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.

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.

RESULTS

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).

Figure 1.

Histogram showing the percentage of TA loci disrupted per gene in the B. abortus strain A19 (A) and S2308 (B) transposon insertion library. The x-axis shows the percentage of TA sites per gene that contain a transposon insertion in the B. abortus mutant library. The y-axis shows the number of genes corresponding to each proportion bin. E: essential based on Gumbel; EB: essential based on binomial; NE: non-essential; S: too short; U: uncertain

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.

Figure 2.

Circular maps of all transposon insertion sites in B. abortus genomes of strains A19 and S2308. The outer to inner circles are scale marks of the genome (Mb), coding sequences on the forward strand, coding sequences on the reverse strand, and number of inserted reads per locus from the input, expressed in lg10. The colors in the status labels represent the types of genomic features in the innermost circle of the circular map. E: essential based on Gumbel; EB: essential based on binomial; NE: non-essential; S: too short; U: uncertain. A,B Transposon insertion sites on B. abortus A19 chromosomes. C,D Transposon insertion sites on B. abortus S2308 chromosomes

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.

Figure 3.

Quantitative distribution of gene functional categories in the B. abortus A19 (A) and S2308 (B) genomes

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.

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).

Table 1. Genes associated with cofactor biosynthesis identified via KEGG enrichment analysis.

Gene_ID	Gene	Annotation
B. abortus A19
DR186_RS02480	fabG	3-Oxoacyl-[acyl-carrier-protein] reductase
DR186_RS02500	fabF	Beta-ketoacyl-ACP synthase II
DR186_RS03970	hemB	Porphobilinogen synthase
DR186_RS04855	-	NAD kinase
DR186_RS05220	-	NAD+ synthase
DR186_RS05310	folP	Dihydropteroate synthase
DR186_RS05315	folB	Dihydroneopterin aldolase
DR186_RS05320	folK	2-Amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase
DR186_RS05560	folE	GTP cyclohydrolase I FolE
DR186_RS05845	lipA	Lipoyl synthase
DR186_RS05980	fabZ	3-Hydroxyacyl-ACP dehydratase FabZ
DR186_RS07155	-	Dihydrofolate reductase
DR186_RS07600	carA	Glutamine-hydrolyzing carbamoyl-phosphate synthase small subunit
DR186_RS07620	carB	Carbamoyl-phosphate synthase large subunit
DR186_RS07930	hemF	Oxygen-dependent coproporphyrinogen oxidase
DR186_RS08585	-	Adenylosuccinate synthase
DR186_RS08925	-	Thiamine diphosphokinase
DR186_RS09320	-	Nicotinate-nucleotide adenylyltransferase
DR186_RS09495	ubiG	Bifunctional 2-polyprenyl-6-hydroxyphenol methylase/3-demethylubiquinol 3-O-methyltransferase UbiG
DR186_RS09555	hemC	Hydroxymethylbilane synthase
DR186_RS10440	hemJ	Protoporphyrinogen oxidase HemJ
DR186_RS10445	hemE	Uroporphyrinogen decarboxylase
DR186_RS10470	coaE	Dephospho-CoA kinase
DR186_RS10555	coaA	Type I pantothenate kinase
DR186_RS10905	metK	Methionine adenosyltransferase
DR186_RS10965	fabB	Beta-ketoacyl-ACP synthase I
DR186_RS10650	-	Folylpolyglutamate synthase/dihydrofolate synthase family protein
DR186_RS01815	hemA	5-Aminolevulinate synthase
DR186_RS06015	pyrH	UMP kinase
DR186_RS09550	-	Uroporphyrinogen-III synthase
DR186_RS03995	glyA	Serine hydroxymethyltransferase
DR186_RS05705	coaD	Pantetheine-phosphate adenylyltransferase
DR186_RS02275	ubiA	4-hydroxybenzoate octaprenyltransferase
DR186_RS04010	-	Riboflavin synthase
DR186_RS09610	-	Ubiquinone biosynthesis hydroxylase
DR186_RS04815	-	Cysteine desulfurase family protein
DR186_RS00585	pncB	Nicotinate phosphoribosyltransferase
DR186_RS03635	ndk	Nucleoside-diphosphate kinase
DR186_RS05895	-	CTP synthase
DR186_RS12010	-	Bifunctional riboflavin kinase/FAD synthetase
DR186_RS13325	folD	Bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase FolD
DR186_RS14195	-	Aspartate carbamoyltransferase catalytic subunit
DR186_RS14245	lipB	Lipoyl(octanoyl) transferase LipB
DR186_RS16105	ubiE	Bifunctional demethylmenaquinone methyltransferase/2-methoxy-6-polyprenyl-1%2C4-benzoquinol methylase UbiE
DR186_RS13420	-	Glutamate--cysteine ligase
DR186_RS11415	hemH	Ferrochelatase
DR186_RS16095	coaBC	Bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate--cysteine ligase CoaBC
B. abortus S2308
BAB_RS16450	pncB	Nicotinate phosphoribosyltransferase
BAB_RS17600	hemA	5-Aminolevulinate synthase
BAB_RS18030	ubiA	4-Hydroxybenzoate octaprenyltransferase
BAB_RS18225	fabG	3-Oxoacyl-[acyl-carrier-protein] reductase
BAB_RS18235	fabF	Beta-ketoacyl-ACP synthase II
BAB_RS19675	glyA	Serine hydroxymethyltransferase
BAB_RS19685	ribD	Bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase RibD
BAB_RS19690	-	Riboflavin synthase
BAB_RS20070	purB	Adenylosuccinate lyase
BAB_RS20505	-	NAD kinase
BAB_RS20875	-	NAD+ synthase
BAB_RS20880	gltX	Glutamate--tRNA ligase
BAB_RS20950	folP	Dihydropteroate synthase
BAB_RS20955	folB	Dihydroneopterin aldolase
BAB_RS20960	folK	2-Amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase
BAB_RS21170	folE	GTP cyclohydrolase I FolE
BAB_RS21285	coaD	Pantetheine-phosphate adenylyltransferase
BAB_RS21415	lipA	Lipoyl synthase
BAB_RS21545	fabZ	3-Hydroxyacyl-ACP dehydratase FabZ
BAB_RS21580	pyrH	UMP kinase
BAB_RS22685	-	Dihydrofolate reductase
BAB_RS23085	carA	Glutamine-hydrolyzing carbamoyl-phosphate synthase small subunit
BAB_RS23105	carB	Carbamoyl-phosphate synthase large subunit
BAB_RS23385	hemF	Oxygen-dependent coproporphyrinogen oxidase
BAB_RS24715	-	Nicotinate-nucleotide adenylyltransferase
BAB_RS24870	ubiG	Bifunctional 2-polyprenyl-6-hydroxyphenol methylase/3-demethylubiquinol 3-O-methyltransferase UbiG
BAB_RS24930	-	Uroporphyrinogen-III synthase
BAB_RS24935	hemC	Hydroxymethylbilane synthase
BAB_RS24990	-	Ubiquinone biosynthesis hydroxylase
BAB_RS25770	hemJ	Protoporphyrinogen oxidase HemJ
BAB_RS25775	hemE	Uroporphyrinogen decarboxylase
BAB_RS25880	coaA	Type I pantothenate kinase
BAB_RS25960	-	Bifunctional folylpolyglutamate synthase/dihydrofolate synthase
BAB_RS26220	metK	Methionine adenosyltransferase
BAB_RS26285	fabB	Beta-ketoacyl-ACP synthase I
BAB_RS27285	-	Bifunctional riboflavin kinase/FAD synthetase
BAB_RS28550	folD	Bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase FolD
BAB_RS29425	lipB	Lipoyl(octanoyl) transferase LipB
BAB_RS31170	coaBC	Bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate--cysteine ligase CoaBC
BAB_RS31180	ubiE	Bifunctional demethylmenaquinone methyltransferase/2-methoxy-6-polyprenyl-1%2C4-benzoquinol methylase UbiE

Table 5. Genes associated with oxidative phosphorylation identified via KEGG enrichment analysis.

Gene_ID	Gene	Annotation
B. abortus A19
DR186_RS02120	-	F0F1 ATP synthase subunit A
DR186_RS02130	-	F0F1 ATP synthase subunit B
DR186_RS04195	-	NADH-quinone oxidoreductase subunit A
DR186_RS04200	-	NADH-quinone oxidoreductase subunit B family protein
DR186_RS04205	-	NADH-quinone oxidoreductase subunit C
DR186_RS04210	-	NADH-quinone oxidoreductase subunit D
DR186_RS04215	nuoE	NADH-quinone oxidoreductase subunit NuoE
DR186_RS04220	nuoF	NADH-quinone oxidoreductase subunit NuoF
DR186_RS04225	nuoG	NADH-quinone oxidoreductase subunit NuoG
DR186_RS04230	nuoH	NADH-quinone oxidoreductase subunit NuoH
DR186_RS04235	nuoI	NADH-quinone oxidoreductase subunit NuoI
DR186_RS04240	-	NADH-quinone oxidoreductase subunit J
DR186_RS04245	nuoK	NADH-quinone oxidoreductase subunit NuoK
DR186_RS04250	nuoL	NADH-quinone oxidoreductase subunit L
DR186_RS04260	nuoN	NADH-quinone oxidoreductase subunit NuoN
DR186_RS09115	-	F0F1 ATP synthase subunit gamma
DR186_RS09630	sdhA	Succinate dehydrogenase flavoprotein subunit
DR186_RS09120	atpA	F0F1 ATP synthase subunit alpha
DR186_RS09625	-	Succinate dehydrogenase iron-sulfur subunit
DR186_RS09110	atpD	F0F1 ATP synthase subunit beta
DR186_RS10090	ppa	Inorganic diphosphatase
DR186_RS09635	sdhD	Succinate dehydrogenase%2C hydrophobic membrane anchor protein
DR186_RS09640	sdhC	Succinate dehydrogenase%2C cytochrome b556 subunit
DR186_RS02125	-	F0F1 ATP synthase subunit C
DR186_RS04255	-	NADH-quinone oxidoreductase subunit M
DR186_RS09125	-	F0F1 ATP synthase subunit delta
B. abortus S2308
BAB_RS17890	-	F0F1 ATP synthase subunit A
BAB_RS17895	-	F0F1 ATP synthase subunit C
BAB_RS17900	-	F0F1 ATP synthase subunit B
BAB_RS19860	-	NuoB/complex I 20 kDa subunit family protein
BAB_RS19865	-	NADH-quinone oxidoreductase subunit C
BAB_RS19870	-	NADH-quinone oxidoreductase subunit D
BAB_RS19875	nuoE	NADH-quinone oxidoreductase subunit NuoE
BAB_RS19880	nuoF	NADH-quinone oxidoreductase subunit NuoF
BAB_RS19885	nuoG	NADH-quinone oxidoreductase subunit NuoG
BAB_RS19890	nuoH	NADH-quinone oxidoreductase subunit NuoH
BAB_RS19895	nuoI	NADH-quinone oxidoreductase subunit NuoI
BAB_RS19900	-	NADH-quinone oxidoreductase subunit J
BAB_RS19905	nuoK	NADH-quinone oxidoreductase subunit NuoK
BAB_RS19910	nuoL	NADH-quinone oxidoreductase subunit L
BAB_RS19915	-	NADH-quinone oxidoreductase subunit M
BAB_RS19920	nuoN	NADH-quinone oxidoreductase subunit NuoN
BAB_RS24515	atpD	F0F1 ATP synthase subunit beta
BAB_RS24520	-	F0F1 ATP synthase subunit gamma
BAB_RS24525	atpA	F0F1 ATP synthase subunit alpha
BAB_RS24530	-	F0F1 ATP synthase subunit delta
BAB_RS25000	-	Succinate dehydrogenase iron-sulfur subunit
BAB_RS25005	sdhA	Succinate dehydrogenase flavoprotein subunit
BAB_RS25015	sdhC	Succinate dehydrogenase%2C cytochrome b556 subunit
BAB_RS25435	ppa	Inorganic diphosphatase

Figure 4.

Functional categorization of putative essential genes in B. abortus strains A19 (A) and S2308 (B) determined through KEGG enrichment analysis

Table 2. Genes associated with ribosomal biogenesis identified via KEGG enrichment analysis.

Gene_ID	Gene	Annotation
B. abortus A19
DR186_RS02465	rpsF	30S ribosomal protein S6
DR186_RS04130	rplM	50S ribosomal protein L13
DR186_RS04320	rpsD	30S ribosomal protein S4
DR186_RS06025	rpsB	30S ribosomal protein S2
DR186_RS06260	rpsM	30S ribosomal protein S13
DR186_RS06285	rpsE	30S ribosomal protein S5
DR186_RS06290	rplR	50S ribosomal protein L18
DR186_RS06295	rplF	50S ribosomal protein L6
DR186_RS06305	rpsN	30S ribosomal protein S14
DR186_RS06310	rplE	50S ribosomal protein L5
DR186_RS06330	rpmC	50S ribosomal protein L29
DR186_RS06335	rplP	50S ribosomal protein L16
DR186_RS06340	rpsC	30S ribosomal protein S3
DR186_RS06345	rplV	50S ribosomal protein L22
DR186_RS06350	rpsS	30S ribosomal protein S19
DR186_RS06355	rplB	50S ribosomal protein L2
DR186_RS06360	-	50S ribosomal protein L23
DR186_RS06365	rplD	50S ribosomal protein L4
DR186_RS06370	rplC	50S ribosomal protein L3
DR186_RS06390	rpsG	30S ribosomal protein S7
DR186_RS06395	rpsL	30S ribosomal protein S12
DR186_RS06420	rplJ	50S ribosomal protein L10
DR186_RS06425	rplA	50S ribosomal protein L1
DR186_RS06430	rplK	50S ribosomal protein L11
DR186_RS07860	-	50S ribosomal protein L25/general stress protein Ctc
DR186_RS09355	rpmA	50S ribosomal protein L27
DR186_RS09360	rplU	50S ribosomal protein L21
DR186_RS10190	rpmB	50S ribosomal protein L28
DR186_RS10945	rpsO	30S ribosomal protein S15
DR186_RS06240	rplQ	50S ribosomal protein L17
DR186_RS06300	rpsH	30S ribosomal protein S8
DR186_RS00145	rpsA	30S ribosomal protein S1
DR186_RS10720	rplT	50S ribosomal protein L20
DR186_RS09660	rplS	50S ribosomal protein L19
DR186_RS06280	rpmD	50S ribosomal protein L30
DR186_RS06315	rplX	50S ribosomal protein L24
DR186_RS06325	rpsQ	30S ribosomal protein S17
DR186_RS06375	rpsJ	30S ribosomal protein S10
DR186_RS09010	rpmF	50S ribosomal protein L32
DR186_RS09225	rpsP	30S ribosomal protein S16
B. abortus S2308
BAB_RS16055	rpsA	30S ribosomal protein S1
BAB_RS19990	rpsD	30S ribosomal protein S4
BAB_RS21590	rpsB	30S ribosomal protein S2
BAB_RS21795	rplQ	50S ribosomal protein L17
BAB_RS21805	rpsK	30S ribosomal protein S11
BAB_RS21810	rpsM	30S ribosomal protein S13
BAB_RS21830	rpmD	50S ribosomal protein L30
BAB_RS21835	rpsE	30S ribosomal protein S5
BAB_RS21840	rplR	50S ribosomal protein L18
BAB_RS21845	rplF	50S ribosomal protein L6
BAB_RS21850	rpsH	30S ribosomal protein S8
BAB_RS21855	rpsN	30S ribosomal protein S14
BAB_RS21860	rplE	50S ribosomal protein L5
BAB_RS21865	rplX	50S ribosomal protein L24
BAB_RS21870	rplN	50S ribosomal protein L14
BAB_RS21875	rpsQ	30S ribosomal protein S17
BAB_RS21885	rplP	50S ribosomal protein L16
BAB_RS21890	rpsC	30S ribosomal protein S3
BAB_RS21895	rplV	50S ribosomal protein L22
BAB_RS21900	rpsS	30S ribosomal protein S19
BAB_RS21905	rplB	50S ribosomal protein L2
BAB_RS21910	-	50S ribosomal protein L23
BAB_RS21920	rplC	50S ribosomal protein L3
BAB_RS21940	rpsG	30S ribosomal protein S7
BAB_RS21945	rpsL	30S ribosomal protein S12
BAB_RS21970	rplJ	50S ribosomal protein L10
BAB_RS21975	rplA	50S ribosomal protein L1
BAB_RS21980	rplK	50S ribosomal protein L11
BAB_RS23315	-	50S ribosomal protein L25/general stress protein Ctc
BAB_RS24420	rpmF	50S ribosomal protein L32
BAB_RS24630	rpsP	30S ribosomal protein S16
BAB_RS25030	rplS	50S ribosomal protein L19
BAB_RS25530	rpmB	50S ribosomal protein L28

Table 3. Genes associated with carbon metabolism identified via KEGG enrichment analysis.

Gene_ID	Gene	Annotation
B. abortus A19
DR186_RS01655	pgi	Glucose-6-phosphate isomerase
DR186_RS04415	rpe	Ribulose-phosphate 3-epimerase
DR186_RS04705	accC	Acetyl-CoA carboxylase biotin carboxylase subunit
DR186_RS04710	accB	Acetyl-CoA carboxylase biotin carboxyl carrier protein
DR186_RS05195	rpiA	Ribose-5-phosphate isomerase RpiA
DR186_RS05870	pdhA	Pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha
DR186_RS05960	gltA	Citrate synthase
DR186_RS07125	serB	Phosphoserine phosphatase SerB
DR186_RS07845	-	Ribose-phosphate pyrophosphokinase
DR186_RS08790	gap	Type I glyceraldehyde-3-phosphate dehydrogenase
DR186_RS09140	fsa	Fructose-6-phosphate aldolase
DR186_RS09630	sdhA	Succinate dehydrogenase flavoprotein subunit
DR186_RS09735	odhB	2-Oxoglutarate dehydrogenase complex dihydrolipoyllysine-residue succinyltransferase
DR186_RS09745	sucD	Succinate--CoA ligase subunit alpha
DR186_RS09750	sucC	ADP-forming succinate--CoA ligase subunit beta
DR186_RS09755	mdh	Malate dehydrogenase
DR186_RS10655	accD	Acetyl-CoA carboxylase%2C carboxyltransferase subunit beta
DR186_RS08785	tkt	Transketolase
DR186_RS09740	-	2-Oxoglutarate dehydrogenase E1 component
DR186_RS06200	-	NADP-dependent isocitrate dehydrogenase
DR186_RS05860	-	Pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase
DR186_RS09625	-	Succinate dehydrogenase iron-sulfur subunit
DR186_RS10275	-	Acetyl-CoA carboxylase carboxyltransferase subunit alpha
DR186_RS00485	acnA	Aconitate hydratase AcnA
DR186_RS05865	-	Pyruvate dehydrogenase complex E1 component subunit beta
DR186_RS05885	eno	Phosphopyruvate hydratase
DR186_RS03995	glyA	Serine hydroxymethyltransferase
DR186_RS09635	sdhD	Succinate dehydrogenase%2C hydrophobic membrane anchor protein
DR186_RS06505	cysE	Serine O-acetyltransferase
DR186_RS09640	sdhC	Succinate dehydrogenase%2C cytochrome b556 subunit
DR186_RS05445	-	Cysteine synthase A
DR186_RS13325	folD	Bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase FolD
DR186_RS13335	pgl	6-Phosphogluconolactonase
DR186_RS13340	zwf	Glucose-6-phosphate dehydrogenase
DR186_RS11575	gndA	NADP-dependent phosphogluconate dehydrogenase
B. abortus S2308
BAB_RS16360	acnA	Aconitate hydratase AcnA
BAB_RS17440	pgi	Glucose-6-phosphate isomerase
BAB_RS19675	glyA	Serine hydroxymethyltransferase
BAB_RS20075	rpe	Ribulose-phosphate 3-epimerase
BAB_RS20350	accC	Acetyl-CoA carboxylase biotin carboxylase subunit
BAB_RS20355	accB	Acetyl-CoA carboxylase biotin carboxyl carrier protein
BAB_RS20890	-	NADP-dependent malic enzyme
BAB_RS21430	-	Pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase
BAB_RS21435	-	Pyruvate dehydrogenase complex E1 component subunit beta
BAB_RS21440	pdhA	Pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha
BAB_RS21455	eno	Phosphopyruvate hydratase
BAB_RS21525	gltA	Citrate synthase
BAB_RS21755	-	NADP-dependent isocitrate dehydrogenase
BAB_RS22655	serB	Phosphoserine phosphatase SerB
BAB_RS23305	-	Ribose-phosphate pyrophosphokinase
BAB_RS24225	tkt	Transketolase
BAB_RS24230	gap	Type I glyceraldehyde-3-phosphate dehydrogenase
BAB_RS25000	-	Succinate dehydrogenase iron-sulfur subunit
BAB_RS25005	sdhA	Succinate dehydrogenase flavoprotein subunit
BAB_RS25015	sdhC	Succinate dehydrogenase%2C cytochrome b556 subunit
BAB_RS25105	odhB	2-Oxoglutarate dehydrogenase complex dihydrolipoyllysine-residue succinyltransferase
BAB_RS25110	-	2-Oxoglutarate dehydrogenase E1 component
BAB_RS25115	sucD	Succinate--CoA ligase subunit alpha
BAB_RS25120	sucC	ADP-forming succinate--CoA ligase subunit beta
BAB_RS25125	mdh	Malate dehydrogenase
BAB_RS25605	-	Acetyl-CoA carboxylase carboxyltransferase subunit alpha
BAB_RS25965	accD	Acetyl-CoA carboxylase%2C carboxyltransferase subunit beta
BAB_RS26870	gndA	NADP-dependent phosphogluconate dehydrogenase
BAB_RS28550	folD	Bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase FolD
BAB_RS28565	zwf	Glucose-6-phosphate dehydrogenase

Table 4. Genes associated with amino acid biosynthesis identified via KEGG enrichment analysis.

Gene_ID	Gene	Annotation
B. abortus A19
DR186_RS00130	aroA	3-Phosphoshikimate 1-carboxyvinyltransferase
DR186_RS01735	argF	Ornithine carbamoyltransferase
DR186_RS02625	thrC	Threonine synthase
DR186_RS03405	dapA	4-Hydroxy-tetrahydrodipicolinate synthase
DR186_RS04415	rpe	Ribulose-phosphate 3-epimerase
DR186_RS04715	aroQ	Type II 3-dehydroquinate dehydratase
DR186_RS05195	rpiA	Ribose-5-phosphate isomerase RpiA
DR186_RS05960	gltA	Citrate synthase
DR186_RS07125	serB	Phosphoserine phosphatase SerB
DR186_RS07650	-	Pyridoxal phosphate-dependent aminotransferase
DR186_RS07845	-	Ribose-phosphate pyrophosphokinase
DR186_RS08790	gap	Type I glyceraldehyde-3-phosphate dehydrogenase
DR186_RS09140	fsa	Fructose-6-phosphate aldolase
DR186_RS09230	-	Chorismate mutase
DR186_RS09325	-	Glutamate-5-semialdehyde dehydrogenase
DR186_RS09490	-	Aspartate kinase
DR186_RS09780	dapF	Diaminopimelate epimerase
DR186_RS10905	metK	Methionine adenosyltransferase
DR186_RS08785	tkt	Transketolase
DR186_RS06200	-	NADP-dependent isocitrate dehydrogenase
DR186_RS07110	-	Acetolactate synthase 3 large subunit
DR186_RS04115	argC	N-acetyl-gamma-glutamyl-phosphate reductase
DR186_RS05170	glnA	Type I glutamate--ammonia ligase
DR186_RS00485	acnA	Aconitate hydratase AcnA
DR186_RS02330	aroC	Chorismate synthase
DR186_RS05885	eno	Phosphopyruvate hydratase
DR186_RS03995	glyA	Serine hydroxymethyltransferase
DR186_RS06505	cysE	Serine O-acetyltransferase
DR186_RS10255	aroB	3-Dehydroquinate synthase
DR186_RS01730	-	Aspartate aminotransferase family protein
DR186_RS05210	-	Class II 3-deoxy-7-phosphoheptulonate synthase
DR186_RS05445	-	Cysteine synthase A
DR186_RS10020	argH	Argininosuccinate lyase
DR186_RS15915	dapD	2%2C3%2C4%2C5-tetrahydropyridine-2%2C6-dicarboxylate N-succinyltransferase
DR186_RS15900	argB	Acetylglutamate kinase
DR186_RS15925	dapE	Succinyl-diaminopimelate desuccinylase
B. abortus S2308
BAB_RS16045	aroA	3-Phosphoshikimate 1-carboxyvinyltransferase
BAB_RS16360	acnA	Aconitate hydratase AcnA
BAB_RS17510	-	Aspartate aminotransferase family protein
BAB_RS18350	thrC	Threonine synthase
BAB_RS19125	dapA	4-Hydroxy-tetrahydrodipicolinate synthase
BAB_RS19675	glyA	Serine hydroxymethyltransferase
BAB_RS20075	rpe	Ribulose-phosphate 3-epimerase
BAB_RS20360	aroQ	Type II 3-dehydroquinate dehydratase
BAB_RS21455	eno	Phosphopyruvate hydratase
BAB_RS21525	gltA	Citrate synthase
BAB_RS21755	-	NADP-dependent isocitrate dehydrogenase
BAB_RS22655	serB	Phosphoserine phosphatase SerB
BAB_RS23305	-	Ribose-phosphate pyrophosphokinase
BAB_RS24225	tkt	Transketolase
BAB_RS24230	gap	Type I glyceraldehyde-3-phosphate dehydrogenase
BAB_RS24635	-	Chorismate mutase
BAB_RS24720	-	Glutamate-5-semialdehyde dehydrogenase
BAB_RS24865	-	Aspartate kinase
BAB_RS25140	dapF	Diaminopimelate epimerase
BAB_RS26220	metK	Methionine adenosyltransferase
BAB_RS28035	-	Aspartate-semialdehyde dehydrogenase
BAB_RS31000	dapD	2%2C3%2C4%2C5-tetrahydropyridine-2%2C6-dicarboxylate N-succinyltransferase

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.

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.

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.

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.

MATERIALS AND METHODS

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.

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).

Figure 5.

Schematic diagram of mutant library construction

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).

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.

Human and animal rights and informed consent

This article does not contain any studies with human or animal subjects performed by any of the authors.