Ae-Ran Kwon

Translational Attenuation and mRNA Stabilization as Mechanisms of erm(B) Induction by Erythromycin

ABSTRACT Translational attenuation has been proposed to be the mechanism by which the erm(B) gene is induced. Here, we report genetic and biochemical evidence, obtained by using erythromycin as the inducing antibiotic, that supports this hypothesis. We also show that erythromycin increases the level of the erm(B) transcript by stalling the ribosome on the leader mRNA and thereby facilitating the stabilization and processing of the mRNA. Erythromycin-induced mRNA stabilization and processing were observed with an ochre stop at codons 11 to 13 of the leader but not with an ochre stop at codon 10. This suggests that erythromycin does not stall the ribosome before codon 11 of the leader reaches the aminoacyl site. Secondary structure analyses of the erm(B) transcripts by in vitro and in vivo chemical probing techniques identified conformational changes in the transcripts that result from induction by erythromycin. These findings demonstrate that stalling of erythromycin-bound ribosomes at leader codon 11 causes the refolding of mRNA into a conformation in which the translational initiation site for the structural gene is unmasked and renders erm(B) translationally active.

ermK leader peptide: Amino acid sequence critical for induction by erythromycin

TheermK gene fromBacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. TheermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure ofermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction ofermK was studied using the PCR-based megaprimer mutation method.ermK methylases with altered leader peptide codons were translationally fused toE. coli β-galactosidase reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutivelyermK gene. Thus, ribosome stalling at codon 7 appears to be important forermK induction.

Role of Disulfide Bond of Arylsulfate Sulfotransferase in the Catalytic Activity

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of sulfate group from a phenyl sulfate ester to a phenolic acceptor. The promoter region and the transcription start sites of Enterobacter amnigenus astA have been determined by primer extension analysis. Northern blot analysis resolved two mRNA species with lengths of 3.3 and 2.0 kb, which correspond to the distances between the transcriptional initiation sites and the two inverted repeat sequences (IRSs). By length, the 3.3 kb RNA could comprise the three-gene (astA with dsbA and dsbB) operon. ASST has three highly conserved cysteine residues. Reducing and non-reducing SDS-PAGE and activity staining showed that disulfide bond is needed for the activity of the enzyme. To identify the cysteine residues responsible for the disulfide bond formation, a series of Cys to Ser mutants has been constructed and the enzymatic activity was measured. Based on the results, we assumed that the first cysteine (Cys349) might be involved in disulfide bond mainly with the second cysteine (Cys445) and result in active conformation.

Cloning, sequence analysis, and characterization of theastA gene encoding an Arylsulfate sulfotransferase fromCitrobacter freundii

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy ofCitrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homology, they were designated asastA anddsbA, respectively. The amino acid sequence ofastA deduced fromC. freundii was highly similar to that of theSalmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, andCampylobacter jejuni, encoded by theastA genes. However, the ASST activity assay revealed different acceptor specificities. Usingp-nitrophenyl sulfate (PNS) as a donor substrate, α-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol,p-acetaminophen, tyramine and tyrosine.

Distribution of bacteria with the arylsulfate sulfotransferase activity

This study is to predict the possible roles of the arylsulfate sulfotransferase (ASST) in the microorganism. At first we studied the spectrum of a distribution of the ASST enzyme through about 1,300 bacteria and the several selected strains were compared withKlebsiella K-36 previously reported in the level of DNA homology using the Southern blot method. From this study, we could predict that this enzyme would not exist in specific bacteria and it might not be a critical enzyme for the life of bacteria.

Bacterial arylsulfate sulfotransferase as a reporter system.

To investigate whether the arylsulfate sulfotransferase (ASST) is suitable as a reporter system for monitoring gene expression, a reporter vector carrying the fragments of the astA coding region without the promoter region was constructed and designated as pSY815. As a test of the ASST reporter systems suitability, the regulatory regions of ermC and lacZ were inserted upstream of the coding region of the reporter gene to generate pSY815‐EC and pSY815‐LZ, respectively. In the absence of the inserted regulatory regions, the plasmids displayed very low background activities in Bacillus subtilis and Escherichia coli. The ASST activity under the control of the ermC regulatory region was increased 4.4‐fold in B. subtilis when induced by 0.1 μgml–1 of erythromycin. These results were consistent with a lacZ reporter gene assay of the ermC regulatory region. Furthermore, we confirmed that the lacZ promoter in E. coli was strongly induced to a 17.9‐fold increase by 0.05 mM of isopropyl‐β‐D‐thiogalactopyranoside (IPTG) in this reporter system. These results indicate that the ASST is a suitable reporter system. The lack of endogenous activity, the simple detection of enzyme activity in the living cell, the commercially available non‐toxic substrates, and the high sensitivity make ASST a useful genetic reporter system for monitoring gene expression and understanding gene regulation.