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B Weisblum

Publications and source records attributed to B Weisblum.

At least 19 recordsLinked to original sources
PubMed2000

Peptide analogues of the VanS catalytic center inhibit VanR binding to its cognate promoter.

The dodecamer peptide SLCHDSVIGWEC, named E12, was selected from a combinatorial peptide library on the basis of its ability to bind to VanR, the two-component signal transduction response regulator which controls expression of vancomycin resistance in Enterococcus faecium. The binding of E12 was localized to the N-terminal, regulatory domain of VanR which contains Asp-55, the residue which accepts the phosphoryl group from His-164 in the activated VanS sensor kinase. E12, along with a related sequence SLAHDSIIGYLS, named E12.1, was found to inhibit the binding of VanR approximately P to a DNA segment which corresponds to its cognate promoter PvanH. With a single gap, both E12 and E12.1 could be aligned with the octadecamer sequence YLAHDIKTPLTSIIGYLS, comprising Tyr-161 through Ser-178, of the catalytic center dimerization domain of VanS, a sequence with which VanR also normally interacts. Alanine substitution analysis of E12.1 identified six amino acids as indispensable for its ability to inhibit VanR approximately P-PvanH DNA complex formation. A similar analysis of the corresponding amino acids in VanS showed a parallel dependence except for the substitutions Leu-162 --> Ala and Gly-175 --> Ala which interfered with the ability of E12.1 to compete with protein-DNA complex formation, but did not inhibit the ability of VanS to bind VanR. Our findings support a model in which E12 mimics the VanS phosphorylatable sequence with which the regulatory domain of VanR interacts, and thus functions as a "minimalist" analogue of VanS. Our results also indicate the usefulness of phage-displayed peptides as a general tool for mimicking the interacting faces of interacting proteins.

Alanine↗
PubMed2000

Modulation of erm methyltransferase activity by peptides derived from phage display.

Combinatorial peptide display on phage M13 protein pIII was used to discover peptide sequences that selectively bind to ErmC' methyltransferase from Bacillus subtilis. One peptide, Ac-LSGVIAT-NH(2), inhibited methylation in vitro with a 50% inhibitory concentration of 20 microM. Interestingly, the set of six peptides which inhibited ErmC' stimulated ErmSF, a homologous methyltransferase from Streptomyces fradiae. Thus, Ac-LSGVIAT-NH(2) may not act directly at the catalytic center of ErmC', but may modulate its activity by binding at a structurally unrelated, but functionally linked, site.

Bacillus subtilis↗
PubMed1999

Dissecting the VanRS signal transduction pathway with specific inhibitors.

The VanRS two-component signal transduction pathway from Enterococcus faecium was reconstituted in vitro from partially purified components and shown to be inhibited by the halophenyl isothiazolone LY-266,400, inhibitor A, a compound shown previously to reduce expression of the AlgR1-AlgR2 two-component signal transduction pathway in Pseudomonas aeruginosa (S. Roychoudhury, N. A. Zielinski, A. J. Ninfa, N. E. Allen, L. N. Jungheim, T. I. Nicas, and A. M. Chakrabarty, Proc. Natl. Acad. Sci. USA 90:965-969, 1993). Inhibitor A attenuates phosphoryl transfer from VanS approximately P to VanR by its action on the ability of VanR to accept. We observed an apparent stimulatory effect of inhibitor A on VanS autophosphorylation which is attributable to the accumulation of VanS approximately P as an intermediate unable to transfer Pi to the inhibited VanR. Thus, inhibitor A acts on the second of two sequential steps which lead to transcriptional activation of the VanHAXYZ gene cluster and the resultant expression of vancomycin resistance.

Bacterial Proteins↗
PubMed1998

Macrolide resistance.

The macrolides have evolved through four chemical generations since erythromycin became available for clinical use in 1952. The first generation, the 14-membered ring macrolide erythromycin, induced resistance and was replaced by the second generation 16-membered ring macrolides which did not. The inability to induce came at the price of mutation, in the pathogenic target strain, to constitutive expression of resistance. A third generation of macrolides improved the acid-stability, and therefore the pharmacokinetics of erythromycin, extending the clinical use of macrolides to Helicobacter pylori and Mycobacterium tuberculosis. Improved pharmacokinetics resulted in the selection of intrinsically resistant mutant strains with rRNA structural alterations. Expression of resistance in these strains was unexpected, explainable by low rRNA gene copy number which made resistance dominant. A fourth generation of macrolides, the 14-membered ring ketolides are the most recent development. Members of this generation are reported to be effective against inducibly resistant strains, and ketolide resistant strains have not yet been reported. In this review we discuss details of the ways in which bacteria have become resistant to the first three generations of macrolides, both with respect to their biochemistry, and the genetic mechanisms by which their expression is regulated.

Journal Article↗
PubMed1997

Induction of ermSV by 16-membered-ring macrolide antibiotics.

The erm family of 23S rRNA adenine-N6-methyltransferases confers resistance to all macrolide-lincosamide-streptograminB (MLS) antibiotics, but not all MLS antibiotics induce synthesis of Erm methyltransferase with equal efficiency in a given organism. The induction efficiency of a test panel of MLS antibiotics was studied by using two translational attenuator-lac reporter gene fusion constructs, one based on ermSV from Streptomyces viridochromogenes NRRL 2860 and the other based on ermC from Staphylococcus aureus RN2442. Four types of responses which were correlated with the macrolide ring size were seen, as follows: group 1, both ermSV and ermC were induced by the 14-membered-ring macrolides erythromycin, lankamycin, and matromycin, as well as by the lincosamide celesticetin; group 2, neither ermSV nor ermC was induced by the 12-membered-ring macrolide methymycin or by the lincosamide lincomycin or the streptogramin type B antibiotic ostreogrycin B; group 3, ermSV was selectively induced over ermC by the 16-membered-ring macrolides carbomycin, chalcomycin, cirramycin, kitasamycin, maridomycin, and tylosin; and group 4, ermC was selectively induced over ermSV by the 14-membered-ring macrolide megalomicin. These data suggest that the leader peptide determines the specificity of induction by different classes of MLS antibiotics and that for a given attenuator, a major factor which determines whether a given macrolide induces resistance is its size.

Anti-Bacterial Agents↗
PubMed1997

Essential role of endogenously synthesized tylosin for induction of ermSF in Streptomyces fradiae.

We compared ermSF induction in wild-type Streptomyces fradiae NRRL B-2702 and that in GS-14, a tylA mutant which cannot synthesize tylosin. Our findings suggest that (i) endogenously synthesized tylosin plays an obligatory role in ermSF induction and (ii) tylosin, or a biosynthetic intermediate beyond tylactone, has an "autocrine" function that induces ErmSF synthesis, thereby enabling S. fradiae to resist higher levels of tylosin.

Anti-Bacterial Agents↗
PubMed1996

A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme.

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan [correction of peptidodoglycan] precursors are wrong.

Anti-Bacterial Agents↗
PubMed1995

Methylation of minimalist 23S rRNA sequences in vitro by ErmSF (TlrA) N-methyltransferase.

ermSF (synonym tlrA) from Streptomyces fradiae NRRL 2702 confers resistance to the macrolide-lincosamide- streptogramin type B (MLS) superfamily of antibiotics. ErmSF specifically methylates Bacillus subtilis 23S rRNA in vitro at A2085 (B. subtilis coordinate, which is equivalent to the Escherichia coli coordinate A2058). In the present studies, partial B. subtilis 23S rRNA sequences containing portions of the peptidyltransferase circle which include A2085 were constructed in order to identify structural requirements needed for RNA to function as substrate of ErmSF. A model methylase substrate based on the 41-nucleotide construct DK111, ggCCUAUCCGUCGCGGGUUCGCCCGCGACAGGACGGA*AAGA, had methyl-acceptor activity. This sequence contains 23S rRNA stem 73 [Stade, K., et al. (1994) Nucleic Acids Res. 22, 1394-1399] underlined, flanking a tetraloop-like (UUCG), and the impaired sequence AAAGA, at the 3' end containing A2085 (A*). A set of systematic alterations introduced into the sequence suggested that the four unpaired nucleotides in stem 73 are necessary for methyl-acceptor activity, whereas inversion of 11 out 13 paired bases in stem 73 conferred no significant reduction in methyl-acceptor activity.

Base Sequence↗
PubMed1994

Regulation of plasmid pE194 replication: control of cop-repF operon transcription by Cop and of repF translation by countertranscript RNA.

The cop-rep region of plasmid pE194 contains two tandem structural genes, cop and repF, as well as the plus and minus origins of replication. The two structural genes comprise an operon whose expression is repressed by the binding of Cop protein to a 28-bp inverted complementary repeat sequence that overlaps the cop-repF promoter. From its position relative to the promoter and the experimentally determined footprint made by the Cop protein, the 28-bp inverted complementary repeat sequence is presumed to function as the cop operator. The intercistronic region between cop and repF is 80 nucleotides (nt) long and is transcribed bidirectionally: in the forward direction as part of the synthesis of the cop-repF message (ca. 900 nt), and in the reverse direction to yield a countertranscript ca. 65 nt long. The proposed countertranscript RNA (ctRNA) can form a single stem-and-loop structure that includes the single SphI sequence of plasmid pE194 as part of the loop-forming segment. Enlargement of the proposed loop from 6 to 14 nt by insertion of a SphI-BamHI adapter at the SphI site or contraction of the proposed loop down to 4 nt, by cutting with SphI followed by blunting with S1 nuclease, yields mutants with an increased copy number. By gel retardation and DNaseI footprinting analysis, Cop protein was shown to bind to the promoter region of cop; no binding by Cop protein at the 5' end of repF was detected. Two major transcripts were synthesized in vitro by using cop-repF region DNA as a template, the tandem cop-repF transcript, and the ctRNA. Addition of purified Cop protein to an vitro transcription reaction mixture reduced only the rate of cop-repF transcription but not that of ctRNA. These observations suggest that regulations of repF occurs at two levels: (i) with Cop protein acting as a repressor of cop-repF mRNA transcription and (ii) with ctRNA acting as a repressor of RepF translation.

Amino Acid Sequence↗
PubMed1994

23S rRNA domain V, a fragment that can be specifically methylated in vitro by the ErmSF (TlrA) methyltransferase.

The DNA sequence that encodes 23S rRNA domain V of Bacillus subtilis, nucleotides 2036 to 2672 (C. J. Green, G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F. Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro. The RNA transcripts served as a substrate in vitro for specific methylation of B. subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics on Streptomyces fradiae NRRL 2702, the host from which it was cloned. Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics.

Adenine↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗