Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5' end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.     

Plasmid vectors based on Tn10 DNA: gene expression regulated by tetracycline

The regulatory region of the tetracycline resistance determinant from transposon Tn10 has been used to construct plasmid vectors for gene expression regulated by tetracycline. Plasmids pRS tetBam-8 and pRS tetBam-16 include the tet regulatory region, the segment coding for the first four amino acids of the tetracycline resistance protein (tetA protein), and a linker region with SalI, HpaII, and BamHI restriction sites for gene fusions. Plasmid pTB-1, a derivative of pRS tetBam-8 and of the beta-galactosidase gene-containing plasmid pMC1403, constitutively expresses a tetA fragment-beta-galactosidase fusion protein. If a multicopy runaway replication plasmid, pMOBglII-16 that includes a 2.7-kb BglII DNA fragment from Tnl10 that provides tetR protein is present along with pTB-1, the expression of beta-galactosidase is reduced eightfold. Tetracycline acts as an inducer of the system and restores the level of beta-galactosidase activity measured in transformants containing pTB-1 alone. Plasmid mutants unable to produce active tetR protein are ineffective in reducing expression. Escherichia coli carrying plasmids that express both tetA protein and tetR protein show an increase in the tetracycline resistance level after incubation with the drug. The observations are consistent with the previously proposed mechanism of regulation of tetracycline resistance in Tn10.     

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.     

Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain.

Deletion of the tetA(L) chromosomal region of Bacillus subtilis in a strain designated JC112 increased the strain's sensitivity to low tetracycline concentrations. It also resulted in phenotypic changes that correlate with the previously found role of TetA(L) in mediating electrogenic NA+/H+ antiport. Growth of JC112 was impaired relative to that of the wild type at both pH 7.0 and 8.3; Na(+)- and K(+)-dependent pH homeostases were impaired at alkaline pH. The phenotype of JC112 was complemented by plasmid-borne tetA(L) and related tet(K) genes; the antiport activity conferred by the tet(K) gene had an apparently higher preference for K+ over Na+ than that conferred by tetA(L). The data were consistent with TetA(L) being the major Na+(K+)/H+ antiporter involved in pH homeostasis in B. subtilis as well as a significant Na+ extrusion system. The phenotype of JC112 was much more pronounced than that of an earlier transposition mutant, JC111, with a disruption in the putative tetA(L) promoter region. Northern (RNA) blot analysis of tetA(L) RNA from wild-type and JC111 strains revealed the same patterns. That JC111 nevertheless exhibited some Na+ and alkali sensitivity may be accounted for by disruption of regulatory features that, in the wild type, allow increased tetA(L) expression under specific conditions of pH and monovalent cation concentration. Evidence for several different regulatory effects emerged from studies of lacZ expression from the transposon of JC111 and from a tetA(L)-lacZ translational fusion introduced into the amyE locus of wild-type and JC112 strains.     

Novel tetracycline resistance determinant isolated from an environmental strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.     

Induction of ermC requires translation of the leader peptide.

ermC confers resistance to macrolide-lincosamide streptogramin B antibiotics by specifying a ribosomal RNA methylase, which results in decreased ribosomal affinity for these antibiotics. ermC expression is induced by exposure to erythromycin. We have previously proposed a translational regulation model in which erythromycin causes stalling of a ribosome, which is translating a leader peptide. Stalling causes a conformation shift in the ermC mRNA which in turn unmasks the methylase ribosomal binding site. A prediction of this translational attenuation model for ermC induction was tested by replacing the second codon of the putative ermC leader peptide coding region by TAA. As expected, the introduction of this mutation resulted in an uninducible phenotype which was suppressible by two ochre suppressor mutations in Bacillus subtilis. It is concluded that translation through the leader peptide coding region, in frame with the predicted leader peptide, is required for ermC induction.     

Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression.

Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth. Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized. All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function. Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr). IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene. Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter. In addition, it appears that E. coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion. These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E. coli. We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.     

Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT

The tetQ-rteA-rteB operon of the Bacteroides conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of tetQ and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for tetQ. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of tetQ. Finally, comparison of the CTnDOT tetQ leader regions with upstream regions of five tetQ genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

In ribosomal protein S12 mutant or L24 mutant the expression of λN gene was depressed at translational level. To study its mechanism the λN gene region of λN -lacZ gene fusion was trimmed from its 5′ end to 3′ end with DNA exonuclease III (DNA cxoIII) in order to alter the TIR (translational initiation region) and the ding region of λN gene. After DNA sequencing 23 species of different λN-lacZ fused genes were obtained. The β-galactosidase activities of these deletants in ribosomal protein mutant were compared with that in wild type strain. The result indicated that (i) S12 mutant could affect 305 subunit’s binding to the TIR of λN gene messenger and cause the difficulty in forming 30s initiation complex and then decrease the efficiency of translational initiation; (ii) in S12 mutant the coding region of λN gene alw affected the expression λN gene; (iii) in L24 mutant the inhibition of λN gene expression was not related to translational initiation and the 5′ end of the coding region of λN gene, but related to the 3′ end of λN gene.     

Intracistronic complementation of the tetracycline resistance membrane protein of Tn10.

The structural gene region for tetracycline resistance on Tn10 consists of two complementation groups, tetA and tetB (M. S. Curiale and S. B. Levy, J. Bacteriol. 151:209-215, 1982). Using a series of deletion mutants, we have determined that the tetA region is 450 to 600 base pairs long and that the tetB region, which is adjacent to tetA, is 600 to 750 base pairs long. Point mutations in either tetA or tetB affected the amount and size of the inducible inner-membrane Tet protein synthesized in Escherichia coli maxicells. Moreover, deletions in these regions led to the synthesis of an appropriately smaller Tet protein. A single tetracycline-inducible RNA of about 1,200 bases was detected that was homologous with the tetracycline resistance structural gene region. These results indicate that the tetA and tetB complementation regions represent two parts of a single gene encoding two domains of the tetracycline resistance protein Tet.     

Escherichia coli ribosomal protein L10 is rapidly degraded when synthesized in excess of ribosomal protein L7/L12.

In Escherichia coli the genes encoding ribosomal proteins L10 and L7/12, rplJ and rplL, respectively, are cotranscribed and subject to translational coupling. Synthesis of both proteins is coordinately regulated at the translational level by binding of L10 or a complex of L10 and L7/L12 to a single target in the mRNA leader region upstream of rplJ. Unexpectedly, small deletions that inactivated the ribosome-binding site of the rplL gene carried on multicopy plasmids exerted a negative effect on expression of the upstream rplJ gene. This effect could be overcome by overproduction of L7/L12 in trans from another plasmid. This apparent stimulation resulted from stabilization of the overproduced L10 protein by L7/L12, presumably because free L10, in contrast to L10 complexed with L7/L12, is subject to rapid proteolytic decay. The contribution of this decay mechanism to the regulation of the rplJL operon is evaluated.     

Dependence of expression of an inducible Staphylococcus aureus cat gene on the translation of its leader sequence

Summary The gene for chloramphenicol (Cm) acetyltransferase (CAT) carried by the staphylococcal plasmid pUB112, whose expression can be stimulated by Cm, is preceded by a regulatory region containing two control elements. One of these consists of a Shine-Dalgarno (SD) sequence followed by an open reading frame coding for a leader peptide of nine amino acids. Previous work has shown that the SD sequence is essential for inducibility of Cm resistance by the antibiotic (Brückner and Matzura 1985). Here we demonstrate that fusion of the leader peptide coding sequence to a truncated 'lacZ gene results in synthesis of a leader peptide--galactosidase fusion protein. Introduction of an ochre nonsense codon into the reading frame of the leader peptide sequence leads to considerable reduction of the basal expression and loss of inducibility of the cat gene. These results reveal that synthesis of the leader peptide is required for the basal and inducible expression of the cat gene and support the model of translational attenuation for its regulation.