Abstract:The maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers after they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific
dif
sites located near the replication terminus.
Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed, as a consequence of horizontal exchange, and the breadth of their functionality in bacterial physiology remains unknown. Here, we characterize a TA system in
Legionella pneumophila
that is highly conserved across
Legionella
species. This system is distantly related to
Escherichia coli
HipBST, and we demonstrate that it is a functional tripartite TA system (denoted HipBST
Lp
). We identify HipBST
Lp
homologs in diverse taxa, yet in the Gammaproteobacteria, these are almost exclusively found in
Legionella
species. Notably, the toxin HipT
Lp
was previously reported to be a pathogenic effector protein that is translocated by
L. pneumophila
into its eukaryotic hosts. Contrary to this, we find no signal of HipT
Lp
translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBST
Lp
, and identify key variations between this system and HipBST
Ec
. Finally, we show that the target of HipT
Lp
is likely not conserved with any characterized HipA or HipT toxin. This work serves as a useful comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.
IMPORTANCE
Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen
Legionella pneumophila
that is highly conserved across
Legionella
species genomes. This system (denoted HipBST
Lp
) is a distant homolog of the recently discovered split-HipA system in
Escherichia coli
(HipBST
Ec
). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipT
Lp
) possesses bifunctionality as an
L. pneumophila
virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.
Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed, as a consequence of horizontal exchange, and the breadth of their functionality in bacterial physiology remains unknown. Here, we characterize a TA system in
Legionella pneumophila
that is highly conserved across
Legionella
species. This system is distantly related to
Escherichia coli
HipBST, and we demonstrate that it is a functional tripartite TA system (denoted HipBST
Lp
). We identify HipBST
Lp
homologs in diverse taxa, yet in the Gammaproteobacteria, these are almost exclusively found in
Legionella
species. Notably, the toxin HipT
Lp
was previously reported to be a pathogenic effector protein that is translocated by
L. pneumophila
into its eukaryotic hosts. Contrary to this, we find no signal of HipT
Lp
translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBST
Lp
, and identify key variations between this system and HipBST
Ec
. Finally, we show that the target of HipT
Lp
is likely not conserved with any characterized HipA or HipT toxin. This work serves as a useful comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.
IMPORTANCE
Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen
Legionella pneumophila
that is highly conserved across
Legionella
species genomes. This system (denoted HipBST
Lp
) is a distant homolog of the recently discovered split-HipA system in
Escherichia coli
(HipBST
Ec
). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipT
Lp
) possesses bifunctionality as an
L. pneumophila
virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.
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