General method for direct cloning of DNA fragments generated by the polymerase chain reaction.
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Base Sequence↗
Biomedical subjects
B Weisblum
Publications and source records attributed to B Weisblum.
Transcriptional attenuation control of ermK, a macrolide-lincosamide-streptogramin B resistance determinant from Bacillus licheniformis.
ermK instructs bacteria to synthesize an erythromycin-inducible 23S rRNA methylase that confers resistance to the macrolide, lincosamide, and streptogramin B antibiotics. Expression of ermK is regulated by transcriptional attenuation, in contrast to other inducible erm genes, previously described, which are regulated translationally. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino-acid leader peptide together with its ribosome binding site. Additionally, the mRNA leader sequence can fold in either of two mutually exclusive conformations, one of which is postulated to form in the absence of induction and to contain two rho factor-independent terminators. Truncated transcription products ca. 210 and 333 nucleotides long were synthesized in the absence of induction, both in vivo and in vitro, as predicted by the transcriptional attenuation model; run-off transcription in vitro with rITP favored the synthesis of the full-length run-off transcript over that of the 210- and 333-nucleotide truncated products. Northern (RNA) blot analysis of transcripts synthesized in vivo in the absence of erythromycin indicated that transcription terminated at either of the two inverted complementary repeat sequences in the leader that were postulated to serve as rho factor-independent terminators; moreover, no full-length transcripts were detectable in the uninduced samples. In contrast, full-length (ca. 1,200-nucleotide) transcripts were only detected in RNA samples synthesized in vivo in the presence of erythromycin. Full-length transcripts formed in the absence of induction from transcriptional readthrough past the two proposed transcription terminators would fold in a way that would sequester the ribosome binding site together with the first two codons of the ErmK methylase, reducing its efficiency in translation. This feature could therefore provide additional control of expression in the absence of induction; however, such regulation, if operative, would act only secondarily, both in time and place, relative to transcriptional control. Analysis by reverse transcriptase mapping of in vivo transcripts from two primers that bracket the transcription terminator responsible for the 210-nucleotide truncated fragment supports the transcriptional attenuation model proposed and suggests further that the synthesis of the ermK message is initiated constitutively upstream of the proposed terminator but completed inductively downstream of this site.
Amino Acid Sequence↗
Replication genes of plasmid pE194-cop and repF: transcripts and encoded proteins.
In vivo transcription of the replication region of plasmid pE194 yeidls two classes of mRNAs that encode Cop and RepF proteins, respectively. These transcripts are oriented 5' to 3' exclusively in the clockwise direction on the standard map. The cop region contains an open reading frame capable of encoding a 55-amino-acid protein that was demonstrated electrophoretically as a 6-kilodalton product synthesized in Bacillus subtilis minicells and chemically by N-terminal sequencing of a 116-kilodalton fusion protein with Escherichia coli beta-galactosidase. Four transcripts derived from the repF region were found, of which the longest, approximately 720 nucleotides, had the length, orientation, and transcription start site necessary to code for the full-length RepF protein (216 amino acid residues), deduced from the DNA sequence. The 5' ends of the shorter repF transcripts fall within the repF open reading frame. We propose that (i) cop specifies a protein rather than an RNA countertranscript, (ii) the Cop protein functions as a negative-acting element in pE194 replication by regulating synthesis of both RepF and of itself, and (iii) increased plasmid copy number can be explained in terms of cop region mutations that either reduce the intrinsic activity of Cop protein or the rate of its synthesis.
Amino Acid Sequence↗
The ermC leader peptide: amino acid alterations leading to differential efficiency of induction by macrolide-lincosamide-streptogramin B antibiotics.
The inducibility of ermC by erythromycin, megalomicin, and celesticetin was tested with both wild-type ermC and several regulatory mutants altered in the 19-amino-acid-residue leader peptide, MGIFSIFVISTVHYQP NKK. In the model test system that was used, the ErmC methylase was translationally fused to beta-galactosidase. Mutational alterations that mapped in the interval encoding Phe-4 through Ile-9 of the leader peptide not only affected induction by individual antibiotics, but did so differentially. The subset of mutations that affected inducibility by the two macrolides erythromycin and megalomicin overlapped and were distinct from the subset of mutations that affected induction by celesticetin. These studies provide a model system for experimentally varying the relative efficiencies with which different antibiotics induce the expression of ermC. The possibility that antibiotics with inducing activity interact directly with the nascent leader peptide was tested by using a chemically synthesized decapeptide, MGIFSIFVIS--, attached at its C-terminus to a solid-phase support. This peptide, however, failed to bind erythromycin in vitro.
Amino Acid Sequence↗
Conformational alterations in the ermC transcript in vivo during induction.
ermC is an inducible antibiotic resistance gene from Staphylococcus aureus, one of several whose expression is regulated at the level of mRNA secondary structure. During induction of ermC, the inhibition of a ribosome active in translation of a short leader peptide by low levels of antibiotic belonging to the macrolide-lincosamide-streptogramin b family is believed to cause a rearrangement in mRNA secondary structure. The resultant conformational isomerization unmasks the methylase ribosome binding site and initiator Met codon, causing increased translation of the ermC transcript. Expression of ermC can also be demonstrated in Bacillus subtilis carrying plasmid pE194. To probe the ermC transcript in vivo during induction, ermC was transferred to B. subtilis by transformation and the resultant transformants were treated with dimethyl sulfate which reacts with N-1 of adenine and N-3 of cytosine residues in a manner that is sensitive to secondary structure. The bases modified in vivo were detected by primer extension with reverse transcriptase using total cellular RNA as template and a complementary ermC-specific oligonucleotide as primer. Physical evidence was obtained for the secondary structural rearrangements predicted by the ermC regulatory model. Additionally, physical evidence was obtained demonstrating that during induction, the stalled ribosome protects codons 9 and 10 of the leader peptide from modification by dimethyl sulfate, in agreement with genetic data obtained previously that identified the integrity of codons 5-9 as critical for induction of ermC by erythromycin.
Amino Acid Sequence↗
ermC leader peptide. Amino acid sequence critical for induction by translational attenuation.
The ermC mRNA leader segment, which encodes a 19 amino acid leader peptide, MGIFSIFVISTVHYQPNKK, plays a key role in regulating expression of the ErmC methylase. The contribution of specific leader peptide amino acid residues to induction of ermC was studied using a model system in which the ErmC methylase was translationally fused to Escherichia coli beta-galactosidase as indicator gene. Codons of the ermC leader peptide were altered systematically by replacement of leader DNA segments with double-stranded DNA constructed from chemically synthesized oligonucleotides. Missense mutations that resulted in reduced efficiency of induction involved codons for amino acid residues 5 to 9 (-SIFVI-). Nonsense mutations causing termination of the leader peptide at codons 10 (-S-) or 12 (-V-) remained inducible. These findings suggest that the codons for residues 5 to 9 of the leader peptide comprise the critical region in which ribosomes stall in the presence of erythromycin.
Amino Acid Sequence↗
Erythromycin-induced ribosome stall in the ermA leader: a barricade to 5'-to-3' nucleolytic cleavage of the ermA transcript.
The Staphylococcus aureus ermA gene, whose product confers resistance to the macrolide-lincosamide-streptogramin B family of antibiotics, is induced at the level of translation by nanomolar concentrations of erythromycin. Erythromycin also specifically stabilizes ermA transcripts, and the induced stabilization requires in-phase translation of at least one of two small leader peptides in the 5' leader region of the transcript. Erythromycin-induced mRNA stabilization was tested in three constructions in which the ermA transcript was elongated by making insertions at the ermA transcription start. Whereas mRNA downstream of the leader peptide is stabilized by erythromycin, mRNA upstream is not. In the presence of erythromycin, specific mRNA decay intermediates in both the extended ermA genes and the wild-type ermA gene were detected by both Northern blotting and S1 nuclease mapping. The 5' ends of the intermediates map to the sequences that encode each of the two ermA leader peptides, suggesting that the intermediates are produced by stalled erythromycin-bound ribosomes acting as barricades to degradation by 5'-to-3' RNases. In addition, whereas erythromycin was found previously to stabilize ermA transcripts only physically, an ermC-cat-86 hybrid transcript was stabilized both physically and functionally by erythromycin.
Amino Acid Sequence↗
Erythromycin-induced stabilization of ermA messenger RNA in Staphylococcus aureus and Bacillus subtilis.
Erythromycin-induced stabilization of ermA mRNA was studied in Staphylococcus aureus, its original host background, and in Bacillus subtilis, subcloned on plasmid vectors. By RNA blot analysis it was shown that 40 nM-erythromycin specifically increased the chemical half-life of ermA mRNA from 2.5 to 17.5 minutes whereas the half-life of cat-86 mRNA was not increased by erythromycin. While expression of ermA has been shown to be induced by erythromycin at the level of translation, our studies with three ermA constitutive mutants demonstrated that mRNA stabilization in growing cells occurred independently of induced gene expression, suggesting that the stabilized mRNA was not functional for protein synthesis. Studies of ermA/lacZ fusions demonstrated that the 5' end of the mRNA was sufficient to confer stabilization. Translation of specific amino acid codons in a leader peptide located at the extreme 5' end of the mRNA was required for the erythromycin-induced stabilization as a frameshift mutation introduced into the leader peptide determinant abolished stabilization. By S1 mapping, no differences were detected in the length of the 5' or 3' end of ermA mRNA with the addition of erythromycin, indicating that the stabilized transcript was not processed at its ends.
Bacillus subtilis↗
Translational attenuation control of ermSF, an inducible resistance determinant encoding rRNA N-methyltransferase from Streptomyces fradiae.
An inducible resistance determinant, ermSF, from the tylosin producer Streptomyces fradiae NRRL 2338 has been cloned, sequenced, and shown to confer inducible macrolide-lincosamide-streptogramin B resistance when transferred to Streptomyces griseofuscus NRRL 23916. From mapping studies with S1 nuclease to locate the site of transcription initiation, the ermSF message contains a 385-nucleotide 5' leader sequence upstream from the 960-nucleotide major open reading frame that encodes the resistance determinant. On the basis of the potential secondary structure that the ermSF leader can assume, a translational attenuation model similar to that for ermC is proposed. The model is supported by mutational analysis involving deletions in the proposed attenuator. By analysis with restriction endonucleases, ermSF is indistinguishable from the tlrA gene described by Birmingham et al. (V. A. Birmingham, K. L. Cox, J. L. Larson, S. E. Fishman, C. L. Hershberger, and E. T. Seno, Mol. Gen. Genet. 204:532-539, 1986) which comprises one of at least three genes from S. fradiae that can confer tylosin resistance when subcloned into S. griseofuscus. When tested for inducibility, ermSF appears to be strongly induced by erythromycin, but not by tylosin.
Amino Acid Sequence↗
Messenger RNA from Staphylococcus aureus that specifies macrolide-lincosamide-streptogramin resistance. Demonstration of its conformations and of the leader peptide it encodes.
The +1 site for transcription initiation of the inducible 23 S rRNA adenine methylase encoded by plasmid pE194 was determined experimentally by nuclease S1 mapping of mRNA synthesized in vivo, and by nuclease T1 mapping of (5'-gamma-32P)-end-labeled transcripts synthesized in vitro. By partial digestion of the in vitro transcripts using S1 and cobra venom nuclease as probes of mRNA conformation, the analysis was extended to reveal single-stranded and double-stranded regions, respectively, which correspond to the critical stems and loops postulated for active and inactive conformations of the nascent mRNA. According to the model for induction, the transition from inactive to active conformation involves disruption of mRNA secondary structure which, in turn, is predicated on protracted occupancy by ribosomes complexed with erythromycin of one of the critical stem sequences. Ribosome occupancy of the critical stem sequence is due to the presence of an open reading frame that encodes part of a 19 amino acid residue "leader" peptide. The existence of this peptide, deduced from the nucleotide sequence of the control region upstream from the methylase structural gene, was demonstrated in vivo as part of a translational fusion with Escherichia coli beta-galactosidase in which the first four amino acid residues of the N-terminal sequence of the fusion protein, analyzed directly by the microsequencing method, were found to comprise N-terminal amino acids 2 through 5, Gly-Ile-Phe-Ser, predicted for the leader peptide.
Amino Acid Sequence↗
Post-transcriptional regulation of chloramphenicol acetyl transferase.
The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) messenger ribonucleic acid (mRNA) encoded by plasmid pC194 was determined experimentally using gamma-32P-ATP-labeled run-off transcripts partially digested with T1 ribonuclease. By partial digestion of the in vitro transcripts with S1-, T1-, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions which contain the complementary sequences CCUCC, and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12 bp inverted complementary repeat sequence (-19.5 kcal/mol). The CAT mRNA was stable to digestion by T1 ribonuclease at the 4 guanosine residues in the Shine and Dalgarno sequence GGAGG, even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194, induced with chloramphenicol (CAM), contain about the same concentration of pulse labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the nonacetylatable CAM analog fluorothiamphenicol was tested using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro using Ba131 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by CAM required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role in regulating its own synthesis.
Acetyltransferases↗
N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci.
The nucleotide sequence of a structural gene ermE for ribosomal RNA (rRNA) N6-amino adenine N-methyl transferase (NMT) of Streptomyces erythraeus, cloned by Thompson et al. [Gene 20 (1982) 51-62], has been determined. The NMT amino acid (aa) sequence deduced from the nucleotide sequence contains extensive homology to aa sequences of cognate NMTs specified by: (1) plasmid pE194 from Staphylococcus aureus, 30% G + C, ermC; (2) plasmid pAM77 from Streptococcus sanguis, 43% G + C; as well as to (3) a chromosomal determinant from Bacillus licheniformis 759, 46% G + C, ermD, cloned in a recombinant plasmid pBD90. These findings suggest that all four NMT structural genes could have evolved from a common progenitor sequence despite the wide range of % G + C of the erm genes reflecting their current respective hosts. Comparison of the four NMT sequences with respect to localized hydrophobicity averaged over a moving window of 11 aa indicates that the common features of localized hydrophobicity that characterize the C-terminal portion of the ermE and ermD proteins are distinguishable from a contrasting pattern of hydrophobicity that characterizes the ermC and pAM77-coded proteins.
Amino Acid Sequence↗
Inducible resistance to macrolides, lincosamides and streptogramin type B antibiotics: the resistance phenotype, its biological diversity, and structural elements that regulate expression--a review.
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Amino Acid Sequence↗
Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.
A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.
Anti-Bacterial Agents↗
23S ribosomal ribonucleic acid of macrolide-producing streptomycetes contains methylated adenine.
Coresistance to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics by a common biochemical mechanism characterizes clinically resistant pathogens. Of 10 streptomycetes tested for resistance to macrolide, lincosamide, and streptogramin B-type antibiotics, only 1, Streptomyces erythreus, the organism used for production of erythromycin, was found resistant to all three classes; moreover, it was the only streptomycete in the series tested found to contain N6-dimethyladenine (m62A) in 23S ribosomal ribonucleic acid, the structural alteration of ribosomal ribonucleic acid associated with clinical resistance. Of the seven streptomycetes tested for the presence of m62A and N6-methyladenine (m6A), two, S. fradiae and S. cirratus, which produce the macrolide antibiotics tylosin and cirramycin, respectively, were found to contain m6A, but not m62A. The remaining strains tested, including strains which produce lincomycin and streptogramins, contained neither m6A nor m62A.
Adenine↗
Deoxyribonucleic acid sequence common to staphylococcal and streptococcal plasmids which specify erythromycin resistance.
Plasmids from erythromycin-resistant Staphylococcus aureus, Streptococcus sanguis, and Streptococcus faecalis show deoxyribonucleic acid sequence homology. The homologous sequences can be localized to specific restriction endonuclease fragments, which in the case of S. aureus plasmid pI258 involves a single fragment from either EcoRI or HindIII digest known to contain the erythromycin resistance determinant. Complementary ribonucleic acid probes prepared from S. aureus plasmid pI258 and S. sanguis plasmid pAM77 also hybridize to specific fragments in restriction endonuclease digests of deoxyribonucleic acid from erythromycin-resistant Streptococcus progenes and Streptococcus pneumoniae. These studies suggest a common origin for a class of erythromycin resistance determinants in unrelated strains of pathogenic bacteria for which exchange of genetic material has not been demonstrated.
Bacillus subtilis↗
Altered methylation of adenine in 23 S ribosomal RNA associated with erythromycin resistance in Streptomyces erythreus and Streptococcus fecalis.
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Adenine↗
Aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in Escherichia coli.
Bacillus circulans NRRL B-3312, a nonpathogenic bacterium that produces the aminoglycoside antibiotic butirosin, is known to contain an aminoglycoside phosphotransferase that is similar to the neomycin phosphotransferases of clinically isolated antibiotic-resistant bacteria. Purified DNAs from B. circulans and the plasmid ColE1-ApR were digested with EcoRI endonuclease and the resulting fragments covalently joined with polynucleotide ligase. The recombined DNA was used to transform E. coli and ampicillin-neomycin resistant colonies were selected. Analysis of several clones indicated that neomycin resistance in the E. coli transformants was due to the presence of the B. circulans phosphotransferase gene. This observation is consistent with the notion that anitbiotic-modifying enzymes from antibiotic-producing organisms may be the sources of antibiotic resistance in plasmid-containing bacteria.
Bacillus↗