Bacterial Toxin HigB Associates with Ribosomes and Mediates Translation-dependent mRNA Cleavage at A-rich Sites

Most pathogenic Proteus species are primarily associated with urinary tract infections, especially in persons with indwelling catheters or functional/anatomic abnormalities of the urinary tract. Urinary tract infections caused by Proteus vulgaris typically form biofilms and are resistant to commonly used antibiotics. The Rts1 conjugative plasmid from a clinical isolate of P. vulgaris carries over 300 predicted open reading frames, including antibiotic resistance genes. The maintenance of the Rts1 plasmid is ensured in part by the HigBA toxin-antitoxin system. We determined the precise mechanism of action of the HigB toxin in vivo, which is distinct from other known toxins. We demonstrate that HigB is an endoribonuclease whose enzymatic activity is dependent on association with ribosomes through the 50 S subunit. Using primer extension analysis of several test mRNAs, we showed that HigB cleaved extensively across the entire length of coding regions only at specific recognition sequences. HigB mediated cleavage of 100% of both in-frame and out-of-frame AAA sequences. In addition, HigB cleaved ∼20% of AA sequences in coding regions and occasionally cut single As. Remarkably, the cleavage specificity of HigB coincided with one of the most frequently used codons in the AT-rich Proteus spp., AAA (lysine). Therefore, the HigB-mediated plasmid maintenance system for the Rts1 plasmid highlights the intimate relationship between host cells and extrachromosomal DNA that enables the dynamic acquisition of genes that impart a spectrum of survival advantages, including those encoding multidrug resistance and virulence factors.Toxin-antitoxin (TA)³/addiction/suicide modules typically include an autoregulated operon encoding a labile antitoxin and a more stable toxic protein (1). TA toxins facilitate stress survival (chromosomal) or plasmid maintenance and post-segregational killing (extrachromosomal; reviewed in Refs. 1, 2). Most chromosomal TA toxins inhibit cell growth by reversibly targeting either protein translation or DNA replication; their cognate antitoxins prevent toxin activity during periods of optimal growth but enable finely tuned control of TA module toxicity during relatively short periods of environmental stress. However, prolonged stress leads to a point of no return and cell death (3–5).There are six confirmed chromosomal TA loci in Escherichia coli K12 cells: dinJ-yafQ, relBE, yefM-yoeB, mazEF, chpBI-BK, and hipBA. The toxins MazF and ChpBK are sequence-specific endoribonucleases that cleave free mRNA (6–10). The RelE toxin interacts with the ribosome and induces mRNA cleavage with a preference for the UAG stop codon (11–13). The YafQ toxin is a ribosome-associated endoribonuclease that cleaves in-frame AAA codons that are followed by either an A or G in the subsequent codon (14). The YoeB toxin inhibits translation at the initiation step, apparently by destabilization of the initiation complex (15). HipA toxin is a kinase whose mechanism of action is not known (16, 17).Although the mechanism of action of many E. coli chromosomal and plasmid-derived toxins has been determined, the precise function of the HigB toxin has not been characterized. The higBA TA module is not present in E. coli K12; it resides on the Rts1 plasmid that typically replicates in Proteus spp. and imparts kanamycin resistance as well as temperature-sensitive post-segregational killing at 42 °C (18, 19). Interestingly, one or more chromosomal counterparts of higBA have been reported for several pathogens, including Vibrio cholerae, Streptococcus pneumoniae, E. coli CFT073, and E. coli O157:H7 (20). Some characterization of the two V. cholerae HigBA modules has been performed. First, one of the two higBA modules was shown to possess the general characteristics of TA systems by demonstration of toxin-antitoxin interaction, module organization/regulation, HigB toxicity, and rescue of toxicity with the cognate HigA antitoxin (21). Overexpression of HigB derived from two individual higBA modules encoded in V. cholerae or from Rts1 leads to inhibition of protein synthesis through translation-dependent mRNA cleavage in a manner similar to, but distinct from, RelE (22).HigB is a member of the RelE family of toxins, including RelE, YafQ, and YoeB (20). In this study, we have identified the precise mode of action of HigB from Rts1. HigB associated with the 50 S ribosomal subunit, and this HigB-ribosome complex cleaved within mRNA coding regions at all AAA triplet sequences, both in-frame and out-of-frame. HigB appeared to be responsible for the mRNA cleavage activity of the HigB-ribosome complex because a HigB H92Q mutant lacked mRNA cleavage activity but remained associated with the ribosome. Finally, the cleavage specificity of HigB on plasmid Rts1 coincided with the sequence (AAA, lysine) of either the most abundant or the second most abundant codon in its Proteus host.