Toxin–antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Shewanella oneidensis. Here, we report the crystal structure and solution conformation of the SO_3166–SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite RX4–6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the RX4–6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure–function analysis provides first insights into the neutralization mechanism of the HEPN–MNT TA family.

Eukaryotic circular Rep encoding single-stranded DNA (CRESS DNA) viruses exhibit high genomic diversity. Four species of circoviruses within the genus Circovirus have been identified in pigs, including the non-pathogenic porcine circovirus type 1 (PCV1), and the pathogenic PCV2, PCV3, and PCV4. In this study, a novel single circular DNA virus was identified in pigs suffering from respiratory, diarrheal, and reproductive failure diseases. The viral Rep and Cap showed low amino acid sequence identities to the four PCV species, with the highest identity of 26.2% to PCV2 Rep and 20.8% to PCV4 Cap. The classification of CRESS DNA viruses primarily relies on Rep, and biochemical analyses of the viral Rep confirm its classification as a CRESS DNA virus. Thus, it was tentatively named porcine circovirus type 5 (PCV5). This tentative designation reflects its host and genomic organization; however, phylogenetic analysis indicates that it clusters outside the family Circoviridae and is most closely related to the fur seal faces-associated circular DNA virus (FSfaCV). The positive detection rate for PCV5 in tested samples was 54.4% (294 out of 540), with 24.3% (17 out of 70) of pig farms in China testing positive for the virus. A close correlation between the copy number of PCV5 and the severity of infectious diseases was observed. Moreover, a PCV5 virus-like particles (VLPs)-based enzyme-linked immunosorbent assay, which was developed, also elucidated that PCV5 has been circulating. We subsequently established an in vitro culture system for PCV5, successfully purifying the virus. Morphological identification revealed that the purified PCV5 has a diameter of approximately 20 nm, and the recombinant PCV5 Cap could assemble into VLPs similar to purified PCV5. These results indicate that PCV5, associated with porcine circovirus-associated disease, commonly circulates within Chinese swine. IMPORTANCE: The single-stranded circular DNA virus, tentatively named porcine circovirus type 5 (PCV5), belongs to the circular Rep encoding single-stranded DNA (CRESS DNA) viruses. Notably, PCV5 is not classified within the same family as the previously reported PCV1–PCV4. PCV5, which was associated with porcine circovirus associated-disease, is widely prevalent in Southern China. PCV5 is characterized by a larger genome (2,904 nt), along with longer Cap (1,182 nt) and Rep (1,056 nt) regions, as well as an extended stem-loop structure (stem: 9 nt and loop: 17 nt). Importantly, we subsequently established an in vitro culture system for PCV5, successfully purifying the virus. Morphological identification revealed that the purified PCV5 has a diameter of approximately 20 nm. Additionally, a variant of the PCV5 Cap, lacking the N-terminal 63 residues, was expressed in Escherichia coli , and purified Cap could assemble into virus-like particles similar to purified PCV5. These results hold significant implications for the study of CRESS DNA viruses. The emergence of PCV5 warrants further study.

HigB-HigA is a bacterial toxin–antitoxin (TA) system in which the antitoxin HigA can mask the endoribonuclease activity of toxin HigB and repress the transcription of the TA operon by binding to its own promoter region. The opportunistic pathogen Pseudomonas aeruginosa HigBA (PaHigBA) is closely associated with the pathogenicity by reducing the production of multiple virulence factors and biofilm formation. However, the molecular mechanism underlying HigBA TA operon transcription by PaHigA remains elusive. Here, we report the crystal structure of PaHigA binding to the promoter region of higBA operon containing two identical palindromic sequences at 3.14 Å resolution. The promoter DNA is bound by two cooperative dimers to essentially encircle the intact palindrome region. The helix-turn-helix (HTH) motifs from the two dimers insert into the major grooves of the DNA at the opposite sides. The DNA adopts a canonical B-DNA conformation and all the hydrogen bonds between protein and DNA are mediated by the DNA phosphate backbone. A higher resolution structure of PaHigA-DNA complex at 2.50 Å further revealed three water molecules bridged the DNA-binding interface and mediated the interactions between the bases of palindromic sequences and PaHigA (Thr40, Asp43, and Arg49). Structure-based mutagenesis confirmed these residues are essential for the specific DNA-binding ability of PaHigA. Our structure–function studies therefore elucidated the cooperative dimer–dimer transcription repression mechanism, and may help to understand the regulation of multiple virulence factors by PaHigA in P. aeruginosa.