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

Abstract

The aminoglycoside antibiotic hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal-subunit formation. Pulse-chase labeling experiments were performed, which verified lower rates of ribosomal-subunit synthesis in drug-treated cells. Hygromycin B exhibited a concentrationdependent inhibitory effect on viable-cell numbers, growth rate, protein synthesis, and 30S and 50S subunit formation. Unlike other aminoglycosides, hygromycin B was a more effective inhibitor of translation than of ribosomal-subunit formation in E. coli. Examination of total RNA from treated cells showed an increase in RNA corresponding to a precursor to the 16S rRNA, while mature 16S rRNA decreased. Northern hybridization to rRNA in cells treated with hygromycin B showed that RNase II-and RNase III-deficient strains of E. coli accumulated 16S rRNA fragments upon treatment with the drug. The results indicate that hygromycin B targets protein synthesis and 30S ribosomal-subunit assembly.Currently, only a limited number of antibiotics are available to treat multidrug-resistant strains of infectious bacteria, and resistance to even the newest antimicrobial agents is appearing (13). Increasing antibiotic resistance in microbial populations has necessitated the search for alternate cellular targets for new and existing antimicrobial agents. There are many antibiotics available that inhibit translation in bacterial cells by binding to the 30S or 50S ribosomal subunit (3). It has been observed that a number of ribosomal antibiotics possess a second inhibitory activity that prevents ribosomal-subunit assembly. The formation of a functional ribosome is a vital cellular process and is therefore 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 antibiotics for their possible effects on inhibition of subunit formation. Identifying and comparing these antimicrobial agents will aid in the search for more suitable targets for future antibiotics. Crystallographic data have been obtained for several antibiotics from the aminoglycoside class (2, 25). These studies have shown that binding occurs through unique base interactions in the decoding center of the 30S ribosome, which effectively halts translation by preventing movements necessary for protein synthesis to occur (8). Recent work with the aminoglycoside compounds neomycin and paromomycin has shown that these antimicrobial agents have a second inhibitory target, which is inhibition of 30S ribosomal-subunit formation (18,19). These drugs are capable of inhibiting 30S ribosomalsubunit formation by stalling assembly at an intermediate phase of 30S biosynthesis.There are many antibiotics that target translation by binding to ribosomal subunits that have not been examined for possible inhibitory effects on ribosomal-subunit assembly. One of those antimicrobial agents is the aminoglycoside hygromycin B, a 30S subunit translational inhibitor. Most aminoglycosid...

Antibacterial to antifungal conversion of neamine aminoglycosides through alkyl modification. Strategy for reviving old drugs into agrofungicides

“…6 However, unusual bicyclic ring-containing aminoglycosides are additionally active against yeasts. [7][8][9] A recent report revealed inhibition of plant pathogenic oomycetes Phytophora and Pythium sp. by neomycin, paramomycin, ribostamycin and streptomycin, although they showed comparatively little activity against several other fungal genera.…”

Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae

“…Over the past two decades, several genetic screens have uncovered mutant collections defective in the CPY-pathway of vacuolar localization including pep (Jones 1977), vps (Bankaitis et al 1986; Rothman and Stevens 1986; Bonangelino et al 2002), vam (Wada et al 1992), and env (Takahashi et al 2008) mutants. Some mutants in these collections have shown sensitivities to low levels of the antibiotic hygromycin B, an aminoglycoside translation inhibitor isolated from Streptomyces hygroscopicus (Mcgaha and Champney 2007). Hygromycin B is an atypical aminoglycoside antibiotic in that it has dual inhibitory effects on translation; it interferes with both ribosomal translocation and with aminoacyl-t-RNA recognition (Cabañas et al 1978).…”

Inhibition of Bacterial Ribosome Assembly: a Suitable Drug Target?

“…These are neomycin and paromomycin in both E. coli and S. aureus (116) and hygromycin B in E. coli alone (114). The data showed a reduced accumulation of both subunits in all cases, with an effect on the 50S subunit that is proportionately greater than that on the 30S subunit in some instances (114,115).…”

“…These are neomycin and paromomycin in both E. coli and S. aureus (116) and hygromycin B in E. coli alone (114). The data showed a reduced accumulation of both subunits in all cases, with an effect on the 50S subunit that is proportionately greater than that on the 30S subunit in some instances (114,115). A putative coupling between the assembly of the 50S subunit and that of the 30S subunit, rather than a restriction of translation, is proposed to account for this finding (28), but such a coupling has not been established.…”