist L , Boinett CJ , Cain AK . Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol, 2013, 10(7): 1161–1169

[4] Bermingham A , Derrick JP . The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. BioEssays, 2002, 24(7): 637–648

[5] Bolla J-M , Alibert-Franco S , Handzlik J , Chevalier J , Mahamoud A , Boyer G , Kieć-Kononowicz K , Pagès J-M . Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria. FEBS Lett, 2011, 585(11): 1682–1690

[6] Cain AK , Barquist L , Goodman AL , Paulsen IT , Parkhill J , van Opijnen T . A decade of advances in transposon-insertion sequencing. Nat Rev Genet, 2020, 21(9): 526–540

[7] Chao MC , Abel S , Davis BM , Waldor MK . The design and analysis of transposon insertion sequencing experiments. Nat Rev Microbiol, 2016, 14(2): 119–128

[8] Cloeckaert A, Grayon M, Grépinet O (2002) Identification of Brucella melitensis vaccine strain Rev. 1 by PCR-RFLP based on a mutation in the rpsL gene. Vaccine 20(19–20): 2546–2550

[9] Conde-Álvarez R , Arce-Gorvel V , Gil-Ramírez Y , Iriarte M , Grilló M-J , Gorvel JP , Moriyón I . Lipopolysaccharide as a target for brucellosis vaccine design. Microb Pathog, 2013, 58: 29–34

[10] Corbel MJ . Brucellosis: an overview. Emerg Infect Dis, 1997, 3(2): 213–221

[11] Craig NL . Target site selection in transposition. Annu Rev Biochem, 1997, 66: 437–474

[12] De Y , Dong C , Cao Y , Wang X , Yang X , Wang N , Zhang C , Wang Z , Lyu Y , Wu Q . Genome-wide sequence transposon insertion sites and analyze the essential genes of Brucella melitensis. Microb Pathog, 2017, 112: 97–102

[13] Debroy R , Ramaiah S . MurC ligase of multi-drug resistant Salmonella Typhi can be inhibited by novel Curcumin derivative: evidence from molecular docking and dynamics simulations. Int J Biochem Cell Biol, 2022, 151: 106279

[14] DeJesus MA , Ambadipudi C , Baker R , Sassetti C , Ioerger TR . TRANSIT-a software tool for Himar1 TnSeq analysis. PLoS Comput Biol, 2015, 11(10): e1004401

[15] Ding S , Wu X , Li G , Han M , Zhuang Y , Xu T . Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell, 2005, 122(3): 473–483

[16] Duysak T , Jeong J-H , Kim K , Kim J-S , Choy HE . Analysis of random mutations in Salmonella Gallinarum dihydropteroate synthase conferring sulfonamide resistance. Arch Microbiol, 2023, 205(12): 363

[17] Friedrich MJ , Rad L , Bronner IF , Strong A , Wang W , Weber J , Mayho M , Ponstingl H , Engleitner T , Grove C , Pfaus A , Saur D , Cadiñanos J , Quail MA , Vassiliou GS , Liu P , Bradley A , Rad R . Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice. Nat Protoc, 2017, 12(2): 289–309

[18] Gray AN , Koo B-M , Shiver AL , Peters JM , Osadnik H , Gross CA . High-throughput bacterial functional genomics in the sequencing era. Curr Opin Microbiol, 2015, 27: 86–95

[19] Green B , Bouchier C , Fairhead C , Craig NL , Cormack BP . Insertion site preference of Mu, Tn5, and Tn7 transposons. Mob DNA, 2012, 3(1): 3

[20] Guo Y , Park JM , Cui B , Humes E , Gangadharan S , Hung S , FitzGerald PC , Hoe K-L , Grewal SIS , Craig NL , Levin HL . Integration profiling of gene function with dense maps of transposon integration. Genetics, 2013, 195(2): 599–609

[21] Hasanjani Roushan MR , Ebrahimpour S . Human brucellosis: an overview. Caspian J Intern Med, 2015, 6(1): 46–47

[22] Hayes F . Transposon-based strategies for microbial functional genomics and proteomics. Annu Rev Genet, 2003, 37: 3–29

[23] Jaroensuk J , Sutthaphirom C , Phonbuppha J , Chinantuya W , Kesornpun C , Akeratchatapan N , Kittipanukul N , Phatinuwat K , Atichartpongkul S , Fuangthong M , Pongtharangkul T , Hollmann F , Chaiyen P . A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways. J Biol Chem, 2024, 300(2): 105598

[24] Jiang S , Li H , Zhang L , Mu W , Zhang Y , Chen T , Wu J , Tang H , Zheng S , Liu Y , Wu Y , Luo X , Xie Y , Ren J . Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic Acids Res, 2025, 53(D1): D1670–D1676

[25] Kleckner N , Chan RK , Tye B-K , Botstein D . Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition. J Mol Biol, 1975, 97(4): 561–575

[26] Lo Sciuto A , Martorana AM , Fernández-Piñar R , Mancone C , Polissi A , Imperi F . Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence. Virulence, 2018, 9(1): 1718–1733

[27] Mojica SA , Salin O , Bastidas RJ , Sunduru N , Hedenstrom M , Andersson CD , Núñez-Otero C , Engström P , Valdivia RH , Elofsson M , Gylfe Å . N-acylated derivatives of sulfamethoxazole block Chlamydia fatty acid synthesis and interact with FabF. Antimicrob Agents Chemother, 2017, 61(10): e00716-17

[28] Morgan RD , Dwinell EA , Bhatia TK , Lang EM , Luyten YA . The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection. Nucleic Acids Res, 2009, 37(15): 5208–5221

[29] Russ WP , Figliuzzi M , Stocker C , Barrat-Charlaix P , Socolich M , Kast P , Hilvert D , Monasson R , Cocco S , Weigt M , Ranganathan R . An evolution-based model for designing chorismate mutase enzymes. Science, 2020, 369(6502): 440–445

[30] Shen W , Le S , Li Y , Hu F . SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS One, 2016, 11(10): e0163962

[31] Shen X , Johnson NV , Kreamer NNK , Barnes SW , Walker JR , Woods AL , Six DA , Dean CR . Defects in efflux (oprM), β-lactamase (ampC), and lipopolysaccharide transport (lptE) genes mediate antibiotic hypersusceptibility of Pseudomonas aeruginosa strain Z61. Antimicrob Agents Chemother, 2019, 63(7): e00784-19

[32] Shimosaka T , Makarova KS , Koonin EV , Atomi H . Identification of dephospho-coenzyme A (dephospho-CoA) kinase in Thermococcus kodakarensis and elucidation of the entire CoA biosynthesis pathway in archaea. mBio, 2019, 10(4): e01146-19

[33] Sun Y , Zhang T , Lu B , Li X , Jiang L . Application of cofactors in the regulation of microbial metabolism: a state of the art review. Front Microbiol, 2023, 14: 1145784

[34] van Neer RHP , Dranchak PK , Aitha M , Liu L , Carlson EK , Jacobsen IE , Battaile K , Fang Y , Tao D , Rai G , Padia J , Lovell S , Suga H , Inglese J . Active- and allosteric-site cyclic peptide inhibitors of secreted M. tuberculosis chorismate mutase. ACS Infect Dis, 2025, 11(3): 703–714

[35] van Opijnen T , Bodi KL , Camilli A . Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods, 2009, 6(10): 767–772

[36] van Opijnen T , Camilli A . Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol, 2013, 11(7): 435–442

[37] Venkatesan M , Fruci M , Verellen LA , Skarina T , Mesa N , Flick R , Pham C , Mahadevan R , Stogios PJ , Savchenko A . Molecular mechanism of plasmid-borne resistance to sulfonamide antibiotics. Nat Commun, 2023, 14(1): 4031

[38] Wang D , Li D , Zhang G , Wang C , Wei G . Disruption of por1 gene in Candida utilis improves co-production of S-adenosylmethionine and glutathione. J Biotechnol, 2019, 290: 16–23

[39] Xiao J, Sun S, Liu Z, Fan C, Zhu B, Zhang D (2023) Analysis of key genes for the survival of Pantoea agglomerans under nutritional stress. Int J Biol Macromol 253(Pt 5): 127059. https://doi.org/10.1016/j.ijbiomac.2023.127059

[40] Yang G , Billings G , Hubbard TP , Park JS , Yin Leung K , Liu Q , Davis BM , Zhang Y , Wang Q , Waldor MK . Time-resolved transposon insertion sequencing reveals genome-wide fitness dynamics during infection. mBio, 2017, 8(5): e01581-17

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