Hygromycin B Inhibition of Protein Synthesis and Ribosome Biogenesis in Escherichia coli

The aminoglycoside antibiotic hygromycin B was examined in Escherichiacoli cells for inhibitory effects on translation and ribosomal-subunitformation. Pulse-chase labeling experiments were performed,which verified lower rates of ribosomal-subunit synthesis indrug-treated cells. Hygromycin B exhibited a concentration-dependentinhibitory effect on viable-cell numbers, growth rate, proteinsynthesis, and 30S and 50S subunit formation. Unlike other aminoglycosides,hygromycin B was a more effective inhibitor of translation thanof ribosomal-subunit formation in E. coli. Examination of totalRNA from treated cells showed an increase in RNA correspondingto a precursor to the 16S rRNA, while mature 16S rRNA decreased.Northern hybridization to rRNA in cells treated with hygromycinB showed that RNase II- and RNase III-deficient strains of E.coli accumulated 16S rRNA fragments upon treatment with thedrug. The results indicate that hygromycin B targets proteinsynthesis and 30S ribosomal-subunit assembly.

INTRODUCTION

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Introduction

Currently, only a limited number of antibiotics are availableto treat multidrug-resistant strains of infectious bacteria,and resistance to even the newest antimicrobial agents is appearing(13). Increasing antibiotic resistance in microbial populationshas necessitated the search for alternate cellular targets fornew and existing antimicrobial agents. There are many antibioticsavailable that inhibit translation in bacterial cells by bindingto the 30S or 50S ribosomal subunit (3). It has been observedthat a number of ribosomal antibiotics possess a second inhibitoryactivity that prevents ribosomal-subunit assembly. The formationof a functional ribosome is a vital cellular process and istherefore also an important cellular target (4).

In order to broaden our understanding of subunit assembly inhibitors,it is important to examine new or poorly studied antibioticsfor their possible effects on inhibition of subunit formation.Identifying and comparing these antimicrobial agents will aidin the search for more suitable targets for future antibiotics.Crystallographic data have been obtained for several antibioticsfrom the aminoglycoside class (2, 25). These studies have shownthat binding occurs through unique base interactions in thedecoding center of the 30S ribosome, which effectively haltstranslation by preventing movements necessary for protein synthesisto occur (8). Recent work with the aminoglycoside compoundsneomycin and paromomycin has shown that these antimicrobialagents have a second inhibitory target, which is inhibitionof 30S ribosomal-subunit formation (18, 19). These drugs arecapable of inhibiting 30S ribosomal-subunit formation by stallingassembly at an intermediate phase of 30S biosynthesis.

There are many antibiotics that target translation by bindingto ribosomal subunits that have not been examined for possibleinhibitory effects on ribosomal-subunit assembly. One of thoseantimicrobial agents is the aminoglycoside hygromycin B, a 30Ssubunit translational inhibitor. Most aminoglycosides have twovariable amino sugar rings that are connected to a 2-deoxystreptaminering (Fig. 1A). The position at which the two variable ringsare attached determines the class of aminoglycoside to whichthe compound belongs. Hygromycin B is unusual in that its structurehas two ether linkages connecting two of its three sugar residues,resulting in a fourth, five-membered ring (Fig. 1B). This extendedstructure has been shown to bind to the 30S ribosomal subunitnear the A, P, and E sites and spans a 13-Å distance onthe small subunit (2, 20).

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FIG. 1. Chemical structures of aminoglycoside antibiotics. (A) A generic 2-deoxystreptamine aminoglycoside. (B) Hygromycin B.

Hygromycin B has been in use since 1957 and was the first additivein livestock feed to be approved by the Food and Drug Administration.It has been used extensively since then as a growth aid andparasite control agent in the production of swine and poultry.It removes parasites by disrupting the egg-laying process andthen killing the adults (1, 17). Hygromycin B has also beenused in the laboratory to select for recombinant cells containinga hygromycin B resistance marker (12).

The structure and mechanism of binding of hygromycin B setsthe compound apart from other aminoglycosides, and thereforeit is of interest to examine the effects these differences haveon the inhibition of subunit assembly and its effects on turnoverof 16S rRNA in hygromycin B-treated cells. Previous work hasshown that RNases II and III and polynucleotide phosphorylase(PNPase) are involved in the turnover of 23S rRNA in the precursorthat forms in the presence of the 50S inhibitor azithromycin(23). The accumulation of 16S rRNA fragments during treatmentwith 30S ribosomal-subunit inhibitors has not been examined.One goal of this study was to determine if the 30S ribosomal-subunitinhibitor hygromycin B had any effect on the accumulation of16S rRNA fragments in E. coli cells with RNase mutations. Theresults indicate that hygromycin B also has two inhibitory targets,preventing protein synthesis and 30S ribosomal-subunit formationwith a stimulation of 16S rRNA turnover.

MATERIALS AND METHODS

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Materials and Methods

Studies were conducted with Escherichia coli strain D10-1 (11).The characteristics of the RNase mutant strains have been describedrecently (23). Hygromycin B was purchased from Sigma. [³H]uridine(45 mCi/mmol) and [³⁵S]methionine (Trans ³⁵S-Label; 1.175 Ci/mmol)were purchased from Perkin-Elmer and MP Biomedical, respectively.

Measurements of cell growth, viability, subunit assembly, and translation rates. The MIC for hygromycin B was determined by a dilution methodas described previously (6). Cells were grown at 37°C intryptic soy broth (TSB) in the presence and absence of the antibiotic.Growth rates were measured by following the increase in celldensity using a Klett-Summerson colorimeter. Cell viabilitywas measured by colony counting after serial dilution. RNA waslabeled by treating the cells with 1 µCi/ml [³H]uridine(1 µg/ml) and allowing the cultures to grow for two doublingsin the presence or absence of hygromycin B. Isotope incorporationwas halted by adding uridine to a final concentration of 50µg/ml, followed by a 15-min chase period. Cells were collectedby centrifugation at 6,500 rpm for 10 min and held at –70°Cuntil lysis. The cells were lysed with a lysozyme freeze-thawmethod as described previously (7). The lysates were spun on5 to 20% sucrose gradients in S buffer (10 mM Tris-HCl, pH 8.0,0.5 mM Mg acetate, 50 mM NH₄Cl) to separate ribosome particles.Fractions were collected from the gradients and mixed with 3ml of Scintisafe Gel (Fisher), and the incorporation of [³H]uridinewas measured by liquid scintillation counting.

Protein synthesis rates were determined by adding [³⁵S]methionine(1 µCi/ml) to the cell culture before the uridine chaseperiod. Samples of 0.4 ml were collected at 5-min intervalsfor 15 min. The collected samples were precipitated with 20%trichloroacetic acid (TCA). The TCA-precipitated material wascollected on Whatman GF/A glass fiber filters, washed with 10%TCA, and measured by liquid scintillation counting.

Uridine pulse-chase labeling. Cells were grown in 25 ml of TSB at 27°C in the presenceor absence of the drug. At a Klett reading of 10, hygromycinB (50 µg/ml) was added to the culture. The control samplewas grown without antibiotic. After growth had continued forone cell doubling, the RNA was pulse-labeled with [³H]uridine(1 µCi/ml) for 3 min and then chased with uridine at 50µg/ml. Samples of 4 ml were taken at six time intervals.Cells were collected by centrifugation, washed in S buffer,and stored at –70°C before lysis and sucrose gradientcentrifugation.

Analysis of total RNA via the Aligent Bioanalyzer. E. coli cells were grown in TSB with various concentrationsof hygromycin B. RNA was extracted from 1 ml of cells usingthe AquaPure RNA isolation kit (Bio-Rad). Total RNA was examinedusing an Aligent Bioanalyzer 2100 and the RNA 6000 "lab on achip": 5 µl (200 ng/µl) of sample from the totalRNA was loaded into each well of the RNA 6000 chip. Sample preparationand the loading procedure and run were carried out accordingto the manufacturer's recommendations for total-RNA analysis.

Northern analysis of 16S rRNA. RNA oligonucleotides were isolated from the top fractions ofsucrose gradients and were denatured and separated on 1.5% gelsas described previously (23). RNA from the gels was blottedonto Nytran nylon membranes using a Turbo blot apparatus (S&S).Construction of biotinylated 16S and 23S rRNA-specific DNA probesand the hybridization of membranes with biotin-labeled probewere performed as described previously (23). The probe was detectedon the membranes using the North2South chemiluminescent hybridizationkit (Pierce Chemical Co.) according to the manufacturer's directions.A low-range, ready-to-use RNA ladder (Fermentas) was biotinlabeled using the Label-It kit (Mirus) according to the manufacturer'sdirections and used to estimate the size of the RNA.

RESULTS

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Results

Aminoglycoside antibiotics are well-known inhibitors of translationin bacterial cells (3). The MIC for hygromycin B in E. colicells growing in TSB was found to be 150 µg/ml. Cellswere grown at a subinhibitory concentration to examine the effectsof the antibiotic on cellular activities. The rate of proteinsynthesis in growing E. coli cells was determined by measuringthe incorporation of [³⁵S]methionine into cellular proteins.Figure 2A shows the relative rate of incorporation of [³⁵S]methioninewith increasing concentrations of hygromycin B as a percentageof control [³⁵S]methionine incorporation. The 50% inhibitoryconcentration (IC₅₀) for inhibition of translation was 16 µg/ml.

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FIG. 2. Inhibition of protein synthesis, growth rate, and cell viability in E. coli cells with increasing concentrations of hygromycin B. (A) Inhibition of protein synthesis by hygromycin B. The arrow indicates the IC₅₀. (B) Percent decrease in growth rate ( ${square}$ ) and viable-cell numbers (•) with increasing concentrations of hygromycin B. The arrow indicates the IC₅₀ for the cell numbers. The results are the means of two determinations.

The inhibition of cell viability and of the cellular growthrate was consistent with the demonstrated inhibitory effectsof hygromycin B on translation. As shown in Fig. 2B, hygromycinB diminished the number of viable cells and increased the doublingtime in a concentration-dependent fashion. Hygromycin B inhibitedviable-cell numbers by 50% at a concentration of 20 µg/ml,and the growth rate was reduced by half at 25 µg/ml.

The effect of hygromycin B on ribosomal-subunit formation ingrowing E. coli cells was also determined. The concentrationdependence of ribosomal-subunit assembly was measured by examiningsucrose gradient profiles of ribosomal subunits labeled with[³H]uridine during growth in the presence and absence of hygromycinB. Figure 3A to C shows sucrose gradient profiles of lysatesfrom cells grown with and without the antibiotic. The 30S ribosomal-subunitamounts were reduced in proportion to the drug concentration(Fig. 3D). An IC₅₀ of 65 µg/ml for inhibition of ribosomal-subunitformation was seen. There was also increased accumulation offragmented RNA in the top fractions of gradients from cellstreated with hygromycin B. The amounts of 50S ribosomal subunitswere also reduced at higher concentrations of the antibiotic.

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FIG. 3. Sucrose gradient profiles of E. coli grown in the presence and absence of hygromycin B. (A) Gradient profile of control cell lysate. (B) Lysate from cells grown in 30 µg/ml hygromycin B. (C) Lysate from cells grown in 60 µg/ml hygromycin B. (D) Concentration-dependent inhibition of ribosomal-subunit assembly in E. coli cells. The percentages of the total gradient cpm were calculated for the 30S and 50S regions for the control and antibiotic-treated gradient samples. Inhibition of 30S assembly (•) and inhibition of 50S assembly ( ${square}$ ) are shown. The arrow indicates the IC₅₀ for 30S ribosomal-subunit formation. The results are the means of two determinations.

Pulse-chase labeling kinetic analysis was also used to measurethe rates of ribosomal-subunit synthesis in growing cells. Figure4A and B shows the pulse-chase kinetic analysis of ribosomal-subunitformation in control and hygromycin B-treated cells. In theabsence of the antibiotic, 30S ribosomal-subunit formation wascomplete in 15 min and 50S subunit formation reached a plateauin 30 min. When cells were treated with hygromycin B, 30S subunitformation did not reach control levels until 60 min and 50Sribosomal subunit amounts were estimated to reach control levelsin 120 min. 30S and 50S ribosomal-subunit formation rates wereinhibited equally relative to the control rates. These resultsare comparable to those observed in previous assembly studieswith the aminoglycosides neomycin and paromomycin (18).

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FIG. 4. [³H]uridine pulse-chase labeling kinetic analysis of ribosomal-subunit formation. (A) Assembly of 30S subunits ( ${square}$ ) and 50S subunits ( ${blacksquare}$ ) without hygromycin B. (B) Assembly of 30S subunits ( ${square}$ ) and 50S subunits ( ${blacksquare}$ ) with hygromycin B. The results are the means of two determinations.

Total RNA was examined in E. coli cells grown with hygromycinB at various concentrations of antibiotic and in control cellsusing the RNA 6000 lab on a chip kit and Bioanalyzer 2100 (Agilent).Figure 5A shows the virtual gel from the Agilent Bioanalyzeranalysis. With increasing concentrations of hygromycin B, therewas an increase in the amount of RNA accumulation correspondingto a 16S rRNA precursor (Fig. 5B). There was also a concentration-dependentdecrease in the amount of 16S rRNA present in the antibiotic-treatedcells, along with an increase in the number of small fragmentsof RNA. A small decrease in 23S rRNA was also seen at the highestconcentration of hygromycin B used. Table 1 lists the percentageof the total area for each peak, corresponding to small RNA,16S rRNA, its precursor, and 23S rRNA.

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FIG. 5. Agilent bioanalyzer analysis of total RNA from E. coli cells treated with hygromycin B. (A) Virtual gel produced by the Agilent software based on the results from a fluorescent chromatograph. 23S and 16S rRNAs and tRNA are indicated. (B) Tracing of lane 4 (75 µg/ml) showing a 16S rRNA precursor peak.

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TABLE 1. Relative amounts of RNA from Agilent RNA analysis

Previous work with the 50S ribosomal-subunit inhibitor azithromycinshowed that 23S rRNA fragments accumulate upon drug treatment(23). They were present in greater numbers in E. coli strainscontaining one or more mutations in specific RNase genes. RNasesII and III and PNPase were shown to be involved in the turnoverof 23S rRNA in 50S subunit precursors. Analysis by Northernhybridization with 16S and 23S rRNA-specific probes was performedon RNAs isolated from the top fractions of sucrose gradientsof lysates from wild-type and RNase mutant strains of E. coligrown with hygromycin B. The RNase mutant strains examined inthis study included strains defective in RNase I, RNase II,RNase III, and PNPase. Figure 6 shows a Northern blot hybridizedwith a 16S rRNA-specific probe. RNase II and III mutant strainscontained substantial numbers of 16S fragments in comparisonwith the control and an RNase I mutant. The sizes of RNA fragmentswere estimated to be between 200 and 900 bases. No accumulationof 23S RNA fragments was visualized in the top gradient fractionsexamined with a 23S rRNA-specific probe (data not shown).

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FIG. 6. Northern hybridization analysis of rRNA from control and RNase mutant strains. RNA was isolated from the top regions of sucrose gradients from cells grown with and without hygromycin B. Lane 1, control RNA from an RNase I strain; lane 2, RNA from an RNase I mutant grown with hygromycin B; lane 3, RNA from an RNase II mutant grown with hygromycin B; lane 4, RNA from a PNPase mutant grown with hygromycin B; lane 5, RNA from an RNase III mutant grown with hygromycin B. The positions of RNA size standards are indicated, along with 16S rRNA added as a marker in lanes 1 and 3.

DISCUSSION

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Discussion

It is known that aminoglycoside antibiotics inhibit translationby binding to the 30S ribosomal subunit. Recent studies haveshown that the aminoglycoside antibiotics neomycin and paromomycinpossess a secondary inhibitory target, preventing the formationof functional 30S ribosomal subunits (18, 19). These previousstudies showed that the antibiotics prevent translation andribosomal-subunit formation with equal effectiveness in growingE. coli cells. A difference was seen between the effects ofhygromycin B on protein synthesis and ribosomal-subunit assembly.Hygromycin B was a better inhibitor of translation than of 30Sribosomal-subunit formation, with IC₅₀s of 16 µg/ml and65 µg/ml, respectively. Inhibition of the growth rateand total viable-cell numbers was in accordance with the reductionof protein synthesis rates.

Hygromycin B has been shown to affect a ribosomal ATPase (RbbA)that is required for protein synthesis (10). RbbA is a proteinthat binds to the 30S subunit near the E site and functionsto aid ejection of tRNA from the subunit (26). Hygromycin Bbinds near the binding site for RbbA, disrupting the bindingof the ATPase and inhibiting 70 to 80% of its activity. Otheraminoglycoside antibiotics did not have this strong inhibitoryeffect. The effects of hygromycin B on this enzyme may contributeto the marked difference seen in its inhibitory effect on translationcompared with the effect on subunit formation.

Inhibition of 50S ribosomal-subunit assembly by hygromycin Bwas also observed. Most 50S protein biosynthesis inhibitorsdo not affect 30S subunit synthesis (4). However, 50S ribosomal-subunitformation is affected by inhibitors of 30S ribosomal-subunitassembly. The aminoglycosides neomycin and paromomycin havebeen shown to cause a reduction in 50S ribosomal-subunit formationin E. coli at higher concentrations of these drugs (18). HygromycinB demonstrated a similar effect on the formation of the 50Sribosomal subunit. The indirect action of these antibioticson the 50S subunit is likely due to a downstream polar effecton 23S rRNA processing. 30S particle biosynthesis precedes 50Sribosomal-subunit synthesis and is dependent on RNase III processingfrom a common precursor RNA (24). Therefore, an inhibitory effecton 30S formation could have a coupled downstream action of slowingor halting 50S biosynthesis. A generalized inhibitory effecton ribosomal-protein synthesis is a less likely explanation,because with inhibitors like the streptogramin A compounds,a parallel decline in the formation of both subunits is observed(5).

Pulse-chase kinetic analysis of ribosomal-subunit formationin cells treated with hygromycin B supports this linkage effecton 50S particle synthesis. In control cells, the time requiredto synthesize a 50S particle was twice as long as 30S synthesis.In drug-treated cells, the rates of synthesis of the 30S and50S subunits were decreased fourfold compared with control rates.The results for the pulse-chase kinetic analysis suggested equalinhibition of the rates of formation of 30S and 50S ribosomalsubunits, consistent with a linkage of 30S particle formationwith 50S synthesis.

An examination of total RNA from hygromycin B-treated E. colicells also indicated a specific inhibition of 16S rRNA processing.Total-RNA analysis showed an increase in small fragments ofRNA and a decrease in the amount of RNA corresponding to 16SrRNA. In addition, the amount of RNA corresponding to the 17SrRNA precursor increased (Table 1 and Fig. 5). The accumulationof the 17S precursor has been observed in ribosomal-subunitchaperone mutants (14-16, 22). 23S rRNA amounts were reducedonly at the highest antibiotic concentration used. This is consistentwith an indirect effect of the stalling of 30S subunit formationon 50S particle synthesis.

Northern blot analysis with a 16S rRNA-specific probe showedincreased accumulation of fragmented 16S rRNA in several RNasemutants (Fig. 6). RNA from RNase II and RNase III mutant strainstreated with drug contained larger amounts of 16S fragmentsthan an RNase I mutant. Previous work with the 50S assemblyinhibitors has also shown these enzymes to be important in theturnover of 23S rRNA in a stalled intermediate (23). This accumulationof rRNA fragments is the result of antibiotic binding to anintermediate particle in ribosomal-subunit assembly. These particlesare degraded by RNases in wild-type cells. In drug-treated RNase-defectivestrains, this process is retarded, and a buildup of rRNA fragmentsoccurs.

The accumulation of 16S rRNA fragments in the RNase II and RNaseIII mutants is an indication of the importance of these enzymesin the breakdown of the ribosomal-subunit precursor. RNase IIIis an important endoribonuclease that is essential for the separationof 16S and 23S rRNAs from a common precursor (24). Hybridizationwith a 23S-specific probe showed no accumulation of 23S fragmentsin any of the drug-treated samples, indicating that hygromycinB specifically targets only the 30S ribosome in assembly andtranslation.

Hygromycin B binds with specificity only to the 30S ribosomalsubunit and inhibits translation by interfering with the A,P, and E sites only on this subunit (2, 20). Hygromycin B hasbeen shown to bind to a part of 16S rRNA helix 44 that changesposition during translocation, and it is believed that hygromycinB binding to helix 44 restricts the movement of the helix duringprotein synthesis (9). It is possible that hygromycin B bindingto the helix could also restrict movements necessary to formfunctional 30S subunits and in so doing prevent conformationalrearrangement required for processing of the 16S rRNA transcriptand subsequent 30S ribosomal-subunit maturation.

This work is a continuation of studies examining possible ribosomal-subunitassembly inhibitors. The formation of novel hybrid aminoglycosidecompounds between neomycin and hygromycin is an indication ofthe importance of these drugs in controlling infections (21,27). More studies such as this one are needed to elucidate thepotential of ribosomal-subunit assembly as an antibiotic targetand to increase our understanding of the mechanism behind specificinhibition of this vital cellular function.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, J. H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614. Phone: (423) 439-2022. Fax: (423) 439-2030. E-mail: Champney{at}etsu.edu.

Published ahead of print on 16 October 2006.

REFERENCES

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