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J Bacteriol. Feb 2009; 191(4): 1191–1199.
Published online Dec 5, 2008. doi:  10.1128/JB.01013-08
PMCID: PMC2631989

HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea[down-pointing small open triangle]

Abstract

Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.

Toxin-antitoxin (TA) loci that encode protein antitoxins share certain characteristics. They are two-component systems that encode two small proteins, a toxin and an antitoxin; the antitoxin neutralizes the toxin by direct protein-protein contact; the antitoxin has a DNA binding domain and autoregulates TA operon transcription; many such toxins cleave mRNA; the toxin families exhibit complex phylogenetic patterns due to extensive horizontal gene transfer; and members of the same family are often detected on phages and plasmids, as well as on chromosomes (3, 14, 33). A previous comprehensive analysis of ≈120 prokaryotic genomes led to the identification of ≈600 loci belonging to the six known TA gene families (relBE, mazEF, vapBC, phd-doc, ccdAB, and parDE). The genes are distributed in a striking pattern: slowly growing free-living organisms particularly have many TA loci, while obligatory intracellular organisms have few or none (27, 33). For example, Mycobacterium tuberculosis has more than 60, and Sulfolobus spp. has ≈30 TA loci, while Mycobacterium leprae and rickettsiae have none.

The RelE and MazF families of toxins inhibit translation by mRNA cleavage (34, 45) and were coined mRNA interferases (45). Doc of P1 also inhibits translation (26). Two other TA families, typified by ccd of plasmid F and parDE of plasmid RK2, encode inhibitors of DNA gyrase (1, 4, 20, 30). The biological functions of chromosome-encoded TA loci have been the subjects of considerable debate. In almost all cases investigated, chromosome-encoded TA loci are induced by nutritional stress, raising the possibility that these TA loci function to help the cells cope with environmental stress (5, 14). In further support of the stress response hypothesis, transcription of TA loci was induced by heat shock in Sulfolobus solfataricus and by exposure to chloroform in Nitrosomonas europaea (18, 40). There is also evidence that a MazF toxin homologue elicits programmed cell death during Myxococcus species development (32). It has been suggested that mazEF of Escherichia coli also mediates programmed cell death (2, 24, 25), although we and others have not been able to reproduce these results (10, 42).

A bicistronic locus closely linked to the pilus gene cluster of Haemophilus influenzae (hif) was called hic (for hif contiguous) (29). In a recent bioinformatic study, it was suggested that the hicAB loci constitute a novel TA gene family with many members in bacteria and archaea (27). Predicted HicA proteins (COG1724) are small and have a double-stranded RNA-binding fold, while predicted HicB proteins (COG1598/4226) have a DNA binding domain fused to a degraded RNase H fold (27). The bioinformatic analyses did not predict with certainty which of the proteins would be the toxin or the antitoxin, although HicB has a DNA binding domain similar to that of known antitoxins.

Here, we present evidence showing that the hicAB genes of E. coli K-12 have properties very similar to those of previously characterized TA loci. Thus, induction of hicA inhibited translation and induced multiple cleavages in three model mRNAs and tmRNA. Induction of hicB neutralized the detrimental effects of HicA, indicating that HicB can function as an antitoxin. Similar to bona fide TA loci, transcription of hicAB was induced by amino acid and carbon starvation by a Lon-dependent mechanism. Finally, we present a comprehensive update of TA gene phylogeny. Our analysis revealed 1,340 TA loci in 218 prokaryotic genomes, 119 of which belong to the new hicAB TA gene family.

MATERIALS AND METHODS

Growth conditions and media.

Cells were grown either in standard Luria-Bertani (LB) broth or in M9 minimal medium at 37°C, supplemented with 0.2% glucose, 1 μg/μl thiamine, and amino acids in defined concentrations. When appropriate, the medium was supplemented with ampicillin (30 or 100 μg/ml) or chloramphenicol (50 μg/ml). When amino acid starvation was induced in M9 minimal medium by the addition of 0.4 mg/ml serine hydroxymate (SHX; Sigma-Aldrich), serine was excluded from the medium. Glucose starvation was induced in M9 minimal medium containing 0.05% glucose by the addition of methyl α-d-glucopyranoside (Sigma-Aldrich). Expression of the PA1/O4/O3 promoter was induced by the addition of 2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and expression of the PBAD promoter was induced by the addition of 0.2% arabinose.

Bacterial strains and plasmids.

Bacterial strains and plasmids used and constructed are listed in Table Table1,1, and DNA primers are listed in Table Table22.

TABLE 1.
Strains and plasmids used and constructed
TABLE 2.
DNA primers

(i) Strain MGJ36.

Strain MG1655ΔhicAB was constructed by deletion of hicAB from the chromosome of MG1655, by the procedure described in reference 11. A PCR product was synthesized with the primers delta hicAB-f and delta hicAB-r, with pKD3 as template. The PCR product was electroporated into strain BW25113/pKD46, and the cells were spread on Luria agar plates containing 25 μg/ml chloramphenicol and incubated at 37°C. The ΔhicAB::cat allele from BW25113ΔhicAB was then transduced into MG1655. The cat gene was removed with the plasmid pCP20, resulting in MG1655ΔhicAB.

(ii) Plasmid pMJ221.

The hicA gene was amplified from MG1655 chromosomal DNA, using the primers hicA-f and hicA-r. The PCR product was digested with BamHI and EcoRI and inserted into pNDM220. This plasmid expresses HicA upon addition of IPTG.

(iii) Plasmid pMJ331.

The hicB gene was amplified from MG1655 chromosomal DNA, using hicB-f and hicB-r as primers. The PCR product was digested with HindIII and XbaI and inserted into pBAD33. Cells carrying this plasmid express hicB upon addition of arabinose. PCR fragments were confirmed by sequencing.

Rates of protein, RNA, and DNA synthesis.

Cells were grown at 37°C in M9 minimal medium with 0.2% glucose and amino acids in defined concentrations to an optical density at 450 nm (OD450) of ≈0.5. The cultures were diluted 10 times, and 2 mM IPTG was added at an OD450 of 0.3. Samples of 0.5 ml were added to 5 μCi of [35S]methionine (protein synthesis), 2 μCi of [methyl-3H]thymidine (DNA synthesis), or 0.1 μCi of [2-14C]uracil (RNA synthesis) plus a 100-fold excess of unlabeled carrier. After 1 min of incorporation, samples were chased for 10 min with 0.5 ml/mg of cold methionine, 0.5 mg/ml thymidine, or 0.5 mg/ml uracil, respectively. The samples were harvested and resuspended in 200 μl cold 20% trichloroacetic acid and centrifuged at 20,000 × g for 30 min at 4°C. The samples were washed twice with 200 μl cold 96% ethanol. Precipitates were transferred to vials, and the amounts of incorporated radioactivity were counted in a liquid scintillation counter.

Primer extension analysis.

Cells were grown in LB medium at 37°C. At an OD450 of 0.5, the cultures were diluted 10 times and grown to an OD450 of 0.5, and transcription of the toxin was induced by the addition of 2 mM IPTG. To inhibit translation, chloramphenicol (50 μg/ml) was added. Primer extension analysis was used to map the hicAB promoter and the cleavage patterns of the dksA, ompA, rpoD, and, ssrA mRNAs, using 32P-labeled primers, following extension by reverse transcriptase. The primer (3 pmol) was labeled with 2 μl of [γ-32P]ATP at a concentration of 6,000 Ci/mmol by addition of 0.4 μl polynucleotide kinase (New England Biolabs Inc.) in polynucleotide kinase buffer and incubated for 1 h at 37°C. Labeled primer was hybridized to 10 to 20 μg total RNA and extended with reverse transcriptase (Finnzymes). The labeled cDNA was fractionated by using a 6% polyacrylamide gel electrophoresis, which was dried and placed on a phosphorimager screen.

Reverse transcription qPCR.

RNA was extracted from all cell samples using an RNeasy mini-kit (Qiagen) according to the manufacturer's instructions. cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative PCR (qPCR) reactions were run in duplicate simultaneously. Briefly, 10 to 20 ng cDNA was mixed with 0.3 μm primers and 10 μl of 2× PCR Master Mix for Sybr green kit from Eurogentec (Seraing, Belgium), and the qPCR was run on a LightCycler 480 real-time PCR system (Roche). Relative expression was analyzed using qBase software (19).

RESULTS

Overproduction of HicA is bacteriostatic.

The hicAB locus of E. coli K-12 is shown schematically in Fig. Fig.1A.1A. The hicA reading frame (formerly yncN) consists of 58 codons and has a GTG start codon and a predicted Shine-Dalgarno sequence (AGGGAGG). The hicB gene (formerly ydcQ) has 145 codons and starts 24 bp downstream of hicA. We used primer extension analysis to map the transcriptional start site upstream of hicA. Interestingly, the hicA mRNA 5′ end was not detectable with total RNA prepared from exponentially growing cells (Fig. (Fig.1B,1B, −2′ sample). However, the addition of chloramphenicol induced the appearance of a distinct cDNA band, consistent with a transcriptional start site located 66 bp upstream of the hicA start codon (Fig. 1A and B). Thus, blockage of translation induced hicAB transcription. No additional promoter was detected in the region between hicA and hicB (data not shown).

FIG. 1.
Genetic organization of the hicAB locus. (A) Genetic map of hicAB. The enlargement shows the hicAB promoter region. (B) The 5′ ends of the hicAB mRNA were mapped by primer extension using a 32P-labeled hicA-20 primer (see Materials and Methods). ...

The hicA gene was PCR amplified and inserted downstream of the IPTG-inducible PA1/03/04 promoter of the low-copy-number vector pNDM220, resulting in pMJ221. Similarly, hicB was inserted downstream of the arabinose-inducible PBAD promoter of pBAD33, resulting in pMJ331. Strains MG1655/pMJ221 (PA1/03/04::hicA) and MG1655/pMJ331 (PBAD::hicB) were grown exponentially in rich medium. Induction of hicA resulted in a rapid cessation of cell growth (Fig. (Fig.2A)2A) and simultaneously inhibited cell proliferation (Fig. (Fig.2B).2B). These results show that ectopic production of HicA is bacteriostatic. By contrast, induction of hicB had no effect on cell growth.

FIG. 2.
hicAB of E. coli is a TA locus. (A) Growth of MG1655 wild-type (wt) cells carrying pMJ221 (PA1/O3/O4::hicA) or pMJ331 (PBAD::hicB) were grown exponentially in LB medium at 37°C. At an OD450 of ≈0.5, transcription of hicA and hicB was induced ...

HicB neutralizes HicA-mediated inhibition of growth.

Next, we deleted the chromosome-encoded copy of the hicAB locus and repeated the hicA induction experiment described above. In this case, HicA also inhibited cell growth (data not shown) and, moreover, reduced the number of colony-forming cells (Fig. (Fig.2B).2B). This result indicated that HicA was more active in the ΔhicAB strain than in the wild-type strain. Therefore, we tested whether HicB could neutralize HicA. Cells containing plasmids carrying the inducible hicA (IPTG) and hicB (arabinose) genes were grown exponentially. As described before, induction of hicA inhibited cell growth (Fig. (Fig.2C).2C). Strikingly, however, later induction of hicB resulted in an immediate resumption of cell growth, showing that HicB can indeed counteract HicA. Moreover, the rapid resumption of cell growth indicated that HicA was bacteriostatic rather than bactericidal, at least for the 90 min of hicA induction used here.

HicA inhibits the global rate of translation.

Since the molecular target of HicA is unknown, we measured the rates of protein, DNA, and RNA syntheses after induction of hicA. As shown in Fig. Fig.3,3, the rate of translation was strongly inhibited (20-fold after 1 h with hicA induction), while replication and transcription continued unaffected during this period.

FIG. 3.
Rates of protein, DNA, and RNA synthesis after induction of hicA. Cells of E. coli MG1655 carrying pMJ221 (PA1/O3/O4::hicA) were grown exponentially in M9 minimal medium, and hicA was induced by addition of IPTG (2 mM) at time zero. Samples were taken ...

Induction of hicA induces mRNA cleavage.

Ectopic production of mRNA interferases, such as RelE and MazF, results in a dramatic inhibition of translation (7, 10). Therefore, we investigated whether ectopic production of HicA would induce mRNA cleavage in three different test mRNAs. The primer extension analysis illustrated in Fig. Fig.44 shows that HicA induced multiple cleavage sites in the ompA, dksA, and rpoD mRNAs. By comparison of the band patterns obtained with RNA prepared from cells treated with chloramphenicol, HicA-mediated cleavages were distinguished from premature terminations of reverse transcriptase (Fig. (Fig.4,4, Cml). The HicA-mediated cleavage patterns in the three test mRNAs did not yield any obvious consensus recognition motifs.

FIG. 4.
HicA induces cleavage of the ompA, dksA, and rpoD mRNAs and tmRNA. Primer extension analysis of the ompA (A), dksA (B), and rpoD (C) mRNAs and tmRNA (D) using the 32P-labeled primers ompA-ctr-ccw, dksA PE1, rpoD PE1, and 10SA-2, respectively, are shown. ...

HicA-induced RNA cleavage does not depend on translation.

Next, we investigated whether HicA also induced cleavages in tmRNA. Figure Figure4D4D shows a primer extension analysis of the coding region of tmRNA before and after induction of hicA. As shown, HicA induced cleavage of wild-type tmRNA at two sites (A↓AAC). Addition of chloramphenicol did not lead to any appreciable cleavage of tmRNA, thus showing that the cleavages were induced by HicA. Primer extension analysis of the nontranslated regions of tmRNA did not reveal additional cleavage sites (data not shown).

Both of the HicA-induced cleavage sites were located within the translated part of tmRNA. Therefore, we tested HicA cleavage of a nontranslated variant of tmRNA that had its GCA resume codon changed to a UAA ochre codon (44). The mutant tmRNA exhibited a cleavage pattern indistinguishable from that of the wild-type tmRNA (Fig. (Fig.4D,4D, third and fourth panels). Thus, the HicA-mediated cleavages in tmRNA were independent of tmRNA translation.

Amino acid and carbon starvation induce hicAB transcription.

We investigated whether hicAB would be induced during nutrient starvation. Amino acid starvation was induced by the addition of SHX to exponentially growing cells, and the relative level of hicAB mRNA was measured by qPCR. As shown in Fig. Fig.5A,5A, SHX induced a rapid and dramatic increase in the level of hicAB transcripts. Sixty minutes after the onset of amino acid starvation, the level of hicAB mRNA was ≈15-fold increased. Assuming that the metabolic stability of hicAB mRNA was not changed by amino acid starvation, as was the case with relBE mRNA (9), we conclude that amino acid starvation activates transcription of hicAB.

FIG. 5.
Nutrient starvation induces hicAB transcription by a Lon-dependent mechanism. (A) Cells were grown exponentially in LB medium, and samples were withdrawn before and after the addition of SHX (1 mg/ml). (B) Cells were grown exponentially in M9 minimal ...

Next, we induced glucose starvation by the addition of α-methyl glucoside to cells growing in glucose minimal medium (Fig. (Fig.5B).5B). In this case, hicAB transcription was also stimulated, although to a lesser degree (≈sixfold). Finally, we measured the effect of chloramphenicol on hicAB transcription (Fig. (Fig.5C).5C). As shown, hicAB transcription was stimulated strongly by chloramphenicol (≈12-fold), consistent with the result shown in Fig. Fig.1B1B.

Activation of hicAB depends on Lon.

The above-described results are consistent with the notion that HicB is an unstable antitoxin that autoregulates hicAB transcription. If so, induction of hicAB transcription might depend on a cellular protease. To test this possibility, we investigated the transcriptional response to amino acid starvation in lon or clpP protease mutant strains. Deletion of clpP did not reduce induction of hicAB during amino acid starvation (Fig. (Fig.5D).5D). However, deletion of lon severely reduced the activation of hicAB transcription during amino acid starvation (Fig. (Fig.5E).5E). This observation suggests that HicB is an autorepressor of hicAB transcription and that Lon degrades HicB during starvation.

Distribution of hicAB and other TA loci in prokaryotic genomes.

Previously, we generated a comprehensive phylogenetic analysis of the six known TA locus families in 126 completely sequenced prokaryotic genomes (RelE, MazF, VapC, Doc, CcdB, and ParE) (33). Here, we extended the previous survey to encompass 218 (22 archaeal and 196 bacterial) genomes and included the hicAB gene family in the analysis. In total, we identified 1,340 TA loci divided among 1,069 loci of bacteria (≈5.4 loci per genome) and 272 loci of archaea (≈12.4 loci per genome), of which 119 were hicAB loci (Table (Table3).3). Figure Figure66 shows the two bacterial and two archaeal genomes with the highest number of hicAB loci together with members of the six other TA families. Treponema denticola, a gram-negative spirochete, has at least 32 TA loci, 9 of which belong to the hicAB family. Photorhabdus luminescens, an enteric insect pathogen, has at least 59 TA loci, 8 of which belong to the hicAB family. Methanosarcina activorans and M. mazei have 21 and 18 TA loci, respectively. In both cases, 8 of these are hicAB loci. Thus, genomes carrying multiple TA loci are common.

FIG. 6.
Bacterial and archaeal genomes with many hicAB loci. The bacterial (two upper diagrams) and archaeal (two lower diagrams) genomes with the highest number of hicAB loci are shown. Other TA loci are included for comparison.
TABLE 3.
Phyletic distribution of seven TA families in 218 prokaryotic genomesa

Table S1 in the supplemental material gives an overview of the TA loci that we identified in 218 prokaryotic genomes. Detailed information for all these genes is given in Table S2 in the supplemental material. Our extensive survey supports conclusions drawn from a previous study that encompassed 118 genomes (33). All archaea, even Nanoarchaeum spp., with its tiny ≈490-kb genome, have at least two TA loci (see Table S1 in the supplemental material), whereas almost all obligate intracellular organisms are devoid of TA loci (49 bacterial species) (see Table S3 in the supplemental material). Thus, the average number of TA loci in free-living bacteria is estimated here to be ≈7.2 per genome. Even with this correction, it appears that archaea have, on average, significantly more TA loci than bacteria. Some organisms, both archaeal and bacterial particularly, have many TA loci. Organisms with more than 10 TA loci are listed in Table S4 in the supplemental material. Many of these organisms are characterized by slow growth (e.g., Mycobacterium tuberculosis and species of Geobacter, Synechocystis, Nostoc, Nitrosomonas, and Caulobacter and many archaea).

DISCUSSION

Here, we show that hicAB of E. coli K-12 has many characteristics in common with a number of well-characterized TA loci such as relBE and mazEF. (i) The hicA gene encodes an inhibitor of translation whose ectopic production leads to mRNA cleavage. (ii) hicB encodes a protein that neutralizes HicA. (iii) Nutritional stress induces hicAB transcription by a Lon-dependent mechanism. (iv) Chloramphenicol also induced transcription of the hicAB locus. Observations (iii) and (iv) are consistent with the proposal that HicB is an unstable transcriptional repressor of hicAB transcription and that Lon degrades HicB. This proposal is consistent with the presence of an HTH DNA binding motif in the C terminus of HicB (6). (v) The phylogenetic pattern of the hicAB locus is similar to that of the relBE and vapBC loci (Table (Table33 and see Table S1 in the supplemental material).

Ectopic production of HicA-induced cleavages in three model mRNAs and in tmRNA.

We do not know if these cleavages are direct or indirect, that is, if HicA overproduction activates an endogenous RNase or if HicA itself is an RNase (Fig. (Fig.4).4). Although many TA loci encode mRNA-cleaving enzymes, there are examples of such translational inhibitors that mediate indirect mRNA cleavage (13), and the final test to determine whether HicA is an RNase awaits biochemical experiments.

The hicA (formerly yncN) gene of E. coli K-12 has not been described before (23), whereas hicB (formerly ydcQ) has been implicated in genetic interaction with σE, the sigma factor of E. coli that responds to stress in the cell membrane and periplasmic space (12). Thus, in wild-type cells, σE is essential. However, suppressor mutations that render σE nonessential are easily obtained, and one class of such suppressors had IS1 elements inserted into hicB (6). Deletion of hicB from wild-type cells resulted in a significant downregulation of extracytoplasmic stress responses (both the σE and the Cpx-dependent responses) and a severe reduction in the formation of outer membrane vesicles (6). The reason(s) for these complex phenotypes is not known. However, we suggest that deletion of hicB (ydcQ) leads to hyperactivation of HicA that, in turn, leads to degradation of mRNAs encoding proteins involved in extracytoplasmic stress. We are now testing this proposal.

Including that of hicAB, seven TA families are now known (Table (Table3).3). Some free-living organisms have a plethora of these gene systems. For example, M. tuberculosis has more than 60 TA loci and N. europaea has at least 49 (see Table S1 and S3 in the supplemental material). The phylogenetic pattern is consistent with the proposal that TA loci are particularly beneficial to slowly growing organisms. The reason for this apparent correlation is not known. E. coli has at least seven TA loci that encode mRNA interferases: four relBE loci (relBE [15], yefM yoeB [16], dinJ yafQ [31], and prlF yhaV [35]), two mazEF loci (mazEF and chpB [28]), and hicAB (this work). In all cases, transcription of these loci is induced by amino acid starvation. Thus, even some rapidly growing organisms have multiple TA loci. The reason for this apparent redundancy is not known.

The literature contains a number of hypotheses that explain the physiological function(s) of TA loci. One group suggested that mazEF mediates programmed cell death during amino acid starvation (2), although we and others (10, 42) have not been able to confirm this. By contrast, we have obtained solid evidence that relBE and mazEF of E. coli are induced during nutrient starvation and reduce the global level of translation without cell killing (8-10). There are several reasons why a reduced global level of translation may be beneficial rather than detrimental to starving cells. Most importantly, it may reduce the level of translational errors by reducing the level of uncharged tRNAs (37, 38). Second, a reduced cellular level of translation may help the cells adapt more rapidly to nutrient starvation. The phylogenetic distribution of TA loci (i.e., their absence from obligate intracellular organisms) is consistent with the proposal that they function as stress response elements that are triggered by nutrient starvation and other forms of environmental stresses. This hypothesis is supported by the observations that TA loci were induced by heat shock in S. solfataricus and by exposure to chloroform in Nitrosomonas europaea (18, 40). However, we are aware that this hypothesis does not explain the apparent massive redundancy of TA loci.

It has also been proposed that TA loci may function to help the cells enter a dormant state called persister cells (22, 36, 43). Persister cell formation can be viewed as an extreme case of translational inhibition, and the phenomenon is compatible with the proposal that TA loci reduce the cellular level of translation during nutritional stress.

Due to the differential metabolic stabilities of the toxins and antitoxins, TA loci may also lead to genetic stabilization of the chromosomal regions to which they are closely linked (39). To test these hypotheses more stringently, we are now analyzing model organisms devoid of all known TA loci.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by the Danish Centre for mRNP Biogenesis and Metabolism and the Wellcome Trust.

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 December 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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