# Comparative study of adenosine 3'-pyrophosphokinase domains of MuF polymorphic toxins

Eloïse Paulet, Julia Bartoli, Atakan Kabtan, Chloé Piras, Audrey Tempier, Eric Cascales, Julie Viala

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Eloïse Paulet, Julia Bartoli, Atakan Kabtan, Chloé Piras, Audrey Tempier, et al. Comparative study of adenosine 3'-pyrophosphokinase domains of MuF polymorphic toxins. FEBS Open Bio, 2025, 15 (7), pp.1103-1112. 10.1002/2211-5463.70038. hal-05293224

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# Comparative study of adenosine 3'-pyrophosphokinase domains of MuF polymorphic toxins

Eloïse M. Paulet, Julia Bartoli, Atakan Kabtan, Chloé M. Piras, Audrey C. Tempier, Eric Cascales and Julie P. Viala ID

Aix Marseille Univ, CNRS, LISM (UMR7255), IMM, Marseille, France

**Keywords**

adenosine 3'-pyrophosphohydrolase;
adenosine 3'-pyrophosphokinase; Apk2;
MuF; (p)ppApp; (p)ppGpp

**Correspondence**

J. P. Viala, Aix Marseille Univ, CNRS, LISM (UMR7255), IMM, 31 Chemin Joseph Aiguier, 13009 Marseille, France
E-mail: julie.viala@univ-amu.fr

Eloïse M. Paulet and Julia Bartoli
contributed equally to this article.

(Received 10 January 2025, revised 12 March 2025, accepted 4 April 2025)

doi:10.1002/2211-5463.70038

Edited by Pierre Cosson

Polymorphic toxins (PT) are multidomain proteins used for interbacterial competition and pathogenesis. The N-terminal domain of PT specifies the mode of transport and names the family, while the variable C-terminal domain carries the toxic activity, which can be counteracted by immunity proteins that protect the PT-producing bacterium. The MuF family of polymorphic toxins is specifically associated with temperate phages, and our recent work showed that the C-terminal domain of a MuF toxin encoded by a *Streptococcus pneumoniae* prophage carries adenosine 3'-pyrophosphokinase activity. This type of toxin, which combines a MuF N-terminal domain and an adenosine 3'-pyrophosphokinase C-terminal domain, is called Apk2 for adenosine 3'-pyrophosphokinase family 2. Here, we extend the characterization of this novel family of toxins by providing information on two new members encoded by prophages of *Mannheimia haemolytica* and *Pasteurella multocida*. Production of their adenosine 3'-pyrophosphokinase domains (Apk2tox) in the heterologous host *Escherichia coli* revealed different levels of toxicity, essentially due to their stability. *In vitro* assays with the purified *M. haemolytica* Apk2tox domain demonstrated that, identically to that of *S. pneumoniae*, it exclusively produces (p)ppApp from ATP. The role of immunity proteins and their interchangeability in cross-protection and protein-protein interaction assays was tested. While the immunity proteins that hydrolyse pppApp to ATP are interchangeable, those that inhibit the toxin by protein-protein interaction are mainly active against their intrastrain partner. Overall, this study highlights the conserved features of these enzymatic domains, such as their toxicity, their specific activity toward ATP, and their universal and specific immunities.

Much studied in the 1970s and then somehow neglected, bacteriophages have been the subject of renewed interest in recent decades. In the context of phagotherapy, lytic phages, which infect and kill bacteria, are considered a possible alternative to

the use of antibiotics [1]. By contrast, temperate phages, which integrate their genetic material into the bacterial chromosome of the host, offer an angle of study for understanding genome evolution [2,3].

**Abbreviations**

AHTC, anhydrotetracycline; Aph1, adenosine 3'-pyrophosphohydrolase; Apk2tox, toxic domain of Apk2; Apk(x), adenosine 3'-pyrophosphokinase family (x); HD, hydrolysis motif; lapK, immunity of adenosine 3'-pyrophosphokinase; IPTG, isopropyl β-b-thiogalactopyranoside; mhao, *Mannheimia haemolytica*; pmv, *Pasteurella multocida*; PT, polymorphic toxin; RBS, ribosome binding site; RSH, RelA/SpoT homolog; SAH, small alarmone hydrolase; SAS, small alarmone synthetase; SAX-HPLC, strong anion exchange high-performance liquid chromatography; snu, *Streptococcus pneumoniae*; syn, synthesis motif; TxSS, type x secretion system.
Phage genomes usually encode structural compo-
nents of the phage and proteins and enzymes that sub-
vert the host functions [4,5]. Recent analyses further
showed that phage genomes can encode putative
toxins. Indeed, a family of polymorphic toxins (PT)
specifically associated with temperate phages has been
identified and has a conserved MuF domain on the
N-terminal side [6,7]. Polymorphic toxins are multido-
main proteins involved in pathogenesis and interbac-
terial competition. They include colicins and
subfamilies of type V (T5SS), type VI (T6SS) and type
VII (T7SS) secretion system effectors [8]. PT are char-
acterized by a conserved N-terminal domain involved
in toxin transport. Each N-terminal domain defines a
family of PT. These toxins also possess a variable
C-terminal domain that carries the toxic activity. Thus,
PT of the MuF family have an N-terminal conserved
MuF domain, named after the GpF protein of the Mu
phage, and a C-terminal extension carrying a predicted
toxic activity. *muf* genes are systematically associated
with the genes encoding the phage head. Little is
known about the MuF proteins except that they are
thought to be present in 2–3 copies in the virion head
and that they bind viral DNA [9,10].

Recently, we characterized a member of the MuF PT
family encoded by a prophage of the Gram-positive bacte-
rium *Streptococcus pneumoniae*. We showed that its
C-terminal extension has (p)ppApp synthetase activity
and confers toxicity when produced in the *Escherichia coli*
heterologous host [11]. This family of proteins that associ-
ates an N-terminal MuF domain with a C-terminal (p)
ppApp synthetase domain is known as Apk2 (adenosine
3'-pyrophosphokinase family 2) [12]. Another family,
called Apk1 (adenosine 3'-pyrophosphokinase family 1),
was also demonstrated to have (p)ppApp synthetase activ-
ity, but is associated with a N-terminal PAAR domain,
which is part of the T6SS tip complex [12,13]. ppApp and
pppApp, collectively written (p)ppApp, are modified
nucleotides resulting from the addition of a pyrophos-
phate group from an ATP molecule to the 3'-hydroxyl
end of an ADP and ATP molecule, respectively. Except
for the adenosine group instead of the guanosine, (p)
ppApp is similar to the (p)ppGpp alarmone, which is syn-
thesized during the stringent response to nutritional stress,
such as amino acid deprivation. The accumulation of (p)
ppGpp downregulates macromolecule synthesis pathways
and upregulates stress control pathways [14,15]. The pro-
duction and degradation of (p)ppGpp are regulated by
three types of enzymes: RSH (RelA/SpoT homolog), SAS
(small alarmone synthetase), and SAH (small alarmone
hydrolase) [14,15]. Like SpoT, well-characterized in
*E. coli*, RSHs possess both catalytic sites associated with
(p)ppGpp synthesis and hydrolysis. However, both sites

are not always functional: RelA in *E. coli* lacks hydrolase
activity due to the substitution of conserved amino acids.
SAS and SAH, on the other hand, contain a single cata-
lytic domain, responsible for synthesis and hydrolysis,
respectively. The synthesis domain contains five syn (syn-
thesis) motifs involved in the coordination of the magne-
sium cofactor, ATP, and GTP/GDP. The hydrolysis
domain contains six HD (hydrolysis) motifs, involved in
the coordination of the manganese cofactor, the guanine
base, and the hydrolysis of (p)ppGpp [14,16]. The (p)
ppApp synthetase domain from Apk2 displays Syn motifs
similar to those of the (p)ppGpp synthetases, with some
sequence variations [11].

In the *S. pneumoniae* prophage genome, the *apk2*
gene is followed by the *iapK* and *aph1* genes, which
encode immunity proteins. The IapK (immunity of
adenosine 3'-pyrophosphokinase) protein inhibits Apk2
toxin activity by occluding the pyrophosphate acceptor
nucleotide-binding site [11,13,17]. Aph1 (adenosine 3'-
pyrophosphohydrolase) hydrolyzes (p)ppApp in a man-
ner similar to (p)ppGpp hydrolases, partially regenerat-
ing the cellular pools of ATP and ADP [11,12]. Here, we
extend the characterization of adenosine 3'-
pyrophosphokinases by providing information on two
new members of the Apk2 family encoded by the pro-
phages of the Gram-negative bacteria *Mannheimia hae-
molytica* and *Pasteurella multocida*. Similar to that of
*S. pneumoniae*, their C-terminal Apk2tox domains con-
ferred toxicity when produced in the heterologous host
*E. coli*. The *M. haemolytica* Apk2tox domain was fur-
ther shown to have (p)ppApp synthetase activity in
vitro. We then unsuccessfully tried to convert Apk2tox to
a (p)ppGpp synthetase through substitution of the Syn
motifs. Finally, the interchangeability of the immunity
proteins was also investigated by cross-neutralization
and protein-protein interaction assays.

**Materials and methods**

**Bacterial strains and media**

*Escherichia coli* strains used in this study are listed in Table S1. Bacteria were grown in 2YT, Lysogeny broth (LB) or MacConkey agar (BD, Sparks, MD, USA), in the presence of antibiotics to maintain plasmids (ampicillin 100 µg·mL⁻¹, kanamycin 50 µg·mL⁻¹ or chloramphenicol 50 µg·mL⁻¹).

Plasmid construction and site-directed mutagenesis

Plasmids and primers used in this study are listed in
Tables S2 and S3, respectively. PCR amplifications were
performed with Phusion High-Fidelity DNA Polymerase (NEB, Evry, France). Site-directed mutagenesis was performed on plasmids following the instructions of the QuickChange site-directed mutagenesis kit (Stratagene). DNA templates corresponding to a portion of *S. pneumoniae* SPNA45, *M. haemolytica* D174, and *P. multocida* subsp. *multocida* HN06 prophage genomic regions were sequence-optimized for *E. coli* and synthesized by IDT. All constructs were checked by DNA sequencing (Eurofins Genomics, Ebersberg, Germany). The sequences of the proteins can be found in the Kyoto Encyclopedia of Genes and Genomes database (https://www.genome.jp/kegg/) using the following organism code and locus tags for *apk2*, *iapK*, and *aph1*, respectively:

snu, SPNA45_00317, SPNA45_00318, and SPNA45_00319 for *S. pneumoniae*;
mhao, J451_00890, J451_00895 and J451_00900 for *M. haemolytica*; and
pmv, PMCN06_2090, PMCN06_2091 and PMCN06_2092 for *P. multocida*.

## Toxicity, survival, and toxicity neutralization assays

These assays were performed as previously described [11]. Toxicity assays: *E. coli* MG1655 cells were transformed with plasmids allowing production of the toxin domain under the control of a PBAD promoter, which is glucose-locked and arabinose-induced. For toxicity assays, serial dilutions of bacterial cultures were spotted on LB agar plates containing either 1% glucose or 0.2% arabinose, and the plates were incubated at 37 °C for 18 h. Survival assay: 0.2% arabinose was added to the culture medium while the bacteria were in the exponential phase. Then, at different time points, aliquots were harvested, washed, and serial dilutions were spotted on LB agar plates containing 1% glucose. Toxicity neutralization assays: *E. coli* MG1655 cells were cotransformed with two plasmids, one allowing the production of the toxin domain under the control of a PBAD promoter and the other allowing the production of the immunity protein under the control of an anhydrotetracycline (AHTC)-inducible PTET promoter. Serial dilutions of bacterial cultures were spotted on LB agar plates containing either 1% glucose or 0.2% arabinose, and 200 ng·mL⁻¹ AHTC, and the plates were incubated at 37 °C for 18 h.

## Immunodetection

Protein samples were analyzed by standard SDS/PAGE and western blot. Immunodetection was performed using primary anti-FLAG mouse antibody (Clone M2; Merck, Darmstadt, Germany), and anti-mouse alkaline phosphatase-conjugated secondary antibody (Jackson ImmunoResearch, Cambridge, UK).

## *In vitro* synthesis of (p)ppApp or (p)ppGpp coupled with HPLC analysis

These experiments were performed as previously described [11,18]. Briefly, the *in vitro* pppApp or pppGpp synthesis reaction was carried out in 10 mM Tris-HCl pH 8, 100 mM NaCl, and 15 mM MgCl₂ containing 5 mM of nucleotide substrates (ATP alone or with GTP) and 1 µM of purified enzyme, and up to 50 µM for enzymes whose Syn motifs had been modified (see Ref. [11] for enzyme purification process). After incubation for 2 h at 37 °C, the reaction mixture was passed through a spin filter column and injected onto an HPLC system equipped with a strong anion exchange analytical column (SAX, Waters Spherisorb). Nucleotide separation was performed using an ionic strength gradient from solvent A (50 mM KH₂PO₄ pH 3.4) to solvent B (1 M KH₂PO₄ pH 3.4).

## Bacterial two-hybrid

Plasmids allowing the production of proteins fused to the T18 or T25 domains of the Bordetella pertussis adenylate cyclase were cotransformed in *E. coli* BTH101. Bacteria were grown overnight in LB supplemented with 0.5 mM IPTG, and 2 µL were spotted on MacConkey agar medium containing 1% maltose.

## Results

### The predicted adenosine 3'-pyrophosphokinase domains encoded by *M. haemolytica* and *P. multocida* prophages are toxic in *E. coli*

Investigation of the distribution of (p)ppApp synthetases revealed the existence of two families. Both families have a C-terminal domain with 3'-adenosine pyrophosphokinase activity but differ in their N-terminal domain: PAAR for the Apk1 family and MuF for the Apk2 family. Eighty-nine Apk2 homologous sequences were found and were distributed across multiple bacterial phyla such as Pseudomonadota, Bacteroidota, Bacillota, and Actinomycetota [12]. Two recent studies characterized the (p)ppApp synthetase activity of the C-terminal domain of Apk2 (Apk2tox) encoded by prophages from *Bacteroides caccae* and from *S. pneumoniae* that belong to the Bacteroidota and Bacillota phyla, respectively [11,12]. In this study, we extended the characterization to two new members encoded by prophages from *M. haemolytica* and *P. multocida* that belong to the Pseudomonadota phylum. To evaluate and compare the toxicity of the Apk2tox domains, corresponding coding sequences of *S. pneumoniae*, *M. haemolytica*, and *P. multocida* (hereafter referred to as Apk2tox-snu, Apk2tox-mhao and
Apk2<sub>tox-pmv</sub>, respectively) were cloned into the pBAD33 vector under the control of the P<sub>BAD</sub> promoter and an attenuated (5G) ribosome binding site (RBS). These domains were then heterologously produced in *E. coli*. Serial dilutions of *E. coli* cultures transformed with the empty vector or with the pBAD33-*apk2*<sub>tox-snu</sub>, pBAD33-*apk2*<sub>tox-mhao</sub>, and pBAD33-*apk2*<sub>tox-pmv</sub> constructs were plated on LB agar containing glucose or arabinose to repress or induce the P<sub>BAD</sub> promoter, respectively (Fig. 1A). While the growth of bacteria transformed by the different plasmids was comparable under repression conditions, the production of Apk2<sub>tox</sub> domains conferred growth inhibition. The Apk2<sub>tox-mhao</sub> domain displayed a high toxic activity, similar to that of the Apk2<sub>tox-snu</sub> domain, whereas the Apk2<sub>tox-pmv</sub> domain was less toxic in *E. coli*.

**Heterologous production of the S. pneumoniae and M. haemolytica Apk2<sub>tox</sub> domains is bactericidal to E. coli**

To test whether the Apk2<sub>tox</sub> toxins have a bactericidal or bacteriostatic effect, a survival assay was performed. For this, samples of *E. coli* cultures producing the Apk2<sub>tox</sub> domains were harvested at different induction times (0–90 min) and spotted on solid medium under repressive conditions. This allowed us to estimate the number of cells that have survived the

induction period. Figure 1B shows that the activity of Apk2<sub>tox-snu</sub> and Apk2<sub>tox-mhao</sub> was bactericidal, since the bacterial growth observed on repressive solid media was reduced as the induction time for domain production increased. In contrast, Apk2<sub>tox-pmv</sub> did not

**Fig. 1.** Toxicity of *Streptococcus pneumoniae*, *Mannheimia haemolytica*, and *Pasteurella multocida* Apk2<sub>tox</sub> domains in *Escherichia coli*. (A) Toxicity assays—*E. coli* transformed with pBAD33 vectors encoding the toxic C-ter domain of Apk2 from *S. pneumoniae*, *M. haemolytica*, or *P. multocida* (Apk2<sub>tox-snu</sub>, Apk2<sub>tox-mhao</sub> or Apk2<sub>tox-pmv</sub>, respectively) were grown to mid-exponential phase, and serial dilutions were spotted on selective medium that repressed or induced *apk2*<sub>tox</sub> expression. Data shown are representative of *n* ≥ 3 experiments. (B) Survival assays—*E. coli* transformed with pBAD33 vectors encoding Apk2<sub>tox-snu</sub>, Apk2<sub>tox-mhao</sub>, or Apk2<sub>tox-pmv</sub> were grown to mid-exponential phase. After induction of the *apk2*<sub>tox</sub> genes for 0–90 min as indicated, culture samples were harvested, washed, serially diluted, and spotted onto selective medium that repressed the expression of these genes. The experiment was performed twice. (C) Protein production—Genes encoding FLAG-tagged catalytic-null variants of Apk2<sub>tox</sub> domains were induced in *E. coli* for 5 h. Proteins from the corresponding cell extracts were separated by SDS/PAGE, and FLAG-tagged Apk2<sub>tox</sub> domains were immunodetected. Full-length form and degradation products are indicated by a black arrowhead and the asterisk, respectively. Molecular weight markers are indicated on the left (in kDa). The panel on bottom shows a piece of membrane stained with Ponceau red as a control for protein loading. Data shown are representative of *n* = 3 experiments.

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Correlation between Apk2tox quantity and toxicity. (A) Sequences of the Apk2tox-snu 5G and 4G suboptimal ribosome binding site (RBS) variants. (B) Protein production—The FLAG-tagged catalytic-null variant of Apk2tox-snu was produced in *Escherichia coli* for 5 h using a 5G or 4G suboptimal RBS. Proteins from the corresponding cell extracts were separated by SDS/PAGE, and the FLAG-tagged Apk2tox-snu domain was immunodetected. The full-length form is indicated by a black arrowhead (expected mass ~ 23 kDa, molecular weight marker on the left). The panel on the bottom shows a piece of membrane stained with Ponceau red as a control for protein loading. Data shown are representative of *n* = 3 experiments. (C) Survival assays—*E. coli* transformed with pBAD33 vectors encoding the Apk2tox-snu gene cloned downstream of a suboptimal 5G or 4G RBS were grown to mid-exponential phase. After induction of the Apk2tox-snu domain for 0–60 min as indicated, culture samples were harvested, washed, serially diluted, and spotted onto selective medium that repressed the expression of *apk2tox-snu*. The experiment was performed twice.</figcaption></figure>

kill *E. coli*, as bacterial growth was comparable to that of *E. coli* cells bearing the empty vector, even after up to 90 min of induction.

## Estimation of Apk2tox domain levels in heterologous host *E. coli*

The previous experiment showed that transient production of Apk2tox of *S. pneumoniae* or *M. haemolytica* in *E. coli* is bactericidal, whereas transient production of Apk2tox of *P. multocida* is not, even though the experimental setup was the same: same promoter, same RBS, and synthetic DNA optimized for *E. coli*. These results echo those of another study in which it was shown that the production of Apk1tox from *Pseudomonas aeruginosa* in the heterologous host *E. coli* had a bactericidal effect, whereas production of Apk2tox from *B. caccae* had a bacteriostatic effect [12]. Taken together, these results raised the question of whether Apktox domains have activities with different efficiencies or whether a production/stability issue occurred despite identical production conditions.

To address this question, the amount of Apk2tox domains in *E. coli* was examined by western blot. Protein extracts were therefore prepared from *E. coli* strains producing FLAG-tagged Apk2tox domains (which displayed toxicity identical to untagged versions, Fig. S1) for 5 h. To avoid toxicity during the

experiments, the key aspartate residues of the catalytic site of Apk2tox domains were substituted by glycine. The proteins were then separated by SDS/PAGE, and the Apk2tox FLAG-tagged domains were immunodetected with the anti-FLAG antibody. The Apk2tox-snu and Apk2tox-mhao tagged domains were detected at the expected size (~ 23 kDa) (Fig. 1C). According to the different replicates, Apk2tox-snu and Apk2tox-mhao were produced in comparable amounts or slightly less for the second one, an observation that is consistent with the results of the survival assays, in which the Apk2tox-mhao was reproducibly slightly less toxic than Apk2tox-snu (Fig. 1B). The Apk2tox-pmv tagged domain was not detected at the expected size, but a degradation fragment was detected at ~ 11 kDa, indicating that Apk2tox-pmv was cleaved and explaining the diminished toxicity observed for Apk2tox-pmv in *E. coli*.

To unambiguously correlate protein level and toxicity without working with different domains, Apk2tox-snu production and toxicity were compared in the heterologous host *E. coli* using the initial attenuated 5G RBS or a further suboptimal (4G) RBS (Fig. 2A). Immunodetection showed a significant difference in the production of Apk2tox-snu under the control of the 4G RBS compared to the 5G (Fig. 2B). In our western blot experiments, we observed that an induction time of 5 h to produce Apk2tox-snu allowed the accumulation of the full-length domain, whereas a
shorter induction time led to the detection of majori-
tarian degradation products. We suggest that a degra-
dation system must be saturated to observe the full-
length form and that using a lower RBS (4G) reduces
production, preventing saturation of the degradation
system and making the full-length form more difficult
to detect compared to the degradation products. This
decrease correlated with an attenuated bactericidal
effect (Fig. 2C), demonstrating that differences in tox-
icity can be explained by differences in heterologous
production or stability.

In vitro synthesis of pppApp by Apk2<sub>tox</sub> domains

To confirm the predicted adenosine 3'-
pyrophosphokinase activity of the Apk2<sub>tox</sub> domains, *in vitro* pppApp synthesis reactions were performed. Purified Apk2<sub>tox-mhao</sub> domain was incubated with ATP for 2 h at 37 °C, and the products were analyzed by strong anion exchange high-performance liquid chromatography (SAX-HPLC). Apk2<sub>tox-snu</sub>, for which the adenosine 3'-pyrophosphokinase activity was previously characterized [11], was used as a positive control. The instability of Apk2<sub>tox-pmv</sub> prevented its production and purification. The chromatograms obtained after SAX-HPLC analysis showed a single peak corresponding to ATP substrate in the control reaction without enzyme, a peak that decreased in the reactions with the Apk2<sub>tox-snu</sub> and Apk2<sub>tox-mhao</sub> domains, reflecting ATP consumption (Fig. 3). Two additional peaks appeared in the same samples, corresponding to AMP and pppApp, both products of the transfer of a pyrophosphate group from one ATP molecule to another. When similar experiments were performed with both ATP and GTP as substrates, no pppGpp was detected (Fig. S2, left panels), confirming that the Apk2<sub>tox-mhao</sub> domain, like the previously characterized Apk2<sub>tox-snu</sub> domain, specifically synthesizes pppApp.

Conversion of the syn2-3-4 motifs of Apk2tox to
syn motifs of guanosine 3'-pyrophosphokinase
does not change nucleotide specificity

Based on a multiple protein sequence alignment
comparing guanosine 3'-pyrophosphokinase RSH
members with predicted or proven adenosine 3'-
pyrophosphokinases, a previous study identified spe-
cific signatures at conserved syn sites that differ from
one family to the other [11]. We thus investigated
whether it was possible to convert a pppApp synthe-
tase into a pppGpp synthetase by introducing substitu-
tions at the syn motifs of the Apk2tox-snu domain. The
Apk2tox-snu syn2, syn3, and syn4 motifs were

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** SAX-HPLC analysis of nucleotides after *in vitro* reactions with Apk2tox domains. Strong anion exchange high-performance liquid chromatography (SAX-HPLC) chromatograms of nucleotide products of *in vitro* synthesis reactions, with ATP as substrate and the indicated purified Apk2tox domain. The control chromatogram (top panel) corresponds to a reaction that did not contain any enzyme. The peaks corresponding to AMP and ATP were determined with control experiments using commercial nucleotides. The experiment was performed twice.</figcaption></figure>

substituted to correspond to consensus sequences asso-
ciated with RSH. The GxN residues were converted to
SxH at the syn3 site, which has been described to rec-
ognize guanosine in RSH [16], while the RYT and YH
residues of the syn2 and syn4 motifs, respectively, were
converted to AVR and IR. The Apk2tox-snu domain
variants were purified and used in vitro synthesis
reactions with SAX-HPLC analysis of the products.
<table><caption>**Table 1.** Toxicity neutralization assays. The evaluation of the toxicity level upon heterologous production in *Escherichia coli* is ranked strong (++) or intermediate (+) (Fig. 1A). The rescue profile based on several experiments is summarized in this table. The evaluation of the neutralization is ranked total (++), intermediate (+, small colonies or visible at lower dilutions than with total neutralization), and no neutralization (−) (Fig. S3).</caption><thead><tr><th rowspan="3">Toxicity</th><th rowspan="3"></th><th rowspan="3"></th><th colspan="6">Neutralization</th></tr><tr><th rowspan="2">Empty plasmid</th><th colspan="3">IapK</th><th colspan="3">Aph1</th></tr><tr><th>snu</th><th>mhao</th><th>pmv</th><th>snu</th><th>mhao</th><th>pmv</th></tr></thead><tbody><tr><td rowspan="3">Apk2<sub>tox</sub></td><td>snu</td><td>++</td><td>—</td><td>++</td><td>—</td><td>++/+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>mhao</td><td>++</td><td>—</td><td>—</td><td>++</td><td>+/-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>pmv</td><td>+</td><td>—</td><td>+/-</td><td>-/+</td><td>++</td><td>++</td><td>++</td><td>++</td></tr></tbody></table>

Introduction of these substitutions resulted in progressive inactivation of the Apk2tox-snu adenosine 3'-pyrophosphokinase activity but did not lead to pppGpp production (Fig. S2, right panels). The failure to convert the Apk2tox-snu (p)ppApp synthetase to a (p)ppGpp synthetase by substituting conserved amino acids in the syn motifs suggests either that additional amino acids are key to control the specificity of the substrate or of the reaction, or that the environment provided by the protein backbone is not suitable for producing a functional enzyme after these substitutions.

## Interchangeability of the Apk2tox immunity proteins

The *apk2* genes are followed by two genes, *iapK* (immunity of apk) and *aph1* (adenosine 3'-pyrophosphohydrolase), which encode immunity proteins. IapK was shown to neutralize Apk2tox by occluding the active site of the toxin, whereas Aph1 cleaves the pyrophosphate group of pppApp, hence regenerating the ATP pool [11,12]. To assess the interchangeability of the immunity proteins whose genes are genetically linked to the *S. pneumoniae*, *M. haemolytica*, and *P. multocida* muf genes, we performed neutralization assays. For this, *E. coli* cells were cotransformed with a plasmid allowing the production of one of the Apk2tox domains (snu, mhao, and pmv) and a plasmid allowing the production of one of the six immunity proteins. Cultures of the cotransformants were then serially diluted and spotted on LB agar supplemented with L-arabinose and AHTC to induce the expression of the cloned toxin and immunity genes. The results demonstrated that, as expected, a nonspecific cross-immunity was provided by the different Aph1 proteins, which hydrolyze pppApp, the product of the reaction catalyzed by the different Apk2tox domains (Table 1 and Fig. S3). However, in our experimental setup, neutralization was only partial, except in the

case of Apk2tox-pmv, whose toxicity is low. By contrast, we observed an orthologous relationship for the IapK immunity proteins: Each Apk2tox domain toxicity was neutralized by its cognate IapK immunity (Table 1 and Fig. S3). However, IapKpmv was able to fully protect cells from Apk2tox-snu toxicity and we noticed a decreasing gradient of versatility: IapKpmv > IapKsnu > IapKmhao.

To further understand the specificity of cross-neutralization between Apk2 toxins and IapK immunities, protein-protein interactions were assessed by bacterial two-hybrid. Figure 4 shows that a strict orthologous relationship exists between cognate pairs. Surprisingly, while the toxicity neutralization assay indicated that IapKpmv protects from Apk2tox-snu toxicity, these two proteins do not interact in the bacterial two-hybrid assay. A possible interpretation could be that in the neutralization experimental setup (i.e., the toxin/immunity ratio is in favor of the immunity

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Apk2tox domain and IapK immunity protein-protein interactions. Bacterial two-hybrid analysis—Drops of cultures of *Escherichia coli* BTH101 reporter cells producing the FLAG-tagged catalytic-null variant of indicated Apk2tox domains and IapK immunity proteins fused to the T25 and T18 domains of the *Bordetella pertussis* adenylate cyclase, respectively, were spotted on McConkey agar medium. The red color of the colony reports the reconstitution of the adenylate cyclase due to the interaction between the tested proteins. Data shown are representative of *n* = 4 experiments.</figcaption></figure>
<table><caption>**Table 2.** Percentage of identity between Apk2<sub>tox</sub> domains and IapK and Aph1 immunities based on Omega Clustal protein sequence alignments.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Apk2<sub>tox</sub></th><th colspan="3">IapK</th><th colspan="3">Aph1</th></tr><tr><th>snu</th><th>mhao</th><th>pmv</th><th>snu</th><th>mhao</th><th>pmv</th><th>snu</th><th>mhao</th><th>pmv</th></tr></thead><tbody><tr><td>snu</td><td>100</td><td>44</td><td>46</td><td>100</td><td>35</td><td>38</td><td>100</td><td>45.2</td><td>42.7</td></tr><tr><td>mhao</td><td>44</td><td>100</td><td>45.5</td><td>35</td><td>100</td><td>36.6</td><td>45.2</td><td>100</td><td>57</td></tr><tr><td>pmv</td><td>46</td><td>45.5</td><td>100</td><td>38</td><td>36.6</td><td>100</td><td>42.7</td><td>57</td><td>100</td></tr></tbody></table>

protein), an immunity protein with low affinity for a toxin could compensate by its higher production level. But, an interaction that would be too labile and/or dynamic may not be captured by the bacterial two-hybrid method, as we have previously observed [19].

## Discussion

Three previous studies characterized polymorphic toxins with adenosine 3'-pyrophosphokinase (Apk) activity from the Apk1 family, which is associated with the PAAR domain of the Type VI secretion system (T6SS) [13] and from the Apk2 family, which is associated with MuF phage proteins [11,12]. This study extends the characterization of this new family of enzymes to include two new members of the Apk2 family encoded by bacteriophages from the Gram-negative bacteria *M. haemolytica* and *P. multocida*. Overall, our results on these Apk domains indicate that (a) heterologous production of these domains in *E. coli* is toxic, (b) protein levels correlate with toxicity, with a potential bactericidal effect, (c) these enzymes catalyze the exclusive synthesis of (p)ppApp, (d) substitution of the conserved syn2, 3, and 4 motifs is not sufficient to change the specificity of the reaction toward (p)ppGpp synthesis, and (e) IapK immunity proteins are orthologous to their cognate Apk2<sub>tox</sub> domains with more or less pronounced stringency.

Due to its instability, we were not able to demonstrate the adenosine 3'-pyrophosphokinase activity of the Apk2<sub>tox</sub> domain of *P. multocida*. However, this potential activity is also supported by the fact that the *aph1* gene, which is genetically linked to it, possesses the typical signatures of (p)ppApp hydrolases and all the conserved active site residues that determine the specificity for (p)ppApp [12].

Aph1 immunity proteins, which are enzymes hydrolyzing the (p)ppApp product, share between 43% and 57% identity (Table 2). On the other hand, the percentage of identity of IapK immunity proteins drops to 35–38% (Table 2) and probably reflects the adaptation of IapK to their cognate toxin, given their mode of

action by protein-protein interaction and occlusion of the active site. Our bacterial two-hybrid results highlight this orthogonality. However, our experimental setup in the toxicity neutralization assays revealed a slight versatility with IapK<sub>pmv</sub>, which provides protection against Apk2<sub>tox-snu</sub>. Does this observation reflect an evolutionary history? *M. haemolytica* and *P. multocida* being quite distant in the bacterial phylogenetic tree from *S. pneumoniae*, one may hypothesize that prophage elements moved from one strain to the other by horizontal gene transfer. While it is difficult to conceive that the prophage elements were acquired by transduction because of the specificity of phages for bacterial species, *Streptococcus* and *Pasteurella* strains are naturally competent and are well known for their capacity to acquire foreign DNA by transformation [20,21].

It is noteworthy that these bacterial species occupy the upper respiratory tract. However, *Mannheimia* and *Pasteurella* contribute to bovine respiratory diseases [20] while *S. pneumoniae* is an opportunistic pathogen of the upper respiratory tract responsible for human infections [21]. Human respiratory infections caused by *Pasteurella* are relatively rare, but some cases were reported in patients with chronic lung diseases [20] and bacteria of the genus *Streptococcus* are present in the bacterial microbiota of the bovine respiratory tract [22]. It would be interesting to decipher the dynamic exchange of genetic material, and notably of prophage elements, between bacterial strains in their natural habitat and to understand the physiological role of these prophages and of MuF polymorphic toxins.

## Conclusions

In conclusion, this study extends our understanding of polymorphic toxins with adenosine 3'-pyrophosphokinase (Apk) activity by analyzing two new members of the Apk2 family. Their integrity/stability must be taken into account when comparing their activity/toxicity in the heterologous host *E. coli*. Two types of immunity proteins can counteract their toxicity, either by protein-protein interaction with the
toxin or by hydrolysing the product and hence restoring the ATP pool. Although very similar to RSH proteins, the conversion of their (p)ppApp synthetase activity to (p)ppGpp synthetase is not straightforward and, if possible, would require further analysis. The physiological role of these polymorphic toxins remains to be established.

## Acknowledgements

We thank Lisa Duchemin for cloning during her training. We thank the members of the Cascales group for fruitful discussions. This work was funded by the ANR (G4PLAST, ANR-17-CE13-0005), the CNRS, and Aix-Marseille Université.

## Conflict of interest

The authors declare no conflict of interest.

## Peer review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/2211-5463.70038.

## Data accessibility

Supporting data are contained in the manuscript in the Supporting Information section.

## Author contributions

EMP, JB, AK, CMP, ACT and JPV acquired and interpreted the data. JPV conceived the project. EMP and JPV wrote the paper. JB and EC edited the paper.

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**Supporting information**

Additional supporting information may be found
online in the Supporting Information section at the end
of the article.

**Fig. S1.** Toxicity of *S. pneumoniae*, *M. haemolytica* and *P. multocida* Apk2tox and Apk2tox FLAG-tagged domains in *E. coli*.

**Fig. S2.** Strong anion exchange HPLC analysis of nucleotides after *in vitro* reactions with Apk2tox domains.

**Fig. S3.** Toxicity neutralization assays and evaluation of cross-immunity.

**Table S1.** Bacterial strains.

**Table S2.** Plasmids.

**Table S3.** Primers.
<figure><img src="image_7.png" /><figcaption>Figure SI 1 - Toxicity of *S. pneumoniae*, *M. haemolytica* and *P. multocida* Apk2tox and Apk2tox FLAG-tagged domains in *E. coli*</figcaption></figure>

*E. coli* transformed with pBAD33 vectors encoding Apk2tox-snu, Apk2tox-mhao or Apk2tox-
pmv or their FLAG-tagged versions were grown to mid-exponential phase and serial dilutions were spotted on selective medium that repressed or induced *apk2tox* expression.

The experiment was performed twice.
<figure><img src="image_8.png" /><figcaption>Figure SI 2</figcaption></figure>
Figure SI 2– **Strong anion exchange HPLC analysis of nucleotides after *in vitro* reactions with Apk2<sub>tox</sub> domains**

Left panels- *In vitro* synthesis reactions were performed with the indicated purified domain and with ATP and GTP as substrates. Rel<sub>seq</sub> is the catalytic N-terminal fragment (residues 1 to 385) of the bifunctional RelA/SpoT homolog from *Streptococcus dysgalactiae* subsp. *Equisimilis*. The control chromatogram corresponds to a reaction that did not contain any enzyme. Right panels- *In vitro* synthesis reactions were performed with the Apk2<sub>tox-snu</sub> purified domains that had amino acid substitutions in the synthesis motifs (syn) as specified in the main text. ATP and GTP were provided as substrates. As no activity was detected for Apk2<sub>tox-snu</sub> with substitutions in the syn motifs when used at 1 µM like in the other assays, *in vitro* reactions were also performed with 50 µM of these variants. Apk2<sub>tox-snu</sub> with substitutions in the syn3 motif was the only one for which a weak residual 3'-adenosine pyrophosphokinase activity was detected in this condition.

The peaks corresponding to regular nucleotides or pppGpp were determined with control experiments using commercial nucleotides.
<figure><img src="image_9.png" /><figcaption>Figure SI 3- Toxicity neutralization assays and evaluation of cross-immunity</figcaption></figure>

E. coli MG1655 cells were co-transformed with two plasmids and grown to mid-exponential phase before to be serially diluted and plated on LB agar plates containing anhydrotetracycline to induce the expression of the indicated (snu, mhao, pmv) *iapK* or *aph1* immunity genes from the first plasmid, and glucose or arabinose to repress or induce the expression of the indicated *apk2* from the second plasmid. Data shown are representative of n ≥3 experiments.
<table><caption>Table SI 1- Bacterial strains</caption><thead><tr><td>Lab code</td><td>Name</td><td>Genotype</td><td>Reference</td></tr></thead><tbody><tr><td>EB3</td><td>BTH101</td><td>F-, cya-99, araD139, galE15, galK16, rpsL1 (Str r), hsdR2, mcrA1, mcrB1</td><td>[1]</td></tr><tr><td>EB70</td><td>DH5α</td><td>fhuA2, Δ(argF-lacZ)U169, phoA, glnV44, Φ80, Δ(lacZ)M15, gyrA96, recA1, relA1, endA1, thi-1, hsdR17</td><td>Lab stock</td></tr><tr><td>EB72</td><td>BL21(DE3) pLys</td><td>F-, ompT, gal, dcm, lon, hsdSB, (rB- mB-), λ(DE3), pLysS(cm<sup>R</sup>)</td><td>Lab stock</td></tr><tr><td>EB944</td><td>MG1655</td><td>F-, λ-, ilvG-, rfb-50, rph-1</td><td>Lab stock</td></tr></tbody></table>
<table><caption>Table SI 2- Plasmids</caption><thead><tr><th>Lab code</th><th>Description</th><th>Reference</th></tr></thead><tbody><tr><td colspan="3">Toxicity, survival and toxicity neutralization assays</td></tr><tr><td>pEB1017</td><td>pBAD33</td><td>[2]</td></tr><tr><td>pJV381</td><td>pBAD33-apk2<sub>tox-snu</sub> (amino acid residues 320 to 506 from the full-length Apk2<sub>snu</sub> under the control of a RBS made of 5G)</td><td>[3]</td></tr><tr><td>pJV383</td><td>pBAD33-apk2<sub>tox-snu</sub> (as above but with a RBS made of 4G)</td><td></td></tr><tr><td>pJV390</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G (D390G if full-length Apk2<sub>snu</sub>) (RBS made of 5G)</td><td>[3]</td></tr><tr><td>pJV391</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G (D390G if full-length Apk2<sub>snu</sub>) (RBS made of 4G)</td><td></td></tr><tr><td>pJV421</td><td>pBAD33-apk2<sub>tox-mhao</sub> (amino acid residues 359 to 553 from the full-length Apk2<sub>mhao</sub> under the control of a RBS made of 5G. PCR product w/ primers ebm2154/2155 at KpnI/SalI restriction sites)</td><td></td></tr><tr><td>pJV422</td><td>pBAD33-apk2<sub>tox-pmv</sub> (amino acid residues 346 to 533 from the full-length Apk2<sub>pmv</sub> under the control of a RBS made of 5G. PCR product w/ primers ebm2152/2153 at KpnI/SalI restriction sites)</td><td></td></tr><tr><td>pJV445</td><td>pBAD33-apk2<sub>tox-snu</sub> 1xFlag (PCRi+SLIC, primers ebm2200/2201, template pJV381)</td><td></td></tr><tr><td>pJV446</td><td>pBAD33-apk2<sub>tox-mhao</sub> 1xFlag (PCRi+SLIC, primers ebm2202/2203, template pJV421)</td><td></td></tr><tr><td>pJV447</td><td>pBAD33-apk2<sub>tox-pmv</sub> 1xFlag (PCRi+SLIC, primers ebm2204/2205, template pJV422)</td><td></td></tr><tr><td>pJV457</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G 1xFlag (RBS made of 5G) (mutagenesis, primers ebm2118/2119, template pJV445)</td><td></td></tr><tr><td>pJV458</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G 1xFlag (RBS made of 4G) (PCRi+SLIC, primers ebm2200/2201, template pJV391)</td><td></td></tr><tr><td>pJV459</td><td>pBAD33-apk2<sub>tox-mhao</sub> D73G 1xFlag (D430G if full-length Apk2<sub>mhao</sub>)(mutagenesis, primers ebm2208/2209, template pJV446)</td><td></td></tr><tr><td>pJV460</td><td>pBAD33-apk2<sub>tox-pmv</sub> D72G 1xFlag (D416G if full-length Apk2<sub>pmv</sub>)(mutagenesis, primers ebm2210/2211, template pJV447)</td><td></td></tr><tr><td>pEB1242</td><td>pASK-IBA37plus</td><td>IBA Lifesciences</td></tr><tr><td>pJV374</td><td>pP<sub>TET</sub>-iapK<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV375</td><td>pP<sub>TET</sub>-aph1<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV419</td><td>pP<sub>TET</sub>-iapK<sub>mhao</sub> (PCR product w/ primers ebm2166/2167 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV420</td><td>pP<sub>TET</sub>-aph1<sub>mhao</sub> (PCR product w/ primers ebm2168/2169 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV423</td><td>pP<sub>TET</sub>-iapK<sub>pmv</sub> (PCR product w/ primers ebm2160/2161 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV424</td><td>pP<sub>TET</sub>-aph1<sub>pmv</sub> (PCR product w/ primers ebm2162/2163 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td colspan="3">Protein Production and Purification</td></tr><tr><td>pEB1520</td><td>pETDuet-1</td><td>Novagen</td></tr><tr><td>pJV403</td><td>pETDuet-1-apk2<sub>tox-snu</sub> iapK<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV435</td><td>pETDuet-1-apk2<sub>tox-mhao</sub> iapK<sub>mhao</sub> (PCR products w/ primers ebm2181/2155 and ebm2179/2180 at EcoRI/SalI and NdeI/XhoI, respectively)</td><td></td></tr><tr><td>pJV439</td><td>pETDuet-1-apk2<sub>tox-snu</sub> syn3<sub>mut</sub> iapK<sub>snu</sub> (mutagenesis G436S N438H according to the coordinates of the full-length protein, primers ebm2134/2135)</td><td></td></tr><tr><td>pJV440</td><td>pETDuet-1-apk2<sub>tox-snu</sub> syn3-syn2<sub>mut</sub> iapK<sub>snu</sub> (further mutagenesis R393A Y394V T395R according to the coordinates of the full-length protein, primers ebm2170/2171)</td><td></td></tr><tr><td>pJV441</td><td>pETDuet-1-apk2<sub>tox-snu</sub> syn3-syn2-syn4<sub>mut</sub> iapK<sub>snu</sub> (further mutagenesis Y452I H453R according to the coordinates of the full-length protein, primers ebm2174/2175)</td><td></td></tr><tr><td colspan="3">Bacterial Two-Hybrid</td></tr><tr><td>pEB354</td><td>pKT25linker</td><td>[4]</td></tr><tr><td>pJV468</td><td>pT25_Apk2<sub>tox-snu</sub> D72G 1xFlag (PCR product w/ primers ebm2122/2212 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV469</td><td>pT25_Apk2<sub>tox-mhao</sub> D73G 1xFlag (PCR product w/ primers ebm2207/2212 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV470</td><td>pT25_Apk2<sub>tox-pmv</sub> D72G 1xFlag (PCR product w/ primers ebm2206/2212 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pEB355</td><td>pUT18Clinker</td><td>[4]</td></tr><tr><td>pJV397</td><td>pT18_lapK<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV455</td><td>pT18_lapK<sub>mhao</sub> (PCR product w/ primers ebm2166/2167 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV456</td><td>pT18_lapK<sub>pmv</sub> (PCR product w/ primers ebm2160/2161 at EcoRI/XhoI restriction sites)</td><td></td></tr></tbody></table>
<table><caption>Table SI 3- Primers</caption><thead><tr><th>Lab code ebm</th><th>5'-3' sequence</th></tr></thead><tbody><tr><td>2118</td><td>AAAGCAGTTAGCAAAATTAACGgCGCTTTACGTTATACAACTATCTTT</td></tr><tr><td>2119</td><td>AAAGATAGTTGTATAACGTAAAGCGcCGTTAATTTTGCTAACTGCTTT</td></tr><tr><td>2122</td><td>gaagaattcatgGCGAAAGCTAAATTCTATAGTGAA</td></tr><tr><td>2134</td><td>GATGGGCCATACAAAaGTGTAcACACGGTTGTCGAGAAAGATGG</td></tr><tr><td>2135</td><td>CCATCTTTCTCGACAACCGTGTgTACACtTTTGTATGGCCCATC</td></tr><tr><td>2152</td><td>ggtggtaccGGGGGcgtctggatgGTTGAGTCAGCTGTAAACGC</td></tr><tr><td>2153</td><td>ctcctcgagCTAGTTGCGGATCTTACCAAT</td></tr><tr><td>2154</td><td>ggtggtaccGGGGGcgtctggatgGTTGATAAAATGGACAGTGTGG</td></tr><tr><td>2155</td><td>ctcctcgagTTACTTTTTTTCCTTGATCAGGT</td></tr><tr><td>2160</td><td>gaagaattcATGGAATACTATTTAGTTTTTCCT</td></tr><tr><td>2161</td><td>ctcctcgagTCACTTGTGTTTGTCAATCCAT</td></tr><tr><td>2162</td><td>gaagaattcATGCACGGTTTGATCAGTATC</td></tr><tr><td>2163</td><td>ctcctcgagTCAGGTTTGAAGAAATTGCTTC</td></tr><tr><td>2166</td><td>ctcctcgagTCAGGTTTGAAGAAATTGCTTC</td></tr><tr><td>2167</td><td>ctcctcgagTTAATGCACTTTAATAAAGCGGA</td></tr><tr><td>2168</td><td>gaagaattcATGTGCCAAGCGGAGCAGT</td></tr><tr><td>2169</td><td>ctcctcgagTCATGCCTGCAAGTATTCAAG</td></tr><tr><td>2170</td><td>GTTAGCAAAATTAACGACGCTTTAgcTgtTcgtACTATCTTTGACTCTGATACTTTTAC</td></tr><tr><td>2171</td><td>GTAAAAGTATCAGAGTCAAAGATAGTacgAacAgcTAAAGCGTCGTTAATTTTGCTAAC</td></tr><tr><td>2174</td><td>GATGGTATCAATTTTGAAATGCAGatCCgCACACAGGAAAGCTTTGACCT</td></tr><tr><td>2175</td><td>AGGTCAAAGCTTTCCTGTGTGcGGatCTGCATTTCAAAATTGATACCATC</td></tr><tr><td>2179</td><td>catcatATGTACCAGTATTACCTGGCG</td></tr><tr><td>2180</td><td>ctcctcgagATGCACTTTAATAAAGCGGATC</td></tr><tr><td>2181</td><td>gaagaattcgATGGTTGATAAAATGGACAGTGTGG</td></tr><tr><td>2200</td><td>tttatcatcgtcgtctttataatcTTTAACACGCTCAATGTTTTTTG</td></tr><tr><td>2201</td><td>gattataaagacgacgatgataaaTGAacagtgcctgcacctgc</td></tr><tr><td>2202</td><td>tttatcatcgtcgtctttataatcCTTTTTTTTCCTTGATCAGGTCG</td></tr><tr><td>2203</td><td>gattataaagacgacgatgataaaTAActgcacctgcaggcatg</td></tr><tr><td>2204</td><td>tttatcatcgtcgtctttataatcGTTGCGGATCTTACCAATGTTC</td></tr><tr><td>2205</td><td>gattataaagacgacgatgataaaTAGctgcacctgcaggcatg</td></tr><tr><td>2206</td><td>gaagaattcATGGTTGAGTCAGCTGTAAACGC</td></tr><tr><td>2207</td><td>gaagaattcATGGTTGATAAAATGGACAGTGTGG</td></tr><tr><td>2208</td><td>CAAGCGATCGCCTCCATCCGCGgCGTGATTCGCTATACGGCGATTCTG</td></tr><tr><td>2209</td><td>CAAGCGATCGCCTCCATCCGCGgCGTGATTCGCTATACGGCGATTCTG</td></tr><tr><td>2210</td><td>GAATCACTTAGTAAAATCACCGgTATTGTGCGTTATACCACAATCTTC</td></tr><tr><td>2211</td><td>GAAGATTGTGGTATAACGCACAATAcCGGTGATTTTACTAAGTGATTC</td></tr><tr><td>2212</td><td>ctcctcgagTTAtttatcatcgtcgtctttataatc</td></tr></tbody></table>
References

1. Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. *Proc Natl Acad Sci USA* 95, 5752–5756

2. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. *J Bacteriol* 177, 4121–4130

3. Bartoli J, Tempier AC, Guzzi NL, Piras CM, Cascales E, Viala JP (2023) Characterization of a (p)ppApp Synthetase Belonging to a New Family of Polymorphic Toxin Associated with Temperate Phages. *Journal of Molecular Biology* 435:1682–282

4. Gully D, Bouveret E (2006) A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli. *Proteomics* 6, 282–293
<figure><img src="image_10.png" /><figcaption>Figure SI 1 - Toxicity of *S. pneumoniae*, *M. haemolytica* and *P. multocida* Apk2<sub>tox</sub> and Apk2<sub>tox</sub> FLAG-tagged domains in *E. coli*</figcaption></figure>

*E. coli* transformed with pBAD33 vectors encoding Apk2<sub>tox-snu</sub>, Apk2<sub>tox-mhao</sub> or Apk2<sub>tox-pmv</sub> or their FLAG-tagged versions were grown to mid-exponential phase and serial dilutions were spotted on selective medium that repressed or induced *apk2<sub>tox</sub>* expression.

The experiment was performed twice.
<figure><img src="image_11.png" /><figcaption>Figure SI 2</figcaption></figure>
Figure SI 2– **Strong anion exchange HPLC analysis of nucleotides after *in vitro* reactions with Apk2<sub>tox</sub> domains**

Left panels- *In vitro* synthesis reactions were performed with the indicated purified domain and with ATP and GTP as substrates. Rel<sub>seq</sub> is the catalytic N-terminal fragment (residues 1 to 385) of the bifunctional RelA/SpoT homolog from *Streptococcus dysgalactiae* subsp. *Equisimilis*. The control chromatogram corresponds to a reaction that did not contain any enzyme. Right panels- *In vitro* synthesis reactions were performed with the Apk2<sub>tox-snu</sub> purified domains that had amino acid substitutions in the synthesis motifs (syn) as specified in the main text. ATP and GTP were provided as substrates. As no activity was detected for Apk2<sub>tox-snu</sub> with substitutions in the syn motifs when used at 1 µM like in the other assays, *in vitro* reactions were also performed with 50 µM of these variants. Apk2<sub>tox-snu</sub> with substitutions in the syn3 motif was the only one for which a weak residual 3'-adenosine pyrophosphokinase activity was detected in this condition.

The peaks corresponding to regular nucleotides or pppGpp were determined with control experiments using commercial nucleotides.
<figure><img src="image_12.png" /><figcaption>Figure SI 3- Toxicity neutralization assays and evaluation of cross-immunity</figcaption></figure>

E. coli MG1655 cells were co-transformed with two plasmids and grown to mid-exponential phase before to be serially diluted and plated on LB agar plates containing anhydrotetracycline to induce the expression of the indicated (snu, mhao, pmv) *iapK* or *aph1* immunity genes from the first plasmid, and glucose or arabinose to repress or induce the expression of the indicated *apk2*<sub>tox</sub> from the second plasmid. Data shown are representative of n ≥3 experiments.
<table><caption>Table SI 1- Bacterial strains</caption><thead><tr><td>Lab code</td><td>Name</td><td>Genotype</td><td>Reference</td></tr></thead><tbody><tr><td>EB3</td><td>BTH101</td><td>F-, cya-99, araD139, galE15, galK16, rpsL1 (Str r), hsdR2, mcrA1, mcrB1</td><td>[1]</td></tr><tr><td>EB70</td><td>DH5α</td><td>fhuA2, Δ(argF-lacZ)U169, phoA, glnV44, Φ80, Δ(lacZ)M15, gyrA96, recA1, relA1, endA1, thi-1, hsdR17</td><td>Lab stock</td></tr><tr><td>EB72</td><td>BL21(DE3) pLys</td><td>F-, ompT, gal, dcm, lon, hsdSB, (rB- mB-), λ(DE3), pLysS(cm<sup>R</sup>)</td><td>Lab stock</td></tr><tr><td>EB944</td><td>MG1655</td><td>F-, λ-, ilvG-, rfb-50, rph-1</td><td>Lab stock</td></tr></tbody></table>
<table><caption>Table SI 2- Plasmids</caption><thead><tr><th>Lab code</th><th>Description</th><th>Reference</th></tr></thead><tbody><tr><td colspan="3">Toxicity, survival and toxicity neutralization assays</td></tr><tr><td>pEB1017</td><td>pBAD33</td><td>[2]</td></tr><tr><td>pJV381</td><td>pBAD33-apk2<sub>tox-snu</sub> (amino acid residues 320 to 506 from the full-length Apk2<sub>snu</sub> under the control of a RBS made of 5G)</td><td>[3]</td></tr><tr><td>pJV383</td><td>pBAD33-apk2<sub>tox-snu</sub> (as above but with a RBS made of 4G)</td><td></td></tr><tr><td>pJV390</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G (D390G if full-length Apk2<sub>snu</sub>) (RBS made of 5G)</td><td>[3]</td></tr><tr><td>pJV391</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G (D390G if full-length Apk2<sub>snu</sub>) (RBS made of 4G)</td><td></td></tr><tr><td>pJV421</td><td>pBAD33-apk2<sub>tox-mhao</sub> (amino acid residues 359 to 553 from the full-length Apk2<sub>mhao</sub> under the control of a RBS made of 5G. PCR product w/ primers ebm2154/2155 at KpnI/SalI restriction sites)</td><td></td></tr><tr><td>pJV422</td><td>pBAD33-apk2<sub>tox-pmv</sub> (amino acid residues 346 to 533 from the full-length Apk2<sub>pmv</sub> under the control of a RBS made of 5G. PCR product w/ primers ebm2152/2153 at KpnI/SalI restriction sites)</td><td></td></tr><tr><td>pJV445</td><td>pBAD33-apk2<sub>tox-snu</sub> 1xFlag (PCRi+SLIC, primers ebm2200/2201, template pJV381)</td><td></td></tr><tr><td>pJV446</td><td>pBAD33-apk2<sub>tox-mhao</sub> 1xFlag (PCRi+SLIC, primers ebm2202/2203, template pJV421)</td><td></td></tr><tr><td>pJV447</td><td>pBAD33-apk2<sub>tox-pmv</sub> 1xFlag (PCRi+SLIC, primers ebm2204/2205, template pJV422)</td><td></td></tr><tr><td>pJV457</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G 1xFlag (RBS made of 5G) (mutagenesis, primers ebm2118/2119, template pJV445)</td><td></td></tr><tr><td>pJV458</td><td>pBAD33-apk2<sub>tox-snu</sub> D72G 1xFlag (RBS made of 4G) (PCRi+SLIC, primers ebm2200/2201, template pJV391)</td><td></td></tr><tr><td>pJV459</td><td>pBAD33-apk2<sub>tox-mhao</sub> D73G 1xFlag (D430G if full-length Apk2<sub>mhao</sub>)(mutagenesis, primers ebm2208/2209, template pJV446)</td><td></td></tr><tr><td>pJV460</td><td>pBAD33-apk2<sub>tox-pmv</sub> D72G 1xFlag (D416G if full-length Apk2<sub>pmv</sub>)(mutagenesis, primers ebm2210/2211, template pJV447)</td><td></td></tr><tr><td>pEB1242</td><td>pASK-IBA37plus</td><td>IBA Lifesciences</td></tr><tr><td>pJV374</td><td>pP<sub>TET</sub>-iapK<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV375</td><td>pP<sub>TET</sub>-aph1<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV419</td><td>pP<sub>TET</sub>-iapK<sub>mhao</sub> (PCR product w/ primers ebm2166/2167 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV420</td><td>pP<sub>TET</sub>-aph1<sub>mhao</sub> (PCR product w/ primers ebm2168/2169 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV423</td><td>pP<sub>TET</sub>-iapK<sub>pmv</sub> (PCR product w/ primers ebm2160/2161 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV424</td><td>pP<sub>TET</sub>-aph1<sub>pmv</sub> (PCR product w/ primers ebm2162/2163 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td colspan="3">Protein Production and Purification</td></tr><tr><td>pEB1520</td><td>pETDuet-1</td><td>Novagen</td></tr><tr><td>pJV403</td><td>pETDuet-1-apk2<sub>tox-snu</sub> iapK<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV435</td><td>pETDuet-1-apk2<sub>tox-mhao</sub> iapK<sub>mhao</sub> (PCR products w/ primers ebm2181/2155 and ebm2179/2180 at EcoRI/SalI and NdeI/XhoI, respectively)</td><td></td></tr><tr><td>pJV439</td><td>pETDuet-1-apk2<sub>tox-snu</sub> syn3<sub>mut</sub> iapK<sub>snu</sub> (mutagenesis G436S N438H according to the coordinates of the full-length protein, primers ebm2134/2135)</td><td></td></tr><tr><td>pJV440</td><td>pETDuet-1-apk2<sub>tox-snu</sub> syn3-syn2<sub>mut</sub> iapK<sub>snu</sub> (further mutagenesis R393A Y394V T395R according to the coordinates of the full-length protein, primers ebm2170/2171)</td><td></td></tr><tr><td>pJV441</td><td>pETDuet-1-apk2<sub>tox-snu</sub> syn3-syn2-syn4<sub>mut</sub> iapK<sub>snu</sub> (further mutagenesis Y452I H453R according to the coordinates of the full-length protein, primers ebm2174/2175)</td><td></td></tr><tr><td colspan="3">Bacterial Two-Hybrid</td></tr><tr><td>pEB354</td><td>pKT25linker</td><td>[4]</td></tr><tr><td>pJV468</td><td>pT25_Apk2<sub>tox-snu</sub> D72G 1xFlag (PCR product w/ primers ebm2122/2212 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV469</td><td>pT25_Apk2<sub>tox-mhao</sub> D73G 1xFlag (PCR product w/ primers ebm2207/2212 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV470</td><td>pT25_Apk2<sub>tox-pmv</sub> D72G 1xFlag (PCR product w/ primers ebm2206/2212 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pEB355</td><td>pUT18Clinker</td><td>[4]</td></tr><tr><td>pJV397</td><td>pT18_lapK<sub>snu</sub></td><td>[3]</td></tr><tr><td>pJV455</td><td>pT18_lapK<sub>mhao</sub> (PCR product w/ primers ebm2166/2167 at EcoRI/XhoI restriction sites)</td><td></td></tr><tr><td>pJV456</td><td>pT18_lapK<sub>pmv</sub> (PCR product w/ primers ebm2160/2161 at EcoRI/XhoI restriction sites)</td><td></td></tr></tbody></table>
<table><caption>Table SI 3- Primers</caption><thead><tr><th>Lab code ebm</th><th>5'-3' sequence</th></tr></thead><tbody><tr><td>2118</td><td>AAAGCAGTTAGCAAAATTAACGgCGCTTTACGTTATACAACTATCTTT</td></tr><tr><td>2119</td><td>AAAGATAGTTGTATAACGTAAAGCGcCGTTAATTTTGCTAACTGCTTT</td></tr><tr><td>2122</td><td>gaagaattcatgGCGAAAGCTAAATTCTATAGTGAA</td></tr><tr><td>2134</td><td>GATGGGCCATACAAAaGTGTAcACACGGTTGTCGAGAAAGATGG</td></tr><tr><td>2135</td><td>CCATCTTTCTCGACAACCGTGTgTACACtTTTGTATGGCCCATC</td></tr><tr><td>2152</td><td>ggtggtaccGGGGGcgtctggatgGTTGAGTCAGCTGTAAACGC</td></tr><tr><td>2153</td><td>ctcctcgagCTAGTTGCGGATCTTACCAAT</td></tr><tr><td>2154</td><td>ggtggtaccGGGGGcgtctggatgGTTGATAAAATGGACAGTGTGG</td></tr><tr><td>2155</td><td>ctcctcgagTTACTTTTTTTCCTTGATCAGGT</td></tr><tr><td>2160</td><td>gaagaattcATGGAATACTATTTAGTTTTTCCT</td></tr><tr><td>2161</td><td>ctcctcgagTCACTTGTGTTTGTCAATCCAT</td></tr><tr><td>2162</td><td>gaagaattcATGCACGGTTTGATCAGTATC</td></tr><tr><td>2163</td><td>ctcctcgagTCAGGTTTGAAGAAATTGCTTC</td></tr><tr><td>2166</td><td>ctcctcgagTCAGGTTTGAAGAAATTGCTTC</td></tr><tr><td>2167</td><td>ctcctcgagTTAATGCACTTTAATAAAGCGGA</td></tr><tr><td>2168</td><td>gaagaattcATGTGCCAAGCGGAGCAGT</td></tr><tr><td>2169</td><td>ctcctcgagTCATGCCTGCAAGTATTCAAG</td></tr><tr><td>2170</td><td>GTTAGCAAAATTAACGACGCTTTAgcTgtTcgtACTATCTTTGACTCTGATACTTTTAC</td></tr><tr><td>2171</td><td>GTAAAAGTATCAGAGTCAAAGATAGTacgAacAgcTAAAGCGTCGTTAATTTTGCTAAC</td></tr><tr><td>2174</td><td>GATGGTATCAATTTTGAAATGCAGatCCgCACACAGGAAAGCTTTGACCT</td></tr><tr><td>2175</td><td>AGGTCAAAGCTTTCCTGTGTGcGGatCTGCATTTCAAAATTGATACCATC</td></tr><tr><td>2179</td><td>catcatATGTACCAGTATTACCTGGCG</td></tr><tr><td>2180</td><td>ctcctcgagATGCACTTTAATAAAGCGGATC</td></tr><tr><td>2181</td><td>gaagaattcgATGGTTGATAAAATGGACAGTGTGG</td></tr><tr><td>2200</td><td>tttatcatcgtcgtctttataatcTTTAACACGCTCAATGTTTTTTG</td></tr><tr><td>2201</td><td>gattataaagacgacgatgataaaTGAacagtgcctgcacctgc</td></tr><tr><td>2202</td><td>tttatcatcgtcgtctttataatcCTTTTTTTTCCTTGATCAGGTCG</td></tr><tr><td>2203</td><td>gattataaagacgacgatgataaaTAActgcacctgcaggcatg</td></tr><tr><td>2204</td><td>tttatcatcgtcgtctttataatcGTTGCGGATCTTACCAATGTTC</td></tr><tr><td>2205</td><td>gattataaagacgacgatgataaaTAGctgcacctgcaggcatg</td></tr><tr><td>2206</td><td>gaagaattcATGGTTGAGTCAGCTGTAAACGC</td></tr><tr><td>2207</td><td>gaagaattcATGGTTGATAAAATGGACAGTGTGG</td></tr><tr><td>2208</td><td>CAAGCGATCGCCTCCATCCGCGgCGTGATTCGCTATACGGCGATTCTG</td></tr><tr><td>2209</td><td>CAAGCGATCGCCTCCATCCGCGgCGTGATTCGCTATACGGCGATTCTG</td></tr><tr><td>2210</td><td>GAATCACTTAGTAAAATCACCGgTATTGTGCGTTATACCACAATCTTC</td></tr><tr><td>2211</td><td>GAAGATTGTGGTATAACGCACAATAcCGGTGATTTTACTAAGTGATTC</td></tr><tr><td>2212</td><td>ctcctcgagTTAtttatcatcgtcgtctttataatc</td></tr></tbody></table>
References

1. Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. *Proc Natl Acad Sci USA* 95, 5752–5756

2. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. *J Bacteriol* 177, 4121–4130

3. Bartoli J, Tempier AC, Guzzi NL, Piras CM, Cascales E, Viala JP (2023) Characterization of a (p)ppApp Synthetase Belonging to a New Family of Polymorphic Toxin Associated with Temperate Phages. *Journal of Molecular Biology* 435:168282

4. Gully D, Bouveret E (2006) A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli. *Proteomics* 6, 282–293