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D Dubnau

Publications and source records attributed to D Dubnau.

At least 73 records · Page 4Linked to original sources

Expression of competence genes in Bacillus subtilis.

A set of competence (com) mutants of Bacillus subtilis was constructed by using Tn917lacZ as a mutagen. In about half of the mutants, the promoterless lacZ element on the transposon was placed under control of putative com promoters. Expression of the mutant com genes was studied by using the beta-galactosidase tag. Two of the mutant genes (those represented by com-124 and com-138) were expressed early in the growth cycle in all of the media tested and were not dependent on the spo0A or spo0H product for expression. The remaining mutants, which represented a minimum of four additional genes, expressed beta-galactosidase in stationary phase during the period in which competence developed. We conclude that expression of com genes is probably regulated transcriptionally and in a growth stage-specific manner. Expression of these genes was also dependent on growth in competence medium and, like competence development, required the presence of glucose and was dependent on the spo0H products. The dependence on the spo0A gene product was partially bypassed by the abrB703 mutation. These effects were qualitatively equivalent to those on competence development. The latter was dependent on spo0A and spo0H, and the spo0A dependency was partially suppressed by abrB703. Several of the mutants were still capable of resolution into light and heavy buoyant density cell fractions when grown in competence medium. All of these expressed beta-galactosidase to a greater extent in the light fraction, showing that expression of these com genes was cell type specific. Development of competence was not markedly affected by mutations in spo0B, spo0E, spo0F, spo0J, or sigB, the structural gene of sigma 37.

Bacillus subtilis↗

Site and substrate specificity of the ermC 23S rRNA methyltransferase.

The purified ermC methyltransferase described here incorporates two methyl groups per Bacillus subtilis 23S rRNA molecule in vitro. The Km for S-adenosyl-L-methionine was 12 microM, and for B. subtilis 23S rRNA the Km was 375 nM. In vivo methylation specified by several related resistance determinants prevented in vitro methylation by the ermC enzyme, suggesting that methylation specified by all of these determinants occurs at homologous sites. Since methyl groups were incorporated in protein-free 23S rRNA molecules, the structure of rRNA alone must contain sufficient information to specify the methylation site.

Bacillus subtilis↗

Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.

Anti-Bacterial Agents↗

Translational autoregulation of ermC 23S rRNA methyltransferase expression in Bacillus subtilis.

ermC specifies an rRNA methyltransferase that confers resistance to erythromycin. The expression of this determinant is induced by the addition of erythromycin. The induction mechanism has been shown to operate posttranscriptionally, and its mechanism has been elucidated. We now show that synthesis of the ermC gene product in Bacillus subtilis is also autoregulated by a mechanism operating on the level of translation. The synthesis of methyltransferase was shown to be gene dosage compensated by Western blot analysis. Several mutants were analyzed that specify altered ermC gene products and are deregulated. Analysis of mutants and of the wild-type strain by Northern blotting demonstrated that autoregulation is posttranscriptional. We suggest a translational repression model in which the ermC methyltransferase binds to its own mRNA, at a region that resembles the methylation target site on 23S rRNA. The overall control of ermC expression is discussed in light of these multiple regulatory mechanisms.

Bacillus subtilis↗

Evidence for the translational attenuation model: ribosome-binding studies and structural analysis with an in vitro run-off transcript of ermC.

Several features of the translational attenuation model of ermC regulation were tested. This model predicts two possible secondary structures for the leader of the ermC transcript and requires that the leader contains two Shine-Dalgarno (SD) sequences. The ribosome binding site for a leader peptide (SD1) is predicted to be accessible, whereas that for the rRNA methylase protein that confers erythromycin (Em) resistance (SD2) is sequestered by base pairing. The model suggests that in the presence of inducer (Em), a ribosome stalls while translating the peptide, altering the mRNA conformation, thereby exposing SD2. The results of our ribosome binding studies demonstrate that SD1 is exposed and binds to ribosomes, whereas SD2 is unavailable. Also, the secondary structure of the 5' region of the ermC transcript was analyzed using methidium propyl-EDTA.Fe (II), T1 nuclease, and nucleases from cobra venom and mung bean sprouts as structure probes. Our results support the previously proposed model for folding of ermC mRNA, and demonstrate that SD1 is single-stranded, while SD2 and its neighboring sequences are largely base paired, consistent with the ribosome-binding results.

Binding Sites↗

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.

Bacterial Proteins↗

Analysis of plasmid deletional instability in Bacillus subtilis.

Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established.

Bacillus subtilis↗

DNA sequence and regulation of ermD, a macrolide-lincosamide-streptogramin B resistance element from Bacillus licheniformis.

The DNA sequence of ermD , a macrolide-lincosamide-streptogramin B (MLS) resistance determinant cloned from the chromosome of Bacillus licheniformis, has been determined. ermD encodes an erythromycin inducible protein of molecular weight 32,796. S1 nuclease mapping of the ermD promoter has revealed the presence of an approximately 354 base leader sequence on the ermD transcript. This leader contains a short open reading frame sufficient to encode a 14 amino acid peptide, which is preceded by a potential ribosomal binding site. The leader sequence has the potential to fold into several base paired structures, in some of which the ribosomal binding site for the ermD product would be sequestered. Deletion analysis demonstrated that the leader contains regulatory sequences. Removal of the ermD promoter and fusion to an upstream promoter did not interfere with induction, strongly suggestion that ermD regulation is posttranscriptional. Based on these features it appears likely that ermD is regulated by a translational attenuation mechanism, analogous to that suggested for ermC , a resistance element from Staphylococcus aureus ( Gryczan et al. 1980; Horinouchi and Weisblum 1980). Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.

Anti-Bacterial Agents↗

Induction of macrolide-lincosamide-streptogramin B resistance requires ribosomes able to bind inducer.

Plasmids were constructed containing the regulatory regions and N-terminal portions of ermC and of ermD , fused in phase with the coding sequence of the Escherichia coli lacZ gene. ermC and ermD are erythromycin (Em) inducible macrolide-lincosamide-streptogramin B resistance elements derived from Staphylococcus aureus and Bacillus licheniformis, respectively. The fusion plasmids were introduced into B. subtilis and used to study ermC and ermD regulation. In both cases, beta-galactosidase synthesis could be induced by low levels of Em. Induction was prevented by introduction of ole-2, a chromosomal mutation which decreases ribosomal affinity for Em. Induction also did not occur in the presence of intact copies of ermC , suggesting that prior or concomitant methylation of 23S rRNA, a treatment known to decrease ribosomal affinity for Em, was capable of interfering with ermC and ermD induction. These experiments are consistent with the translational attenuation model of ermC regulation, and together with other evidence, suggest that ermD is regulated by a similar mechanism.

Anti-Bacterial Agents↗

Evolutionary relationships of the Bacillus licheniformis macrolide-lincosamide-streptogramin B resistance elements.

Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD , an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD . The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD . It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis, another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yield a variety of related Staphylococcal and Streptococcal MLS determinants.

Anti-Bacterial Agents↗

Translational attenuation: the regulation of bacterial resistance to the macrolide-lincosamide-streptogramin B antibiotics.

The regulation of ermC is described in detail as an example of regulation on the level of translation. ermC specifies a ribosomal RNA methylase which confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics. Synthesis of the ermC gene product is induced by erythromycin, a macrolide antibiotic. Stimulation of methylase synthesis is mediated by binding of erythromycin to an unmethylated ribosome. The translational attenuation model, supported by sequencing data and by mutational analysis, proposes that binding of erythromycin causes stalling of a ribosome during translation of a "leader peptide", resulting in isomerization of the ermC transcript from an inactive to an active conformer. The ermC system is analogous to the transcriptional attenuation systems described for certain biosynthetic operons. ermC is unique in that interaction with a small molecule inducer mediates regulation on the translational level. However, it is but one example of nontranscriptional -level control of protein synthesis. Other systems are discussed in which control is also exerted through alterations of RNA conformation and an attempt is made to understand ermC in this more general context. Finally, other positive examples of translational attenuation are presented.

Amino Acid Sequence↗

Plasmid marker rescue transformation in Bacillus subtilis.

We constructed an 18-megadalton plasmid (pBD221) carrying resistance determinants for kanamycin, chloramphenicol, and erythromycin, as well as the hisH determinant from the Bacillus licheniformis chromosome. This plasmid has a copy number of about one and can be stably maintained in Bacillus subtilis. Linear fragments of pBD221 DNA were used to transform competent cultures carrying mutant variants of the same plasmid. Rescue transformation did not proceed by recircularization and replication of the donor DNA. Rescue transformation exhibited first-order dependence on DNA concentration, and the concentration dependence curve was virtually identical to the curve obtained with chromosomal DNA. The donor DNA molecular weight dependence of plasmid marker rescue transformation obtained by using restriction fragments was not distinguishable from previously published data obtained by using fractionated sheared chromosomal DNA. Plasmid rescue transformation, like chromosomal transformation, was dependent on the recE, recA, recB, and recD gene products. Plasmid rescue transformation, like chromosomal transformation, proceeded with few exchanges. Linkage data obtained with the plasmid rescue system fit a quantitative model based on studies with chromosomal transformation. We conclude that plasmid marker rescue transformation probably proceeds by a mechanism similar to the mechanism used during the formation of chromosomal transformants and hence may be considered an appropriate general model for the study of transformational recombination.

Anti-Bacterial Agents↗

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.

Bacillus subtilis↗

Translational attenuation of ermC: a deletion analysis.

ermC is a plasmid gene which specifies resistance to macrolide-lincosamide-streptogramin B antibiotics. The product of ermC was previously shown to be an inducible rRNA methylase, which is regulated translationally, and a mechanism for this regulation, termed the translational attenuation model, has been proposed. This model postulates that alternative inactive and active conformational states of the ermC mRNA are modulated by erythromycin-induced ribosome-stalling during translation of a leader peptide. In the present study the translational attenuation model was tested by constructing a series of deletants missing the ermC promoter and portions of the regulatory (leading) region. In these mutants, ermC transcription is dependent on fusion to an upstream promoter. Depending on the terminus of each deletion within the regulatory region, determined by DNA sequencing, ermC expression is observed to be either high level and inducible (like the wild-type), high level and noninducible, or low level and noninducible. The translational attenuation model predicts that as the deletions extend deeper into the leader region, successively masking and unmasking sequences required for translation of the methylase, an alternation of high and low level methylase expression will be observed. These predictions are confirmed. Based on this and other information, the model is refined and extended, and both direct translational activation and kinetic trapping of a metastable active intermediate during transcription are proposed to explain basal synthesis of methylase and to rationalize the effects of certain regulatory mutants.

Bacillus subtilis↗

Direct selection of recombinant plasmids in Bacillus subtilis.

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thyA thyB host. Thy- strains are resistant to trimethoprim (Tmp), and Thy+ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cmr Tmpr clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRI, BclI, PvuII, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed.

Bacillus subtilis↗

Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis.

pE194, a 3.5-kilobase multicopy plasmid, confers resistance to the macrolide-lincosamide-streptogramin B antibiotics in Bacillus subtilis. By molecular cloning and deletion analysis we have identified a replication segment on the physical map of this plasmid, which consists of about 900 to 1,000 base pairs. This segment contains the replication origin. It also specifies a trans-acting function (rep) required for the stable replication of pE194 and a negatively acting copy control function which is the product of the cop gene. The target sites for the rep and cop gene products are also within this region. Two incompatibility determinants have been mapped on the pE194 genome and their properties are described. One (incA) resides within the replication region and may be identical to cop. incB, not located in the replication region, expresses incompatibility toward a copy control mutant (cop-6) but not toward the wild-type replicon.

Bacillus subtilis↗

Characterization of a plasmid-specified ribosome methylase associated with macrolide resistance.

The ermC gene of plasmid pE194 specifies resistance to the macrolidelincosamide-streptogramin B antibiotics. This resistance, as well as synthesis of the 29,000 dalton protein product of ermC, has been shown to be induced by erythromycin. Weisblum and his colleagues have established that macrolide resistance is associated with a specific dimethylation of adenine in 23 S rRNA. We show that pE194 specifies an RNA methylase that can utilize either 50 S ribosomes or 23 S rRNA as substrates. Synthesis of this methylase is induced by low concentrations of erythromycin, and the enzyme is produced in elevated amounts by strains carrying a high copy number mutant of pE194. The methylase comigrates with the 29K ermC product on polyacrylamide gels. The purification and some properties of this methylase are described.

Amino Sugars↗