Bacterial Toxin HigB Associates with Ribosomes and Mediates Translation-dependent mRNA Cleavage at A-rich Sites (2009)

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. 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) 3The abbreviations used are: TAtoxin-antitoxinORFopen reading frame./addiction/suicide modules typically include an autoregulated operon encoding a labile antitoxin and a more stable toxic protein (1.Gerdes K. Christensen S.K. Løbner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (886) Google Scholar). TA toxins facilitate stress survival (chromosomal) or plasmid maintenance and post-segregational killing (extrachromosomal; reviewed in Refs. 1.Gerdes K. Christensen S.K. Løbner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (886) Google Scholar, 2.Engelberg-Kulka H. Glaser G. Annu. Rev. Microbiol. 1999; 53: 43-70Crossref PubMed Scopus (319) Google Scholar). 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.Amitai S. Yassin Y. Engelberg-Kulka H. J. Bacteriol. 2004; 186: 8295-8300Crossref PubMed Scopus (138) Google Scholar, 4.Engelberg-Kulka H. Hazan R. Amitai S. J. Cell Sci. 2005; 118: 4327-4332Crossref PubMed Scopus (144) Google Scholar, 5.Pedersen K. Christensen S.K. Gerdes K. Mol. Microbiol. 2002; 45: 501-510Crossref PubMed Scopus (311) Google Scholar). toxin-antitoxin open reading frame. 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.Christensen S.K. Pedersen K. Hansen F.G. Gerdes K. J. Mol. Biol. 2003; 332: 809-819Crossref PubMed Scopus (320) Google Scholar, 7.Muñoz-Gómez A.J. Santos-Sierra S. Berzal-Herranz A. Lemonnier M. Díaz-Orejas R. FEBS Lett. 2004; 567: 316-320Crossref PubMed Scopus (78) Google Scholar, 8.Zhang Y. Zhang J. Hara H. Kato I. Inouye M. J. Biol. Chem. 2005; 280: 3143-3150Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 9.Zhang Y. Zhang J. Hoeflich K.P. Ikura M. Qing G. Inouye M. Mol. Cell. 2003; 12: 913-923Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 10.Zhang Y. Zhu L. Zhang J. Inouye M. J. Biol. Chem. 2005; 280: 26080-26088Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The RelE toxin interacts with the ribosome and induces mRNA cleavage with a preference for the UAG stop codon (11.Christensen S.K. Gerdes K. Mol. Microbiol. 2003; 48: 1389-1400Crossref PubMed Scopus (282) Google Scholar, 12.Christensen S.K. Mikkelsen M. Pedersen K. Gerdes K. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 14328-14333Crossref PubMed Scopus (380) Google Scholar, 13.Pedersen K. Zavialov A.V. Pavlov M.Y. Elf J. Gerdes K. Ehrenberg M. Cell. 2003; 112: 131-140Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). 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.Prysak M.H. Mozdzierz C.J. Cook A.M. Zhu L. Zhang Y. Inouye M. Woychik N.A. Mol. Microbiol. 2009; 71: 1071-1087Crossref PubMed Scopus (131) Google Scholar). The YoeB toxin inhibits translation at the initiation step, apparently by destabilization of the initiation complex (15.Zhang Y. Inouye M. J. Biol. Chem. 2009; 284: 6627-6638Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). HipA toxin is a kinase whose mechanism of action is not known (16.Correia F.F. D'Onofrio A. Rejtar T. Li L. Karger B.L. Makarova K. Koonin E.V. Lewis K. J. Bacteriol. 2006; 188: 8360-8367Crossref PubMed Scopus (163) Google Scholar, 17.Schumacher M.A. Piro K.M. Xu W. Hansen S. Lewis K. Brennan R.G. Science. 2009; 323: 396-401Crossref PubMed Scopus (275) Google Scholar). 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.Tian Q.B. Hayashi T. Murata T. Terawaki Y. Biochem. Biophys. Res. Commun. 1996; 225: 679-684Crossref PubMed Scopus (32) Google Scholar, 19.Tian Q.B. Ohnishi M. Tabuchi A. Terawaki Y. Biochem. Biophys. Res. Commun. 1996; 220: 280-284Crossref PubMed Scopus (69) Google Scholar). 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.Pandey D.P. Gerdes K. Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (740) Google Scholar). 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.Budde P.P. Davis B.M. Yuan J. Waldor M.K. J. Bacteriol. 2007; 189: 491-500Crossref PubMed Scopus (90) Google Scholar). 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.Christensen-Dalsgaard M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google Scholar). HigB is a member of the RelE family of toxins, including RelE, YafQ, and YoeB (20.Pandey D.P. Gerdes K. Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (740) Google Scholar). 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. The E. coli strains BL21(DE3) (F− ompT hsdSβ(rβ-mβ) dcm gal (DE3) tonA) (Novagen) and BW25113 (lacIq rrnBT14 Δlac-ZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) were used for all protein expression and toxicity studies. E. coli K12 Mach1 T1 cells (ΔrecA1398 endA1 tonA Φ80ΔlacM15 ΔlacX74 hsdR(rk+mk+); Invitrogen) were used for all cloning experiments. The higBA module was cloned from the Rts1 plasmid (a generous gift from Dr. Roger Woodgate, National Institutes of Health). The E. coli BW25113 strain with deleted chpBI-BK, hipBA, mazEF, relBE, yefM-yoeB, and dinJ-yafQ modules (BW25113 Δ6) was a generous gift from Qian Tan and Yoshi Yamaguchi of the M. Inouye laboratory. Plasmids used in this study include pBAD24 (23.Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (4031) Google Scholar), pET28a (Novagen), and pBAD/Myc-HisA (Invitrogen). The higBA operon was PCR-amplified from the Rts1 vector with 5′ BamHI/HindIII and cloned the of pET28a to with the The was PCR-amplified from the Rts1 vector with 5′ and cloned the of pBAD24 to The was PCR-amplified from BW25113 cells with 5′ and cloned the of pBAD24 to The was PCR-amplified from the Rts1 vector with 5′ BamHI/HindIII and cloned the of pET28a to The was PCR-amplified from the Rts1 vector with 5′ and an on the 5′ to was cloned the of pBAD/Myc-HisA to The was PCR-amplified from the Rts1 vector with 5′ and a H92Q and cloned the of pBAD24 to were in with either or at The of and were and The of the DNA sequences of used for cloning was confirmed by DNA sequence used for analysis were in the HigBA complex as and A. Inouye M. PubMed Scopus (32) Google Scholar), and (22.Christensen-Dalsgaard M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google were as was the as P. S. M. Cell. Full Text PDF PubMed Scopus Google Scholar). The DNA used for analysis were derived from including open reading of the E. coli genes protein protein protein or were with a and all were to their control the were as (14.Prysak M.H. Mozdzierz C.J. Cook A.M. Zhu L. Zhang Y. Inouye M. Woychik N.A. Mol. Microbiol. 2009; 71: 1071-1087Crossref PubMed Scopus (131) Google of from were extension analysis was as (14.Prysak M.H. Mozdzierz C.J. Cook A.M. Zhu L. Zhang Y. Inouye M. Woychik N.A. Mol. Microbiol. 2009; 71: 1071-1087Crossref PubMed Scopus (131) Google Scholar). used were as and The and were BL21(DE3) and BW25113 and with or for were a the protein were to and the were as by The protein of was by followed by protein synthesis was with the E. coli system for DNA the HigA HigB and of of or of of The were for at °C to the of and the for an at was from BW25113 cells as of was to a of HigB in Although it has been reported that HigB inhibits protein synthesis through translation-dependent mRNA cleavage (22.Christensen-Dalsgaard M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google Scholar), we the enzymatic activity of HigB from the Rts1 plasmid in more and its to family member We or in the pBAD24 We that the toxicity of either RelE or HigB the of a stable strain either toxin in pBAD24 with as the Therefore, in this study we used and for to toxin an all were with strains that were with the plasmid to the of of test this to was we growth of or at of either or not in growth by Interestingly, both toxins growth at the in were toxin RelE expression to an of translation of within a more in protein synthesis was HigB only at the point the of HigB translation to of and (22.Christensen-Dalsgaard M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google are with a RelE and HigB growth by In in of of DNA or that expression of HigB not to in DNA or HigB or RelE expression in of mRNA only the codon is (11.Christensen S.K. Gerdes K. Mol. Microbiol. 2003; 48: 1389-1400Crossref PubMed Scopus (282) Google Scholar, M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google Scholar). HigB the of several we analysis for E. coli and Although no was of HigB was of all were or In the of the control the plasmid but not were were the mRNA in of the that the mRNA we HigB was not to of other TA toxins from the stress of HigB we HigB in an E. coli strain in which of the six known chromosomal TA modules has been deleted relBE, chpBI-BK, hipBA, yefM-yoeB, and The of and were in and BW25113 cells and of the We no in the of of the in the two strains HigB The were with RelE, and no was Therefore, HigB or RelE expression to to of mRNA in vivo, and this not the action of other toxins. of the codon to mRNA in vivo, that the action of HigB is translation-dependent is family member (22.Christensen-Dalsgaard M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google Scholar). that HigB through with the ribosome. Therefore, we ribosome from cells HigB for cells were and mRNA in was to toxin expression the A and We used analysis of the to we were to an association of HigB with the ribosome We not or in 50 or S However, HigB to the in the the S because we a of S with both 50 S ribosomal HigB A and In the HigB associated primarily with S but it was with the 50 S ribosomal and However, HigB was not associated with the S ribosomal In addition, of the S ribosome to its confirmed that HigB with only the 50 S ribosomal and not the S. HigB with the ribosome through the 50 S ribosomal in of the RelE YafQ (14.Prysak M.H. Mozdzierz C.J. Cook A.M. Zhu L. Zhang Y. Inouye M. Woychik N.A. Mol. Microbiol. 2009; 71: 1071-1087Crossref PubMed Scopus (131) Google and YoeB (15.Zhang Y. Inouye M. J. Biol. Chem. 2009; 284: 6627-6638Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), with ribosomes through the 50 S subunit. Therefore, we the ribosome for RelE association with as we for HigB to this is a of RelE family A and The ribosome for RelE was similar to that for with a of S to S and as with the other RelE family it with the ribosome through the 50 S and HigB associated with the ribosome and mRNA we determined the sequence specificity and of its cleavage activity and recognition We from HigB and cells and primer extension analysis on the and that were HigB expression in We used a primer of the 5′ of the translation for mRNA and primer extension to enable of and In we cleavage all were relatively abundant cleavage and were whose were that of the that HigB is a we used to to expression of the toxin HigB-mediated cleavage was in the Therefore, we were to only those primer extension that with to the of the sequence and of all cleavage several of HigB-mediated cleavage as is the recognition sequence in and in in We that HigB cleaved 100% of the AAA sequences in primer extension The regions we AAA and we cut for all of and HigB both in-frame and out-of-frame AAA or sequences for cleavage sequences in HigB cleaved of or more HigB cleaved within the in or in six of the we The of the cleavage in the AAA the in or was not but a preference for cleavage the second were and HigB cleaves the length of the mRNA for with no for and HigB AA sequences with of the cut open in and occasionally cut at single A sequences open of the cleavage in not cut at an A the sequences and have to A.J. PubMed Scopus Google Scholar, Y. C.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is a single endoribonuclease for mRNA and in E. Therefore, HigB cleavage of and other be by and the A.J. Annu. Rev. Microbiol. 2007; PubMed Scopus Google Scholar). We not cleavage in the regions of the mRNAs, HigB ribosome association and that mRNA have an codon for HigB-mediated (11.Christensen S.K. Gerdes K. Mol. Microbiol. 2003; 48: 1389-1400Crossref PubMed Scopus (282) Google Scholar). the mechanism of HigB-mediated mRNA we HigB endoribonuclease activity in a step, we HigB toxin and confirmed that it was a in with or the of HigB toxin or with both HigB HigB in and the of HigB were by of the antitoxin HigA Therefore, were We the HigB was to cleave from E. coli cleavage was in either mRNA or a of with of There was no on the the HigA antitoxin was to the HigB and not the was to we not that the HigB-mediated in cleavage activity is dependent with the ribosome. of RelE, and K. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Y. T. M. I. M. Nat. Mol. Biol. 2005; 12: PubMed Scopus Google a their as for their However, subsequent on YoeB that of this not to a in toxicity or a in its function as an of translation initiation (15.Zhang Y. Inouye M. J. Biol. Chem. 2009; 284: 6627-6638Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In of the in YafQ R. J. A. E. Microbiol. Lett. 2007; PubMed Scopus Google YafQ toxicity and endoribonuclease activity in (14.Prysak M.H. Mozdzierz C.J. Cook A.M. Zhu L. Zhang Y. Inouye M. Woychik N.A. Mol. Microbiol. 2009; 71: 1071-1087Crossref PubMed Scopus (131) Google Scholar). HigB is in the RelE we all family and that HigB from the Rts1 plasmid and E. coli a for Rts1 and for E. coli at the of their sequences. HigB not activity in it mRNA cleavage only association with the ribosome in as for RelE K. Zavialov A.V. Pavlov M.Y. Elf J. Gerdes K. Ehrenberg M. Cell. 2003; 112: 131-140Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar), was a that the HigB mRNA cleavage activity we in was because of an of the mRNA cleavage activity of the ribosome Mol. Cell. 2003; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, to between of we an H92Q mutant in Rts1 HigB and the growth of the mutant strain of of no in growth that is for HigB-mediated We determined the of expression on the of and and no with the control the plasmid but not mRNA was no in cells the Finally, ribosome analysis that the mutant toxin its to with the ribosome and Therefore, the endoribonuclease activity of the HigB-ribosome complex to be by and this activity The HigBA TA system is for plasmid maintenance of the Rts1 plasmid Q.B. Ohnishi M. Tabuchi A. Terawaki Y. Biochem. Biophys. Res. Commun. 1996; 220: 280-284Crossref PubMed Scopus (69) Google Scholar). the E. coli K12 only a its pathogenic E. coli and E. coli O157:H7 to possess one or more of the entire module (20.Pandey D.P. Gerdes K. Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (740) Google Scholar). In the of many pathogenic one or more higBA this is for many with growth or periods (20.Pandey D.P. Gerdes K. Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (740) Google Scholar). a of this toxin imparts mRNA a in protein and growth (21.Budde P.P. Davis B.M. Yuan J. Waldor M.K. J. Bacteriol. 2007; 189: 491-500Crossref PubMed Scopus (90) Google Scholar, M. Gerdes K. Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (145) Google Scholar), we determined that the complex mRNA for cleavage at sequences. HigB 100% of in-frame and out-of-frame AAA sequences in the coding regions of the of of or more HigB AA sequences but with and only occasionally at single As. The conjugative Rts1 plasmid was from a clinical isolate of Proteus vulgaris Y. H. T. J. Bacteriol. PubMed Google Scholar). of the codon for P. vulgaris coding the genes of the Rts1 that HigB cleaves at a sequence that to the most AAA The second most an AA HigB cleavage However, Rts1 in Proteus Y. R. J. Bacteriol. PubMed Google Scholar), which the of Proteus urinary tract of the codon for the of P. that HigB cleaves at a sequence that to the second most encoding for the P. is the most used codon at Although is the most codon in E. coli K12 AAA is at in P. Although the sequence of P. vulgaris has not been it is AT-rich S. J. Bacteriol. PubMed Google as is the of P. M. M.A. T. H. K. S. K. H. E. D. S. J. J. Bacteriol. PubMed Scopus Google Scholar). the E. coli K12 has a G. V. M. J. J. Davis M.A. Y. Science. PubMed Scopus Google Scholar). the P. strain a plasmid distinct from Rts1 that is only is AAA the most codon in this it is as abundant with its host Rts1 has to have a a Proteus strain both Rts1 and that free HigB mRNA of its only Rts1 is not only the stable HigB toxin host it the plasmid more an for of Rts1 over In that the HigB cleavage the of its host sequences both in-frame or to its to plasmid and post-segregational in E. coli K12 have the of an AAA codon at to translation initiation P. J. Bacteriol. 2007; 189: 501-510Crossref PubMed Scopus Google Scholar, H. 2001; PubMed Scopus Google Scholar). AAA is the most codon at in of all E. coli K12 or codons H. 2001; PubMed Scopus Google Scholar). sequences, especially are with single regions in S Nucleic Acids Res. 2002; PubMed Scopus Google Scholar). the sequences are of the translation be to the of mRNA The of the P. vulgaris and P. that strains a of AAA codons in the In we open reading from the P. and an of at E. coli Therefore, HigB on of its AT-rich host First, it one of the most abundant codons of its host cell for it cleaves all sequences of frame. it cleaves specific of the and occasionally cleaves specific single As. targeting AAA at the of In this mechanism and post-segregational killing of that the Rts1 The analysis of the mechanism of HigB the characterization of all of the RelE family as RelE, YafQ, and YoeB from E. coli K12 and HigB from plasmid Rts1. family through the association with the 50 S ribosomal of the S ribosome. However, imparts toxicity through distinct YafQ activity in and in but its sequence-specific activity only association with the ribosome in (14.Prysak M.H. Mozdzierz C.J. Cook A.M. Zhu L. Zhang Y. Inouye M. Woychik N.A. Mol. Microbiol. 2009; 71: 1071-1087Crossref PubMed Scopus (131) Google Scholar). HigB only activity association with the ribosome and cleaves its recognition sequences on both and out-of-frame. In RelE YoeB endoribonuclease activity in K. Zavialov A.V. Pavlov M.Y. Elf J. Gerdes K. Ehrenberg M. Cell. 2003; 112: 131-140Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, Y. Inouye M. J. Biol. Chem. 2009; 284: 6627-6638Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and mRNA cleavage is only for inhibition of translation initiation in (15.Zhang Y. Inouye M. J. Biol. Chem. 2009; 284: 6627-6638Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The in mRNA cleavage activity associated with RelE has been to the activity of the ribosome Mol. Cell. 2003; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). We the of HigB-mediated post-segregational killing we HigB-mediated cleavage the length of HigB to with S ribosomes through the 50 S ribosomal subunit. HigB with the 50 S ribosomal as it a S on the cleave mRNA at the second codon of and it was in the 50 S in The of HigB with ribosomes to an in HigB that its endoribonuclease HigB with the ribosome and cleaves the mRNA it the or not enable to HigB cleaves at the it or subsequent cleavage in mRNA and inhibition of translation a the system for ribosomes is in ribosome by the S we as well as the of HigB in the in ribosomes HigB not be to the ribosome at the HigB cut The of the Proteus host are to the mechanism of HigB action and the of a similar for chromosomal HigB We Roger for the Rts1 plasmid and Yoshi Yamaguchi for the and of the BW25113 We the of the for Zhang for the for and Inouye for and with