Genome-wide identification of essential genes in the invasive Streptococcus anginosus strain

Abstract

Streptococcus anginosus, part of the Streptococcus anginosus group (SAG), is a human commensal increasingly recognized as an opportunistic pathogen responsible for abscesses formation and infections, also invasive ones. Despite its growing clinical importance, the genetic determinants of its pathogenicity remain poorly understood. This study aimed to identify essential genes in S. anginosus 980/01, a bloodstream isolate, under nutrient-rich laboratory conditions using a transposon mutagenesis combined with Transposon-Directed Insertion Site Sequencing (TraDIS). A mutant library was generated using the ISS1 transposon delivered via the thermosensitive plasmid pGh9:ISS1. Following transposition, insertions were mapped using Illumina sequencing and subsequently analyzed. Essential genes were identified based on the absence of insertions and statistical filtering. The library exhibited 98% genome saturation with over 130,000 unique insertion sites. Among 1825 genes, 348 (19.1%) were essential, 1446 non-essential, and 30 non-conclusive. Comparative analyses were performed with S. pyogenes MGAS5005 and S. agalactiae A909. Similarly to the latter, essential genes were enriched in functions related to translation, transcription, and cell wall biosynthesis. However, 40 genes uniquely essential to S. anginosus 980/01 were identified, suggesting unique survival strategies in S. anginosus. This study presents the first genome-wide identification of essential genes for S. anginosus 980/01, highlighting conserved and unique essential genes. These findings provide a basis for understanding its physiology and key genetic determinants of bacterial viability, and may help to uncover the pathogenic potential of S. anginosus in future studies.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-18002-0.

Introduction

Streptococcus anginosus, together with S. constellatus and S. intermedius, constitute the Streptococcus anginosus group (SAG) commonly found on mucosal membranes in the healthy human microbiota, particularly in the oral cavity, gastrointestinal tract, and urogenital tracts¹. Despite this, SAG species are increasingly recognized as opportunistic pathogens, especially in immunocompromised or cystic fibrosis patients^2–4. Among SAGs, S. anginosus is frequently isolated from abscesses, bloodstream infections, and polymicrobial infections, and is more commonly associated with invasive infections than other SAG members, also in individuals without any underlying diseases^2,5–8.

Interestingly, S. anginosus strains exhibit variable hemolytic activity and can express different Lancefield antigens, complicating diagnosis and classification. These strains can also produce diverse virulence factors^9,10. However, the molecular mechanisms underlying the S. anginosus pathogenesis remain poorly understood, with many of its genes still annotated as hypothetical^7,11,12. To assess the essentiality and fitness of each gene within the bacterial genome, the most efficient strategies integrate the next-generation sequencing (NGS) technologies with large-scale transposon mutagenesis^13–17. This approach enables the identification of the chromosomal sequences adjacent to transposon integration sites. However, genes required for bacterial growth and survival under specific experimental conditions, termed essential genes, cannot be disrupted. A saturated transposon mutant library contains a collection of viable mutants with a transposon inserted into every non-essential gene, while insertions into essential genes are lethal. Moreover, such a library can be challenged in specific conditions, such as human blood or antibiotics, to distinguish non-essential genes that are necessary for bacterial survival and growth in this particular environment.

For mutagenesis of Gram-positive bacteria, transposons are typically based either on mariner^18–21 or ISS1^22–26, both utilizing a copy-out–paste-in mechanism. While the mariner-family transposons preferentially integrate at TA dinucleotide sequences²⁷, the ISS1 does not exhibit such specificity²². Essential genes of several streptococcal species have been analysed in the ISS1 Transposon-Directed Insertion Site Sequencing (TraDIS), including S. uberis 0140J¹⁷, S. equi 4047²⁴, and S. agalactiae CNCTC 10/84²⁸. Meanwhile, for S. pyogenes M1T1 5448¹⁸, S. agalactiae A909²⁹, and S. suis SC19³⁰, the mariner-based Krmit or Himar1 have been used. The identification of essential genes provides promising targets for the development of novel antimicrobial therapies³¹.

In this study, we performed an ISS1 TraDIS approach to identify genes essential for the growth of S. anginosus 980/01¹¹, an invasive strain isolated from sepsis, under nutrient-rich laboratory conditions. As the first genome-wide transposon mutagenesis study in S. anginosus, this analysis provides an initial framework for exploring its essential gene set. The TraDIS data for S. anginosus were finally compared to those of S. pyogenes and S. agalactiae to identify conserved essential genes and determine the unique set of genes essential to S. anginosus 980/01. The transposon-based methods, such as TraDIS, have become powerful tools for defining the genetic determinants of bacterial survival and virulence.

Materials and methods

The S. anginosus 980/01 strain is part of the Collection of the National Institute of Medicines, Warsaw, Poland¹¹. It was isolated from the bloodstream of a 67-year-old patient in 2001 in Poland. The strain exhibits α-hemolytic activity and expresses the Lancefield group F antigen on its surface.

S. anginosus cultures

S. anginosus was grown on BHI agar (VWR chemicals) plates or in BHI broth (VWR chemicals) supplemented, if necessary, with erythromycin (Acros Organics) in a concentration of 5 × 10⁻³ g/L. Bacteria were grown at 37 °C, unless otherwise stated, in the presence of a CO₂ generator, CO₂Gen (Thermo Fisher Scientific, Waltham, USA). For extended storage, S. anginosus 980/01 was frozen in BHI with 15% glycerol at − 80 °C.

S. anginosus electrocompetent cells preparation

S. anginosus 980/01 was grown on BHI agar for 24 h. After that, the 25-fold diluted culture was grown in BHI to OD₆₆₀ ~ 0.3, harvested by centrifugation (5000 × g, 4 °C), washed three times with ice-cold 0.5 M sucrose, and resuspended in 0.5 M sucrose with 15% glycerol. Aliquots (50 μL) were stored at − 80 °C.

Transformation of S. anginosus with the pGh9:ISS1 plasmid

The pGh9:ISS1 plasmid²² with thermosensitive replication was used as a donor of the ISS1 transposon. Electrocompetent S. anginosus 980/01 cells were transformed with 50 ng of pGh9:ISS1 DNA via electroporation (2.5 kV/cm, 200 Ω, 25 μF, 5 ms pulse; Bio-Rad Gene Pulser). Cells were recovered in BHI at 28 °C for 4 h; after 1 h of incubation, erythromycin in a sublethal concentration was added to induce resistance expression. Transformants were selected on BHI with erythromycin (BHIE) at 28 °C after 48 h-incubation.

The S. anginosus 980/01 mutant library construction

A single colony of S. anginosus 980/01 carrying pGh9:ISS1 was grown in 10 mL BHIE at 28 °C for 24 h, then heat-shocked at 40 °C for 2.5 h to stop plasmid replication and induce ISS1 transposition. Transposants were selected after overnight growth on 100 BHIE plates (~ 6500 colonies/plate) at 40 °C, in an atmosphere with 5% CO₂. Colonies were harvested into BHI with 15% glycerol and stored at − 80 °C.

PCR detection of pGh9:ISS1

To confirm the presence of pGh9:ISS1 in selected clones, PCR was performed using DreamTaq DNA polymerase and dNTPs (Thermo Fisher Scientific), with FwISS1 and RvISS1 primers (Supplemental File S1). The reaction conditions: initial denaturation at 95 °C for 3 min; 30 cycles of 95 °C for 30 s (denaturation), 50 °C for 30 s (annealing), and 72 °C for 4 min (extension); followed by a final extension at 72 °C for 7 min. PCR products were analyzed by agarose gel electrophoresis.

Identification of ISS1 insertion sites in mutants

To verify the ISS1 transposon insertion site in an individual mutant, genomic DNA was extracted and digested with FastDigest HindIII (Thermo Fisher Scientific). The resulting fragments were ligated using T4 DNA Ligase (Thermo Fisher Scientific), and served as templates for PCR amplification with DreamTaq DNA Polymerase (Thermo Fisher Scientific) using primers FwSAGISS1 and RvSAGISS1 (Supplemental File S1). The PCR products were separated by agarose gel electrophoresis, the bands were excised, purified (MicroElute Gel Extraction Kit, OMEGA Bio-tek, USA), and Sanger sequenced using FwSAGISS1 (detailed results are provided in Supplemental File S1).

ISS1 transposon mutant libraries preparation for Illumina sequencing

The portion of ISS1 transposon mutant library was regrown to OD₆₆₀ = 0.3 in BHIE, spread onto 25 BHIE plates each, pooled, and harvested prior DNA extraction. Genomic DNA was isolated from the bacterial mutant pools using the SDS/Phenol method as described previously^32,33. DNA quality control was performed by measuring the absorbance at 260/230, template concentration was determined using the Qubit fluorimeter (Thermo Fisher Scientific). DNA integrity was analyzed by 0.8% agarose gel electrophoresis.

The ISS1 transposon mutant libraries were constructed according to the protocol (Transposon insertion sequencing (Tn-seq) library preparation protocol—includes Unique Molecular Identifiers (UMI) for PCR duplicate removal: https://www.protocols.io/view/transposon-insertion-sequencing-tn-seq-library-pre-rm7vzn6d5vx1/v1) with minor modifications. In brief, DNA was mechanically sheared using Covaris (Covaris, MA, USA) into 300–500 bp fragment sizes, followed by end repair and TA-adaptor ligation using NEB Ultra II End Repair and Ligation Modules (New England Biolabs, Beverly, USA). Ligation reaction was purified by Ampure XP magnetic beads (Beckman Coulter, Brea, USA) and library restriction digest was performed using SmaI (Thermo Fisher Scientific) for 2 h at 25 °C to cleave the pGh9:ISS1 plasmid 33 bp upstream of the sequence encoding ISS1 to minimise the amount of TnSeq reads mapping to plasmid. The digested library was purified using Ampure XP beads, and the amount of DNA recovered was quantified using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. One hundred nanograms of library DNA was PCR amplified for 20 cycles according to the NEBNext Ultra II DNA library prep kit protocol. Library amplification utilized the specific ISS1 primer containing Nextera XT 5’ overhang (P5) with barcode index sequence and a TA–adaptor compatible unique indexing Nextera XT PCR (P7) primer per library, which facilitated the attachment of the final product to the sequencing flow cell (primer sequences are provided in Supplemental File S1). The libraries were quality-checked using the KAPA Library Quantification kit (KAPA-Roche, Basel, Switzerland), pooled in equimolar ratio, and sequenced on a NextSeq 550 instrument using the NextSeq HighOutput reagent v2.5 (150 cycle) chemistry kit (Illumina, San Diego, USA).

TnSeq data analysis

Raw demultiplexed fastq files were analysed using the TnSeq UMI scripts (https://github.com/nppalani/TnSeq/ and https://github.com/apredeus/TRADIS) modified to handle ISS1 transposon sequence containing data. Initially, the single command pipeline script, fastqtoreadcount_umi.sh, was run. The pipeline performed UMI-based PCR duplication removal, filtered and removed reads according to the transposon tag. After tag removal, the remaining reads were mapped to the S. anginosus 980/01 reference genome. Finally, raw read counts per insertion position output files were generated. Transposon insertions were viewed in Integrative Genomics Viewer³⁴. TnSeq data analysis was further performed using Transit v.3.2.3³⁵. Gene essentiality was calculated using the Tn5Gaps method (https://transit.readthedocs.io/en/latest/method_tn5gaps.html), designed for transposons with random genome insertion. To estimate gene essentiality, the method identifies the longest uninterrupted regions within genes lacking insertions. The ISS1 insertion index was calculated for each gene as the number of unique insertion sites divided by the gene length (in base pairs). Based on this index, genes were categorized as essential, non-essential, or non-conclusive. For genes classified as essential, statistical significance was assessed using a threshold of p < 0.05, following correction for multiple testing using the false discovery rate (FDR) method to reduce the likelihood of false positives.

Metabolic pathway reconstruction and KEGG categorization

The metabolic pathways of S. anginosus 980/01 were reconstructed using the KEGG Mapper tool provided by the Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp)^36,37. Genome annotation files for S. anginosus 980/01 were first uploaded to BlastKOALA to assign KEGG Orthology (KO) identifiers to predicted protein-coding genes. Default parameters were used for KO assignment, including the “Prokaryotes” taxonomic group setting. The resulting KO-annotated gene sets were visualized and mapped to metabolic pathways using KEGG Mapper’s “Reconstruct Pathway” tool. Metabolic reconstructions were compared with annotated reference genomes of S. pyogenes MGAS5005 and S. agalactiae A909, both obtained from the KEGG database. Differences in core metabolism were identified based on the presence or absence of pathway-specific enzymes.

Ortholog identification and essential gene comparison

To identify orthologous genes and assess conservation of essentiality across species, we used OrthoFinder v3.0.1b1 (https://github.com/davidemms/OrthoFinder)³⁸. Protein sequences from S. anginosus 980/01, S. pyogenes MGAS5005, and S. agalactiae A909 were used as input. OrthoFinder was run with default parameters, which include the use of DIAMOND for sequence similarity searches and MCL clustering for orthogroup inference. Only orthologs found in one-to-one orthogroups were considered for essentiality comparisons to avoid ambiguity due to paralogy.

KEGG pathway enrichment analysis

This analysis was performed to identify biological processes overrepresented among essential genes identified by TnSeq. Protein annotations for the complete genome were obtained using eggNOG-mapper (v2.1), (http://eggnog-mapper.embl.de/) and KEGG Orthology (KO) identifiers and pathway memberships (https://www.genome.jp/kegg/kegg2.html)³⁷ were extracted. Gene identifiers were matched using 5-digit locus suffixes. Each gene was labelled as essential or non-essential. For each KEGG pathway, a 2 × 2 contingency table was constructed to compare the number of essential and non-essential genes present or absent in the pathway. Significance of enrichment was assessed using Fisher’s exact test, followed by Bonferroni correction for multiple testing. Pathways with adjusted p-values below 0.05 were considered significantly enriched.

Phylogenetic analysis of S. anginosus 980/01

Phylogenetic analysis of S. anginosus 980/01 was conducted using genome sequences from 76 S. anginosus isolates published by Prasad et al.³⁹ in Table S2. These included both complete and draft genome assemblies representative of the species’ genomic diversity. A reference-free phylogenetic analysis was performed using kSNP4⁴⁰, which detects core single nucleotide polymorphisms (SNPs) across genome sequences without requiring alignment or a reference genome. A maximum-likelihood phylogenetic tree was inferred from the core SNP matrix and visualized using the Microreact (https://microreact.org/)⁴¹, an interactive platform for exploring phylogenetic data alongside associated metadata.

Results

Genome structure and mobile genetic elements of S. anginosus 980/01

The S. anginosus 980/01 genomic DNA was previously sequenced using both short-reads of Illumina and the long-reads of Oxford Nanopore technology to obtain the complete physical map; the sequencing of this strain is a part of the larger project and will be described elsewhere. It consists of 1,883,767 bps chromosome, with no plasmids. The sequence is deposited in GenBank under accession number CP183189, and as BioProject No.PRJNA1228600. The mean %G + C content is 39%. Genome annotation using DFAST v.1.2.18 software (https://github.com/nigyta/dfast_core) revealed that the genome contains 1825 genes coding for proteins, of which 415 are of unknown function. No antimicrobial resistance genes were detected using ResFinder v.4.3.3 (https://orbit.dtu.dk/en/publications/resfinder-an-open-online-resource-for-identification-of-antimicro). Nine putative streptococcal virulence factors were identified using ABRicate v.1.0.1 (https://github.com/tseemann/abricate) with the Virulence Factor Database (https://www.mgc.ac.cn/VFs/), retaining only hits with > 80% both sequence identity and target coverage (Supplemental File S1). Detection of mobile genetic elements revealed: a/ an intact single prophage, 38.9 kb in size (position 591,476–630,426), detected using Phastest (https://phastest.ca/), b/ two putative integrative and conjugative elements (ICE) as well as c/ a single putative integrative and mobilizable element (IME); ICEs and IME were identified using both ICEfinder (https://bioinfo-mml.sjtu.edu.cn/ICEfinder/ICEfinder.html) and ICEscreen v1.3.3 (https://icescreen.migale.inrae.fr/) (Supplemental File S1).

Phylogenetic analysis of S. anginosus 980/01

To investigate the genomic relationship of S. anginosus strain 980/01, we constructed a core-SNP phylogenetic tree based on the genomic sequences of 76 S. anginosus strains³⁹. These included representatives of two subspecies (S. anginosus subsp. anginosus and S. anginosus subsp. whileyi), as well as two proposed genomosubspecies (S. anginosus genomosubsp. AJ1 and S. anginosus genomosubsp. vellorensis), as previously defined by multilocus sequence analysis (MLSA) (see Supplemental File S1, worksheet tab “Strains for core-SNP tree”).

In the resulting phylogenetic tree (Fig. 1), strain 980/01 clustered within the S. anginosus subsp. anginosus clade, grouping closely with 11 other strains and showing more distant relationships to an additional 25 strains within the same subspecies. This clustering indicates that S. anginosus 980/01 shares a recent common ancestor with these 11 strains and belongs to a well-supported sub-lineage of S. anginosus subsp. anginosus. Therefore, we conclude that S. anginosus 980/01 is a representative member of the this subspecies and may serve as a suitable reference strain for comparative studies involving this sub-lineage.

Fig. 1.

Maximum-likelihood phylogenetic tree of S. anginosus 980/01 and related S. anginosus isolates based on core genome single-nucleotide polymorphisms (SNPs). Coloured boxes indicate the S. anginosus subspecies groups. SNPs were identified using kSNP4. All included strains are listed in Supplemental File S1.

The S. anginosus 980/01 mutant library construction

To construct the S. anginosus 980/01 transposon mutant library, the pGh9:ISS1 plasmid of thermosensitive replication origin was used as a donor of the ISS1 transposon. To enable ISS1 transposition, the bacterial culture was incubated at a restrictive temperature as described in Materials and Methods section. Due to replicative ISS1 transposition into the random chromosomal site, the plasmid was incorporated between two duplicated ISS1 transposon sequences (Fig. 2). The resulting library contained 5.3 × 10⁹ clones, with the ISS1 transposition frequency of 0.29% (calculated as CFU on BHIE versus BHI).

Fig. 2.

Schematic representation of ISS1 transposition into the S. anginosus 980/01 chromosome. The S.anginosus chromosome is shown as a thick black line, while pGh9:ISS1 as a thin black line. Arrows indicate gene localisation.

The S. anginosus 980/01 mutant library validation

The ISS1 integration sites in 30 randomly chosen erythromycin-resistant clones from the ISS1 S. anginosus 980/01 mutant library were analysed. The sequencing results revealed that in 28 out of 30 clones, ISS1 was integrated into unique sites, whereas in two mutants (6.7%), ISS1 was detected at redundant genomic locations. Nevertheless, the library predominantly comprises unique clones, with 93.3% showing distinct integration sites (Table 1).

Table 1.

Transposon insertion sites in 30 mutants

No of mutant	Insertion site (bp)	Encoded function
1	51909	Phosphoenolpyruvate-dihydroxyacetone phosphotransferase (EC 2.7.1.121), subunit DhaM; DHA-specific IIA component
2	61149	Tagatose 1,5-bisphosphate aldolase (EC 4.1.2.40)
3	62441	Holliday junction DNA helicase RuvB
4	112778	Copper chaperone
5	250131	Glutamyl aminopeptidase (EC 3.4.11.7)
6	326163	Translation initiation factor 2
7	327112	Phosphate transport system regulatory protein PhoU
8	335194	hypothetical protein
9	339133	Glycyl-tRNA synthetase beta chain (EC 6.1.1.14)
10	467565	Permease/hypothetical protein
11*	486888	Cystathionine gamma-synthase (EC 2.5.1.48)
12*	486888	Cystathionine gamma-synthase (EC 2.5.1.48)
13	522974	Methionine ABC transporter ATP-binding protein
14	556391	thioesterase family protein
15	711094	PTS system, cellobiose-specific IIC component (EC 2.7.1.69)
16	921484	hypothetical protein
17	845413	Phosphate ABC transporter, periplasmic phosphate-binding protein PstS
18	968611	Maltose O-acetyltransferase (EC 2.3.1.79)
19	1061362	Late competence protein ComEC, DNA transport
20	986233	Signal recognition particle associated protein
21	1254429	Lipoteichoic acid synthase LtaS Type IIc
22	1322410	Fibronectin/fibrinogen-binding protein
23	1420899	Cationic amino acid transporter—APC Superfamily
24	1502700	Hydrolase (HAD superfamily)
25*	1536265	contains glycosyl transferase family 2 region
26*	1536265	contains glycosyl transferase family 2 region
27	1537722	hypothetical protein
28	1577977	Mobile element protein
29	1654917	PTS system, cellobiose-specific IIA component (EC 2.7.1.69)
30	1680876	Para-aminobenzoate synthase, amidotransferase component (EC 2.6.1.85)

The numbers of the redundant mutants are marked with an asterisk (*).

The S. anginosus 980/01 mutant library characteristics

For sequencing, three independent replicates of the ISS1 transposon mutant library (BHI_A, BHI_B, and BHI_C) were regrown and prepared for DNA extraction. After sequencing, each of them was analyzed using the Transit software with the Tn5gaps method³⁵.

Analysis revealed 132,000–175,000 single insertion sites of ISS1 in the genome, on average every 10.7–14.3 nt. This corresponds to a library saturation of 98% (1,796 disrupted genes of 1,825 annotated), with an average of 64.7 insertions per disrupted gene and a mean of 2,718 sequencing reads per gene (Fig. 3, Supplemental Files S1 and S2). The ISS1 insertion index calculated for each gene was a base to consider a given gene essential, non-essential, and non-conclusive (Supplemental File S1). For genes classified as essential in S. anginosus 980/01 statistical significance was assessed using a false discovery rate (FDR) correction, with a significance threshold of p < 0.05.

Fig. 3.

Location of all ISS1 transposon insertion sites mapped to the S. anginosus 980/01 genome. The outer rings in red and cyan show coding sequences on the (+) and (−) strands, respectively. The innermost black histogram displays the location of each transposon insertion with the height of each bar representing the detection frequency of each insertion. Genome coordinates are shown on the outermost ring.

Based on these parameters, 348 of 1825 (19.1%) of the S. anginosus 980/01 genes were classified as essential, 1446 (79.2%) were non-essential, while 30 (1.7%) were non-conclusive. The functional categories analysis of essential genes is presented in Fig. 3. The main category encompasses genes involved in translation, transcription, and cell wall biogenesis.

Reconstruction of metabolic pathways

Based on the whole-genome analysis of S. anginosus 980/01, metabolic pathways were reconstructed using the KEGG Mapper of Kyoto Encyclopedia of Genes and Genomes^36,37. They were compared with data for S. pyogenes M1T1 5448⁴² and S. agalactiae A909⁴³, other human pathogenic streptococci. Like S. anginosus 980/01, these strains were originally isolated from the blood of patients with sepsis. Notably, the study on S. pyogenes M1T1 5448 relied on the genome annotation of S. pyogenes MGAS5005, as the genome sequence of M1T1 5448 had not yet been published.

The comparison of KEGG data indicates that elements involved in DNA replication and repair, transcription, and translation, as well as the major metabolic pathways, are similar to those of S. pyogenes MGAS5005 and S. agalactiae A909. So, for ATP production, S. anginosus 980/01 relies on fermentation and substrate phosphorylation.

In predicted carbohydrate metabolism, due to the presence of specific genes, S. anginosus 980/01 can carry out the citrate cycle reactions facilitated by citrate synthase, aconitate hydratase, and isocitrate dehydrogenase. Those genes are absent in S. agalactiae A909 and S. pyogenes MGAS5005, but present in e.g., S. suis and S. mutans⁴⁴. However, these genes are not essential for S. anginosus 980/01 under the tested growth conditions.

Comparison of essential gene sets of S. anginosus 980/01 with S. pyogenes and S. agalactiae

To identify conserved and species-specific essential genes, we compared the S. anginosus 980/01 dataset with published essential gene sets from other human pathogenic streptococci, S. pyogenes M1T1 5448⁴² and S. agalactiae A909⁴³. Like S. anginosus 980/01, these strains were originally isolated from the blood of patients with sepsis.

The analysis was conducted using data provided by Le Breton et al. and Hooven et al.^18,29. S. pyogenes, and S. agalactiae as well as S. anginosus 980/01 were cultivated at 37ºC, under nutrient-rich growth conditions. S. pyogenes was grown in Todd-Hewitt broth supplemented with 0.2% yeast extract¹⁸, while S. anginosus 980/01—in Brain Heart Infusion broth. In contrast, S. agalactiae was grown on Tryptic Soy Agar²⁹, a comparatively less nutrient-rich medium than the other two.

Genes from S. pyogenes and S. agalactiae were categorized by the authors as essential, critical, non-essential, or not-defined/inconclusive. Orthologs were identified using OrthoFinder and comparisons were made between: a/S. anginosus 980/01 and S. pyogenes MGAS5005, b/ S. anginosus 980/01 and S. agalactiae A909, and c/ S. pyogenes MGAS5005 and S. agalactiae A909.

The essentiality classifications of each orthologous pair were compared, excluding genes with the essentiality unknown or inconclusive. The results are summarized in Fig. 4a and Supplemental File S1.

Fig. 4.

Functional and comparative analysis of essential genes in S. anginosus and related streptococci. (a/) Functional classification of 348 essential genes, including 40 strain-specific, in S. anginosus 980/01. (b/) Comparison of essential genes identified in S. anginosus 980/01, S. pyogenes MGAS5005, and S. agalactiae A909. The Venn diagram illustrates shared and unique essential genes. The bottom-most value reflect numbers of genes conserved across all three species. (c/) Top enriched KEGG pathways among essential genes identified by TnSeq data analysis. Bars represent the number of essential genes mapped to each KEGG pathway. Only the top 5 pathways (based on adjusted p-value < 0.05) are shown. Enrichment significance was calculated using Fisher’s exact test with Bonferroni correction.

The proportion of essential genes in S. anginosus (19%) is comparable to that in S. pyogenes MGAS5005 (298 essential genes of 1866; 16%)¹⁸ and S. agalactiae A909 (317 of 2136; 15%)²⁹. Among the genes classified as essential in all three species, 178 (53.5%) were shared; between S. anginosus and S. pyogenes it was 59.9%, and 62.4% between S. anginosus and S. agalactiae (Fig. 4b). These 178 genes essential for 3 strains were enriched in categories such as translation (53 genes), cell wall biogenesis (19), lipid transport and metabolism (16), replication and amino acid transport and metabolism (13 each), carbohydrate transport and metabolism (12), coenzyme transport and metabolism (9), transcription (8), and energy production and conversion (6). Other functional groups included posttranslational modification, protein turnover, and chaperones, inorganic ion transport and metabolism, intracellular trafficking and nucleotide transport and metabolism (5 genes each), cell division, secretion, and vesicular transport (4 each), as well as hypothetical proteins (3) and signal transduction mechanisms (2) (Fig. 4c; Supplemental File S1, worksheet tab “TnSeq KEGG enrichment”).

Interestingly, 8 genes encoding glycolytic pathway enzymes were essential in all three species. One gene encoding 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase was classified as non-conclusive in S. pyogenes MGAS5005¹⁸. The essential genes include eno and gapA, encoding enolase and glyceraldehyde 3-phosphate dehydrogenase, respectively. These enzymes not only have metabolic roles but also act as moonlighting proteins that bind host plasminogen and contribute to virulence in other streptococci^45,46. The subcellular localisation of these enzymes in S. anginosus 980/01 will be investigated further.

Although the bulk of genes essential under the tested laboratory conditions are common for the three strains, we identified 40 essential genes unique to S. anginosus 980/01. Most of these genes encode transcription–related proteins and hypothetical proteins with unknown function (Fig. 4a, Supplemental File S3). However, this group also includes:

• sodA, encoding superoxide dismutase, a key enzyme in the oxygen defence system,

• ssaC encoding the substrate-binding component of a manganese ABC transporter (SsaABC), essential for manganese acquisition, oxidative stress resistance, and full virulence in streptococci^47,48,

• purB, encoding adenylosuccinate lyase, catalyzing key reactions in the de novo purine biosynthesis pathway, is indispensable for nucleotide production and cellular proliferation,

• clpC and clpX, encoding ATPase subunits of the Clp protease complex, as well as mecA, encoding an adaptor enabling ClpC activity, and ctsR, a regulator of the clp genes; all genes shown to be involved in adaptive response in B. subtilis^49,50.

• ldh, encoding L-lactate dehydrogenase, which catalyzes the conversion of pyruvate to lactate while regenerating NAD⁺, a critical step for maintaining glycolytic flux under anaerobic or microaerophilic conditions.

Discussion

This study provides the genome-wide map of essential genes in S. anginosus 980/01, an invasive strain isolated from sepsis, with the use of high–density transposon mutagenesis (TraDIS) approach. Due to considerable genetic and phenotypic heterogeneity observed among S. anginosus strains^8,10, there is a clear need for molecular characterization to better understand both conserved and strain-specific requirements of this opportunistic pathogen. Despite growing recognition of the clinical relevance of the S. anginosus and other members of the S. anginosus group, functional genomic data for this species remain limited. Our findings contribute to filling this gap. The phylogenetic placement of strain 980/01 within the sub-lineage of S. anginosus subsp. anginosus group supports the relevance of its essential gene set. These genes likely represent conserved functions across related isolates, highlighting their potential broader significance.

We identified 348 genes essential for growth under laboratory nutrient-rich, CO₂-enriched aerobic conditions. Many of these genes are conserved across S. pyogenes MGAS5005¹⁸ and S. agalactiae A909²⁹, reflecting core metabolic and cellular processes essential to streptococcal viability. Although compositional differences between media used in these studies may influence the essentiality of individual genes, the observed overlap in core essential genes indicates conservation of fundamental cellular functions. Notably, 40 genes were uniquely essential in S. anginosus 980/01, highlighting species-specific physiological traits and potential therapeutic targets.

Among these, sodA, encoding a sole superoxide dismutase in S. anginosus, was identified. This correlates with the need for a CO₂-enriched atmosphere to support colony formation, suggesting limited oxidative stress tolerance. A similar dependence on sodA has been observed in S. thermophilus, where its disruption leads to oxygen hypersensitivity which can be rescued by manganese ions⁵¹.

Also uniquely essential was ssaC, encoding the substrate-binding component of the SsaACB, the manganese transporter. Manganese is a key cofactor for enzymes involved in oxidative stress defence, including superoxide dismutase, however, it plays a role beyond catalytic functions (for review⁵²). The essentiality of ssaC under nutrient-rich conditions highlights low manganese bioavailability, necessitating high-affinity uptake systems⁵¹.

Genes involved in protein quality control, clpC and clpX encoding ATPases of the Clp protease complex, and together with mecA and a putative ctsR gene were also found to be essential in S. anginosus 980/01, and dispensable in S. pyogenes MGAS5005 and S. agalactiae A909. In S. pneumoniae R6, clpX, but not clpC, is also essential⁵³. In many bacteria, impairment of the Clp protease has pleiotropic effects on cell wall composition or virulence (for review⁵⁴). Notably, in B. subtilis, all four genes are involved in the heat shock response^49,50. Given that the ISS1-transposon mutant library in this study was generated at 40 °C, a temperature that may trigger proteotoxic stress, the observed essentiality could reflect a condition-dependent requirement for these genes.

We also found purB, encoding adenylosuccinate lyase, to be essential. This indicates a dependence on de novo purine synthesis, which may reflect adaptation to purine-limited environments such as mucosal surfaces or abscesses⁵⁵. Notably, S. agalactiae A909 encodes two purB homologs, potentially providing functional redundancy and explaining its non-essentiality in that species. Given the limited data on purine availability in such niches, purB may represent a metabolic vulnerability worth further investigation.

Finally, ldh, encoding L-lactate dehydrogenase, was also uniquely essential in S. anginosus 980/01, reflecting its reliance on homolactic fermentation for NAD⁺ regeneration. In contrast, S. pyogenes and S. agalactiae, can employ alternative fermentation pathway as mixed-acids fermentation⁴⁴.

In summary, this study identifies essential genes in S. anginosus 980/01, providing insight into its metabolic constraints and potential vulnerabilities. These findings enhance our understanding of S. anginosus physiology, particularly within S. anginosus subsp anginosus group, and establish the basis for future research on its pathogenic potential. Comparative analysis across multiple bloodstream isolates could help identify conserved targets for specific drug discovery.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AK performed the research, analyzed the data, and wrote the manuscript; KŻ performed the research; JG analyzed the data, and wrote the manuscript; RG analyzed the data; IKZ supervised the project, analyzed the data, and wrote the manuscript.

Funding

The study was funded by the National Science Centre, Poland (grant number 2018/29/B/NZ6/00624).

Data availability

The *S. anginosus* 980/01 genome sequence is available under GenBank accession number: CP183189. The datasets presented in this study have been deposited in the BioProject database under accession number PRJNA1228600.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The strain was collected by the National Institute of Medicines (Warsaw, Poland) as continuous surveillance in accordance with the World Health Medical Association 1966 Declaration of Helsinki and the EU rules of Good Clinical Practice.