The type VI secretion system of the emerging pathogen Stenotrophomonas maltophilia complex has antibacterial properties

ABSTRACT Antagonistic behaviors between bacterial cells can have profound effects on microbial populations and disease outcomes. Polymicrobial interactions may be mediated by contact-dependent proteins with antibacterial properties. The type VI secretion system (T6SS) is a macromolecular weapon used by Gram-negative bacteria to translocate proteins into adjacent cells. Many pathogens deploy the T6SS to escape immune cells, eliminate commensal bacteria, and facilitate infection. Stenotrophomonas maltophilia complex is a Gram-negative opportunistic pathogen that primarily affects immunocompromised people and infects the lungs of patients with cystic fibrosis. Infections with the bacterium can be deadly and are challenging to treat because many isolates are multidrug-resistant. We found that globally dispersed S. maltophilia complex clinical and environmental strains possess T6SS genes. We demonstrate that the T6SS of an S. maltophilia complex patient isolate is active and can eliminate other bacteria. We provide evidence that the T6SS contributes to the competitive fitness of S. maltophilia complex against a co-infecting Pseudomonas aeruginosa isolate, and the T6SS alters the organization of S. maltophilia and P. aeruginosa co-cultures. This study expands our knowledge of the mechanisms employed by S. maltophilia complex to secrete antibacterial proteins and compete against other bacteria. IMPORTANCE Infections with the opportunistic pathogen Stenotrophomonas maltophilia complex can be fatal for immunocompromised patients. The mechanisms used by the bacterium to compete against other prokaryotes are not well understood. We found that the type VI secretion system (T6SS) allows S. maltophilia complex to eliminate other bacteria and contributes to the competitive fitness against a co-infecting isolate. The presence of T6SS genes in isolates across the globe highlights the importance of this apparatus as a weapon in the antibacterial arsenal of S. maltophilia complex. The T6SS may confer survival advantages to S. maltophilia complex isolates in polymicrobial communities in both environmental settings and during infections.

listed S. maltophilia as an important pathogen for which novel antibiotic treatments are urgently needed (6,7).
Immunocompromised people and cancer patients are especially susceptible to S. maltophilia (4,9).Cystic fibrosis (CF), a genetic disease that affects more than 150,000 people worldwide, leads to chronic pulmonary bacterial infections (10).Approximately 10%-30% of CF patients harbor S. maltophilia in their lungs at least once during their life (11,12).CF pulmonary S. maltophilia infections can be associated with up to threefold higher mortality, more severe exacerbations, and an increased risk of requiring lung transplants (13).The bacterium is also detected in sputum from COVID-19 patients and has the highest rates of multidrug resistance among bacterial species from this population (14)(15)(16).
S. maltophilia is ubiquitously found in water and soil environments and has been isolated from hospital surfaces and medical devices (17,18).The species exhibits extensive genomic diversity and has been subdivided into 23 monophyletic lineages (19).Due to its diversity, the phylogenetic classification of the species is problematic, and Gröschel et al. proposed the term "S.maltophilia complex" for isolates that are identified as S. maltophilia by diagnostic procedures (19).
In this study, we performed bioinformatic searches using NCBI databases and identified S. maltophilia complex isolates from both patient and environmental sources that possess T6SS-encoding genes.We found that a clinical S. maltophilia complex strain encodes a T6SS that is active under standard laboratory conditions and can eliminate other bacteria like Escherichia coli and Burkholderia cenocepacia.We also observed that the T6SS confers a competitive advantage to this S. maltophilia against a co-infecting P. aeruginosa strain obtained from the same patient.We used confocal microscopy to determine that the T6SS alters the spatial organization of co-cultures containing both S. maltophilia and P. aeruginosa.The presence of T6SS-encoding genes in S. maltophilia complex isolates across the globe highlights the importance of this apparatus as a weapon in the antibacterial arsenal of S. maltophilia.The T6SS may confer survival advantages to S. maltophilia complex strains in polymicrobial communities in environ mental settings and during infections.

T6SS genes are found globally distributed in S. maltophilia complex patient and environmental isolates
To analyze the distribution of T6SS-encoding genes in S. maltophilia isolates, we used the sequence of the TssC sheath protein from Xanthomonas citri to search among approxi mately 1,000 S. maltophilia complex genomes from RefSeq and GenBank databases for homologous proteins (52,53).X. citri belongs to the same Xanthomonadaceae family as S. maltophilia and possesses a T6SS that contributes to resistance against eukaryotic predators (54).We discovered that 61 S. maltophilia complex isolates from animal, human, plant, and environmental sources encode at least one copy of the TssC protein (Fig. 1A; Table S1).Strains harboring TssC-encoding genes are dispersed across multiple countries from Africa, Asia, Australia, Europe, and America, and some were obtained from patient lung, blood, urine, and wound samples (Fig. 1B; Table S1).Three CF isolates (B4, B5, and H_59_creteil) possess TssC-encoding genes (Fig. 1A; Table S1).
Based on their amino acid sequence, S. maltophilia complex TssC proteins cluster into three distinct phylogenetic groups: group 1, group 2, or group 3 (Fig. 1A).Group 1 TssC proteins are the most common and share homology to the TssC of B. cenocepacia and Acinetobacter baumannii, whose TssC sequences belong to the i4b subtype (34,36,55,56).By contrast, TssC proteins from group 2 cluster with TssC1 from P. aeruginosa, which belongs to the i3 T6SS subtype, and TssC from X. citri (26,37,54).Four S. maltophilia isolates harbor only a single TssC from group 2 and no TssC from group 1 (Fig. 1; Table FIG 1 Patient and environmental S. maltophilia complex isolates from diverse geographic locations harbor T6SS-encoding genes.(A) A BlastP search was conducted to identify S. maltophilia complex TssC proteins.TssC sequences of the indicated strains were aligned, and a phylogenetic tree was constructed.
Branch titles designate the strain name that encodes the respective TssC homolog.Branch numbers indicate support values.Strains that harbor a TssC from groups 1 and 2 are indicated by a "−1" or "−2, " respectively, after the strain name.(B) The number of S. maltophilia complex isolates with T6SS-encoding genes from each country is displayed.S2).Finally, S. maltophilia complex TssC proteins from group 3 are clustered with TssC2 from P. aeruginosa and TssC from Vibrio cholerae, which are part of the i1 subtype (34).
We surveyed across S. maltophilia complex strains that encode TssC proteins to determine if they also encode VirB10 and VirD4 proteins, which are essential for the antibacterial T4SS in S. maltophilia K279a.All S. maltophilia complex isolates that encode a TssC protein from group 3, as well as isolate MDMC339 (which encodes a TssC from group 2) and isolate AS012546 (which encodes a TssC from group 1), also encode proteins with 50% or greater homology to the K279a VirB10 and VirD4 proteins (Fig. 2).We next performed an average nucleotide identity (ANI) analysis using S. maltophilia complex genomes that encode T6SS proteins.We also included the K279a genome (which encodes T4SS, but not T6SS, proteins) in this ANI analysis (Fig. 2).Most genomes that encode both TssC and Vir proteins cluster near K279a, while many isolates encod ing TssC proteins from groups 1 and 2 form a separate group (Fig. 2).In conclusion, S. maltophilia complex isolates from both clinical and environmental sources encode diverse TssC proteins that cluster into three distinct groups.
FIG 2 ANI between 61 S. maltophilia complex strains (and S. maltophilia K279a) encoding TssC, VirB10, and VirD4 proteins.Genomic nucleotide sequences of the indicated S. maltophilia complex isolates were compared using FastANI, and a matrix was created from the obtained values.Strains were considered to encode VirB10 and VirD4 T4SS proteins (which are essential for the antibacterial activity of the T4SS) if they shared >50% similarity to their respective homologs from K279a.TssC groups are defined in Fig. 1.

STEN00241 possesses essential T6SS-encoding genes and a repertoire of predicted toxins
To further understand the organization and function of T6SS-encoding genes in S. maltophilia complex, we analyzed sputum isolate STEN00241, which harbors a single TssC from group 1. STEN00241 possesses a main operon with T6SS-encoding genes (referred to as T6SS-1) predicted to encode proteins that form the membrane complex, baseplate, inner tube, sheath, and the tip of the apparatus (Fig. 3A).Additionally, we discovered eight genes encoding VgrG proteins distributed throughout the genome (Fig. 3A and B).vgrG-1, vgrG-2, vgrG-3, and vgrG-4 are found within the main T6SS operon (Fig. 3A).Both vgrG-2 and vgrG-3 are near genes predicted to encode chaperones (DUF4123), phospholipases (with a DUF2235), and immunity proteins (DUF3304) (46,57,58).vgrG-4 is located near a gene predicted to encode a protein with a lysozyme-like function.
Unlike vgrG1-4, other vgrG genes are found at locations distal to the main T6SS operon (Fig. 3B).Downstream of vgrg-5, a gene encoding a colicin/pyocin-like protein is found, while a putative endopeptidase is encoded downstream of vgrG-6.Similarly, a gene predicted to encode a phospholipase is located downstream of vgrG-7.Three genes encoding Rhs proteins are also found in the genome of STEN00241: one is located downstream of vgrG-8, while the other two are found near each other but distant from a vgrG gene.These findings demonstrate that S. maltophilia complex STEN00241 harbors essential T6SS-encoding genes as well as a diverse array of putative effector-encoding genes.

The STEN00241 T6SS is active and displays antibacterial properties
Since we observed multiple encoded toxins with putative antibacterial properties in the genome of STEN00241, we hypothesized that the T6SS is used to eliminate competitor bacteria.We engineered a T6SS deficient mutant of STEN00241 by deleting the tssM gene (ΔtssM), which is essential for the function of T6SS in other bacteria (55,59).The ΔtssM strain has a similar growth rate to the wild type (WT) strain in liquid LB medium (Fig. S1).We co-cultured WT or ΔtssM STEN00241 with E. coli cells and observed that the WT strain robustly eliminates E. coli at 37°C (Fig. 4A) and 25°C (Fig. S2).By contrast, the ΔtssM mutant is significantly impaired at killing E. coli (Fig. 4A; Fig. S2).Approximately the same number of WT and ΔtssM STEN00241 cells are recovered following co-culture with E. coli (Fig. 4B).Secretion of the Hcp protein in the supernatant has been previously used to demonstrate active T6SS in other bacterial species (55,60).We observed that WT S. maltophilia secretes Hcp (~18 kDa) in the supernatant, while the ΔtssM mutant does not (Fig. 4C).
STEN00241 also eliminates B. cenocepacia strain K56-2 in a T6SS-dependent manner (Fig. 5A).By contrast, the T6SS does not contribute to killing of the P. aeruginosa PA14 laboratory strain or a P. aeruginosa CF isolate (PA32; Fig. 5B and C).Similarly, the T6SS does not affect the ability of STEN00241 to eliminate the Staphylococcus aureus JE2 laboratory strain (Fig. 5D).These results demonstrate that STEN00241 can utilize the T6SS to eliminate some heterologous bacterial species.

The T6SS contributes to the competitive fitness of STEN00241 against a co-infecting P. aeruginosa isolate
Interactions that occur between two bacterial species co-isolated from the same patient can have distinct outcomes compared to interactions that occur between strains obtained from different sources (61,62).STEN00241 was co-isolated from the same patient as P. aeruginosa strain PSA01136 (63).We competed STEN00241 and PSA01136 with one another and observed their dynamics.Approximately 10-fold fewer PSA01136 cells are recovered when the strain is competed against WT STEN00241 compared to the ΔtssM mutant (Fig. 6A).By contrast, the number of recovered PSA01136 cells is not significantly affected by the T6SS when co-cultures are performed in liquid conditions, which allow only minimal contact to occur between cells (Fig. S3).Since we observed that the STEN00241 T6SS has a significant impact on the survival of the co-infecting PSA01136 strain, we wondered whether the T6SS also influences the survival of STEN00241 during co-cultures with PSA01136.We observed that the number of recovered STEN00241 ΔtssM cells is significantly lower compared to the number of recovered WT S. maltophilia cells when co-cultured on a solid medium with PSA01136, suggesting that the T6SS also contributes to the survival of STEN00241 against the co-infecting PSA01136 isolate (Fig. 6B).
To determine the impact of the STEN00241 T6SS on the community structure when mixed with the co-infecting PSA01136 isolate, we used confocal microscopy to visualize co-cultures of STEN00241 (WT or ΔtssM) expressing mCherry and PSA01136 express ing green fluorescent protein.When PSA01136 is co-cultured with WT STEN00241, P. aeruginosa forms large, distinct clusters from which STEN00241 cells are mostly excluded (Fig. 6E; Fig. S4A).By contrast, when PSA01136 is co-cultured with STEN00241 ΔtssM, the two strains form a mixed, interwoven pattern (Fig. 6F; Fig. S4B).Taken together, these results provide evidence that the T6SS contributes to the competitive fitness of STEN00241 when co-cultured with a co-infecting P. aeruginosa isolate and alters interactions between the two bacteria.

T4SS-and T6SS-encoding genes are found at distinct genomic locations in S. maltophilia complex strains
Previous work showed that S. maltophilia K279a uses a T4SS to eliminate bacteria (20,21,24), and here, we provide evidence that the T6SS can also display antibacterial proper ties.We wondered whether the two systems are found at the same genomic locations in different strains.We analyzed the complete genomes of four S. maltophilia isolates to compare the genomic locations of T6SS and T4SS gene clusters.For this comparison, we used strain STEN00241, which encodes a TssC from group 1 (within the T6SS-1 cluster), strain K279a, which possesses a T4SS gene cluster but no T6SS-encoding genes, strain SJTL3, which encodes both a TssC from group 1 (within a T6SS-1 cluster) and a TssC from group 2 (within a T6SS-2 cluster), and strain T50-20, which encodes a TssC from group 3 (within a T6SS-3 cluster) and a T4SS (Fig. 7).
From this analysis, we found that the S. maltophilia isolates that lack genes encod ing T6SS or T4SS elements possess upstream and downstream regions of the missing sequences at approximately the same genomic locations as strains that harbor T6SS or T4SS genes (Fig. 7).SJTL3 and STEN00241 appear to have genes encoding a putative (p)ppGpp synthase/hydrolase and a toxin from a toxin-antitoxin system, where T4SS genes are found in K279a (Fig. 7).Both T50-20 and K279a encode a putative decarboxy lase in place of the T6SS-1 cluster.The GC content of the T4SS and T6SS-3 clusters is 61%-62% GC compared to the 66% overall genomic GC content of K279a and T50-20.We did not detect phage-related sequences in the immediate vicinity (~50,000 nucleotides) of T4SS or T6SS modules (Fig. 7, yellow diamonds).

DISCUSSION
The study presented here describes the T6SS as an antibacterial weapon in the arsenal of S. maltophilia complex.In both host and environmental settings, bacteria like S. maltophilia live in polymicrobial communities, where interactions with other cells influence the ability of a species to persist (65).These interactions are often antagonistic and may be mediated by diffusible antibacterial small molecules or by contact-depend ent proteins that are transferred between cells to intoxicate competitors (25,65).
We report here that T6SS-encoding genes are found in geographically diverse S. maltophilia complex isolates obtained from patient and non-patient sources, suggesting that the T6SS might confer a competitive advantage against other bacteria during infection as well as for survival in the environment.This hypothesis is supported by our finding that the T6SS is important for the elimination of E. coli at both 25°C and 37°C.P. aeruginosa and B. cenocepacia also possess active T6SS clusters that may contribute to pathogenicity and allow those pathogens to outcompete other bacteria (25,27).P. aeruginosa Hcp proteins and antibodies against Hcp have been detected in the sputum of CF patients (26).P. aeruginosa CF isolates with loss-of-function mutations in T6SS regulator genes become susceptible to killing by species from the Burkholderia cepacia complex (27).It is currently unclear if the T6SS of S. maltophilia is important in infections such as those seen in CF patients.
P. aeruginosa and S. maltophilia complex species are frequently isolated from the same patients, and both cooperative and antagonistic interactions between the two pathogens have been described (20)(21)(22).During mouse lung infections, P. aeruginosa can increase S. maltophilia proliferation and associated immune responses (22).Patient isolates of P. aeruginosa and S. maltophilia have been observed to share genomic sequences, suggesting that inter-species horizontal gene transfer is common among these organisms (66).However, competitive interactions can also occur between these two pathogens.S. maltophilia K279a uses the T4SS to deliver toxins with predicted lipase and lysozyme-like activity to intoxicate and kill P. aeruginosa strains (20,21,24).We provide evidence that an S. maltophilia complex pulmonary isolate can utilize the T6SS to eliminate heterologous bacteria like E. coli and B. cenocepacia and to compete against a co-infecting P. aeruginosa isolate.
Even though the T6SS contributes to the competitive fitness of STEN00241 against its co-infecting P. aeruginosa PSA01136 strain, it does not play a significant role in competition against the lab strain PA14 or the CF isolate PA32.Perault et al. observed that P. aeruginosa isolates from adult patients harbor mutations in the gacS, gacA, retS, fha1, and pppA genes that encode T6SS regulators in P. aeruginosa (27).We also observed mutations in these genes in P. aeruginosa PSA01136 when compared to PA14.However, the impact played by those mutations in mediating PSA01136 T6SS activity is unclear.P. aeruginosa employs defense mechanisms against T6SS attacks, such as the Arc immunity pathways and stress response systems (66,67).We suspect that variable expression of defensive and offensive systems in the co-infecting PSA01136 isolate could explain the differences we observed in their competitive fitness against STEN00241 compared to PA14.Although some exceptions have been recently identified, most T6SS are not effective at eliminating Gram-positive bacteria (68).Competition with STEN00241 reduced the number of recovered S. aureus during co-cultures compared to monocultures, but the T6SS did not affect the survival of S. aureus.
The distribution and organization of T6SS-encoding genes across bacterial genomes are diverse.Species from the Burkholderia genus may harbor up to six distinct T6SS clusters, while P. aeruginosa strains generally possess three T6SS clusters (26,69).V. cholerae strains employ a single T6SS operon that encodes structural and regulatory proteins, as well as orphan vgrG gene loci that contain effectors with diverse functions (70).We found that S. maltophilia complex strains can encode TssC proteins from groups 1 and 2, but all isolates that encode a TssC from group 3 only encode a single TssC protein.All S. maltophilia complex strains with a TssC from group 3 also harbor genes encoding T4SS essential components similar to the ones used by K279a to eliminate bacteria (20,21,24).In other bacteria that harbor multiple T6SS clusters, each system can play a role in mediating virulence, obtaining nutrients, and conferring competitive advantages against other bacteria (71)(72)(73).It is unclear why some S. maltophilia complex isolates harbor T4SS genes while others possess T6SS components.Based on the four complete genomes analyzed in Fig. 7, T4SS or T6SS modules appear at conserved but distinct genomic locations, suggesting that they are not a part of interchanged mobile elements.We propose that S. maltophilia complex strains have separate genomic locations which serve as an armory that accommodates different molecular weapons.We speculate that evolutionary pressures dictated by the environment, like competition with eukaryotic or prokaryotic cells, the energy cost of assembling and firing a T4SS or T6SS in different conditions, and the efficacy of secreted toxins, might play key roles in determining which secretion system is acquired and maintained by a S. maltophilia complex strain.Future work will determine whether different S. maltophilia T6SS are required for different processes.
Results presented here enhance our understanding of the secretion systems and antibacterial weapons employed by an important emerging pathogen.We propose that strains harboring T6SS-encoding genes have competitive fitness advantages during survival in both infections and on hospital or environmental surfaces.Additional studies will elucidate how the S. maltophilia complex T6SS is regulated, which components of the apparatus are required for activity, which toxins are important in mediating the observed competitive fitness advantages, and how the system might affect virulence.
Understanding the mechanisms of the antibacterial T6SS in S. maltophilia complex could lead to the development of treatment strategies against strains resistant to antibiotics.

Bacterial strains and growth conditions
S. maltophilia complex STEN00241 and P. aeruginosa strain PSA01136 were previ ously isolated and sequenced as a part of a bacterial whole genome sequencing surveillance study (63).E. coli DH5α, B. cenocepacia K56-2, P. aeruginosa PA14, and S. aureus JE2 were used for co-culture assays with STEN00241.P. aeruginosa PA32 (CFBR509_Pae_20170525_S_EBPa32) was isolated from the sputum of a CF patient (61).E. coli SM10 was used to conjugate plasmids into S. maltophilia.Strains were routinely grown in LB medium at 37°C unless otherwise indicated.The following antibiotic concentrations were used where appropriate: chloramphenicol (20 µg/mL), imipenem (20 µg/mL or 32 µg/mL for imipenem monohydrate), gentamicin (75 µg/mL), and tetracycline (20 µg/mL).All strains used in this study are listed in Table S2.

Construction of phylogenetic trees and ANI matrix
S. maltophilia complex protein sequences from both RefSeq and Genbank NCBI databases were retrieved in June 2022 and used to create a local BLAST database.A local BlastP search with a maximum E-value of 0.05 and a BLOSUM62 matrix was conducted using the TssC protein sequence of X. citri.Incomplete hits were eliminated from further analyses.S. maltophilia complex TssC sequences and TssC sequences from X. citri, P. aeruginosa PAO1 (TssC1 and TssC2), V. cholerae V52, B. cenocepacia K56-2, and A. baumannii DSM 30011 were aligned using MUSCLE (74).Alignments were used to construct a phylogenetic tree in PhyML with a Bayesian Information Criterion Smart Model Selection (75,76).Branch supports were calculated using the aLRT SH-like fast likelihood-based method (75).The final tree was created with iTOL (77).S. maltophilia genomes encoding a TssC protein were also confirmed to encode TssB and TssM T6SS proteins.The ANI of S. maltophilia complex genomes harboring T6SS-encoding genes was calculated using FastANI, and a pairwise ANI matrix was built using ANIClustermap (78).

Molecular biology
Standard molecular biology techniques were used to construct plasmids and PCR products.All restriction enzymes, DNA polymerases, and Gibson mixes were utilized according to manufacturer instructions.Plasmid constructs and PCR products were sequenced by GENEWIZ (Azenta Life Sciences, Chelmsford, MA, USA).

Genetic manipulation of S. maltophilia
To engineer the S. maltophilia complex STEN00241 ΔtssM mutant, a similar allelic exchange method to the one described by Welker et al. was used (79).Briefly, 1,000 bp upstream and downstream of the tssM gene (including the start and stop codons) were assembled on the pEX18Tc suicide vector using Gibson assembly.The pEX18Tc-ΔtssM construct was transformed into E. coli SM10 and then conjugated into STEN00241.Conjugants were identified by growth on tetracycline and imipenem and then plated onto freshly made 15% sucrose LB plates (with no NaCl).Colonies were screened by PCR and confirmed by Sanger sequencing.Plasmids used in this study are listed in Table S2, and primers are listed in Table S3.

Co-culture assays
Overnight cultures of the indicated strains were made from single colonies and were diluted 1:100 in fresh LB medium.Cultures were grown for 4 hours and set to an OD 600 = 1.0.For E. coli DH5α carrying the tetracycline-resistant pEX18Tc plasmid, tetracycline was added to the growth media, and cultures were washed with fresh LB before co-culturing with S. maltophilia.A 1× volume of target strains was mixed with a 10× volume S. maltophilia and centrifuged, and mixtures were resuspended in a 1× volume of LB. 5 µL of the mixed strains or of the indicated strains alone were spotted on LB plates (or added to 3 mL of liquid LB).Co-cultures were then incubated at 37°C (or 25°C where indicated) for 20 hours.For co-cultures spotted on LB plates, colonies were excised, placed in 1 mL of liquid LB, and vortexed for 30 seconds.Cells were then serially diluted and plated on the following selective plates: tetracycline (20 µg/mL) to select for E. coli, chloramphenicol (20 µg/mL) to select for P. aeruginosa, gentamicin (75 µg/mL) to select for B. cenocepacia, Staphylococcus isolation agar (Trypticase soy agar with 7.5% NaCl) to select for S. aureus, and imipenem to select for S. maltophilia.

Confocal microscopy
Plasmid pMRP9-1 (GFP+) (80) was introduced into P. aeruginosa PSA01136, and plasmid pMP7605 (mCherry) (81) was introduced into S. maltophilia complex STEN00241 (both WT and ΔtssM) by electroporation.STEN00241 WT and ΔtssM expressing mCherry and P. aeruginosa PSA01136 expressing GFP+ were inoculated into LB medium from single colonies.Overnight cultures were diluted 1:100 in fresh LB medium, grown for 4 hours, washed with LB, and set to an OD 600 = 1.0.A 1× volume of PSA01136 expressing GFP+ was mixed with a 10× volume STEN00241 mCherry, centrifuged, and resuspended in 1× LB volume.Five microliters of the mixed strains or the indicated strains alone was spotted on dry LB plates and grown overnight.Colonies were visualized without a cover slip from the same LB plates on which they were spotted using a Zeiss LSM 710 upright microscope equipped with a Plan-Apochromat 10× objective.A 488 nm laser was used to observe P. aeruginosa PSA01136 expressing GFP+, and a 555 nm laser was used to observe S. maltophilia expressing mCherry.Images were analyzed in FIJI.Representative images of three biological replicates are shown.Representative fields of view were taken from the colonies of each biological replicate.

Hcp immunoblotting
Polyclonal rabbit antibodies against the STEN00241 Hcp peptide N-LLQPRSATASTSGG-C were generated by Genescript.Overnight cultures of WT or ΔtssM STEN00241 were washed with LB and back-diluted to an OD 600 = 0.01.Strains were grown at 37°C to an OD 600 ≈ 0.7.For cell fractions, 300 µL of cultures were centrifuged, and cell pellets were resuspended in 100 µL of 1× Laemmli buffer.Samples were incubated at 99°C for 30 minutes.For supernatant fractions, cultures were centrifuged for 10 minutes at 2,000 × g, supernatants were filter sterilized twice using 0.22 µm filters, and 700 µL of the filtered supernatants were incubated with trichloroacetic acid at a final concentration of 20% overnight at 4°C.Samples were then centrifuged for 10 minutes and washed three times with cold acetone.Precipitated proteins were resuspended in 150 µL of 1× Laemmli buffer in sodium dodecyl sulfate (SDS) buffer and incubated at 99°C for 60 minutes.Proteins from both cellular and supernatant fractions were separated on an SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane.The membrane was probed with primary antibodies against Hcp (1:1,000) and RNA polymerase subunit alpha (1:2,000) and secondary antibodies against rabbit (for Hcp) and mouse (for RNA polymerase subunit alpha) (1:5,000, LI-COR Biosciences).The membrane was imaged using a Bio-Rad ChemiDoc imager and analyzed in FIJI.

FIG 4
FIG 4 Deletion of the tssM gene in STEN00241 abolishes the killing of E. coli and secretion of Hcp.(A) E. coli resistant to tetracycline was grown alone or in the presence of STEN00241 (WT or ΔtssM) on solid LB medium at a 10:1 (STEN00241:E.coli) ratio for 20 hours at 37°C.The number of surviving E. coli cells was determined by plating mixtures on tetracycline plates.(B) Same as in panel A, but the number of surviving STEN00241 cells was determined by plating mixtures on imipenem plates.Three independent biological replicates were performed.For panel A, a one-way ANOVA with post-hoc Tukey honestly significant difference (HSD) was used to determine statistical significance.For panel B, Welch's unequal variances t-test was used to determine statistical significance.****P < 0.0001; NS, not significant (P > 0.05).D.L., detection limit.(C) Whole cells and supernatants from STEN00241 WT and ΔtssM were probed with antibodies against Hcp and RNA polymerase subunit alpha (cell lysis control).Some non-specific bands, which are likely due to the cross-reactivity of the Hcp antibody with other cytoplasmic proteins, were observed for cellular fractions.

FIG 5
FIG 5 The STEN00241 T6SS contributes to the elimination of B. cenocepacia, but not P. aeruginosa PA14, PA32, or S. aureus.STEN00241 was mixed with target cells at a ratio of 10:1 (STEN00241:target cells) on solid LB medium and incubated for 20 hours at 37°C.(A) The number of surviving B. cenocepacia target cells was determined by plating mixtures on gentamicin plates.(B) The number of surviving P. aeruginosa PA14 target cells was determined by plating mixtures on chloramphenicol plates.(C) The number of surviving P. aeruginosa PA32 target cells was determined by plating mixtures on chloramphenicol plates.(D) The number of surviving S. aureus target cells was determined by plating mixtures on Staphylococcus isolation agar.Three independent biological replicates were performed.A one-way ANOVA with post-hoc Tukey HSD was used to determine statistical significance.**P < 0.01; NS, not significant (P > 0.05).D.L., detection limit.

FIG 6
FIG 6 STEN00241 uses the T6SS to compete against a P. aeruginosa co-infecting isolate.(A) P. aeruginosa PSA01136 was grown alone or in the presence of STEN00241 (WT or ΔtssM) at a 10:1 (STEN00241:PSA01136) ratio on solid LB medium and incubated for 20 hours at 37°C.The number of surviving P. aeruginosa PSA01136 cells was determined by plating mixtures on chloramphenicol plates.(B) Same as above, but the number of surviving STEN00241 cells was determined by plating mixtures on imipenem plates.Three independent biological replicates were performed.For panel A, a one-way ANOVA with post-hoc Tukey HSD was used to determine statistical significance.For panel B, Welch's unequal variances t-test was used to determine statistical significance.**P < 0.01, *P < 0.05, NS, not significant (P > 0.05).D.L., detection limit.For panels C to F, STEN00241 WT alone expressing mCherry (C), STEN00241 ΔtssM alone expressing mCherry (D), co-cultures between P. aeruginosa PSA01136 expressing green fluorescent protein (GFP) and STEN00241 WT expressing mCherry (E), or co-cultures between P. aeruginosa PSA01136 expressing GFP and STEN00241 ΔtssM expressing mCherry (F) spotted onto LB plates were visualized without a cover slip using a Zeiss LSM 710 upright microscope.Images were analyzed in FIJI and are representative of three independent biological replicates.The scale bar represents 100 µm.

FIG 7
FIG 7 Genes coding for T4SS and T6SS components are found at conserved locations in S. maltophilia complex genomes.T4SS (purple), T6SS-1 (blue), T6SS-2 (red), and T6SS-3 (green) genomic sequences were mapped on the genomes of S. maltophilia K279a, STEN00241, SJTL3, and T50-20.When sequences were missing, upstream and downstream genomic regions were mapped instead.Dashed lines and names below the solid black lines indicate the putative locations of elements that are missing from the genomes.Rectangles on the black line, gene representations, and names above the solid black line represent elements present in the genomes.Numbers below the lines correspond to genomic locations indicated by the black tick marks in each genome.GC percentages for each gene cluster are displayed below their respective names, and whole genome GC percentages are displayed to the right of each genome.Yellow diamonds represent putative phage elements predicted by PHASTER (64).