Identification and characterization of a HEPN-MNT family type II toxin–antitoxin in Shewanella oneidensis

Toxin–antitoxin (TA) systems are prevalent in bacteria and archaea. However, related studies in the ecologically and bioelectrochemically important strain Shewanella oneidensis are limited. Here, we show that SO_3166, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S. oneidensis and Escherichia coli. SO_3165, a putative minimal nucleotidyltransferase (MNT), neutralized the toxicity of SO_3166. Gene SO_3165 lies upstream of SO_3166, and they are co-transcribed. Moreover, the SO_3165 and SO_3166 proteins interact with each other directly in vivo, and antitoxin SO_3165 bound to the promoter of the TA operon and repressed its activity. Finally, the conserved Rx4-6H domain in HEPN family was identified in SO_3166. Mutating either the R or H abolished SO_3166 toxicity, confirming that Rx4-6H domain is critical for SO_3166 activity. Taken together, these results demonstrate that SO_3166 and SO_3165 in S. oneidensis form a typical type II TA pair. This TA pair plays a critical role in regulating bacterial functions because its disruption led to impaired cell motility in S. oneidensis. Thus, we demonstrated for the first time that HEPN-MNT can function as a TA system, thereby providing important insights into the understanding of the function and regulation of HEPNs and MNTs in prokaryotes.     

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf 4 i nhibition t oxin and Rorf0727 be named PfiA for Pf iT a ntitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′-AATTC N₅GTTAA -3′) overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.     

Identification and functional analysis of two toxin–antitoxin systems in Campylobacter jejuni

Toxin–antitoxin (TA) systems are widely distributed in bacteria and play an important role in maintaining plasmid stability. The leading foodborne pathogen, Campylobacter jejuni, can carry multiple plasmids associated with antibiotic resistance or virulence. Previously a virulence plasmid named pVir was identified in C. jejuni 81‐176 and IA3902, but determining the role of pVir in pathogenesis has been hampered because the plasmid cannot be cured. In this study, we report the identification of two TA systems that are located on the pVir plasmid in 81‐176 and IA3902, respectively. The virA (proteic antitoxin)/virT (proteic toxin) pair in IA3902 belongs to a Type II TA system, while the cjrA (RNA antitoxin)/cjpT (proteic toxin) pair in 81‐176 belongs to a Type I TA system. Notably, cjrA (antitoxin) represents the first noncoding small RNA demonstrated to play a functional role in Campylobacter physiology to date. By inactivating the TA systems, pVir was readily cured from Campylobacter, indicating their functionality in Campylobacter. Using pVir‐cured IA3902, we demonstrated that pVir is not required for abortion induction in the guinea pig model. These findings establish the key role of the TA systems in maintaining plasmid stability and provide a means to evaluate the function of pVir in Campylobacter pathobiology.     

The RES domain toxins of RES‐Xre toxin‐antitoxin modules induce cell stasis by degrading NAD+

Type II toxin‐antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res‐xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2 Å crystal structure of the intact Pseudomonas putida RES‐Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix‐turn‐helix DNA‐binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP‐ribosylating enzymes such as diphtheria toxin but has an atypical NAD⁺‐binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD⁺ levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated.     

Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes

Prokaryotic chromosomes code for toxin–antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory ‘toxin’, encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.     

A cis‐acting antitoxin domain within the chromosomal toxin–antitoxin module EzeT of Escherichia coli quenches toxin activity

Toxin–antitoxin (TA) systems are widespread genetic modules in the genomes of bacteria and archaea emerging as key players that modulate bacterial physiology. They consist of two parts, a toxic component that blocks an essential cellular process and an antitoxin that inhibits this toxic activity during normal growth. According to the nature of the antitoxin and the mode of inhibition, TA systems are subdivided into different types. Here, we describe the characterization of a type II‐like TA system in Escherichia coli called EzeT. While in conventional type II systems the antitoxin is expressed in trans to form an inactive protein–protein complex, EzeT consists of two domains combining toxin and cis‐acting antitoxin functionalities in a single polypeptide chain. We show that the C‐terminal domain of EzeT is homologous to zeta toxins and is toxic in vivo. The lytic phenotype could be attributed to UDP‐N‐acetylglucosamine phosphorylation, so far only described for type II epsilon/zeta systems from Gram‐positive streptococci. Presence of the N‐terminal domain inhibits toxicity in vivo and strongly attenuates kinase activity. Autoinhibition by a cis‐acting antitoxin as described here for EzeT‐type TA systems can explain the occurrence of single or unusually large toxins, further expanding our understanding of the TA system network.     

Shewanella oneidensis cytochrome c maturation component CcmI is essential for heme attachment at the non‐canonical motif of nitrite reductase NrfA

Shewanella oneidensis is renowned for its respiratory versatility, which is largely due to abundant c‐type cytochromes. Maturation of these proteins depends on a Ccm system encoded by genes in an unusual chromosomal arrangement, but the detailed mechanism is not understood. In this study, we identify SO0265 as CcmI, an apocytochrome c chaperone that is important and essential for maturation of c‐type cytochromes with the canonical heme binding motif(s) (HBM; CX₂CH) and nitrite reductase NrfA carrying a non‐canonical CX₂CK motif respectively. We show that the N‐terminal transmembrane segment of CcmI, CcmI‐1, is sufficient for maturation of the former but the entire protein is required for maturation of the latter. Although S. oneidensis possesses a heme lyase, SirEFG, dedicated for non‐canonical HBMs, it is specific for SirA, a sulfite reductase with a CX₁₅CH motif. By presenting evidence that the periplasmic portion of CcmI, CcmI‐2, interacts with NrfA, we suggest that CcmI also takes the role of Escherichia coli NrfG for chaperoning apo‐NrfA for maturation at CX₂CK. Moreover, intact CcmI is required for maturation of NrfA, presumably by ensuring that heme attachment at canonical HBMs occurs before apoprotein degradation.     

Identification and characterization of a nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> putative toxin-antitoxin locus

Background  

Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapD _Hi , encoded by gene HI0450). Both vapD _Hi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapD _Hi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapX _Hi for the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections.     

The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin–antitoxin proteins HipA_SO and HipB_SO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin–antitoxin protein module. Here we show that phosphorylated HipA_SO can engage in an unexpected ternary complex with HipB_SO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipB_SO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipA_SO in the ternary complex, indicative of its role in binding HipA_SO to abolish its function in persistence. The structure of HipA_SO in complex with a non-hydrolyzable ATP analogue shows that HipA_SO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipA_SO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.     

Co-evolution of quaternary organization and novel RNA tertiary interactions revealed in the crystal structure of a bacterial protein–RNA toxin–antitoxin system

Genes encoding toxin–antitoxin (TA) systems are near ubiquitous in bacterial genomes and they play key roles in important aspects of bacterial physiology, including genomic stability, formation of persister cells under antibiotic stress, and resistance to phage infection. The CptIN locus from Eubacterium rectale is a member of the recently-discovered Type III class of TA systems, defined by a protein toxin suppressed by direct interaction with a structured RNA antitoxin. Here, we present the crystal structure of the CptIN protein–RNA complex to 2.2 Å resolution. The structure reveals a new heterotetrameric quaternary organization for the Type III TA class, and the RNA antitoxin bears a novel structural feature of an extended A-twist motif within the pseudoknot fold. The retention of a conserved ribonuclease active site as well as traits normally associated with TA systems, such as plasmid maintenance, implicates a wider functional role for Type III TA systems. We present evidence for the co-variation of the Type III component pair, highlighting a distinctive evolutionary process in which an enzyme and its substrate co-evolve.