Phylogeny Reveals Novel HipA-Homologous Kinase Families and Toxin-Antitoxin Gene Organizations

Abstract

Toxin-antitoxin modules function in the genetic stability of mobile genetic elements, bacteriophage defense, and antibiotic tolerance. A gain-of-function mutation of the Escherichia coli K-12 hipBA module can induce antibiotic tolerance in a subpopulation of bacterial cells, a phenomenon known as persistence. HipA is a Ser/Thr kinase that phosphorylates and inactivates glutamyl tRNA synthetase, inhibiting cellular translation and inducing the stringent response. Additional characterized HipA homologues include HipT from pathogenic E. coli O127 and YjjJ of E. coli K-12, which are encoded by tricistronic hipBST and monocistronic operons, respectively. The apparent diversity of HipA homologues in bacterial genomes inspired us to investigate overall phylogeny. Here, we present a comprehensive phylogenetic analysis of the Hip kinases in bacteria and archaea that expands on this diversity by revealing seven novel kinase families. Kinases of one family, encoded by monocistronic operons, consist of an N-terminal core kinase domain, a HipS-like domain, and a HIRAN (HIP116 Rad5p N-terminal) domain. HIRAN domains bind single- or double-stranded DNA ends. Moreover, five types of bicistronic kinase operons encode putative antitoxins with HipS-HIRAN, HipS, γδ-resolvase, or Stl repressor-like domains. Finally, our analysis indicates that reversion of hipBA gene order happened independently several times during evolution. IMPORTANCE Bacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipA, encoding the kinase toxin of the hipBA toxin-antitoxin (TA) module of E. coli. HipA-homologous kinases phosphorylate and thereby inactivate specific tRNA synthetases, thus inhibiting protein translation and cell proliferation. Here, we present a comprehensive phylogenetic analysis of bacterial Hip kinases and discover seven new families with novel operon structures and domains. Overall, Hip kinases are encoded by TA modules with at least 10 different genetic organizations, 7 of which have not been described before. These results open up exciting avenues for the experimental analysis of the superfamily of Hip kinases.

Keywords: GltX; HIRAN; HipB; HipS; HipT; Stl; TrpS; high persister A; kinase.

FIG 1

Overview of the hipBA, hipBST, and yjjJ operons and their protein products. (A) hipBA encodes the antitoxin HipB (blue) and toxin kinase HipA (red/orange) that form an inactive HipB₂HipA₂ complex that can bind to operators in the promoter region via HTH DNA-binding domains in HipB and thereby autoregulate transcription (dashed line) (28). Upon HipB degradation by Lon protease (88) and activation of HipA, HipA phosphorylates and inhibits glutamine tRNA synthetase (GltX/GltRS), thereby halting translation and inducing the stringent response (51, 52, 54). (B) hipBST encodes three proteins, HipB (blue), HipS (orange), and HipT (red) that form an inactive HipBST complex (59). HipS is homologous to the N-subdomain-1 of HipA and functions as the antitoxin that neutralizes HipT (59). Like HipB of HipBA, HipB of HipBST has an HTH domain and augments the inhibition of HipT by HipS but does not function as an antitoxin on its own. Free HipT phosphorylates and inhibits tryptophan tRNA synthetase (TrpS) and thereby halts translation in a similar fashion as HipA. (C) yjjJ is a single cistron operon that encodes a HipA-homologous kinase YjjJ (green) that, when overproduced, inhibits cell growth (60). YjjJ, that we coin HipH, has an HTH-domain in its N terminus that may function to bind DNA.

FIG 2

Phylogeny and genetic contexts of HipA-homologous kinases. (A) Simplified phylogenetic tree covering 1,239 HipA-homologous kinases (the “Hip Tree”) (see Fig. S2 in the supplemental material for details). The Hip Tree was divided into 11 main clades I to XI. The coloring of the Hip Tree reflects the genetic contexts that encode the kinases such that each color corresponds to a distinct kinase family encoded by a distinct type of TA module. All main clades are monophyletic except clade XI that consists of six different kinase families. Small blue triangles within the red triangles of main clades VI and VII symbolize subclades of kinases encoded by TA modules with a reversed gene order relative to hipBA—that is—with the gene order hipAB. The Hip Tree was visualized by iTOL (82). (B) Genetic organizations of the TA modules encoding the 1,239 HipA-homologous kinases. The various types of genetic organization were obtained by manual inspection of the genes upstream and downstream of the kinase genes listed in Table S1 in the supplemental material. The coloring of the Hip kinases in panel B follows the coloring of the clades in panel A. Putative antitoxins with helix-turn-helix (HTH) domains are colored light green. Stl/HTH, putative antitoxins containing HTH domain and a domain with structural similarity to the “polyamorous” repressor Stl encoded by Staphylococcus aureus; HipS, HipS-like; HIRAN, HIP116 Rad5p N-terminal domain.

FIG 3

Structural mapping of insertions and deletions observed in characterized HipA kinases. (A) Schematic overview of HipA showing the conserved regions (the Gly-rich loop, activation loop, catalytic motif, and Mg-binding motif) in red as well as insertions (ω [green]) and deletions (Δ [blue]) in C. crescentus HipA₁ and HipA₂ relative to HipA from E. coli K-12 based the sequence alignment shown in Fig. S3. An insertion in HipA₁ relative to HipA is denoted ω5′, while insertions in HipA₂ relative to HipA are marked ω1″, ω2″, ω3″, ω4″, and ω6″. Deletions in HipA₁ and HipA₂ relative to HipA are marked Δ1, Δ2, and Δ3. Regions of high sequence divergence are shown in yellow. (B) Mapping of the insertions and deletions in HipA₁ onto the structure of HipA of E. coli K-12 (PDB accession no. 3FBR) using the same nomenclature and color scheme as in panel A (45). ATP is shown with colored sticks. (C) Mapping of the insertions and deletions in HipA₂ onto the structure of HipA. Note that the insertions ω1″ and ω3″ in HipA₂ are located close to the region that in HipA interacts with the C terminus of HipB (yellow sticks) and could perhaps affect antitoxin activity.

FIG 4

Alignment of HipH and HipT kinases. Sequence alignment of subclades containing HipT of E. coli O127 and HipH of E. coli K-12. Deletions (Δ) and insertions (ω) relative to the HipH_{E coli K-12} subclade are indicated, as well as the four conserved kinase motifs (Gly-rich loop, activation loop, catalytic motif, and Mg²⁺-binding motif). HTH domains in the N terminus of HipH kinases are boxed in red.

FIG 5

Comparative analysis of HIRAN domains. The HIRAN domains of HipL and HipN have sequence motifs required for DNA binding. (A) Schematic overview of the HIRAN domain found in human helicase-like transcription factor (HLTF) showing the relative positions and sequences involved in ssDNA and dsDNA binding (72). (B) Structure of the human HLTF HIRAN domain with the four regions necessary for DNA binding shown as sticks (PDB accession no. 4XZF) (72). (C) Overview of sequence motifs present (+) or absent (−) in homologues of HipL and HipN.