Conjugative type iv secretion systems enable bacterial antagonism that operates independently of plasmid transfer

ABSTRACT Bacterial cooperation and antagonism mediated by secretion systems are among the ways in which bacteria interact with one another. Here we report the discovery of an antagonistic

property of a type IV secretion system (T4SS) sourced from a conjugative plasmid, RP4, using engineering approaches. We scrutinized the genetic determinants and suggested that this

antagonistic activity is independent of molecular cargos, while we also elucidated the resistance genes. We further showed that a range of Gram-negative bacteria and a mixed bacterial

population can be eliminated by this T4SS-dependent antagonism. Finally, we showed that such an antagonistic property is not limited to T4SS sourced from RP4, rather it can also be observed

in a T4SS originated from another conjugative plasmid, namely R388. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on the

genetic level and provide the foundation for future mechanistic studies. SIMILAR CONTENT BEING VIEWED BY OTHERS INNER MEMBRANE COMPONENTS OF THE PLASMID PKM101 TYPE IV SECRETION SYSTEM TRAE

AND TRAD ARE DNA-BINDING PROTEINS Article Open access 04 March 2025 SYSTEMATIC INVESTIGATION OF RECIPIENT CELL GENETIC REQUIREMENTS REVEALS IMPORTANT SURFACE RECEPTORS FOR CONJUGATIVE

TRANSFER OF INCI2 PLASMIDS Article Open access 16 November 2023 GENOMIC DIVERSITY OF _MCR_-CARRYING PLASMIDS AND THE ROLE OF TYPE IV SECRETION SYSTEMS IN INCI2 PLASMIDS CONJUGATION Article

Open access 01 March 2025 INTRODUCTION Microbial populations are a dynamic, diverse, and critically important component of any ecological system1,2,3. The ubiquity of microbes cannot be

understated, as they are present in a myriad of environments, ranging from bodies of water to soils, to even plants and animal systems3,4,5. Regardless of the environmental setting, however,

bacterial interaction plays an essential part in shaping entire communities, as microbes can either cooperate amongst themselves or antagonize each other6,7,8,9,10,11,12. Strategies for

bacterial antagonism can either be non-contact-dependent or contact-dependent. For non-contact dependent, bacteria can: (1) produce specialized metabolites for the inhibition of competitor

cells (e.g., antibiotic production by _Streptomyces_), (2) deplete essential nutrients (e.g., siderophores sequestering iron), or (3) produce enzymes that interfere with competitor cell’s

life cycle (e.g., Esp production by _Staphylococcus epidermidis_ blocks _Staphylococcus aureus_ ability to form biofilms)10,11,12,13,14. On the other hand, contact-dependent strategies can:

(1) deliver toxins to opposing cell membrane receptors (e.g., the _E. coli_ EC93 CDI system) or (2) use specialized secretion systems to directly inject toxins inside competing bacteria

(e.g., type VI secretion system (T6SS) or type IV secretion systems (T4SSs)12,15,16,17,18. Moreover, the antagonistic capabilities of bacterial secretion systems have piqued the interest of

researchers looking to exploit them for antibacterial technologies, such as the programmable and targeted killing of cells in mixed populations using a T6SS, the use of conjugation to

deliver lethal cargo that encodes antimicrobial molecules (e.g., CRISPR-Cas-based nucleases and toxin-intein systems) with great efficacy, as well as a recently uncovered type IVB secretion

system (T4BSS) on _Pseudomonas putida_ that can inject toxins into competitor cells in a contact-dependent manner with potential applications in biocontrol19,20,21,22. These advances

highlight the versatility of secretion systems, either natural or synthetic, that bacteria utilize, to compete against other members of their community. A major family of secretion systems

are the T4SSs involved in bacterial conjugation-dependent horizontal gene transfer. T4SS-mediated conjugation is a well-known mechanism for bacterial cooperation. For example, conjugation

can disseminate antibiotic resistance genes within bacterial communities to allow them to thrive under antibiotic stress. It can also contribute to the spread of hypervirulence plasmids to

allow the recipient population to colonize bodily tissues23,24,25,26. While the role of conjugative T4SSs in cooperative interactions has been well-studied, their potential in bacterial

antagonism has been overlooked for decades. Although there have been reports about bacteria-killing T4SS, such as _Xanthomonas_, _Lysobacter, Stenotrophomonas_, and even _P. putida_

delivering toxic payloads to target cells, it should be noted that these rely on translocated effectors and are not strictly conjugative in nature17,18,22,27,28,29. On the other hand, recent

work leveraging conjugative T4SS has shown that conjugative T4SSs can enable interbacterial antagonism by transferring DNA-based cargo into target cells19,20,21,30. However, all of the

examples mentioned, whether natural or artificial, are dependent on the effective delivery and/or expression of some type of molecular cargo. Another overlooked example of antagonism

mediated by conjugative systems was reported in the 1950’s31. Characterized by a steep decline in viable recipient cells after exposure to an excessive amount of high-frequency recombination

(Hfr) donor cells, this deadly event was dubbed lethal zygosis. The features of the lethal event based on the F system include (a) the dependence on close cell-to-cell contact (b) the

formation of a mating pair using the conjugative apparatus, (c) independence of horizontal gene transfer as indicated by the failure of detecting viable F- plasmid after the exposure to F+

plasmid when the horizontal gene transfer was blocked by nalidixic acid32, (d) the collateral transfer of genetic material that prevents recipient cell death as seen by the few surviving

transconjugants32. These characteristics of this lethal effect suggest that the mating pair formation system of F enables bacterial antagonism in a manner independent of horizontal gene

transfer. Of note is that this phenomenon alludes only to F-based systems and, to the best of our knowledge, has not been shown on any other conjugative platform. In addition, genetic

determinants of the lethal phenomenon observed in the F system remain unclear to this day31,32,33,34,35. Nevertheless, because almost all conjugative plasmids encode the mating pair

formation system (referred to as T4SS) to enable close cell-to-cell contact, we hypothesize that conjugative T4SS may harbor a potential for enabling bacterial antagonism. Here, we have

expanded upon this phenomenon by uncovering the antagonistic property of T4SS originated from conjugative plasmid RP4 using engineering approaches. RP4 (also known as RK2) is a well-studied

plasmid that belongs to a different incompatibility group (IncP) than the F system (IncF), but the antagonism by its T4SS has gone unnoticed since the first isolation of plasmid RP4 in the

1960s36,37,38,39,40,41. Our engineering efforts on this conjugative system imply that the antagonistic activity of T4SS from RP4 is, as far as we know, independent of molecular cargo being

delivered into targeted cells. Moreover, we scrutinized the genes conferring resistance to this T4SS-dependent antagonism and showed its dependency on cell-to-cell contact. Furthermore, the

lethal phenotype was observed to be effective against Gram-negative bacteria in both mono- and mixed-culture settings. Finally, we demonstrated that such an antagonistic property is not

limited to T4SS sourced from RP4, rather it can also be observed in a T4SS derived from another conjugative plasmid belonging to another incompatibility group (i.e., IncW), namely

R38842,43,44. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on a genetic level and provide the foundation for future

mechanistic studies. RESULTS AN RP4 DERIVATIVE INCAPABLE OF TRANSFER ENDOWS THE HOST CELL WITH ANTAGONISTIC PROPERTIES To evaluate the antagonistic potential of conjugative T4SSs, we chose

RP4 as our model system. RP4 is a well-known conjugative plasmid that expresses all the requisite genes for a T4SS, which is ancestrally related to the T4SS found in _Agrobacterium

tumefaciens_ (Figs. 1a and b)37,39,45,46,47,48,49. This machine is a complex macromolecular secretion system, that grants RP4 the ability to self-transfer across a broad range of

Gram-negative bacteria37,39,41,45,46,47. Since RP4 encodes both T4SS and DNA-processing machinery for self-transmissibility, an indispensable step to probe the antagonistic property of T4SS

is to decouple it from the self-transfer function. To this end, we first incorporated GFP onto RP4, denoted as RP4-GFP, followed by the disruption of the DNA-processing machinery39,50

(_oriT_ and _traLKJX_) to generate RP4-GPF-_ΔoriT_ (Fig. 1c). The incorporation of GFP onto the plasmids is to better visualize plasmid transfer based on fluorescence. By transforming the

corresponding plasmid into an _E. coli_ NEB® 10-beta, we prepared four types of donors containing RP4, RP4-GFP, RP4-GFP-_ΔoriT_, and no plasmid, respectively. In addition to these, we also

incorporated pUZ8002, an RP4 derivative with a deficient _oriT_ commonly used in _Streptomyces_ genetics51,52. To evaluate the donors’ antagonistic capabilities, these were incubated

individually with _E. coli_ DA32838 as the recipient strain (Fig. 1d)53. Briefly, we mixed the donor and recipient _E. coli_ and spotted the mixture on cellulose acetate filters (0.45 µm

pore size) placed on an agar medium. Cells were harvested at both 0 and after 3 hours of incubation. Since donor and recipient cells, and the plasmids all possess unique antibiotic markers,

we quantified the colony-forming units (CFUs) of donors, recipients, and transconjugants (i.e., recipient cells that have acquired the plasmid from the donor cells due to bacterial

conjugation) by plating the bacterial cell mixture on appropriate antibiotic agar media. As expected, after 3 hours of incubation, donors containing RP4-GFP-_ΔoriT_ or pUZ8002 led to no

transconjugants in contrast to those containing RP4-GFP or RP4 (Supplementary Fig. 1a). Strikingly, compared to the treatment with the donors containing RP4-GFP, RP4, or no plasmid, there

was a ~100-fold reduction in viable recipient CFU after the exposure to the donor containing RP4-GFP-_∆oriT_ (Fig. 1e). The lethal effect is not due to the overgrowth of the donor as shown

by the similar CFU as the other types of donors after the 3-hour treatment (Supplementary Fig. 1b). Moreover, not only can pUZ8002 display a similar lethal phenotype (Fig. 1e) and be

non-self-transmissible (Supplementary Fig. 1a), but it is also able to transfer mobilizable plasmids (i.e., pIB139-based plasmid) that carry an _oriT_ (Supplementary Table 2). This result

rules out the possibility that the killing observed in the RP4-GFP-_∆oriT_ plasmid is due to the incorrect assembly of the T4SS caused by the elimination of _traXJKL_ during the plasmid

construction of RP4-GFP-_ΔoriT_. To further support this statement, we knocked out _traK_ in plasmid RP4, which is a gene involved in _oriT_ recognition that is essential for DNA

transfer37,40,46. Results show that the removal of _traK_ was sufficient to replicate the lethal phenotype observed in the RP4-GFP_-∆oriT_ strain (Supplementary Fig. 2). Overall, these

results demonstrate that the antagonistic activity is independent of plasmid transfer and are not the result of incorrect or malfunctioning T4SS. Additionally, donors containing RP4 and

RP4-GFP, which were fully capable of self-transfer (Supplementary Fig. 1a), resulted in an almost negligible reduction in recipient CFU in comparison with the plasmid-free donor (Fig. 1e),

suggesting that disrupting the DNA-processing machinery involved in plasmid transfer is essential for the lethal effect. A hypothesis for this phenomenon is that RP4 and RP4-GFP carry a

resistance mechanism that can only be acquired by the recipient through plasmid transfer. The rest of this work is devoted to characterizing the antagonism and identifying cognate resistance

genes towards it. THE ANTAGONISTIC PHENOTYPE IS DEPENDENT ON T4SS To characterize the antagonistic phenotype displayed by RP4-GFP-_∆oriT_, we sought to determine the genetic factors behind

it. With our knowledge of conjugation machinery and its ancestral relationship to T4SS54,55, we focused our efforts on the _tra2_ operon and _traF_ from the _tra1_ operon of RP4. _TraF_ and

at least ten additional genes (i.e., _trbBCDEFGHIJL_) in the _tra2_ operon are known to be essential for the assembly and function of the RP4-T4SS machinery39 with reported homologues on a

representative T4SS (i.e., _virB/virD4_ T4SS from _Agrobacterium_)49,56,57 (Fig. 1a). To probe their roles in promoting bacterial antagonism, we generated individual mutants of these T4SS

genes on RP4-GFP-_∆oriT_ by replacing the target gene with a spectinomycin resistance marker (_aadA_). These mutants were individually transformed into donors to treat recipient _E. coli_

DA32838. The antimicrobial ability was abolished in all these mutants (Fig. 2a). The antagonistic phenotype of the mutants, including _∆traF, ∆trbF, I, J_, and _L_, could be restored through

genetic complementation assays where the wild-type (WT) gene was re-introduced on the pETDuet-1 vector to the corresponding mutant (Supplementary Fig. 3), demonstrating the crucial roles of

these genes. However, the antagonistic phenotype of other mutants (i.e., _∆trbB_, _C_, _D_, E_, G_, and _H_) could not be restored through complementation, possibly due to the polar effects

caused by the inserted _aadA_ marker in these mutants. We later demonstrated the crucial roles of _trbBCDEGH_ using complementation assays based on the reconstitution approach as discussed

in the paragraph after the next. Moreover, we disrupted four extra genes in the _tra2_ operon of RP4-GFP-_∆oriT_ (i.e., _trbK_, _trbM_, _trbN_, and _trbO_) that are not known to form part of

the T4SS but are in the same operon41,45,46,58. The _∆trbK_, _∆trbN_, and _∆trbO_ mutants still possessed the antagonistic potential after removal, making them dispensable for the

antibacterial effect (Fig. 2a). Interestingly, the removal of _trbM_ eradicated the antagonistic capability, despite not being previously reported as an essential part of the T4SS (Fig. 

2a)37,40,41,46. To further probe its potential function, we disrupted _trbM_ from the fully-transferable RP4-GFP construct. This resulted in an abolishment of conjugative transfer that could

be restored by complementing the mutant with the WT _trbM_ gene on a pETDuet-1 vector, strongly suggesting that _trbM_ is crucial for conjugative transfer (Supplementary Fig. 4). Aside from

these and to rule out the native plasmid addiction system _parDE_59,60,61 contributing to the lethal phenotype, we disrupted the _parE_ toxin gene in RP4-GFP-_∆oriT_ and showed that it has

no effect in the killing efficiency (Fig. 2a). Overall, these gene disruption assays demonstrate that: (1) the T4SS is indeed involved in the antimicrobial effect, (2) _trbM_ plays an

important role in both the observed the phenotype and conjugative transfer, and (3) the RP4 _parDE_ toxin-antitoxin system does not contribute to the observed lethal effect. To determine

whether T4SS genes (namely _traF_ and _trbBCDEFGHIJKL_) and _trbM_ are sufficient for the antimicrobial effect, we reconstituted them under the constitutive expression by the a_mpR_ promoter

on the pCOLADuet-1 cloning vector (Fig. 2b). Gene _trbK_ was included for cloning convenience. Here thereafter, whenever T4SS genes are mentioned, it also includes _trbK_ unless stated

otherwise. The co-expression of these proteins endowed the donor strain with a strong antagonistic phenotype, as demonstrated by the CFU reduction of the recipient after 3-hours of exposure

compared to the control where a donor carried an empty vector was used (Fig. 2c). Moreover, to rule out _E. coli_ strain-specificity contributing to the killing observed in the reconstituted

system, we treated a recipient _E. coli_ DA32838 strain with two additional _E. coli_ donor strains (i.e., DA32838 and XL1-Blue) equipped with either the reconstituted T4SS + TrbM or an

empty pCOLADuet-1 vector control. Results show that different _E. coli_ donors share comparable killing activity against the recipient strain, ruling out strain-specificity as a contributing

factor to the observed killing (Supplementary Fig. 5). As mentioned previously, to further determine the essential roles of _trbBCDEGH_ that failed to complement the corresponding mutants

of RP4-GFP-_∆oriT_ (Supplementary Fig. 3), we removed these individual genes from the reconstituted system. These knockouts abolished the antimicrobial phenotype, which could be restored by

complementation with the WT gene (Supplementary Fig. 6), thus demonstrating their indispensable roles. Additionally, reports of T4SSs capable of transferring plasmids containing conjugative

machinery belonging to other T4SSs suggest that crosstalk between different T4SS is possible, hinting at the possibility that components from a related T4SS could complement a knockout of

its respective homologue62,63. To test this hypothesis, we transformed the donor strains carrying either _∆trbB_ or _∆trbJK_ mutant of T4SS + TrbM with their respective _virB/D4_ homologues,

namely _virB11_ and _virB5_48,49. Due to difficulties in obtaining a proper _trbJ_ knockout on the reconstituted T4SS + TrbM, a _trbJK_ knockout was used here instead. Fortunately, the

removal of _trbK_ does not hamper the killing activity of either the RP4-GFP_-∆oriT_ construct (Fig. 2a) or on the reconstituted T4SS (Supplementary Fig. 7a). Next, we performed

complementation assays as done for the reconstitution experiments done for Supplementary Fig. 3. Results show that both _∆trbB_ and _∆trbJK_ were unable to be complemented by their

respective _virB/D4_ homologue (Supplementary Fig. 7b). This suggests that the interactions between the essential components of the RP4-T4SS are specific and have diverged greatly from the

_virB/D4_ T4SS. NON-T4SS GENES CAN ALTER THE ANTAGONISTIC PHENOTYPE OF THE DONOR Next, we set out to explore how non-T4SS genes within the same operon, namely _trbM_, _N_, _O_, and _P_,

could affect the lethality displayed by the donor. These four genes are not known to be part of the core region necessary for plasmid transfer between bacteria, with very limited reports

available regarding their function. What we do know is that _trbM_ is most likely exported across the cell membrane, due to the presence of a signal peptide within its sequence64.

Nonetheless, the general consensus is that _trbM_ is a non-essential protein for intraspecific mating in _E. coli_40,65. This is supported by past work showing that _trbM_ on plasmid R751, a

homologue of _trbM_ found in plasmid RP4 (76.73% amino acid identity and E-value 4e-90 using BLASTp), is not crucial for conjugative transfer to take place66. However, our results from our

_trbM_ knockout and complementation assays contradict these notions and show that it is essential for conjugative transfer in plasmid RP4 (Supplementary Fig. 4). On the other hand, _trbN_ is

a homologue of _virB1_ in the _A. tumefaciens_ system, which codes for a murein-degrading enzyme that is non-essential for conjugative transfer of DNA49. Regarding _trbO_ and _trbP_,

virtually no information is available regarding their function, with the only noteworthy report of _trbP_ being that it appears to be a homologue of _traX_ of the F plasmid, which is a pilin

acetylase45,67. Nonetheless, neither _trbO_ nor _trbP_ have been reported to be part of or contribute to the conjugative transfer of plasmid RP443,45,58,68. Since the successful

reconstitution of the lethality on the pCOLADuet-1 vector provides a more convenient and simplified cloning platform, we decided to investigate the impact of _trbM_, _N_, _O_, _P_ on the

lethal phenotype based on the reconstituted T4SS. We first removed _trbM_ from the reconstituted system. Next, we performed experiments using a two-plasmid system in which the T4SS genes

were on pCOLADuet-1 and either _trbM_, _N_, _O_, or _P_ were on pETDuet-1. Surprisingly, results show that T4SS by itself, without _trbM_, is enough to kill (Fig. 2d). The addition of _trbO_

or _trbP_ both abolished the lethality of the donor (Fig. 2d). The addition of _trbM_ into the system, either on the initial reconstituted plasmid that included _trbM_ in the same operon as

T4SS genes or in the complementation plasmid, enhanced the killing (Fig. 2d). Although the findings regarding _trbM_ seemed inconsistent with our initial gene disruption of _trbM_ on the

RP4-GFP-_∆oriT_ shown in Fig. 2a and Supplementary Fig. 4, in which _trbM_ was shown to be essential for both lethal phenotype and conjugation, it could possibly be explained by the

co-existence of T4SS genes alongside _trbO_ or _trbP_ on the _∆trbM_ mutant of RP4-GFP-_∆oriT_. The enhancing effect of _trbM_ on the lethal phenotype is possibly due to its role in

strengthening intercellular contact, as previous reports on its homologues have revealed their roles in efficient conjugative DNA transfer by enhancing contact66,69. Unlike _trbMOP_, the

addition of _trbN_ had no detectable impact on the lethal effect. Next, we investigated whether _trbM_ could overcome the impact of the expression of _trbO_ or _trbP_ by introducing them

individually on a pETDuet-1-based-plasmid together with a reconstituted pCOLADuet-1 based plasmid that contained both T4SS-genes and _trbM_ into donor cells. From our assays, when compared

to the recipients treated with donors containing the T4SS + TrbM, the antagonistic phenotype was preserved if both _trbM_ and _trbP_ were present but lost when _trbO_ was expressed even

though _trbM_ was also in the reconstituted system (Fig. 2d). We further confirmed that the observed phenotypes were not due to the poor expression of _trbM_, _trbO_ or _trbP_ by using LacZ

fusion assays. Specifically, the expression levels of the LacZ fused to the TrbM in the strains cotransformed with T4SS + TrbM and pETDuet, trbO, or trbP were comparable, showing that the

presence of TrbO or TrbP has no discernible impact on the expression TrbM in the T4SS + TrbM construct (Supplementary Fig. 8a). Expression levels of both TrbO and TrbP complementation

plasmids remained at similar levels as well in the presence of the reconstituted T4SS + TrbM construct (Supplementary Fig. 8b). However, it should be noted that our reconstitution

experiments cannot exclude the possibility that other genes and regulation systems found in RP4 could also play a role in the interactions between T4SS, _trbM_, _N_, _O_, and _P_. Since the

expression of _trbM_ can enhance the antimicrobial activity of T4SS and even overcome the negative impact of _trbP_ in the reconstituted system, we decided to include _trbM_ in the donor

containing the reconstituted T4SS genes hereafter to further characterize the antagonism unless otherwise stated. CONJUGATIVE T4SS ITSELF ENCODES COGNATE RESISTANCE TO THE LETHAL PHENOTYPE

Having scrutinized the genetic determinants for the antagonism, the next step was to elucidate its resistance mechanism. Since RP4-T4SS is known for being tied to conjugation, we focused on

membrane proteins (i.e., TrbJ and TrbK) that have been previously reported to prevent the transfer of RP4 to other cells via entry exclusion70,71,72. TrbJ, is a homologue of the VirB5

protein in the _Agrobacterium tumefaciens_ T4SS, a component of the T4SS that is crucial for conjugative transfer of DNA in said microbe73,74. Studies have shown that VirB5 localizes to the

tip of the T4SS pilus and could play a role in mediating host recognition and adhesion73,75. On the other hand, TrbK is a small lipoprotein that has been reported to be involved in entry

exclusion. This protein is processed and localized to the cytoplasmic membrane of the cell where it exerts entry exclusion72. Due to the role of these proteins in blocking conjugation, we

hypothesized that these proteins would also confer resistance to the antagonistic phenotype. As such we expressed them individually on pETDuet-1, as well as together, in a recipient _E.

coli_ DA32838 strain. For rigorousness, we also cloned other genes from the _tra2_ operon, as well as _traF_, on a cloning vector. We then expressed them individually in the recipient strain

and tested them for resistance. Consistent with our hypothesis and past work70,72, only _trbJ_ and _trbK_ expression on the cloning vector allowed the recipient cell to resist exposure to

the donor (Fig. 2e). These results are further supported by the observation that recipient cells carrying a derivative of the reconstituted T4SS+TrbM in which the entry exclusion proteins

TrbJ and TrbK were removed were susceptible to the lethality of the donor (Supplementary Fig. 9). Moreover, we wondered if natural conjugative plasmids that contain entry exclusion genes

could confer resistance against our reconstituted antimicrobial. For this, we introduced natural conjugative plasmids, RP4, R388, and R6K, individually into a recipient _E. coli_

strain76,77. Results showed that our reconstituted antimicrobial based on RP4-T4SS was indeed hampered by RP4 that carries _trbJK_. The other conjugative plasmids tested did not increase

recipient cell survival against our reconstituted antimicrobial compared to the susceptible wild-type recipient cell, suggesting that the resistance and its associated systems are specific

to the T4SS that encodes it (Supplementary Fig. 10). THE OBSERVED LETHAL EFFECT IS CONTACT-DEPENDENT AND IS NOT DUE TO HOST-SECRETED MOLECULES Because the T4SS involved in conjugation works

via cell-to-cell contact54,78,79, we wondered if the antimicrobial functions in a similar way. To answer this, we performed chlorophenol red-β-D-galactopyranoside (CPRG) assays. For these

experiments, we paired our recipient _E. coli_ DA32838 lab strain against a donor _E. coli_ NEB 10-beta strain possessing either an empty pCOLADuet-1 vector or T4SS genes + TrbM directly on

agar (no filter paper used) supplemented with CPRG (Fig. 3a). CPRG has been widely used as a colorimetric substrate for LacZ to assess membrane permeability80 or cell lysis in situ81. Since

only the recipient strain has a fully functional LacZ that can be released onto the medium when its membrane is compromised, the appearance of a red color due to the LacZ-catalyzed

hydrolysis of CPRG implies membrane damage and lysis of recipient cells. In comparison to the empty pCOLADuet-1 vector control, we observed a considerable color change when the recipient was

treated with the donor carrying T4SS + TrbM, as noticed by the absorbance reading and the naked eye (Fig. 3b), thus suggesting that the lethal effect is associated with membrane damage or

cell lysis caused by the conjugative machinery. However, this assay alone is insufficient to demonstrate a dependency on cell-to-cell contact. Therefore, we performed a modified experiment

in which donor-to-recipient contact was disrupted by the addition of an extra membrane filter (0.45 µm pore size) between the strains (Fig. 3c), as well as liquid culture assays with

constant shaking in which conjugation has been reported to be much less frequent due to transient cell-to-cell contact82. In both conditions, the phenotype was abolished (Fig. 3d). This

demonstrates that the lethal phenotype is dependent on intercellular contact. To further support this conclusion, we performed contact-dependent killing (CDK) assays in which the donor and

recipient strains were fluorescently tagged, and part of the recipient colony covered the donor colony. Having shown the dependency on close intercellular contact, we expect a substantial

lack of fluorescent signal corresponding to the recipient (red) only in the area in which it is directly contacting the donor (green). As shown in the fluorescent images, the area occupied

by the T4SS + TrbM donor is devoid of red fluorescence when compared to the empty vector control (Fig. 3e, Supplementary Figs. 11–13). This serves as further proof that the lethal phenotype

is reliant on cell-to-cell contact. Additionally, the intense red fluorescence signal present in the rest of the colony rules out the possibility of the killing being the result of

host-secreted molecules diffusing through the media. Nevertheless, this does not omit host-secreted molecules being delivered directly into the recipient via the T4SS as a contributor to the

phenotype. To address the concern of whether the antagonistic effect is linked to chromosome-associated molecules being transported across the reconstituted T4SS and into the target strain.

We used the same parent _E. coli_ strain as both the donor and the recipient. The donor strain expressed the reconstituted T4SS and TrbM on a cloning vector, while the recipient strain

carried an empty cloning vector expressing a different antibiotic resistance marker for strain differentiation. If there is a chromosome-associated T4SS-transferrable molecule essential for

the killing effect, we would expect that the donor should also contain chromosomal genes to protect itself from being killed in the first place. Since both donor and recipient _E. coli_ have

the same genetic background except the plasmid carried, the recipient is expected to also have immunity and hence will not be killed by the donor. From our data, the recipient _E. coli_ can

still be eliminated by the donor (Supplementary Fig. 14), implying that chromosome-associated molecules being passed through T4SS, if there are any, do not contribute to recipient cell

death. This, alongside the other experiments performed, supports that the mode of killing: (1) is dependent on close cell-to-cell contact, (2) does not depend on host-secreted molecules

diffusing through the media or being delivered into the recipient through the T4SS80,81. THE LETHAL EFFECT IS DEPENDENT ON THE NUMBER OF DONOR CELLS AND IS NOT LIMITED TO _E. COLI_

RECIPIENTS We next sought to determine the antagonistic efficacy of the conjugative T4SS by revealing the minimal initial donor-to-recipient ratios needed for effective inhibition and

susceptible bacterial species. We first employed different ratios (ranging from ~0.3:1 to ~50:1) and performed the experiments against _E. coli_ DA32838 lab strain on filter papers as

described above. The donor NEB 10-beta _E. coli_ strains used for these assays either had T4SS + TrbM or had an empty pCOLADuet-1 vector as a control. Results showed an association between

donor concentration and increased lethality, with ~3:1 being the minimal ratio for a ~100-fold reduction in viable recipient cells in comparison to the control treated with the donor

containing the empty pCOLADuet-1 vector (Fig. 4a). The reliance on the amount of donor cells present is most likely due to there being a higher likelihood of a recipient cell encountering a

donor cell and forming cell-to-cell contact in populations with more donor strains. Next, we determined the antagonistic efficacy against Gram-negative bacterial species, including _E. coli_

New Delhi metallo-β-lactamase-1 (NDM-1)-producing strain (ATCC BAA-2452), _Enterobacter cloacae_ NDM-1-producing strain (ATCC BAA-2468), _Klebsiella pneumoniae subsp. pneumoniae_ (derived

from ATCC BAA-2524), _Pseudomonas aeruginosa_ PAO1-LAC (ATCC 47085), and _Pseudomonas putida KT2440_ (ATCC 47054) (Fig. 4b). All bacterial species tested except for _P. aeruginosa_ were

susceptible, as demonstrated by the CFU reduction of the recipients compared to the corresponding controls in which the strains were treated with the donor containing an empty pCOLADuet-1

vector. The inefficacy against _P. aeruginosa_ could possibly be explained by the interference caused by its native H1-T6SS, which has been reported to defend and kill donor _E. coli_ that

attempt to initiate T4SS-dependent conjugative transfer of RP483. Interestingly, despite attacking with its own secretion system, the donor was not significantly inhibited by _P. aeruginosa_

PAO1 H1-T6SS as shown by the CFU of the T4SS + TrbM donor being similar to the CFU of the control donor carrying the empty pCOLADuet-1 vector (Supplementary Fig. 15). To confirm that the

H1-T6SS could protect _P. aeruginosa_ PAO1 from the T4SS + TrbM donor, we performed experiments in which _Pseudomonas aeruginosa_ MPAO1 and _Pseudomonas aeruginosa_ MPAO1 _∆retS_ (PW9164)

were treated with donors carrying either the reconstituted T4SS + TrbM or an empty pCOLADuet-1 vector. We opted for _P. aeruginosa_ MPAO1 as the recipient strain of choice due to the lack of

commercially available _Pseudomonas aeruginosa_ PAO1-LAC mutants84. Results from the assays show that, in stark contrast to _Pseudomonas aeruginosa_ PAO1-LAC, the WT _Pseudomonas

aeruginosa_ MPAO1 was susceptible to killing. On the other hand, _Pseudomonas aeruginosa_ MPAO1 _∆retS_ (PW9164), which has an upregulated H1-T6SS83, showed a resistant phenotype similar to

_Pseudomonas aeruginosa_ PAO1-LAC (Supplementary Fig. 16). The discrepancy between _Pseudomonas aeruginosa_ PAO1-LAC and _Pseudomonas aeruginosa_ MPAO1 can be attributed genomic differences

between the two strains and these genomic differences possibly translating to different regulation and/or expression levels of the H1-T6SS85. Lastly, we asked if the T4SS + TrbM was capable

of inhibiting multiple recipients in a mixed-population setting. The lethality of the repurposed RP4-T4SS was further demonstrated by its ability to inhibit a two-strain recipient system

composed of _E. coli_ DA32838 and _P. putida_ KT2440 together (Figs. 4c–4e). This shows that the lethal phenotype is capable of indiscriminate killing and that the presence of more than one

strain will not result in the survival of the other strain. T4SS ORIGINATED FROM CONJUGATIVE PLASMID R388 AND ALSO ENABLES INTERBACTERIAL ANTAGONISM Since RP4-derived conjugative T4SS

enables interbacterial antagonism, we asked whether the phenotype could also be observed using other conjugative T4SS. We focused on T4SS originated from a well-studied conjugative plasmid

belonging to another incompatibility group (i.e., IncW), namely R388, due to its high degree of similarity with T4SS from RP4 (Fig. 5a). We reconstituted the T4SS-containing operon from R388

(Supplementary Data 4) on a cloning vector and showed that an _E. coli_ strain harboring this construct was indeed able to antagonize an _E. coli_ DA32838 lab strain (Fig. 5b). These

results further hint that conjugative plasmids in Gram-negative bacteria may be a hidden source for antagonistic conjugative T4SS. DISCUSSION The work done here showcases direct evidence of

a conjugative T4SS encoded by conjugative plasmid RP4, using engineering approaches, being capable of granting a strong antagonistic property to a host _E. coli_ strain. The mode of killing

is not clear, but speculation leads us to propose that, unlike previously reported examples of bacteria-killing T4SS, the recipient’s death could be the result of catastrophic membrane

damage due to excessive attempts at conjugation. Another interesting question that arises is the role of TrbM in enhancing the killing and how it interacts with other components in the T4SS,

particularly protein TrbP and TrbO. In addition, with many technologies already having been developed based on cell-to-cell contact, the possibility of harnessing antagonistic conjugative

T4SS for a similar purpose seems like an obtainable goal19,20,21,86,87,88,89,90,91,92. Future endeavors could be devoted to further characterizing this phenomenon by (1) assessing the type

of and the extent of the damage the recipient suffers, (2) obtaining optimal conditions for increased lethality, (3) engineering the system for better efficiency and the ability to overcome

the resistance, (4) elucidate the mechanism of killing and (5) uncovering the protein-protein interactions responsible for the phenotype. More excitingly, the widespread existence of a

plethora of conjugative plasmids for both Gram-negative and Gram-positive organisms strongly suggests that there may exist an untapped reservoir of potentially antagonistic conjugative

T4SS93,94,95,96,97. Even though RP4 and R388 are two well-studied conjugative plasmids, our study is the first demonstration of the antagonistic properties of their encoded conjugative T4SS.

In addition, our data showed that the cognate resistance mechanism is specific to the entry exclusion genes carried by the T4SS itself, suggesting that a conjugative T4SS from a different

source (non-RP4) could antagonize recipients that are carrying RP4. It is our hope that the work done here sets the foundation for the exploration and development of antimicrobial

technologies based on the lethal phenotype showcased in this study. METHODS REAGENTS & MATERIALS Platinum™ SuperFi II PCR Master Mix (Invitrogen™) and FastDigest Restriction Enzymes were

purchased from Thermo Fisher Scientific. Enzymes for Gibson assembly were from New England BioLabs or Thermo Fisher Scientific. Luria–Bertani (LB) agar and LB broth medium were from BD

Difco™. Kanamycin mono sulfate, streptomycin sulfate, chloramphenicol, and tetracycline hydrochloride were from Fisher Bioreagents by Thermo Fisher Scientific; while carbenicillin disodium

salt, spectinomycin dihydrochloride pentahydrate, apramycin sulfate [nebramycin II], and Isopropyl-beta-D-thiogalactopyranoside (IPTG) were bought from Research Products International Corp.

L-arabinose and trimethoprim were from TCI AmericaTM. Chlorophenol red-β-D-galactopyranoside (CPRG) was purchased from Millipore Sigma. Ampicillin sodium salt was obtained from Alfa Aesar.

LB agar plates with 6% sucrose without sodium chloride were from Teknova. Whatman™ cellulose acetate membrane filters were purchased from GE Healthcare Sciences (CAT. No. 10404006, 0.45 µm

pore size). VWR Porous Adhesive Film for Culture Plates (CAT. No. 60941-086) was purchased from VWR. Cuvettes used for electroporation were Gene Pulser/MicroPulser Electroporation Cuvettes

by Bio-Rad (Cat. No. 165-2089). Isolation of plasmid species was done with ZyppyTM Plasmid Miniprep Kit (Zymo Research) and ZR BAC DNA Miniprep Kit (Zymo Research). Mix & Go! _E. coli_

Transformation Buffer Set (Zymo Research) was used for making chemically competent _E. coli_ cells. BACTERIAL STRAINS & GROWTH CONDITIONS NEB® 10-beta _E. coli_ cells were used for

cloning. _E. coli_ DA3283853 was used as the main recipient strain during this study. _E. coli_ NEB® 10-beta and _E. coli_ DA32838 are intrinsically resistant to streptomycin and

chloramphenicol, respectively. Heat shock, electroporation, or bacterial conjugation were used to introduce plasmids into _E. coli_ cells. _E. coli NDM-1_ (ATCC BAA-2452)_, Enterobacter

cloacae_ (ATCC BAA-2468)_, Pseudomonas putida_ KT2440 (ATCC 47054), and _Pseudomonas aeruginosa_ PAO1-LAC (ATCC 47085) were purchased from American Type Culture Collection (ATCC).

_Pseudomonas aeruginosa_ MPAO1 parent strain and _Pseudomonas aeruginosa_ MPAO1 _∆retS_ (PW9164 _retS_-A02::ISphoA/hah) were purchased from the Manoil Lab from the University of

Washington84. _Klebsiella pneumoniae_ subsp. _pneumoniae_ derived from ATCC BAA-2524 was purchased from Microbiologics. _E. coli_ strain BW25113 bearing pIJ79051,98, and _E. coli_ DY33199

were used for λ red recombineering. _E. coli_ ET12567 (pUZ8002) was used for obtaining pUZ8002 plasmid51,100. _E. coli_ XL1-Blue was purchased from Agilent. LB media and 37 °C were used for

the propagation of all bacteria, except for _P. aeruginosa_ strains, which were cultured in TSB. _P. putida_ KT2440 was cultured at 30 °C in LB. Antibiotics concentrations used in the media

when appropriate were kanamycin (50 µg/mL), streptomycin (100 µg/mL), chloramphenicol (25 µg/mL), carbenicillin (100 µg/mL), spectinomycin (100 µg/mL), ampicillin (100 µg/mL), apramycin (50 

µg/mL), trimethoprim (10 µg/mL). PLASMID CONSTRUCTION FOR EXPRESSION IN _E. COLI_ Plasmid construction was achieved using standard cloning techniques unless stated otherwise, whereas plasmid

design made use of SnapGene software. The oligonucleotide primers used in this study were purchased from MilliporeSigma. Amplified PCR products were recovered using the ZymocleanTM Gel DNA

Recovery Kit. Cloning vectors used in this study were pETDuet-1, pCOLADuet-1, or pCDFDuet-1. Plasmid RP4 was used as the model plasmid from which all the studied genes were sourced from.

Isolation of plasmid species was done with ZyppyTM Plasmid Miniprep Kit (Zymo Research) and ZR BAC DNA Miniprep Kit (Zymo Research). Plasmids were confirmed by DNA sequencing

(Azenta/Genewiz, Plasmidsaurus, or Texas A&M Institute for Genome Sciences and Society). To construct RP4-GFP (pXJ47), we first sequentially cloned PJ23119-gfp and aac(3)IV

(apramycin-resistant gene) to pCDFDuet-1. A linear DNA fragment of PJ23119-gfp-_aac(3)IV_ flanked by ~40 bp homology to _bla_ of RP4 at each end was then amplified from the plasmid by PCR

and introduced to RP4 backbone using λ Red homologous recombineering to result in pXJ47. Briefly, _E. coli_ strain BW25113 bearing pIJ790 (a plasmid containing λ red genes)51,100 and RP4 was

grown overnight at 30 °C and at 250 rpm in LB supplemented with kanamycin and chloramphenicol. The resulting strain was then sub-cultured with 10 Mm L-arabinose to induce the expression of

λ Red genes at 30 °C to the log phase, washed twice with 10% glycerol, then electroporated with the linear DNA fragment. Once electroporated, the cells were recovered and plated on

antibiotic antibiotic-selective plate at 37 °C. Plasmids were mini-prepped using the kit described above and transformed into _E. coli_ NEB® 10-beta. To construct RP4-GFP-ΔoriT (pXJ70), a

two-step process of selection and counter-selection was used98,99. Briefly, _E. coli_ DY331 was used and cultured at 30 °C unless said otherwise. After the induction of λ Red genes in _E.

coli_ DY331 bearing RP4-GFP (pXJ47) using 42 °C, electrocompetent cells were prepared and electroporated with a linear _cat-sacB_ cassette flanked by homology to _traLK-oriT-traJX_ at each

end to disrupt _traLK-oriT-traJX_. A second round of λ Red homologous recombineering was used to replace _cat-sacB_ with a DNA fragment that did not contain the antibiotic resistance gene

and _sacB_. The resulting plasmid (pXJ70) was transformed to _E. coli_ NEB® 10-beta and confirmed by DNA sequencing. To inactivate genes on pXJ70, _E. coli_ strain BW25113 bearing pIJ790 and

pXJ70 was used. The recombineering protocol was similar as used for the construction of pXJ47, except that the DNA fragment to be electroporated into the strain contained _aadA_ gene

(spectinomycin resistance gene, amplified from pCDFDuet-1) flanked at each end by ~40 bp homology to the gene to be disrupted. Plasmids, primers, and DNA sequences used are shown in

Supplementary Table 1 and Supplementary Data 1–4. ANTIMICROBIAL ASSAYS The donor strains used in this study were primarily derived from _E. coli_ NEB® 10-beta. XL1-1 Blue and DA32838 _E.

coli_ strains were used as alternative donors for Supplementary Fig. 5. The recipient strains mainly used in this study were _E. coli_ DA32838 or its derived strain carrying a pETDuet-1

derived plasmid (pXJZ11, pXJZ45, pXJZ69-82, or pLGV67), or pCOLADuet-1 derived plasmid (pXJZ60 or pLGV96) or a conjugative plasmid (RP4, R388, or R6K). _E. coli NDM-1, E. cloacae, K.

pneumoniae_, _P. putida KT2440_ and _P. aeruginosa_ strains (PAO1-LAC, WT MPAO1, and MPAO1 ∆_retS_) were also used as recipients. Donor and recipient cells were grown overnight at 37 °C and

250 rpm in LB broth (TSB for _P. aeruginosa_ strains) supplemented with the appropriate antibiotics. _P. putida_ was grown at 30 °C. Specifically, _E. coli_ NEB® 10-beta cells without any

plasmid were grown in LB supplemented with streptomycin. LB supplemented with kanamycin was used for growing _E. coli_ NEB® 10-beta containing RP4, pXJ70, pXJ47, pUZ8002, or any pCOLADuet-1

derived plasmid. Kanamycin was also used for growing _E. coli_ XL1-Blue and _E. coli_ DA32838 that carried pCOLADuet-1 derived plasmids for Supplementary Fig. 5. Spectinomycin and kanamycin

were used for _E. coli_ NEB® 10-beta containing plasmid mutants derived from pXJ70; kanamycin and carbenicillin were used when the cells contained both pXJ70-derived plasmid mutants and

pETDuet-1 derived plasmids or when the cells contained both pCOLADuet-1 and pETDuet-1 derived plasmids; spectinomycin was used for _E. coli_ NEB® 10-beta containing pCDFDuet-1 derived

plasmid. _E. coli_ DA32838 without plasmid was grown in LB supplemented with chloramphenicol. _E. coli_ DA32838 strains containing pETDuet-1 derived plasmids were grown in LB supplemented

with chloramphenicol and carbenicillin; kanamycin and chloramphenicol were used for _E. coli_ DA32838 strain containing RP4; trimethoprim and carbenicillin were used for _E. coli_ DA32838

strains containing R388 and R6K, respectively. _E. coli NDM-1, E. cloacae, K. pneumoniae_ and _P. putida_ KT2440 were grown in LB supplemented with carbenicillin. _P. aeruginosa_ strains

were grown in TSB supplemented with ampicillin. Afterwards, 40 µL of the overnight culture was used to inoculate 4 mL of LB broth with appropriate antibiotics as mentioned above, and the

subculture was grown at 37 °C and 250 rpm for two hours. In the case of _Pseudomonas putida_, the subculture was prepared by adding 80 µL of overnight culture into 4 mL of LB with

appropriate antibiotics, and the subculture was grown at 30 °C for 2 hours_. Pseudomonas aeruginosa_ strains (PAO1-LAC, WT MPAO1, and MPAO1 ∆_retS_ PW9164) were not sub-cultured because of

their slow growth rate, and its overnight culture was directly used in the following experiment. Next, the donor and recipient were washed twice of pure LB broth to remove any residual

antibiotics that may affect downstream experiments. Antimicrobial assays on membrane filters: Donor and recipient were mixed in different ratios depending on the experiment and 10 µL of each

mating mix was spotted on two separate Whatman™ cellulose acetate membrane filters (0.45 µm pore size) placed on LB agar plate. Once dried, the membrane filters were immediately harvested

to quantify the initial CFUs of donors and recipients (denoted as 0 hour), while the other filter papers were harvested after the 3-hour treatment at 37 °C. The harvested membrane filters

were resuspended in 1 mL of pure LB broth and vigorously vortexed to detach cells from the filter. The resulting suspension was transferred to a 1.5 mL tube and centrifuged at 17,000 _g_ for

1 minute. The supernatant was discarded, and the pellet was resuspended in 50 µL of pure LB. Then, 10 µL of the resuspension was used to perform 10-fold serial dilutions. Selective agar

media were spotted using 5 µL from the original 50 µL suspension and from the 10-fold serial dilutions. The agar plates were then incubated overnight at 30 °C or 37 °C to allow bacterial

growth. LB agar plates supplemented with appropriate antibiotics were used for counting strains based on _E. coli_ NEB® 10-beta or recipient strains based on _E. coli_ DA32838. LB agar

plates supplemented chloramphenicol and kanamycin were used for counting _E. coli_ DA32838 transconjugants in Supplementary Fig. 1a. While LB agar plates supplemented chloramphenicol and

carbenicillin were used for counting _E. coli_ DA32838 transconjugants for Supplementary Table 2. LB agar plates supplemented with carbenicillin were used for counting _E. coli NDM-1, E.

cloacae, K. pneumoniae_ and _P. putida_ KT2440, while LB agar plate with ampicillin was used for counting _P. aeruginosa_ strains. Colonies were counted in the dilution that allowed for

distinguishable colonies, and the number of dilutions performed that allowed the countable colonies was recorded. The CFUs were determined using the formula: [(number of colonies counted ×

10number of dilutions performed)/5 µL] × 50 µL. Mobilizable plasmid transfer assays: _E. coli_ NEB® 10-beta donors co-transformed with pLGV146 (plasmid derived from pIB139101 that had the

apramycin selective marker replaced with carbenicillin resistance marker) and either pXJ70, pXJ47, pUZ8002 or no plasmid were grown overnight in LB supplemented with carbenicillin at 37 °C,

while the recipient _E. coli_ DA32838 was grown on LB supplemented with chloramphenicol. After subculturing according to the conditions in the Antimicrobial assays section, cells were then

prepared in accordance with the Antimicrobial assays on the membrane filters section. Both the donor and recipient CFU were adjusted to approximately 106. Transconjugants were selected using

LB plates supplemented with both carbenicillin and chloramphenicol. Antimicrobial assays when the donor-to-recipient contact was blocked by an additional membrane filter and in liquid

conditions: _E. coli_ NEB® 10-beta donor containing either an empty vector (pCOLADuet-1) or the reconstituted T4SS + TrbM (pXJZ60) were grown overnight in LB supplemented with kanamycin. The

recipient strain, _E. coli_ DA32838, was grown in LB supplemented with chloramphenicol. Both strains were grown at 37 °C at 250 rpm, after which the strains were subcultured for 2 hours,

and the mating mix was prepared as described above in which the initial donor CFU was set to approximately to the order of 107. For testing under blocked cell-cell contact, 10 µL of donor

only was spotted on a piece of filter paper (0.45 µm pore size). Afterward, an additional piece (0.45 µm pore size) was placed directly on top of the donor spot. Then 2.5 µL of the recipient

cell was spotted directly on top of where the donor would be. For assaying under liquid conditions, 80 µL of donor was mixed with 20 µL of recipient cells. The resulting 100 µL of mating

mix was transferred to a 14 mL culture tube and incubated at 37 °C and 250 rpm for 3 hours. After treatment, pairs in both conditions were harvested by resuspending in 1 mL of LB and

spinning down to collect the cell pellet. After which serial dilutions were performed as described previously. Antimicrobial assays for mixed recipient populations: Overnight cultures of

donor _E. coli_ NEB® 10-beta containing either an empty vector (pCOLADuet-1) or T4SS + TrbM (pXJZ60), recipient _E. coli_ DA32838 containing pCOLADuet-1 and recipient _P. putida_ KT2440 were

grown on LB supplemented with kanamycin and streptomycin, kanamycin, and chloramphenicol, and carbenicillin respectively. Both _E. coli_ strains were incubated overnight at 250 rpm at 37 

°C, whereas _P. putida_ was incubated at 30 °C and also shaken at 250 rpm. Afterwards, 40 µL of an overnight culture of _E. coli_ was used to inoculate 4 mL of LB with appropriate

antibiotics, and the subculture was grown for 2 hours at 37 °C at 250 rpm. In the case of _P. putida_, the subculture was prepared by adding 80 µL of overnight culture into 4 mL of LB with

appropriate antibiotics, and the subculture was grown at 30 °C for 2 hours. Next, the subcultures were washed with pure LB to remove trace antibiotics, and the cells were resuspended in the

following ways: (1) 4 mL of donor _E. coli_ NEB® 10-beta were washed and resuspended in 150 µL of LB, and (2) 1 mL of _E. coli_ DA32838 and _P. putida_ were washed and resuspended in 1 mL of

pure LB. Later, the strains were paired up by adding 40 µL of donor strain and 5 µL of each recipient strain (10 µL total) to obtain the mixed recipient population. Then, 10 µL of the

mating mix was spotted on filter papers (0.45 µm pore size), with cells being harvested at 0 hours and 3 hours after incubation at 30 °C. Harvesting and serial dilution proceeded as detailed

previously. COLORIMETRIC CPRG ASSAYS The donor strains used in this study were _E. coli_ NEB® 10-beta carrying pXJZ60 or an empty vector pCOLADuet-1. The recipient strain used was _E. coli_

DA32838. Donor and recipient cells were grown overnight at 37 °C and at 250 rpm in LB broth supplemented with appropriate antibiotics (kanamycin used for donors, chloramphenicol used for

recipients). Afterward, both strains were sub-cultured for two hours in 4 mL of LB broth supplemented with 1 mM IPTG and appropriate antibiotics as mentioned above. 4 mL of donor strains and

1 mL of recipient were washed with 1 mL of pure LB broth twice to remove any residual antibiotics, then resuspended in 150 µL and 1 mL of pure LB broth, respectively. LB agar was

supplemented with CPRG to a final concentration of 200 µg/mL. A 96-well plate was used in which the wells were filled with 300 µL of LB agar with CPRG. Any unused wells were instead filled

with sterile water to prevent the agar from drying up. Donor and recipient were mixed and 5 µL of the mixture was spotted on the agar in the well; the mating pair ratio used was

approximately 1:1, in which the initial CFUs of the recipient were ~105. The controls used were blank media + donor only and blank media + recipient only; as well as wells supplemented with

carbenicillin to kill both strains. VWR Porous Adhesive Film was used to seal the plates. Plates were incubated for three hours at 37 °C, followed by 13 hours at 30 °C in a Biotek Epoch 2

microplate reader. 30 °C was used in this assay to avoid background color development on the CPRG agar as mentioned by Paradis-Bleau80. The wavelength was set at 574 nm, and readings were

taken every thirty minutes for the duration of the incubation period. The readings after three hours at 30 °C were used for plotting in Fig. 3b. CONTACT-DEPENDENT KILLING (CDK) ASSAYS

Fluorescently tagged donor (sfGFP) and recipient (dTomato) cells of the same genomic background (_E. coli_ NEB® 10-beta) were subcultured in the conditions mentioned in the Bacterial strains

& growth conditions section of the supplemental material. Both strains were then washed twice with plain LB to remove residual antibiotics. Strains carrying either pXJZ38 (pCOLADuet-1 +

sfGFP) or pXJZ39 (T4SS + TrbM + sfGFP) served as the donors, while the recipient strain carried pETomatola (pETDuet-ColE1::ColA + dTomato). The donor initial CFU was adjusted to ~105 while

the initial CFU of the recipient was adjusted to ~104. Afterwards, 3 µL of the donor was spotted on plain LB agar and allowed to dry. Once the donor spot was dry, it was then covered by 10 

µL of the recipient resuspension that was spotted immediately adjacent to it and allowed to dry once more at room temperature. After both donor and recipient spots were dried, these were

incubated at 37 °C for 3 hours, then 30 °C for 3 hours, and then overnight at 30 °C, with images being taken at the end of each incubation period. The iBright™ FL1500 Imaging System from

Thermo Fisher Scientific was used to obtain fluorescent images of donors (Excitation Filter 455 – 485 nm/Emission Filter 508–557 nm) and recipients (Excitation Filter 515 – 545/Emission

Filter 568–617 nm) using the Smart Exposure tool to obtain optimal exposure values. The scale bar was added using the (Fiji Is Just) ImageJ (version 1.54 f) software. 100 ×15 mm Petri dishes

from VWR (CAT. No. 25384-088) were used for the assays. T4SS-LACZ FUSION ASSAYS NEB® 10-beta cells containing the LacZ fusions of the T4SS + TrbM, TrbO, and TrbP or their respective

controls were subcultured in the conditions mentioned in the Bacterial strains and growth conditions section of the supplemental material. All strains were then washed twice with plain LB to

remove residual antibiotics and were then resuspended in 200 µL of LB supplemented with CPRG (200 µg/mL). Next, we aliquoted 100 µL of each strain into wells of Nunc MicroWell 96-Well

Optical-Bottom Plates from Thermo Scientific™ that were previously filled with 100 µL of LB supplemented with CPRG (200 µg/mL) and six 2-fold serial dilutions of all the strains were

performed. Next, the plate was placed inside the Agilent BioTek Synergy H1 Hybrid Multi-Mode Reader pre-warmed to 37 °C, and absorbance readings at 574 nm were taken every 20 minutes for 16 

hours. For Supplementary Fig. 8a, the values corresponding to the readings after 16 hours were used. Due to reading overflow, the values corresponding to the readings of 2 hours were used

for Supplementary Fig. 8b. HOMOLOGY DETECTION USING HHPRED Amino acid sequences for the RP4 and R388 genes known to be involved in conjugation were subjected to a global pairwise alignment

using HHpred102. Default parameters were applied, these were: (1) HHblits = >UniRef30 MSA generation method, (2) 3 MSA generation iterations, (3) an E-value cutoff of 1e−3, (4) minimum

sequence identity of MSA hits with the query of 0%, (5) a minimum coverage of MSA hits of 20%, and (6) alignment mode of global: realign. Homology was determined using three major factors:

(1) A probability value > 87%, (2) E-value < 1.0e−8, and (3) an identity value > 15%. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio

Reporting Summary linked to this article. DATA AVAILABILITY All additional data that support the conclusions from this manuscript are available in the Supplementary Information. Source data

for the main figures, oligonucleotide primers used in plasmid construction, DNA fragments used in λ Red homologous recombineering, genes tested for the RP4 and R388 T4SS antimicrobial

reconstitutions are supplied as Excel files. The sequences used for the design and construction of the plasmids derived from either RP4 or R388 were based on the available nucleotide

sequences found in the National Center for Biotechnology Information (NCBI), under accession numbers BN000925.1 and BR000038.1, respectively. Any additional information is available upon

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Bacterial Type IV Secretion Systems. _PLoS One_ 6, e14780 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This research was financially

supported by Texas A&M Engineering Experiment Station (TEES) and Chemical Engineering Department (TAMU) start-up funds, T3 grant (TAMU), and NIH grant (Award No. R35GM146984) to X. Zhu.

Texas A&M University Graduate Diversity Excellence Fellowship in Genetics supports L. Gordils-Valentin partially. We acknowledge James J. Collins and Allison J. Lopatkin for the early

collaboration on this project and for gifts of RP4, R388, R6K, and _E. coli_ DA32838. We thank Wenjun Zhang for the generous gift of E. coli BW25113/pIJ790, pUZ8002, and pIB139. All data

were plotted using GraphPad Prism, while Figs. 1b–d, 2b, 3a, 3c, and 4e were created with BioRender.com (https://www.biorender.com/). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department

of Chemical Engineering, Texas A&M University, College Station, 77843, TX, US Lois Gordils-Valentin, Huanrong Ouyang, Liangyu Qian & Xuejun Zhu * Interdisciplinary Graduate Program

in Genetics & Genomics, Texas A&M University, College Station, 77843, TX, US Lois Gordils-Valentin & Xuejun Zhu * Department of Biology, Texas A&M University, College

Station, 77843, TX, US Joshua Hong Authors * Lois Gordils-Valentin View author publications You can also search for this author inPubMed Google Scholar * Huanrong Ouyang View author

publications You can also search for this author inPubMed Google Scholar * Liangyu Qian View author publications You can also search for this author inPubMed Google Scholar * Joshua Hong

View author publications You can also search for this author inPubMed Google Scholar * Xuejun Zhu View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS L.G.-V. and X.Z. designed and performed the experiments, analyzed the data, and wrote the manuscript. H.O., L.Q., and J.H. assisted with the experiments, provided technical

expertise, helped with data analysis, provided valuable feedback, and actively participated in manuscript revisions. CORRESPONDING AUTHOR Correspondence to Xuejun Zhu. ETHICS DECLARATIONS

STATISTICS AND REPRODUCIBILITY Statistical analyses for all figures were carried out as described in the figure captions. A confidence interval (CI) of 95% was used and statistical

significance is denoted as *_P_ < 0.05; **_P_ < 0.01; ***_P_ < 0.001; ****_P_ < 0.0001; ns, not significant. All statistical analyses were performed with GraphPad Prism software.

All assays were replicated on independent experiments at least three times and technical replicates were avoided. COMPETING INTERESTS The authors declare no competing interest. PEER REVIEW

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CITE THIS ARTICLE Gordils-Valentin, L., Ouyang, H., Qian, L. _et al._ Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer.

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