Skip to main content
NCBI home page
Applied Microbiology and Biotechnology logoLink to Applied Microbiology and Biotechnology
. 2026 May 23;110:219. doi: 10.1007/s00253-026-13870-6

Neisseria subflava Type 6 Secretion System competition with bacterial and fungal species

Alan Calder 1, Lori A S Snyder 1,✉
PMCID: PMC13375697  PMID: 42176003

Abstract

Abstract

The aim of this study was to assess whether a Type VI Secretion System (T6SS) present in the commensal, non-pathogenic Neisseria subflava strain KU1003-01 plays a role in competition between these bacteria and other commensal and pathogenic Gram-negative microorganisms and the yeast Candida albicans. Using an overlap extension PCR strategy, the core T6SS gene encoding membrane protein TssM was knocked out for N. subflava strain KU1003-01. Co-culture competition assays were conducted with either the N. subflava or N. subflava tssM knock-out strains as the attackers and N. gonorrhoeae strain NCCP11945, N. subflava strain KU1003-02, or C. albicans strain 3153 as the targets. There was a significant difference in recoverable target cell CFUs observed following co-culture with the T6SS knock-out strain in comparison to the parent strain. TssM has been identified as critical to T6SS function for a number of species, and our findings indicate that the T6SS in N. subflava contributes to competition with these other species.

Key points

• Neisseria subflava Type 6 Secretion System is functional

• Neisseria subflava attacks other microbes of the same niche

• Neisseria subflava is competent for transformation

Supplementary information

The online version contains supplementary material available at 10.1007/s00253-026-13870-6.

Keywords: Type 6 Secretion System, Type VI Secretion System, T6SS, Toxin antitoxin system, Neisseria spp., Commensal Neisseria

Introduction

Type VI Secretion Systems (T6SS) are complex multi-protein systems that are estimated to be present in at least one quarter of all Gram-negative bacterial species (Bingle et al. 2008; Boyer et al. 2009), including Neisseria subflava and other non-pathogenic Neisseria spp. (Calder and Snyder 2023). Although it has been speculated that the T6SS may have evolved as a means to increase competitive fitness (Cianfanelli et al. 2016), T6SS can contribute to virulence for some pathogenic species (Hopf et al. 2014; Ma et al. 2014; Clemens et al. 2018). Other potential roles of the T6SS include host cell adhesion (Lertpiriyapong et al. 2012; Liu et al. 2015; Liaw et al. 2019), nutrient acquisition (Wang et al. 2015; Lin et al. 2017; Si et al. 2017; Li et al. 2022), motility (Montenegro et al. 2021), and biofilm formation (Kim et al. 2017). T6SSs are well known for contact-dependent toxin secretion; however, more recent studies have shown that T6SS effectors can also be secreted through mechanisms that are independent of cell-to-cell contact (Song et al. 2021).

It was once thought that T6SS could only act against Gram-negative species, due to the thick cell walls of Gram-positive bacterial species being considered unbreachable by the T6SS spike. It is now known that T6SS can act against Gram-positive bacteria (Le et al. 2021; Pei et al. 2022) as well as fungal species (Trunk et al. 2018, 2019; Luo et al. 2023). In regard to anti-fungal T6SS, Pseudomonas syringae was the first species reported to have a T6SS that acted against yeast cells (Haapalainen et al. 2012).

T6SSs are usually composed of 13 core proteins: TssA; TssB; TssC; TssD (Hcp); TssE; TssF; TssG; TssH (ClpV); TssI (VgrG); TssJ; TssK; TssL; and TssM (Bingle et al. 2008; Coulthurst 2013; Morgado and Vicente 2022). The core proteins are divided into three groups based on the secretion system subunit they co-assemble. TssJLM forms the membrane complex, TssAEFGK forms the baseplate complex, and TssBC forms the needle sheath of the injection apparatus (Yang et al. 2018). When fully assembled, T6SS forms peptidoglycan-anchored trans-envelope channels (Santin et al. 2019) that have similarities to inverted bacteriophage tails (Coulthurst 2013).

In a functioning T6SS, contraction of the TssBC sheath drives a nanotube structure formed of TssD (Hcp) “rings” (Ballister et al. 2008) topped with a TssI, a valine-glycine repeat (VgrG) “spike” protein (Alcoforado et al. 2015) into target cells. VgrG facilitates the delivery of effector proteins into target cells (Cianfanelli et al. 2016), and following delivery, the contracted TssBC sheaths are disassembled by the T6SS AAA + ATPase, TssH (ClpV) (Bönemann et al. 2009; Zoued et al. 2014).

The ability of the T6SS to kill competitor target cells has been demonstrated through mutation or knock-out of key T6SS genes; mutants have been shown to have reduced virulence or an inability to kill competitors (Custodio et al. 2021; Stietz et al. 2020; Wang et al. 2021). The effects of tssM mutants on T6SS function have been documented for a range of species that include Acidovorax citrulli (Fei et al. 2022; Pei et al. 2022), A. baumannii (Repizo et al. 2015; Li et al. 2019; Le et al. 2021), Burkholderia cenocepacia (Aubert et al. 2015), Campylobacter jejuni (Bleumink-Pluym et al. 2013), E. coli (de Pace et al. 2011), Pseudomonas aeruginosa (Lin et al. 2015), Ralstonia solanacearum (Zhang et al. 2012; Asolkar and Ramesh 2020), Vibrio cholerae (Stietz et al. 2020), and Neisseria cinerea (Custodio et al. 2021). For some species, tssM mutants have been used experimentally as T6SS-deficient controls (Fei et al. 2022).

TssM is a key structural component of the T6SS involved in forming the trans-envelope membrane complex (MC) (Felisberto-Rodrigues et al. 2011; Zoued et al. 2016) through associations with the inner membrane lipoprotein TssL and the outer membrane lipoprotein TssJ (Durand et al. 2015; Cianfanelli et al. 2016). Once formed, the MC docks with the T6SS baseplate complex (BC); the cytoplasmic domains of TssM and TssL, located at the base of the membrane complex, interact with the TssG/TssK and TssK/TssE baseplate subunits (Logger et al. 2016; Zoued et al. 2018; Liebl et al. 2019). In addition to TssM being required for production of the T6SS MC, this protein may have other roles including the formation of transient outer membrane pores (Durand et al. 2015) and providing ATPase activity required for assembly of the TssM-TssL-Hcp Complex (Ma et al. 2012).

When functioning as a contact-dependent secretion system, the T6SS “injects” toxins into neighbouring cells (Ben-Yaakov and Salomon 2019). In regard to Neisseria spp., a number of the commensal and pathogenic Neisseria spp. are specific to human hosts and therefore have the potential to interact with one another (Higashi et al. 2011; Baerentsen et al. 2022). Although Neisseria spp. were once considered to have their own niche environments, this being the oro-nasopharynx for the commensals and N. meningitidis (Johnson 1983; Uwamino et al. 2017; Weyand, 2017; Diallo et al. 2019; Dorey et al. 2019) and the genital tract for N. gonorrhoeae (Spinosa et al. 2007), N. subflava has been found to colonise genitourinary areas (Baraldes et al. 2000) as well as the gastrointestinal tract (Niikura et al. 2023). Similarly, over the past few decades, N. gonorrhoeae and N. meningitidis have been isolated from other host sites including the oropharynx, rectum and eye (Morris et al. 2006; Mitchell et al. 2008; Bissessor et al. 2015: Tao et al. 2016; Ladhani et al. 2020; Tsakalos et al. 2021; Butler et al. 2022).

In regard to C. albicans, like Neisseria spp., this can also colonise the oral cavity, genital areas, and gastrointestinal tract including the rectum (Kumamoto 2011; Shirvani et al. 2021). Neisseria spp. and C. albicans are both occasionally isolated from the same clinical sample and these species most likely exist together at specific anatomical sites. While this has been shown to be the case for C. albicans and N. gonorrhoeae (Hipp et al. 1975), this is also likely to be the case for N. subflava and C. albicans. Unlike Candida, Neisseria spp. are considered to be primary colonisers within the oral cavity (Sedghi et al. 2021) and Candida may require the presence of established, primary colonisers in order to adhere (Sultan et al. 2018).

Social interactions between bacterial species within a niche are a common phenomenon (Copeland et al. 2024) and horizontal gene transfer (HGT) between species is a form of social cooperation (Lee et al. 2022). T6SSs are known to have a diverse range of effectors (Smith et al. 2025) and genes encoding these components have been identified on mobile as well as integrative conjugative elements (Coyne et al. 2016; García-Bayona et al. 2021). Auxiliary T6SS gene clusters are thought to be horizontally shared (Jana et al. 2022), and although diversification of T6SS toxin arrays may help prevent the evolution of resistance in competitors (Smith et al. 2025), diversification can also lead to social incompatibilities within a niche. It has been shown that social compatibility between parent strains and offspring can be abolished upon introduction of unique T6SS toxins (Vassallo et al. 2020).

In this study, we generate a tssM knock-out strain of N. subflava strain KU1003-01 and demonstrate that the T6SS plays a role in competition against both commensal and pathogenic bacterial species as well as yeast. Although the antibacterial activity of T6SS was first described over a decade ago, to our knowledge, this is the first report documenting the role of the commensal N. subflava T6SS in competition with other bacterial and fungal species.

Methods

Strains and growth conditions

The cells used for this study were grown from a freezer stock of the original isolate. Neisseria subflava strain KU1003-01 and N. subflava strain KU1003-02 were first isolated in the Spring of 2012 following throat sampling of 64 student volunteers on three separate occasions (Calder et al. 2020). Neisseria gonorrhoeae strain NCCP11945 was received from the National Institute of Health, Korea Centres for Disease Control and Prevention (Chung et al. 2008). C. albicans strain 3153 was donated by Dr. Suzy Moody of Kingston University. All bacterial and fungal species were grown at 37 °C with 5% CO2 on GC agar (Oxoid) supplemented with Kellogg’s (Kellogg et al. 1963) and 5% Fe (NO3)3. For selection of T6SS knock-out mutants, the GC agar was supplemented with 100 µg/ml kanamycin (Sigma-Aldrich). To select for surviving target cells following competition, the growth medium was supplemented with 2 µg/ml ciprofloxacin (Sigma-Aldrich). N. subflava KU1003-01 is sensitive to ciprofloxacin, whilst the target cells were all resistant at this concentration.

Construction of N. subflava T6SS mutant

A splice overlap extension PCR (SOE PCR) (Horton et al. 1989; Wörmann et al. 2016) was used to create a tssM knock-out strain. The oligonucleotide primers were ordered from Sigma-Aldrich (Supplementary Table 1). Genomic DNA was extracted from the N. subflava strain KU1003-01 using a Gentra Puregene Kit (Qiagen). Using a Q5 High-Fidelity 2X Master Mix kit (New England Biolabs), primer pairs HA1 Fwd with HA1 Rev and HA2 Fwd with HA2 Rev were then used to amplify a 196 bp region (HA1) upstream and a 213 bp region downstream (HA2) of tssM. A third 851 bp fragment representing the coding sequence for the kanamycin resistance gene (kan) was amplified using primer pair Kan-Fwd and Kan-Rev using the 1221 bp EZ-Tn5™ < KAN-2 > Transposon (Lucigen) as a template. The HA1, HA2, and Kan PCR products were added to splice overlap extension (SOE) PCR as follows: HA1 and Kan were added along with primer pair HA1-Fwd and Kan-Rev to generate a 1014 bp fragment (HA1 + Kan). The HA1 + Kan and HA2 PCR products were then used as templates along with the primer pairs HA1-Fwd and HA2-Rev to yield the final 1188 bp SOE construct, HA1 + Kan + HA2. At all stages, PCR products were first sized on a 2% agarose E-Gel (Invitrogen), and bands of the expected size were excised and cleaned up using a Wizard® SV Gel and PCR Clean-Up System (Promega).

Transformation of N. subflava strain KU1003-01

The final PCR construct was introduced into N. subflava strain KU1003-01 using a variation of the method of Gunn and Stein (1996) as follows: 10 µl suspensions of piliated N. subflava strain KU1003-01 were spotted onto GC agar (Oxoid) supplemented with Kellogg’s supplement (Kellogg et al. 1963), 5% Fe(NO3)3, and magnesium chloride (Sigma-Aldrich) at a final concentration of 2mM. The spots were allowed to dry before the PCR construct was added at around 1 µg DNA per spot. The plates were incubated right side up at 37 °C with 5% CO2 for 5 h, after which, growth was scraped from the spots using a sterile loop and passed onto fresh GC agar containing kanamycin at 100 µg/ml.

Knockout confirmation using PCR

Following transformation, all kanamycin-resistant colonies of N. subflava strain KU1003-01 were first passed onto fresh GC agar (Oxoid) supplemented with Kellogg’s (Kellogg et al. 1963), 5% Fe(NO3)3, and 100 µg/ml kanamycin and incubated overnight at 37 °C in 5% CO2. The presence of kanamycin cassettes in the resistant isolates was first confirmed by PCR using primers HA1-Fwd and HA2-Rev following genomic DNA extraction using a Gentra Puregene Kit (Qiagen). Sizing of PCR products was carried out using the precast 2% E-Gel electrophoresis system (Invitrogen).

N. subflava strain KU1003-01 parent and knock-out strain sequencing

A chosen isolate identified as producing a PCR product of around 1188 bp, indicative of tssM knock-out, and the parent strain were used for genomic DNA extraction using the Gentra Puregene Kit (Qiagen). The parent and mutant genomic DNA were sent to MicrobesNG (Birmingham, UK) for Illumina whole genome sequencing and de novo read assembly. After assembly of the sequence data by MicrobesNG, the parent strain assembled into 70 contigs (https://www.ncbi.nlm.nih.gov/nuccore/JBQLII000000000) and the tssM mutant strain into 71 contigs (https://www.ncbi.nlm.nih.gov/nuccore/JBQLIJ000000000).

Bacterial and fungal competition assays

The competition assay was adapted from Murdoch et al. (2011) and is detailed as follows. Cells were taken from overnight growth on GC agar and suspended in sterile 1 × phosphate-buffered saline (PBS) (Sigma-Aldrich). The cell suspensions were then adjusted to an OD of 0.5 at 600 nm. A 1:1 (attacker: target) ratio was used for all competition assays, with the attackers being either the N. subflava KU1003-01 parent or tssM knock-out strains and the targets being N. gonorrhoeae strain NCCP11945, N. subflava strain KU1003-02, or C. albicans strain 3153. Using a 5-mm-diameter cork borer, rings were aseptically marked on the surface of the GC agar competition plates. Into each ring, 10 µl volumes of target cells were first spotted onto GC agar (Oxoid) supplemented with Kellogg’s (Kellogg et al. 1963), 5% Fe(NO3)3, and the spots were allowed to dry at 37 °C for 15 min before the addition of either parent or tssM knock-out strains of N. subflava KU1003-01. The co-culture spots were grown on GC agar at 37 °C in 5% CO2 for 24 h after which the spots were aseptically scraped from the plates and suspended in sterile 1 × PBS (Sigma-Aldrich). Surviving colony-forming units (CFUs) of target cells were determined by serial dilution and growth on GC media containing ciprofloxacin at 2 µg/ml.

Calculating recovered CFU/mL for target cells following competition

These were estimated using the formula CFU/mL = (number of colonies × DF)/V, where DF represents the dilution factor and V represents the volume inoculated in mL.

Statistical analysis

Unless stated, experiments were performed in triplicate, and the data were analysed using SPSS (version 28) with P-values of < 0.05 being considered significant. The competition data is presented as the arithmetic mean of replicates ± SD.

RNA-seq

RNA was extracted from the N. subflava strain KU1003-01 and the tssM mutant using the RNAprotect Bacteria Reagent kit (QIAGEN) using Protocols 6 and 7. RNA was stored at −80 °C. RNA integrity was determined using the Agilent 2100 Bioanalyzer with RNA 6000 Nano kit to produce an RNA Integrity Number (RIN). RNA was quantified using Qubit 2.0 Fluorometer with Qubit RNA Broad Range assay kits (Molecular Probes) according to the manufacturer’s instructions. RNA purity was assessed on a BioDrop Duo spectrophotometer at 230, 260, and 280 nm. RNA-seq was conducted by Novogene. Reads were visualised against the genome sequence using Integrative Genomics Viewer (IGV; Robinson et al. 2011).

Results

Generation of N. subflava strain KU1003-01 lacking tssM

N. subflava strain KU1003-01 was transformed with a PCR construct designed to knock out tssM of the Type 6 Secretion System and replace it with a kanamycin resistance cassette (Fig. 1). Successful recovery of kanamycin-resistant colonies after transformation with the PCR construct demonstrates transformability of the commensal N. subflava KU1003-01. Antibiotic resistance testing identified the N. subflava KU1003-01 parental strain as having a kanamycin MIC of around 25 µg/ml. Following transformation, this increased to 100 µg/ml for the transformed strain. Transformation with the PCR construct was further confirmed via PCR, generating a product of the desired size to have knocked-out tssM and inserted the kanamycin resistance cassette. Growth curve analysis revealed no significant differences between the parent and mutant under normal growth conditions (data not presented), as has been observed for other species (Li et al. 2019; Song et al. 2020; De Oliveira et al. 2021; Montenegro Benavides et al. 2021).

Fig. 1.

Fig. 1

Open in a new tab

A A graphical representation of T6SS core cluster A located between positions 1,387,897 and 1,396,664 in N. subflava strain KU1003-01. Genes involved in membrane complex formation are shown in red, and the baseplate complex in gold. Hypothetical non-T6SS genes (hypo) are shown in purple. B A representation of the tssM knock-out showing insertion of the kanamycin resistance cassette. Regions with light-green shading joining A and B highlight sequences identified with 100% identity between the parent and tssM knock-out sequences. C A portion of the Illumina sequence data for the tssM knock-out strain with the sequence for the kanamycin resistance gene highlighted in blue and the first 337 nucleotides of tssA highlighted in gold. Start and stop codons for each of the genes are underlined and the location of the 11mer DUSvar1 sequence included in the PCR construct is shown in red text

Confirmation of the tssM knock-out using whole genome sequencing

To further confirm the generation of the mutant, Illumina sequencing was conducted on the parent and knock-out strains. According to the Illumina sequencing data for the knock-out strain (MicrobesNG), the PCR construct was integrated into the correct genomic location and successfully replaced tssM. To further investigate differences between the parent and knock-out genome sequences, as well as the MinION-enhanced genome sequence from N. subflava KU1003-01 (Calder and Snyder 2023), these were aligned using Mauve (Rissman et al. 2009), and SNP files were generated. No polymorphisms within any of the coding or non-coding regions could be identified between the parent strain, tssM knock-out strain, and the MinION-enhanced genome sequences, other than the intended, engineered knock-out sequence.

RNA-seq demonstrates expression of tssM in the parent strain but not in the mutant

Extracted RNA from the N. subflava strain KU1003-1 parent strain had a RIN of 9.5 and a concentration of 79 ng/µl. The tssM mutant RNA had a RIN of 7.1 and a concentration of 111 ng/µl. These are within the Novogene parameters of ≥ 6.0 and ≥ 50 ng/µl. RNA-seq QC from Novogene indicated 2.1 Gbp of clean bases in the parent and 3.6 Gbp in the mutant. The Q20 scores were 98.62% for the parent and 98.81% for the mutant. There were a total of 14,104,422 reads in the parent strain, of which 13,753,003 mapped to the N. subflava KU1003-01 reference genome sequence, a mapping rate of 97.51%. In the mutant, there were 23,710,014 reads in total and 23,298,097 mapped to the genome sequence, a rate of 98.26%. Gene expression analysis showed that the tssM gene is expressed in the parent strain, but not in the mutant (Fig. 2; Table 1). Table 1 shows RNA-seq expression data for the core T6SS cluster.

Fig. 2.

Fig. 2

Open in a new tab

Output from the Integrative Genomics Viewer (IGV; Robinson et al. 2011) showing all RNA-seq reads mapped to the parent (WT) and tssM knock-out (KO) of N. subflava KU1003-01. Reads are shown in grey, here illustrating that there is no expression of tssM in the knock-out mutant strain

Table 1.

RNA-seq data for the core T6SS cluster in N. subflava KU1003-01 parent (WT) and tssM mutant (KO)

Gene Read counts FPKM1 KO vs WT
Name Start End Strand WT KO WT KO log2 FC2 P-value padj
vgrG1 1018014 1020497 − 299 1512 40.7032 95.1925 1.24 0.004 0.040
clpV 1020642 1023455 − 23 84 2.7638 4.6683 0.77 0.175 0.428
hcp 1023527 1024015 − 6 3 4.1491 0.9594 −2.04 0.144 0.378
tagL 1024086 1025714 − 4 13 0.8303 1.2480 0.59 0.653 0.846
tssL 1025732 1026394 − 3 12 1.5301 2.8305 0.88 0.624 0.836
tssK 1026396 1027811 − 6 27 1.4328 2.9820 1.06 0.171 0.419
tssB 1027902 1028402  +  7 19 4.7246 5.9309 0.34 0.732 0.893
tssC 1028441 1029976  +  38 195 8.3657 19.8539 1.26 0.010 0.076
paar 1030029 1030292 − 264 665 338.1492 393.9310 0.23 0.586 0.816
tssM 1387897 1391172  +  3628 294 374.4827 14.0348 −4.72 1.77E−22 4.35E−19
tssA 1391175 1392797  +  1579 4156 328.9819 400.4603 0.30 0.473 0.734
tssF 1392807 1394573  +  2059 4160 394.0290 368.1792 −0.08 0.838 0.946
tssG 1394537 1395646  +  1053 3225 320.7848 454.3695 0.52 0.214 0.475
tssJ 1395666 1396205  +  1072 1915 671.2889 554.5970 −0.26 0.528 0.773
tssE 1396445 1396618  +  370 511 719.0530 459.2764 −0.63 0.136 0.367
Open in a new tab

1FPKM, fragments per kilobase of transcript sequence per million base pair sequenced. Normalised read counts

2log2 FC, log2 fold change

Data provided by Novogene

TssM is a known key structural protein that is critical to T6SS assembly and function (Felisberto-Rodrigues et al. 2011). Even when other core T6SS genes are expressed, without TssM, the secretion system will not form (Custodio et al. 2021). Numerous other researchers have shown that without TssM, Hcp secretion is diminished (Bleumink-Pluym et al. 2013) and the TssBC sheath does not assemble (Liebl et al. 2019; Stietz et al. 2020). TssM knock-outs have been shown to be less able to kill prey species (Pei et al. 2022; Fei et al. 2022; Repizo et al. 2015; Li et al. 2019; Aubert et al. 2015; Bleumink-Pluym et al. 2013; de Pace et al. 2011; Lin et al. 2015; Zhang et al. 2012; Asolkar and Ramesh, 2020; Stietz et al. 2020; Custodio et al. 2021).

Demonstration of the function of the Type 6 Secretion System in competition against other microbes

The parent and knock-out strains were used as attacker strains in competition assays versus the target bacterial pathogen N. gonorrhoeae, another strain of N. subflava that was isolated from the same host niche (KU1003-02), and the fungal pathogen C. albicans. Significant differences are observable in recovered target cell CFUs following co-culture with either the N. subflava strain KU1003-01 parent or T6SS knock-out strains (Fig. 3). Greater killing by the parental strain with its intact T6SS demonstrates that this system plays a role in competition between N. subflava strain KU1003-01 and other microbes of the nasopharyngeal niche. Demonstration of competition between the knock-out attacker and the target microbes suggests there may be other systems involved as well.

Fig. 3.

Fig. 3

Open in a new tab

Surviving CFU/ml for N. gonorrhoeae strain NCCP11945 (Green), N. subflava strain KU1003-02 (Yellow), and C. albicans strain 3153 (Purple) following competition with either the N. subflava KU1003-01 parent (WT) or tssM knock-out (KO) attacker strains. Assays were performed in triplicate on three separate occasions (n = 9) with each assay triplicate including no-competition controls (NC) containing only target cells. Mean values are represented by bars within each data point cluster. Data from the experimental replicates were analysed using an unpaired two-tailed Student’s t-test for pairwise comparison with the calculated P-values shown on the graph

Discussion

N. subflava strain KU1003-01 is competent for transformation

There are only a few published studies that have confirmed commensal Neisseria spp. including Neisseria elongata, Neisseria mucosa, Neisseria sicca, Neisseria cinerea, Neisseria musculi, and Neisseria macaque take up and transform exogenous DNA (Gunn and Stein 1996; Dillard 2011; Harris-Jones et al. 2024). Most Neisseria spp. transformation investigations carried out to date have focussed on the role of the Type IV pilus and acquisition of antimicrobial resistance factors by the pathogens from commensal Neisseria spp. (Bowler et al. 1994; Higashi et al. 2011; Chen et al. 2020; Manoharan-Basil et al. 2021).

For Neisseria gonorrhoeae, only piliated cells are naturally transformable (Dillard and Chan 2024) and functioning pili (Fussenegger et al. 1997; Wolfgang et al. 1998) as well as the presence of short, conserved DNA Uptake Sequences (DUS) of between 10 and 12 nt in exogenous DNA enhance the likelihood for its uptake and transformation (Goodman and Scocca 1988; Ambur et al. 2007; Duffin and Seifert 2010; Hepp et al. 2016). The overall dominant DUS type within the genome sequences of the pathogens is known as “classical” DUS or AT-DUS and consists of ATGCCGTCTGAA (Berry et al. 2013; Frye et al. 2013). For Neisseria subflava strain KU1003-01, as well as other N. subflava biovar flavescens, and N. elongata, the overall dominant type is known as DUSvar1 or AG-DUS that consists of AGGCCGTCTGAA. For N. mucosa and N. sicca, the dominant type is known as DUSvar2 or AG-mucDUS and consists of AGGTCGTCTGAA (Berry et al. 2013; Frye et al. 2013). Copies of DUS associated with penA in N. subflava have been used to enhance the DNA uptake of the region into N. gonorrhoeae (Kanesaka et al. 2022). In this case, when the penA gene was used without DUS in the donor DNA, no transformants were obtained, emphasizing the need for DUS.

Linear DNA cannot be used to transform most bacteria, and constructs inserted into circular plasmids are usually introduced to recipient cells using methods like electroporation (Heap et al. 2012). Unlike electroporation or transformation methods that are carried out in liquid culture, piliated N. gonorrhoeae are more efficient at taking up DNA from purified PCR products or linearised plasmids added to solid media at high concentrations (Dillard and Chan 2024). Under situations where DUS are lacking or where large plasmids are to be introduced, other methods of transformation can be used and protocols used for the transformation of both commensal and pathogenic Neisseria spp. have been documented (Dillard 2011; Dillard and Chan 2024).

All Neisseria spp. are considered naturally competent for transformation; however, the commensals are documented as less transformable than N. gonorrhoeae under standard laboratory conditions (Dillard 2011). The commensals are however thought to have similar requirements to the pathogens for DNA uptake and transformation (Berry et al. 2013). Based on available evidence that the DUS enhances DNA uptake and transformation (Goodman and Scocca 1988; Ambur et al. 2007; Duffin and Seifert 2010; Hepp et al. 2016; Berry et al. 2013; Frye et al. 2013; Kanesaka et al. 2022), the PCR construct for the tssM knock-out strain includes a commensal DUSvar1 (Fig. 1). Successful recovery of kanamycin-resistant colonies that generated the expected PCR product size demonstrates that N. subflava strain KU1003-01 is naturally competent for transformation as are other Neisseria spp.

Illumina whole genome sequencing for the tssM parental and knock-out strains

Active T6SS can have fitness effects that vary depending on species (Salomon 2016; Kostiuk et al. 2021; Li et al. 2021), and studies investigating knockouts for systems other than the T6SS have shown that where gene knockouts cause alterations to overall fitness, bacteria can adapt and evolve compensatory mutations (Hottes et al., 2013; McCloskey et al. 2018; Patel and Matange 2021). While mutations that alter regulatory mechanisms can occur quickly, other mutations occur over longer timescales (Carroll and Marx 2013; Patel and Matange 2021). Few genetic complementation tools exist for Neisseria spp., and all were developed for N. gonorrhoeae (Wuckelt et al. 2024; Ramsey et al. 2012). Due to the incompatibility of complementation plasmids, a few chromosomal sites in N. gonorrhoeae have been used for gene complementation following knock-out (Ramsey et al. 2012); however, these sites have not yet been characterised for N. subflava (Davey and Valdivia 2020). It was therefore important that whole genome sequencing of the parental and knock-out strains was conducted and compared to one another and to the original N. subflava strain KU1003-01 complete circular genome generated with MinION and Illumina (Calder and Snyder 2023). With no changes noted between the sequences, further analyses of the parent and T6SS knock-out could be investigated.

The T6SS of N. subflava strain KU1003-01 contributes to competition with other bacterial as well as fungal species

The T6SS contributes to competition between N. subflava strain KU1003-01 and the pathogen N. gonorrhoeae strain NCCP11945 as well as the commensal, N. subflava strain KU1003-02, and the yeast C. albicans strain 3153 (Fig. 3). These were specifically chosen for the competition assay due to their ability to colonise the human throat. N. subflava KU1003-01 was isolated from a throat swab of a healthy volunteer (Calder et al. 2020). N. subflava KU1003-02 came from the same sample of the same volunteer. Pathogens that may colonise the pharynx include N. gonorrhoeae (Takahashi et al. 2008) and C. albicans (Bertolini et al. 2021). Therefore, these three are ideal prey for the competition assay as they are able to colonise the same niche.

For Neisseria spp., positioning within mixed communities can be influenced by type IV pili, and target species or strains may segregate from their attackers, moving to the expanding edge of colonies grown on solid media to improve their chances of survival (Oldewurtel et al. 2015; Zöllner et al. 2017; Custodio et al. 2021). In regard to our competition assays, attacker and target cells were inoculated into defined areas on the competition plates at concentrations suitable to produce a “lawn growth”. The addition of target cells to the plates before the N. subflava KU1003-01 attacker strains was done to prevent separation of the species into microcolonies, as seen when competition spots were inoculated using lower cell concentrations; this also limited the possibility of target cells “escaping” to the outer boundaries of the competition spots. Examples of competition spots both with and without defined 5-mm boundaries as well as a competition spot consisting of an attacker N. subflava KU1003-01 and a target N. gonorrhoeae inoculated from a 1:1 mixture containing cells at low concentrations are shown in Supplementary Fig. 1.

In regard to co-culture incubation times, these vary across different studies. For example, for this study, competition spots consisting of attacker and target cells were grown for a period of 24 h. This is in contrast to harvesting competition spots after only 4 h for N. cinerea (Custodio et al. 2021). For reference strains of N. gonorrhoeae, lag phases have been shown to last up to 4 h, and a steep decline in growth is seen only after 36 h (Foerster et al. 2016). Allowing competition between the attacker and target cells to proceed over a longer time period eliminated difficulties in effectively recovering cells from the competition spots after a short period of growth. In addition, while the i3 type T6SS in N. cinerea is thought to be constitutively active (Custodio et al. 2021), the phase of growth where the i2 type T6SS (T6SS-A) in N. subflava strain KU1003-01 (Calder and Snyder 2023) is most active is not yet known.

The competition environment is artificial, and it is not likely that N. subflava strain KU1003-01 would interact in the same way naturally with other strains of N. subflava, N. gonorrhoeae, or C. albicans. As a contact-dependent system, the T6SS has a short range with activity that can become self-limiting. Generally, it is thought that during competition, dead target cells can accumulate and form an interface that can prevent further attacks (Smith et al. 2020). It is possible this may be a contributing factor to any variations seen in surviving target cell CFUs across the assays.

Competition may be multifactorial

The data from the competition assays suggest the T6SS N. subflava strain KU1003-01 is involved in competition with N. gonorrhoeae, other N. subflava, and C. albicans (Fig. 3). The T6SS however may not be the only factor responsible for the reduction in recovered CFUs. While a significant difference exists in recovered target cell CFU following competition with either the T6SS parent or the tssM knock-out strains, a significant difference also exists between CFU recovered from the no-competition controls and the parent and knock-out competition spots. It is notable that T6SS investigations do not tend to report a no-competition control; this may therefore be a typical result for these systems, or it may be that there are other factors involved in N. subflava competition with other microorganisms.

Data from RNA-seq (Table 1) shows that some of the T6SS core genes are still transcribed (tssAEFGJ, paar, and vgrG1). This may account for the results (Fig. 3). The gene tssJ encodes a protein that is part of the membrane complex with TssM and TssL, whilst tssAEFG are parts of the baseplate complex with TssK. It may be that the continued expression of these genes creates a means for the effector proteins to target competitor cells, although this has not been seen in other species (de Pace et al. 2011; Zhang et al. 2012; Bleumink-Pluym et al. 2013; Aubert et al. 2015; Lin et al. 2015; Repizo et al. 2015; Li et al. 2019; Asolkar and Ramesh 2020; Stietz et al. 2020; Custodio et al. 2021; Le et al. 2021; Fei et al. 2022; Pei et al. 2022). It is also possible that the continued expression of vgrG genes in the core and elsewhere on the chromosome enables the partial function of effectors released from the cell during the natural process of autolysis known in this species (Lesher et al. 1977).

Competition between bacterial species is often multifactorial, and individual species are thought to use a range of mechanisms including combinations of both exploitative and interference mechanisms (Stubbendieck and Straight 2016). While exploitative competition can provide benefits through enhanced nutrient acquisition or efficient surface binding, interference competition mechanisms used by commensals usually involve the secretion of antimicrobial toxins (Abt and Pamer 2014; Khare and Tavazoie 2015).

In regard to possible alternative mechanisms used by N. subflava strain KU1003-01 for interference competition, these could include toxin secretion by a contact-dependent secretion system other than the T6SS or through toxin secretion into the extracellular space via a contact-independent secretion system (Hibbing et al. 2010; Blanchard et al. 2014; Bauer et al. 2018; Ramamoorthy et al. 2024; Song et al. 2024).

One example of diffusible contact-independent toxins is bacteriocins (Sharp et al. 2017; Darbandi et al. 2022), and early studies into bacteriocins produced by Neisseria spp. indicated that some strains of N. gonorrhoeae produced gonocins (Flynn and McEntegart 1972; Lawton et al. 1976) and N. meningitidis produced meningocins; both of these bacteriocin types were found to inhibit growth of other bacterial species (Kingsbury et al. 1966; Allunans et al. 2008). Although most bacterial species are thought to be able to produce at least one bacteriocin type (Sharp et al. 2017; Darbandi et al. 2022), it is not thought that N. subflava strain KU1003-01 secretes diffusible toxins for competition. Using a simple “spot on the lawn” assay (Van Reenen et al. 1998; Brown et al. 2011), N. subflava strain KU1003-01 did not produce zones of inhibition on a lawn of N. gonorrhoeae (Supplementary Fig. 1).

Genes encoding a putative Type V Secretion System (T5SS) are present in N. subflava strain KU1003-01; like the T6SS, these are also contact-dependent inhibition (CDI) systems (Fan et al. 2016) and known to regulate growth of neighbouring cells (Jamet et al. 2015). The inhibitory effect of CDIs can be attributed to C-terminal toxin domains of CdiA (CdiA-CT) (Ikryannikova et al. 2020), and for the N. subflava strain KU1003-01, a putative CdiA, Ntox50 C-terminal domain protein is encoded between positions 2,165,738 and 2,165,866. The immunity protein (CdiI) is encoded between positions 2,166,228 and 2,166,527.

CDI systems are also present in N. gonorrhoeae strain NCCP11945 (Jamet et al. 2015) as well as other strains of N. meningitidis and N. gonorrhoeae. For N. meningitidis, CDIs are involved in competition and niche adaptation (Jamet and Nassif 2015; Jamet et al. 2015). For the Neisseria spp. pathogens, CDIs are arranged as polymorphic maf loci with some strains having multiple CDI-associated toxin/immunity genes (Tan et al. 2015).

Although toxin secretion within mixed bacterial populations can be induced through competition for space and nutrients (Cornforth and Foster 2013; Li et al. 2022), it is not clear from this work whether specific signals are responsible for T6SS regulation in N. subflava strain KU103-01. The T6SS-A in N. subflava is similar to the SecReT6 T6SS subtype i2 (Calder and Snyder 2023), and i2 T6SS types in other species are predicted to be regulated by specific signals, for example pH and oxidative stress for the i2-type T6SS in Klebsiella spp. (Storey et al. 2020). Signals for T6SS regulation in other species also include oxidative stress (DeShazer 2019; Storey et al. 2020), alterations to pH or temperature (Zhang et al. 2023), and plasma membrane perturbations (Basler et al. 2013; Lin et al. 2019).

Interactions between commensal Neisseria and other microorganisms

Both N. subflava and the Neisseria spp. pathogens readily autoaggregate (Ledder et al. 2008; Bonazzi et al. 2018); however, N. subflava does not co-aggregate readily with other oral bacterial species (Ledder et al. 2008). In regard to N. subflava within mixed biofilm communities, these are usually found as “islands” within confluent high-biomass regions while other commensal Neisseria spp. appear dispersed (Palmer 2017).

Currently, there are only a limited number of studies published on the interactions between commensal and pathogenic Neisseria spp. as well as interactions between Neisseria spp. and fungi. While studies have focussed on the interactions between N. gonorrhoeae and Candida spp. (Hipp et al. 1975), interactions between commensal and pathogenic Neisseria spp. have only been visualised between N. elongata subspecies glycolytica (ATCC29315) and N. gonorrhoeae strain MS11. These two species have been shown to form intimate connections through their Type 4 pili (Higashi et al. 2011).

Conclusion

This is the first study to show that the N. subflava KU1003-01 T6SS previously identified through genomic analysis (Calder et al. 2020; Calder and Snyder 2023) is biologically active and involved in competition with other organisms that colonise the same niche. Future work will involve the analysis of effector protein genes and generation of knock-out mutants of these effector protein genes with the aim of finding out which effectors or combination of effectors are secreted by the T6SS of N. subflava KU1003-01 to target either bacterial or fungal cells. It may be possible that the neisserial T6SSs could be exploited in the future as a biotechnological means to control or prevent infections by antibiotic-resistant pathogenic Neisseria spp. and/or other pathogenic bacterial or fungal species.

Supplementary information

Below is the link to the electronic supplementary material.

ESM 1 (1.7MB, jpg)

(JPG 1.65 MB)

ESM 2 (1.6MB, jpg)

(JPG 1.63 MB)

ESM 3 (1.5MB, jpg)

(JPG 1.54 MB)

ESM 4 (2MB, jpg)

(JPG 1.96 MB)

ESM 5 (1.7MB, jpg)

(JPG 1.69 MB)

ESM 6 (1.6MB, jpg)

(JPG 1.60 MB)

ESM 7 (1.6MB, jpg)

(JPG 1.59 MB)

ESM 8 (310.6KB, pdf)

(PDF 310 KB)

Author contribution

A.C. data acquisition; A.C. & L.A.S.S. data analysis and interpretation, writing the manuscript.

Funding

This research is funded by the School of Life Sciences, Pharmacy, and Chemistry, Kingston University.

Data availability

All data is presented here with the exception of genome sequencing data, which is available via the accession information in the Methods.

Declarations

Ethical approval and consent to participate

Ethical approval for the collection of bacterial samples from human volunteers from the previous publication (Calder et al. 2020) was granted by the Kingston University ethics committee. Informed consent was obtained from all volunteers.

Conflict of interest

Lori A S Snyder is a guest editor of Applied Microbiology and Biotechnology and was not involved in the peer review process or the editorial decision-making for this manuscript. Alan Calder declares no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Abt MC, Pamer EG (2014) Commensal bacteria mediated defenses against pathogens. Curr Opin Immunol 29(1):16–22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Diniz AJ, Liu YC, Coulthurst SJ (2015) Molecular weaponry: diverse effectors delivered by the type VI secretion system. Cell Microbiol 17(12):1742–1751 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allunans J, Kristiansen KI, Assalkhou R, Bjørås M (2008May 21) Bacteriocins (meningocins) in Norwegian isolates of Neisseria meningitidis: possible role in the course of a meningococcal epidemic. APMIS 116(5):333–344 [DOI] [PubMed] [Google Scholar]
  4. Ambur OH, Frye SA, Tønjum T (2007) New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators. J Bacteriol 189(5):2077–2085 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Asolkar T, Ramesh R (2020) The involvement of the type six secretion system (T6SS) in the virulence of Ralstonia solanacearum on brinjal. 3 Biotech 10(7):1–10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aubert DF, Hu S, Valvano MA (2015) Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia. Microbiology (United Kingdom) 161(11):2161–2173 [DOI] [PubMed] [Google Scholar]
  7. Baerentsen R, Tang CM, Exley RM (2022) Et tu, Neisseria? Conflicts of interest between Neisseria species. Front Cell Infect Microbiol 12(June):1–13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ballister ER, Lai AH, Zuckermann RN, Cheng Y, Mougous JD (2008) In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc Natl Acad Sci U S A 105(10):3733–8 [DOI] [PMC free article] [PubMed]
  9. Baraldes MA, Domingo P, Barrio JL, Pericas R, Gurgui M, Vazquez G (2000) Meningitis due to Neisseria subflava: case report and review. Clin Infect Dis 30(3):615–617 [DOI] [PubMed] [Google Scholar]
  10. Basler M, Ho BT, Mekalanos JJ (2013) Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152(4):884–894 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bauer MA, Kainz K, Carmona-Gutierrez D, Madeo F (2018) Microbial wars: competition in ecological niches and within the microbiome. Microb Cell 5(5):215–219 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ben-Yaakov R, Salomon D (2019) The regulatory network of Vibrio parahaemolyticus type VI secretion system 1. Environ Microbiol 21:2248–2260 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Berry JL, Cehovin A, McDowell MA, Lea SM, Pelicic V (2013) Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species. PLoS Genet 9(12):e1004014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Bertolini M, Vazquez Munoz R, Archambault L, Shah S, Souza JGS, Costa RC, Thompson A, Zhou Y, Sobue T, Dongari-Bagtzoglou A (2021) Mucosal bacteria modulate Candida albicans virulence in oropharyngeal candidiasis. mBio 12(4):e0193721. 10.1128/mBio.01937-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Bingle LE, Bailey CM, Pallen MJ (2008) Type VI secretion: a beginner’s guide. Curr Opin Microbiol 11(1):3–8 [DOI] [PubMed] [Google Scholar]
  16. Bissessor M, Whiley DM, Lee DM, Snow AF, Fairley CK, Peel J, Bradshaw CS, Hocking JS, Lahra MM, Chem MY (2015) Detection of Neisseria gonorrhoeae isolates from tonsils and posterior oropharynx. J Clin Microbiol 53(11):3624–3626 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Blanchard AE, Celik V, Lu T (2014) Extinction, coexistence, and localized patterns of a bacterial population with contact-dependent inhibition. BMC Syst Biol 8(1):23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Bleumink-Pluym NMC, van Alphen LB, Bouwman LI, Wösten MMSM, van Putten JPM (2013) Identification of a functional Type VI secretion system in Campylobacter jejuni conferring capsule polysaccharide sensitive cytotoxicity. PLoS Pathog 9(5):16–18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Bonazzi D, Lo Schiavo V, Machata S, Djafer-Cherif I, Nivoit P, Manriquez V, Tanimoto H, Husson J, Henry N, Chaté H, Voituriez R, Duménil G (2018) Intermittent pili-mediated forces fluidize Neisseria meningitidis aggregates promoting vascular colonization. Cell 174(1):143-155.e16 [DOI] [PubMed] [Google Scholar]
  20. Bönemann G, Pietrosiuk A, Diemand A, Zentgraf H, Mogk A (2009) Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J 28(4):315–325 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Bowler LD, Zhang QY, Riou JY, Spratt BG (1994) Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation. J Bacteriol 176(2):333–7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I (2009) Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics. 10.1186/1471-2164-10-104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Brown CT, Fishwick LK, Chokshi BM, Cuff MA, Jackson JM, Oglesby T, Rioux AT, Rodriguez E, Stupp GS, Trupp AH, Woollcombe-Clarke JS, Wright TN, Zaragoza WJ, Drew JC, Triplett EW, Nicholson WL (2011) Whole-genome sequencing and phenotypic analysis of Bacillus subtilis mutants following evolution under conditions of relaxed selection for sporulation. Appl Environ Microbiol 77(19):6867–6877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Butler L, Shah M, Cottom L, Winter AJ, Lockington D (2022) Five-year review of ocular Neisseria gonorrhoeae infections presenting to ophthalmology departments in Greater Glasgow & Clyde. Scotland. Eye 36(7):1442–1447 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Calder A, Menkiti CJ, Çağdaş A, Lisboa Santos J, Streich R, Wong A, Avini AH, Bojang E, Yogamanoharan K, Sivanesan N, Ali B, Ashrafi M, Issa A, Kaur T, Latif A, Mohamed HAS, Maqsood A, Tamang L, Swager E, Stringer AJ, Snyder LAS (2020) Virulence genes and previously unexplored gene clusters in four commensal Neisseria spp. isolated from the human throat expand the neisserial gene repertoire. Microb Genom 6(9):mgen000423 [DOI] [PMC free article] [PubMed]
  26. Calder A, Snyder LAS (2023) Diversity of the type VI secretion systems in the Neisseria spp. Microb Genom 9(4):mgen000986 [DOI] [PMC free article] [PubMed]
  27. Carroll SM, Marx CJ (2013) Evolution after introduction of a novel metabolic pathway consistently leads to restoration of wild-type physiology. PLoS Genet 9(4):1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Chen M, Zhang C, Zhang X, Chen M (2020) Meningococcal quinolone resistance originated from several commensal Neisseria species. Antimicrob Agents Chemother 64(2):1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Chung G, Yoo J, Oh H, Lee Y (2008) Complete genome sequence of Neisseria gonorrhoeae NCCP11945. J Bacteriol 190(17):6035–6036 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Cianfanelli FR, Monlezun L, Coulthurst SJ (2016) Aim, load, fire: the Type VI Secretion System, a bacterial nanoweapon. Trends Microbiol 24(1):51–62 [DOI] [PubMed] [Google Scholar]
  31. Clemens DL, Lee BY, Horwitz MA (2018) The Francisella Type VI Secretion System. Front Cell Infect Microbiol 8(APR):1–20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Copeland R, Zhang C, Hammer BK, Yunker PJ (2024) Spatial constraints and stochastic seeding subvert microbial arms race. PLoS Comput Biol 20(1):1–18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Cornforth DM, Foster KR (2013) Competition sensing: the social side of bacterial stress responses. Nat Rev Microbiol 11(4):285–293 [DOI] [PubMed] [Google Scholar]
  34. Coulthurst SJ (2013) The Type VI Secretion System - a widespread and versatile cell targeting system. Res Microbiol 164(6):640–654 [DOI] [PubMed] [Google Scholar]
  35. Coyne MJ, Roelofs KG, Comstock LE (2016) Type VI Secretion Systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements. BMC Genomics 17(1):1–21 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Custodio R, Ford RM, Ellison CJ, Liu G, Mickute G, Tang CM, Exley R (2021) Type VI secretion system killing by commensal Neisseria is influenced by expression of type four pili. Elife 10:1–31 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Darbandi A, Asadi A, Mahdizade Ari M, Ohadi E, Talebi M, Halaj Zadeh M, Emamie AD, Ghanavati R, Kakanj M (2022) Bacteriocins: properties and potential use as antimicrobials. J Clin Lab Anal 36(1):1–40 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Davey L, Valdivia RH (2020) Bacterial genetics and molecular pathogenesis in the age of high throughput DNA sequencing. Curr Opin Microbiol 54:59–66. 10.1016/j.mib.2020.01.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. De Oliveira AL, Barbieri NL, Newman DM, Young MM, Nolan LK, Logue CM (2021) Characterizing the Type 6 Secretion System (T6SS) and its role in the virulence of avian pathogenic Escherichia coli strain APECO18. Peer J 9:1–22 [DOI] [PMC free article] [PubMed]
  40. de Pace F, de Paiva JB, Nakazato G, Lancellotti M, Sirsili MP, Stehling EG, da Silveira WD, Sperandio V (2011) Characterization of icmF of the type VI secretion system in an avian pathogenic Escherichia coli (APEC) strain. Microbiology (n Y) 157(10):2954–2962 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. DeShazer D (2019) A novel contact-independent T6SS that maintains redox homeostasis via Zn2+ and Mn2+ acquisition is conserved in the Burkholderia pseudomallei complex. Microbiol Res 226(March):48–54 [DOI] [PubMed] [Google Scholar]
  42. Diallo K, MacLennan J, Harrison OB, Msefula C, Sow SO, Daugla DM, Johnson E, Trotter C, MacLennan CA, Parkhill J, Borrow R, Greenwood BM, Maiden MCJ (2019) Genomic characterization of novel Neisseria species. Sci Rep 9(1):13742 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Dillard JP (2011) Genetic manipulation of Neisseria gonorrhoeae. Curr Protoc Microbiol 23(1):1–8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Dillard JP, Chan JM (2024) Genetic manipulation of Neisseria gonorrhoeae and commensal Neisseria species. Curr Protoc 4(9):1–33 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Dorey RB, Theodosiou AA, Read RC, Jones CE (2019) The nonpathogenic commensal Neisseria: friends and foes in infectious disease. Curr Opin Infect Dis 32(5):490–496 [DOI] [PubMed] [Google Scholar]
  46. Duffin PM, Seifert HS (2010) DNA uptake sequence-mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent. J Bacteriol 192(17):4436–4444 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Durand E, Nguyen VS, Zoued A, Logger L, Péhau-Arnaudet G, Aschtgen MS, Spinelli S, Desmyter A, Bardiaux B, Dujeancourt A, Roussel A, Cambillau C, Cascales E, Fronzes R (2015) Biogenesis and structure of a type VI secretion membrane core complex. Nature 523(7562):555–560 [DOI] [PubMed] [Google Scholar]
  48. Fan E, Chauhan N, Gupta Udatha DBRK, Leo JC, Linke D (2016) Type V secretion systems in bacteria. Virulence Mechanisms of Bacterial Pathogens 2:305–335 [Google Scholar]
  49. Fei N, Ji W, Yang L, Yu C, Qiao P, Yan J, Guan W, Yang Y, Zhao T (2022) Hcp of the type VI secretion system (T6SS) in Acidovorax citrulli group II strain Aac5 has a dual role as a core structural protein and an effector protein in colonization, growth ability, competition, biofilm formation, and ferric iron absorption. Int J Mol Sci. 10.3390/ijms23179632 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Felisberto-Rodrigues C, Durand E, Aschtgen MS, Blangy S, Ortiz-Lombardia M, Douzi B, Cambillau C, Cascales E (2011) Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. PLoS Pathog 7(11):1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Flynn J, McEntegart MG (1972) Bacteriocins from Neisseria gonorrhoeae and their possible role in epidemiological studies. J Clin Pathol 25(1):60–61 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Foerster S, Unemo M, Hathaway LJ, Low N, Althaus CL (2016) Time-kill curve analysis and pharmacodynamic modelling for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae. BMC Microbiol 16(1):1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Frye SA, Nilsen M, Tønjum T, Ambur OH (2013) Dialects of the DNA uptake sequence in Neisseriaceae. PLoS Genet 9(4):e1003458 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Fussenegger M, Rudel T, Barten R, Ryll R, Meyer TF (1997) Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae–a review. Gene 192(1):125–134 [DOI] [PubMed] [Google Scholar]
  55. García-Bayona L, Coyne MJ, Comstock LE (2021) Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering. PLoS Genet 17(4):1–25 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Goodman SD, Scocca JJ (1988) Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 85(18):6982–6986 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Gunn JS, Stein DC (1996) Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant of Neisseria gonorrhoeae. Mol Gen Genet 251(5):509–17 [DOI] [PubMed]
  58. Haapalainen M, Mosorin H, Dorati F, Wu RF, Roine E, Taira S, Nissinen R, Mattinen L, Jackson R, Pirhonen M, Lin N-C (2012) Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for fitness for competition against bacteria and yeasts. J Bacteriol 194(18):4810–4822 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Harris-Jones TN, Chan JM, Hackett KT, Weyand NJ, Schaub RE, Dillard JP (2024) Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals. Infect Immun 92(5):e0000424 [DOI] [PMC free article] [PubMed]
  60. Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton NP (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res 40(8):e59–e59 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Hepp C, Gangel H, Henseler K, Günther N, Maier B (2016) Single-stranded DNA uptake during gonococcal transformation. J Bacteriol 198(18):2515–2523 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Hibbing ME, Fuqua C, Parsek MR, Peterson SB (2010) Bacterial competition: surviving and thriving in the microbial jungle. Nat Rev Microbiol 8(1):15–25 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Higashi DL, Biais N, Weyand NJ, Agellon A, Sisko JL, Brown LM, So M (2011) N. elongata produces type IV pili that mediate interspecies gene transfer with N. gonorrhoeae. PLoS One 6(6):e21373 [DOI] [PMC free article] [PubMed]
  64. Hipp SS, Lawton WD, Savage M, Gaafar HA (1975) Selective interaction of Neisseria gonorrhoeae and Candida albicans and its possible role in clinical specimens. J Clin Microbiol 1(5):476–477 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Hopf V, Göhler A, Eske-Pogodda K, Bast A, Steinmetz I, Breitbach K (2014) BPSS1504, a cluster 1 Type VI secretion gene, is involved in intracellular survival and virulence of Burkholderia pseudomallei. Infect Immun 82(5):2006–2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 10.1016/0378-1119(89)90359-4 [DOI] [PubMed] [Google Scholar]
  67. Hottes AK, Freddolino PL, Khare A, Donnell ZN, Liu JC, Tavazoie S (2013) Bacterial adaptation through loss of function. PLoS Genet. 10.1371/journal.pgen.1003617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Ikryannikova LN, Kurbatov LK, Gorokhovets N V., Zamyatnin AA (2020) Contact-dependent growth inhibition in bacteria: do not get too close!. Int J Mol Sci 21(21):7990 [DOI] [PMC free article] [PubMed]
  69. Jamet A, Nassif X (2015) Characterization of the Maf family of polymorphic toxins in pathogenic Neisseria species. Microb Cell 2(3):88–90 [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Jamet A, Jousset AB, Euphrasie D, Mukorako P, Boucharlat A, Ducousso A, Charbit A, Nassif X (2015) A new family of secreted toxins in pathogenic Neisseria species. PLoS Pathog 11(1):1–16 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Jana B, Keppel K, Fridman CM, Bosis E, Salomon D (2022) Multiple T6SSs, mobile auxiliary modules, and effectors revealed in a systematic analysis of the Vibrio parahaemolyticus pan-genome. mSystems 7(6):1–18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Johnson A (1983) The pathogenic potential of commensal species of Neisseria. J Clin Pathol 36(2):213–223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Kanesaka I, Ohno A, Katsuse AK, Takahashi H, Kobayashi I (2022) The emergence of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone by transfer of resistance from an oral Neisseria subflava reservoir of resistance. J Antimicrob Chemother 77(2):364–373 [DOI] [PubMed]
  74. Khare A, Tavazoie S (2015) Multifactorial competition and resistance in a two-species bacterial system. PLoS Genet 11(12):1–21 [DOI] [PMC free article] [PubMed]
  75. Kellogg DS, Peacock WL, Deacon WE, Brown L, Pirkle DI (1963) Neisseris gonorrhoeae: 1. virulence genetically linked to clonal variation. J Bacteriol 85:1274–1279 [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Kim J, Lee JY, Lee H, Choi JY, Kim DH, Wi YM, Peck KR, Ko KS (2017) Microbiological features and clinical impact of the Type VI secretion system (T6SS) in Acinetobacter baumannii isolates causing bacteremia. Virulence 8(7):1378–1389 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Kingsbury DT (1966) Bacteriocin production by strains of Neisseria meningitidis. J Bacteriol 91(5):1696–1699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Kostiuk B, Santoriello FJ, Diaz-Satizabal L, Bisaro F, Lee KJ, Dhody AN, Provenzano D, Unterweger D, Pukatzki S (2021) Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype. Nat Commun 12(1):1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Kumamoto CA (2011) Inflammation and gastrointestinal Candida colonization. Curr Opin Microbiol 14(4):386–391 [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Ladhani SN, Lucidarme J, Parikh SR, Campbell H, Borrow R, Ramsay ME (2020) Meningococcal disease and sexual transmission: urogenital and anorectal infections and invasive disease due to Neisseria meningitidis. Lancet 395(10240):1865–1877 [DOI] [PubMed] [Google Scholar]
  81. Lawton WD, Bellinger MA, Schling M, Gaafar HA (1976) Bacteriocin production by Neisseria gonorrhoeae. Antimicrob Agents Chemother 10(3):417–420 [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Le NH, Pinedo V, Lopez J, Cava F, Feldman MF (2021) Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector. Proc Natl Acad Sci U S A 118(40):6–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Ledder RG, Timperley AS, Friswell MK, Macfarlane S, McBain AJ (2008) Coaggregation between and among human intestinal and oral bacteria. FEMS Microbiol Ecol 66(3):630–636 [DOI] [PubMed] [Google Scholar]
  84. Lee IPA, Eldakar OT, Gogarten JP, Andam CP (2022) Bacterial cooperation through horizontal gene transfer. Trends Ecol Evol 37(3):223–232 [DOI] [PubMed] [Google Scholar]
  85. Lertpiriyapong K, Gamazon ER, Feng Y, Park DS, Pang J, Botka G, Graffam ME, Ge Z, Fox JG (2012) Campylobacter jejuni Type VI secretion system: roles in adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization. PLoS One. 10.1371/journal.pone.0042842 [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Lesher RJ, Bender GR, Marquis RE (1977) Bacteriolytic action of fluoride ions. Antimicrob Agents Chemother 12(3):339–45 [DOI] [PMC free article] [PubMed]
  87. Li C, Zhu L, Wang D, Wei Z, Hao X, Wang Z, Li T, Zhang L, Lu Z, Long M, Wang Y, Wei G, Shen X (2022) T6SS secretes an LPS-binding effector to recruit OMVs for exploitative competition and horizontal gene transfer. ISME J 16(2):500–510 [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Li J, Xie L, Qian S, Tang Y, Shen M, Li S, Wang J, Xiong L, Lu J, Zhong W (2021) A Type VI Secretion System facilitates fitness, homeostasis, and competitive advantages for environmental adaptability and efficient nicotine biodegradation. Appl Environ Microbiol 87(9):1–20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Li L, Wang YN, Jia HB, Wang P, Dong JF, Deng J, Lu F-M, Zou Q-H (2019) The Type VI secretion system protein AsaA in Acinetobacter baumannii is a periplasmic protein physically interacting with TssM and required for T6SS assembly. Sci Rep 9(1):1–10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Liaw J, Hong G, Davies C, Elmi A, Sima F, Stratakos A, Stef L, Pet I, Hachani A, Corcionivoschi N, Wren BW, Gundogdu O, Dorrell N (2019) The Campylobacter jejuni Type VI Secretion System enhances the oxidative stress response and host colonization. Front Microbiol 10(December):1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Liebl D, Robert-Genthon M, Job V, Cogoni V, Attrée I (2019) Baseplate component TssK and spatio-temporal assembly of T6SS in Pseudomonas aeruginosa. Front Microbiol 10(JULY):1–15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Lin J, Cheng J, Chen K, Guo C, Zhang W, Yang X, Ding W, Ma L, Wang Y, Shen X (2015) The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1. Front Cell Infect Microbiol 5(OCT):1–15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Lin L, Lezan E, Schmidt A, Basler M (2019) Abundance of bacterial Type VI Secretion System components measured by targeted proteomics. Nat Commun 10(1):1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Lin J, Zhang W, Cheng J, Yang X, Zhu K, Wang Y, Wei G, Qian P-Y, Luo Z-Q, Shen X (2017) A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition. Nat Commun 8:1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Liu L, Hao S, Lan R, Wang G, Xiao D, Sun H, Xu J (2015) The Type VI Secretion System modulates flagellar gene expression and secretion in Citrobacter freundii and contributes to adhesion and cytotoxicity to host cells. Infect Immun 83(7):2596–2604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Logger L, Aschtgen MS, Guérin M, Cascales E, Durand E (2016) Molecular dissection of the interface between the Type VI Secretion TssM cytoplasmic domain and the TssG baseplate component. J Mol Biol 428(22):4424–4437 [DOI] [PubMed] [Google Scholar]
  97. Luo J, Chu X, Jie J, Sun Y, Guan Q, Li D, Luo Z-Q, Song L (2023) Acinetobacter baumannii kills fungi via a Type VI DNase effector. mBio 14(1):1–16 [DOI] [PMC free article] [PubMed] [Google Scholar]
  98. Ma J, Bao Y, Sun M, Dong W, Pan Z, Zhang W, Lu C, Yao H (2014) Two functional Type VI Secretion Systems in avian pathogenic Escherichia coli are involved in different pathogenic pathways. Infect Immun 82(9):3867–3879 [DOI] [PMC free article] [PubMed] [Google Scholar]
  99. Ma LS, Narberhaus F, Lai EM (2012) IcmF family protein TssM exhibits ATPase activity and energizes Type VI secretion. J Biol Chem 287(19):15610–15621 [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Manoharan-Basil SS, Laumen JGE, Van Dijck C, De Block T, De Baetselier I, Kenyon C (2021) Evidence of horizontal gene transfer of 50S ribosomal genes rplB, rplD, and rplY in Neisseria gonorrhoeae. Front Microbiol 12:1–17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  101. McCloskey D, Xu S, Sandberg TE, Brunk E, Hefner Y, Szubin R, Feist AM, Palsson BO (2018) Evolution of gene knockout strains of E. coli reveal regulatory architectures governed by metabolism. Nat Commun. 10.1038/s41467-018-06219-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Mitchell L, Coley K, Morgan J (2008) An unexpected increase in Neisseria meningitidis genital isolates among sexual health clinic attendees, Hamilton, New Zealand. Sex Transm Dis 35(5):469–471 [DOI] [PubMed] [Google Scholar]
  103. Montenegro Benavides NA, Alvarez B A, Arrieta-Ortiz ML, Rodriguez-R LM, Botero D, Tabima JF, Castiblanco L, Trujillo C, Restrepo S, Bernal A (2021) The Type VI secretion system of Xanthomonas phaseoli pv. manihotis is involved in virulence and in vitro motility. BMC Microbiol. 10.1186/s12866-020-02066-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  104. Morgado S, Vicente AC (2022) Diversity and distribution of Type VI secretion system gene clusters in bacterial plasmids. Sci Rep 12(1):1–9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Morris SR, Klausner JD, Buchbinder SP, Wheeler SL, Koblin B, Coates T, Chesney M, Colfax GN (2006) Prevalence and incidence of pharyngeal gonorrhea in a longitudinal sample of men who have sex with men: the EXPLORE study. Clin Infect Dis 43(10):1284–1289 [DOI] [PubMed] [Google Scholar]
  106. Murdoch SL, Trunk K, English G, Fritsch MJ, Pourkarimi E, Coulthurst SJ (2011) The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors. J Bacteriol 193(21):6057–6069 [DOI] [PMC free article] [PubMed] [Google Scholar]
  107. Niikura R, Hayakawa Y, Nagata N, Miyoshi-Akiayama T, Miyabayashi K, Tsuboi M, Suzuki N, Hata M, Arai J, Kurokawa K, Abe S, Uekura C, Miyoshi K, Ihara S, Hirata Y, Yamada A, Fujiwara H, Ushiku T, Woods SL, Worthley DL, Hatakeyama M, Han YW, Wang TC, Kawai T, Fujishiro M (2023) Non-Helicobacter pylori gastric microbiome modulates prooncogenic responses and is associated with gastric cancer risk. Gastro Hep Adv 2(5):684–700 [DOI] [PMC free article] [PubMed] [Google Scholar]
  108. Oldewurtel ER, Kouzel N, Dewenter L, Henseler K, Maier B (2015) Differential interaction forces govern bacterial sorting in early biofilms. Elife 4(September):1–21 [DOI] [PMC free article] [PubMed] [Google Scholar]
  109. Palmer R (2017) Interbacterial adhesion networks within early oral biofilms of single human hosts. Appl Environ Microbiol 83(11):1–16 [DOI] [PMC free article] [PubMed] [Google Scholar]
  110. Patel V, Matange N (2021) Adaptation and compensation in a bacterial gene regulatory network evolving under antibiotic selection. Elife 10:1–27 [DOI] [PMC free article] [PubMed] [Google Scholar]
  111. Pei TT, Kan Y, Wang ZH, Tang MX, Li H, Yan S, Cui Y, Zheng HY, Luo H, Liang X, Dong T (2022) Delivery of an Rhs-family nuclease effector reveals direct penetration of the gram-positive cell envelope by a type VI secretion system in Acidovorax citrulli. mLife 1(1):66–78 [DOI] [PMC free article] [PubMed]
  112. Ramamoorthy S, Pena M, Ghosh P, Liao YY, Paret M, Jones JB, Potnis N (2024) Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism. Microbiol Spectr. 10.1128/spectrum.02852-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  113. Ramsey ME, Hackett KT, Kotha C, Dillard JP (2012) New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis. Appl Environ Microbiol 78(9):3068–3078. 10.1128/AEM.07871-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  114. Repizo GD, Gagné S, Foucault-Grunenwald ML, Borges V, Charpentier X, Limansky AS, Gomes JP, Viale AM, Salcedo SP (2015) Differential role of the T6SS in Acinetobacter baumannii virulence. PLoS One 10(9):e0138265 [DOI] [PMC free article] [PubMed]
  115. Rissman AI, Mau B, Biehl BS, Darling AE, Glasner JD, Perna NT (2009) Reordering contigs of draft genomes using the Mauve Aligner. Bioinformatics 25(16):2071–2073 [DOI] [PMC free article] [PubMed] [Google Scholar]
  116. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26. 10.1038/nbt.1754 [DOI] [PMC free article] [PubMed] [Google Scholar]
  117. Salomon D (2016) MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness. Mob Genet Elements 6(1):e1123796 [DOI] [PMC free article] [PubMed] [Google Scholar]
  118. Santin YG, Camy CE, Zoued A, Doan T, Aschtgen MS, Cascales E (2019) Role and recruitment of the TAGL peptidoglycan-binding protein during type VI secretion system biogenesis. J Bacteriol 201(12):1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  119. Sedghi L, DiMassa V, Harrington A, Lynch SV, Kapila YL (2021) The oral microbiome: role of key organisms and complex networks in oral health and disease. Periodontol 2000 87(1):107–31 [DOI] [PMC free article] [PubMed] [Google Scholar]
  120. Sharp C, Bray J, Housden NG, Maiden MCJ, Kleanthous C (2017) Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using hidden Markov models. PLoS Comput Biol 13(7):e1005652 [DOI] [PMC free article] [PubMed] [Google Scholar]
  121. Shirvani F, Behzad A, Abdollahi N, Mohkam M, Sharifian M, Esfandiar N, Fallah F (2021) Frequency and co-colonization of vancomycin-resistant Enterococci and Candida in ICU-hospitalized children. New Microbes New Infect 41:100881 [DOI] [PMC free article] [PubMed] [Google Scholar]
  122. Si M, Zhao C, Burkinshaw B, Zhang B, Wei D, Wang Y, Dong TG, Shen X (2017) Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis. Proc Natl Acad Sci U S A 114(11):E2233–E2242 [DOI] [PMC free article] [PubMed] [Google Scholar]
  123. Smith WPJ, Vettiger A, Winter J, Ryser T, Comstock LE, Basler M, Foster KR (2020) The evolution of the type VI secretion system as a disintegration weapon. PLoS Biol 18(5):1–26 [DOI] [PMC free article] [PubMed] [Google Scholar]
  124. Smith WPJ, Armstrong-Bond E, Coyte KZ, Knight CG, Basler M, Brockhurst MA (2025) Multiplicity of type 6 secretion system toxins limits the evolution of resistance. Proceedings of the National Academy of Sciences 122(2):2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  125. Song H, Kang Y, Qian A, Shan X, Li Y, Zhang L, Zhang H, Sun W (2020) Inactivation of the T6SS inner membrane protein DotU results in severe attenuation and decreased pathogenicity of Aeromonas veronii TH0426. BMC Microbiol 20(1):1–11 [DOI] [PMC free article] [PubMed]
  126. Song L, Pan J, Yang Y, Zhang Z, Cui R, Jia S, Wang Z, Yang C, Xu L, Dong TG, Wang Y, Shen X (2021) Contact-independent killing mediated by a T6SS effector with intrinsic cell-entry properties. Nat Commun. 10.1038/s41467-020-20726-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  127. Song L, Xu L, Wu T, Shi Z, Kareem HA, Wang Z, Dai Q, Guo C, Pan J, Yang M, Wei X, Wang Y, Wei G, Shen X (2024) Trojan horselike T6SS effector TepC mediates both interference competition and exploitative competition. ISME J 18(1):1–13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  128. Spinosa MR, Progida C, Talà A, Cogli L, Alifano P, Bucci C (2007) The Neisseria meningitidis capsule is important for intracellular survival in human cells. Infect Immun 75(7):3594–3603 [DOI] [PMC free article] [PubMed] [Google Scholar]
  129. Stietz MS, Liang X, Li H, Zhang X, Dong TG (2020) TssA–TssM–TagA interaction modulates type VI secretion system sheath-tube assembly in Vibrio cholerae. Nat Commun 11(1):1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  130. Storey D, McNally A, Åstrand M, Santos JPG, Rodriguez-Escudero I, Elmore B, Palacios L, Marshall H, Hobley L, Molina M, Cid VJ, Salminen TA, Bengoechea JA (2020) Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent. PLoS Pathog 16:1–54 [DOI] [PMC free article] [PubMed] [Google Scholar]
  131. Stubbendieck RM, Straight PD (2016) Multifaceted interfaces of bacterial competition. J Bacteriol 198(16):2145–2155 [DOI] [PMC free article] [PubMed] [Google Scholar]
  132. Sultan AS, Kong EF, Rizk AM, Jabra-Rizk MA (2018) The oral microbiome: a lesson in coexistence. PLoS Pathog 14(1):e1006719 [DOI] [PMC free article] [PubMed] [Google Scholar]
  133. Takahashi S, Kurimura Y, Hashimoto J, Takeyama K, Koroku M, Tanda H, Nishimura M, Tsukamoto T (2008) Pharyngeal Neisseria gonorrhoeae detection in oral-throat wash specimens of male patients with urethritis. J Infect Chemother 14(6):442–444. 10.1007/s10156-008-0649-5 [DOI] [PubMed] [Google Scholar]
  134. Tan K, Johnson PM, Stols L, Boubion B, Eschenfeldt W, Babnigg G, Hayes CS, Joachimiak A, Goulding CW (2015) The structure of a contact-dependent growth-inhibition (CDI) immunity protein from Neisseria meningitidis MC58. Acta Crystallogr F Struct Biol Commun 71(Cdi):702–709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  135. Tao G, Hoover KW, Nye MB, Peters P, Gift TL, Peruvemba R, Body BA (2016) Rectal infection with Neisseria gonorrhoeae and Chlamydia trachomatis in men in the United States. Clin Infect Dis 63(10):1325–1331 [DOI] [PubMed] [Google Scholar]
  136. Trunk K, Coulthurst SJ, Quinn J (2019) A new front in microbial warfare-delivery of antifungal effectors by the type VI secretion system. J Fungi. 10.3390/jof5020050 [DOI] [PMC free article] [PubMed] [Google Scholar]
  137. Trunk K, Peltier J, Liu YC, Dill BD, Walker L, Gow NAR, Stark MJR, Quinn J, Strahl H, Trost M, Coulthurst SJ (2018) The type VI secretion system deploys antifungal effectors against microbial competitors. Nat Microbiol 3(8):920–931 [DOI] [PMC free article] [PubMed] [Google Scholar]
  138. Tsakalos A, Xirogianni A, Ekonomou G, Papandreou A, Prokopiou E, Vagiakou E, Tzanakaki G (2021) Symptomatic female genital tract infections due to Neisseria meningitidis in Athens, Greece. Diagnostics Basel 11(7):1265 [DOI] [PMC free article] [PubMed] [Google Scholar]
  139. Uwamino Y, Sugita K, Iwasaki E, Fujiwara H, Nishimura T, Hasegawa N, Iwata S (2017) The first case report of acute cholangitis and bacteremia due to Neisseria subflava. Intern Med 56(2):221–223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  140. Van Reenen CA, Dicks LMT, Chikindas ML (1998) Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum. J Appl Microbiol 84(6):1131–1137 [DOI] [PubMed] [Google Scholar]
  141. Vassallo CN, Troselj V, Weltzer ML, Wall D (2020) Rapid diversification of wild social groups driven by toxin-immunity loci on mobile genetic elements. ISME J 14(10):2474–2487 [DOI] [PMC free article] [PubMed] [Google Scholar]
  142. Wang N, Han N, Tian R, Chen J, Gao X, Wu Z, Liu Y, Huang L (2021) Role of the type VI secretion system in the pathogenicity of Pseudomonas syringae pv. actinidiae, the causative agent of kiwifruit bacterial canker. Front Microbiol 12(February):1–15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  143. Wang T, Si M, Song Y, Zhu W, Gao F, Wang Y, Zhang L, Zhang W, Wei G, Luo Z-Q, Shen X (2015) Type VI secretion system transports Zn to combat multiple stresses and host immunity. PLoS Pathog 11(7):1–25 [DOI] [PMC free article] [PubMed] [Google Scholar]
  144. Weyand NJ (2017) Neisseria models of infection and persistence in the upper respiratory tract. Pathog Dis 75(3):1–13 [DOI] [PubMed] [Google Scholar]
  145. Wolfgang M, Lauer P, Park HS, Brossay L, Hébert J, Koomey M (1998) PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol 29(1):321–330 [DOI] [PubMed] [Google Scholar]
  146. Wörmann ME, Horien CL, Johnson E, Liu G, Aho E, Tang CM, Exley R (2016) Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms. Microbiology (n Y) 162(3):487–502 [DOI] [PMC free article] [PubMed] [Google Scholar]
  147. Wuckelt M, Laurent A, Mouville C, Meyer J, Jamet A, Lecuyer H, Nassif X, Bille E, Pelicic V, Coureuil M (2024) Expanding the genetic toolbox for Neisseria meningitidis with efficient tools for unmarked gene editing, complementation, and labelling. Appl Environ Microbiol 90(9):e0088024. 10.1128/aem.00880-24 [DOI] [PMC free article] [PubMed] [Google Scholar]
  148. Yang X, Pan J, Wang Y, Shen X (2018) Type VI secretion systems present new insights on pathogenic Yersinia. Front Cell Infect Microbiol 8(July):260 [DOI] [PMC free article] [PubMed] [Google Scholar]
  149. Zhang L, Xu J, Xu J, Zhang H, He L, Feng J (2012) TssM is essential for virulence and required for type VI secretion system in Ralstonia solanacearum. Microb Pathog 119(4):125–134 [DOI] [PubMed] [Google Scholar]
  150. Zhang Y, Huang Y, Ding H, Ma J, Tong X, Zhang Y, Tao Z, Wang Q (2023) A σE-mediated temperature gauge orchestrates type VI secretion system, biofilm formation and cell invasion in pathogen Pseudomonas plecoglossicida. Microbiol Res 266(October 2022):127220 [DOI] [PubMed] [Google Scholar]
  151. Zöllner R, Oldewurtel ER, Kouzel N, Maier B (2017) Phase and antigenic variation govern competition dynamics through positioning in bacterial colonies. Sci Rep 7(1):12151 [DOI] [PMC free article] [PubMed] [Google Scholar]
  152. Zoued A, Brunet YR, Durand E, Aschtgen MS, Logger L, Douzi B, Journet L, Cambillau C, Cascales E (2014) Architecture and assembly of the type VI secretion system. Biochim Biophys Acta Mol Cell Res 1843(8):1664–1673 [DOI] [PubMed] [Google Scholar]
  153. Zoued A, Cassaro CJ, Durand E, Douzi B, España AP, Cambillau C, Journet L, Cascales E (2016) Structure–function analysis of the TssL cytoplasmic domain reveals a new interaction between the type VI secretion baseplate and membrane complexes. J Mol Biol 428(22):4413–4423 [DOI] [PubMed] [Google Scholar]
  154. Zoued A, Duneau JP, Durand E, España AP, Journet L, Guerlesquin F, Cascales E (2018) Tryptophan-mediated dimerization of the TssL transmembrane anchor is required for type VI secretion system activity. J Mol Biol 430(7):987–1003 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ESM 1 (1.7MB, jpg)

(JPG 1.65 MB)

ESM 2 (1.6MB, jpg)

(JPG 1.63 MB)

ESM 3 (1.5MB, jpg)

(JPG 1.54 MB)

ESM 4 (2MB, jpg)

(JPG 1.96 MB)

ESM 5 (1.7MB, jpg)

(JPG 1.69 MB)

ESM 6 (1.6MB, jpg)

(JPG 1.60 MB)

ESM 7 (1.6MB, jpg)

(JPG 1.59 MB)

ESM 8 (310.6KB, pdf)

(PDF 310 KB)

Data Availability Statement

All data is presented here with the exception of genome sequencing data, which is available via the accession information in the Methods.

Articles from Applied Microbiology and Biotechnology are provided here courtesy of Springer

RESOURCES