Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1990
The sporulation operon spolIA of Bacillus subtilis consists of three cistrons called spoIIAA, spo... more The sporulation operon spolIA of Bacillus subtilis consists of three cistrons called spoIIAA, spollAB, and spollAC. Little is known about the function of spoIIAA and spollAB, but spollAC encodes a a, factor called aF, which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spollA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of Spoil-AB) and SpoIlAB is, in turn, an antagonist of SpoIIAC (0,F). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits crF-directed gene expression, (ii) a mutation in spoIIAB stimulates aF-directed gene expression, (iii) a mutation in spoIIAA blocks CF-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in aF-directed gene expression caused by a mutation in spoIIAA. The SpoIl-AA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of crF-directed gene expression and/or could be responsible for restricting aF-directed gene expression to the forespore chamber of the sporangium. Gene expression during the process of endospore formation in the Gram-positive soil bacterium Bacillus subtilis is governed in part by five developmentally specific RNA polymerase ofactors, .H, OF, a.E, aG and 0.K (1, 2). These factors come into play in an ordered sequence that is temporally and spatially correlated with the morphological stages of spore formation. Thus 0JH controls gene expression at the onset of sporulation. Next, o-acts during the stage of septum formation, at which time the sporangium is partitioned into separate mother-cell and forespore compartments. 0.E, which is produced after the septum is formed, is, in turn, active during the engulfment stage of sporulation. Finally, the compartment-specific factors 0oG and cuK direct gene expression in the forespore and mother-cell chambers of the sporangium, respectively. a.F is of central importance because it helps to govern the transition from a single-celled sporangium to one that consists of two cellular compartments ofdivergent developmental fates.
Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth a... more Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P 1 ′ site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N -alkyl urea at the P 1 ′ site. Compounds with MICs of ≤4 μg/ml against gram-positive and gram-negative pathogens, including Staphylococcus aureus , Streptococcus pneumoniae , and Haemophilus influenzae , have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC 50 s) for Escherichia coli Ni-PDF were ≤0.1 μM, demonstrating the specificity of the inhibitors. In addition, these compounds were very selective for PDF, with IC 50 s of consistently >200 μM for matrilysin and other mammalian metalloproteases....
The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell divi... more The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible inv...
We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis. In extension... more We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis. In extension of previous nucleotide sequence analysis, our results show that the order of genes in the vicinity of spoIIB is valS folC comC spoIIB orfA orfB mreB mreC mreD minC minD spoIVFA spoIVFB L20 orfX L24 spoOB obg pheB pheA. All 20 genes have the same orientation; the direction of transcription is from valS to pheA. We show that spoIIB is a 332-codon-long open reading frame whose transcription is under sporulation control. The deduced amino acid sequence of the spoIIB gene product, a 36-kDa polypeptide, is highly charged and contains a stretch of uncharged amino acids that could correspond to a transmembrane segment. Surprisingly, mutations in spoIIB, including an in vitro-constructed null mutation, cause only a mild impairment of spore formation in certain otherwise wild-type bacteria. However, when combined with mutations in another sporulation gene, spoVG, mutations in spoIIB cause a sever...
A method for the selective tetra-Boc-protection of polymyxin B nonapeptide (PMBN) has been develo... more A method for the selective tetra-Boc-protection of polymyxin B nonapeptide (PMBN) has been developed. Boc-ON selectively protects the amino side chains of the four diaminobutyric acid (Dab) residues in the presence of the N-terminal free amine.
Early in the process of spore formation in Bacillus subtilis a septum is formed that partitions t... more Early in the process of spore formation in Bacillus subtilis a septum is formed that partitions the sporangium into daughter cells called the forespore and the mother cell. The daughter cells each have their own chromosome but follow dissimilar programs of gene expression. Differential gene expression in the forespore is now shown to be established by the compartmentalized activity of the transcription factor sigma F. The sigma F factor is produced prior to septation, but is active only in the forespore compartment of the post-septation sporangium. The sigma F factor is controlled by the products of sporulation operons spoIIA and spoIIE, which may be responsible for confining its activity to one of the daughter cells.
Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making ... more Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics. We have identified actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor. The dissociation constant for this compound was 0.3 × 10-9 M against Ni-PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value. Microbiological evaluation revealed that actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms. The PDF gene, def, was placed under control of P BAD in E. coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration. The susceptibility of this strain to actinonin increases with decreased levels of PDF expression, indicating that actinonin inhibits bacterial growth by targeting this enzyme. Actinonin provides an excellent starting point from which to derive a more potent PDF inhibitor that has a broader spectrum of antibacterial activity.
Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due ... more Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due to inactivation of transformylase activity. Knockout experiments in Streptococcus pneumoniae R6x indicate that the transformylase ( fmt ) and deformylase ( defB ) genes are essential and that a def paralog ( defA ) is not. Actinonin-resistant mutants of S. pneumoniae ATCC 49619 harbor mutations in defB but not in fmt . Reintroduction of the mutated defB gene into wild-type S. pneumoniae R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.
Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two ... more Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB , were identified in Staphylococcus aureus . The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB , was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB , but not defA , correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. The defB gene could not be disrupted in wild-type S. aureus , suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, the defA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not sh...
Faced with a wealth of antibacterial drug discovery targets as a result of bacterial genomics, we... more Faced with a wealth of antibacterial drug discovery targets as a result of bacterial genomics, we need to carefully select which ones to work with. Choosing bacterial metalloenzymes is one possible approach that can increase the probability of success. Metalloenzymes can be identified through specific motif searches of bacterial genomes. Current state-of-the-art medicinal chemistry allows for the design of inhibitor libraries targeting metalloenzymes and the efficient optimization of leads identified. This approach has been successfully applied to the discovery of in vivo active antibacterial agents that are inhibitors of bacterial peptide deformylase and UDP-3-O-(R-3hydroxymyristoyl)-N-acetylglucosamine deacetylase. Other bacterial metalloenzymes are open to the same approach.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1990
The sporulation operon spolIA of Bacillus subtilis consists of three cistrons called spoIIAA, spo... more The sporulation operon spolIA of Bacillus subtilis consists of three cistrons called spoIIAA, spollAB, and spollAC. Little is known about the function of spoIIAA and spollAB, but spollAC encodes a a, factor called aF, which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spollA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of Spoil-AB) and SpoIlAB is, in turn, an antagonist of SpoIIAC (0,F). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits crF-directed gene expression, (ii) a mutation in spoIIAB stimulates aF-directed gene expression, (iii) a mutation in spoIIAA blocks CF-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in aF-directed gene expression caused by a mutation in spoIIAA. The SpoIl-AA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of crF-directed gene expression and/or could be responsible for restricting aF-directed gene expression to the forespore chamber of the sporangium. Gene expression during the process of endospore formation in the Gram-positive soil bacterium Bacillus subtilis is governed in part by five developmentally specific RNA polymerase ofactors, .H, OF, a.E, aG and 0.K (1, 2). These factors come into play in an ordered sequence that is temporally and spatially correlated with the morphological stages of spore formation. Thus 0JH controls gene expression at the onset of sporulation. Next, o-acts during the stage of septum formation, at which time the sporangium is partitioned into separate mother-cell and forespore compartments. 0.E, which is produced after the septum is formed, is, in turn, active during the engulfment stage of sporulation. Finally, the compartment-specific factors 0oG and cuK direct gene expression in the forespore and mother-cell chambers of the sporangium, respectively. a.F is of central importance because it helps to govern the transition from a single-celled sporangium to one that consists of two cellular compartments ofdivergent developmental fates.
Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth a... more Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P 1 ′ site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N -alkyl urea at the P 1 ′ site. Compounds with MICs of ≤4 μg/ml against gram-positive and gram-negative pathogens, including Staphylococcus aureus , Streptococcus pneumoniae , and Haemophilus influenzae , have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC 50 s) for Escherichia coli Ni-PDF were ≤0.1 μM, demonstrating the specificity of the inhibitors. In addition, these compounds were very selective for PDF, with IC 50 s of consistently >200 μM for matrilysin and other mammalian metalloproteases....
The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell divi... more The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible inv...
We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis. In extension... more We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis. In extension of previous nucleotide sequence analysis, our results show that the order of genes in the vicinity of spoIIB is valS folC comC spoIIB orfA orfB mreB mreC mreD minC minD spoIVFA spoIVFB L20 orfX L24 spoOB obg pheB pheA. All 20 genes have the same orientation; the direction of transcription is from valS to pheA. We show that spoIIB is a 332-codon-long open reading frame whose transcription is under sporulation control. The deduced amino acid sequence of the spoIIB gene product, a 36-kDa polypeptide, is highly charged and contains a stretch of uncharged amino acids that could correspond to a transmembrane segment. Surprisingly, mutations in spoIIB, including an in vitro-constructed null mutation, cause only a mild impairment of spore formation in certain otherwise wild-type bacteria. However, when combined with mutations in another sporulation gene, spoVG, mutations in spoIIB cause a sever...
A method for the selective tetra-Boc-protection of polymyxin B nonapeptide (PMBN) has been develo... more A method for the selective tetra-Boc-protection of polymyxin B nonapeptide (PMBN) has been developed. Boc-ON selectively protects the amino side chains of the four diaminobutyric acid (Dab) residues in the presence of the N-terminal free amine.
Early in the process of spore formation in Bacillus subtilis a septum is formed that partitions t... more Early in the process of spore formation in Bacillus subtilis a septum is formed that partitions the sporangium into daughter cells called the forespore and the mother cell. The daughter cells each have their own chromosome but follow dissimilar programs of gene expression. Differential gene expression in the forespore is now shown to be established by the compartmentalized activity of the transcription factor sigma F. The sigma F factor is produced prior to septation, but is active only in the forespore compartment of the post-septation sporangium. The sigma F factor is controlled by the products of sporulation operons spoIIA and spoIIE, which may be responsible for confining its activity to one of the daughter cells.
Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making ... more Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics. We have identified actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor. The dissociation constant for this compound was 0.3 × 10-9 M against Ni-PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value. Microbiological evaluation revealed that actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms. The PDF gene, def, was placed under control of P BAD in E. coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration. The susceptibility of this strain to actinonin increases with decreased levels of PDF expression, indicating that actinonin inhibits bacterial growth by targeting this enzyme. Actinonin provides an excellent starting point from which to derive a more potent PDF inhibitor that has a broader spectrum of antibacterial activity.
Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due ... more Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due to inactivation of transformylase activity. Knockout experiments in Streptococcus pneumoniae R6x indicate that the transformylase ( fmt ) and deformylase ( defB ) genes are essential and that a def paralog ( defA ) is not. Actinonin-resistant mutants of S. pneumoniae ATCC 49619 harbor mutations in defB but not in fmt . Reintroduction of the mutated defB gene into wild-type S. pneumoniae R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.
Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two ... more Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB , were identified in Staphylococcus aureus . The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB , was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB , but not defA , correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. The defB gene could not be disrupted in wild-type S. aureus , suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, the defA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not sh...
Faced with a wealth of antibacterial drug discovery targets as a result of bacterial genomics, we... more Faced with a wealth of antibacterial drug discovery targets as a result of bacterial genomics, we need to carefully select which ones to work with. Choosing bacterial metalloenzymes is one possible approach that can increase the probability of success. Metalloenzymes can be identified through specific motif searches of bacterial genomes. Current state-of-the-art medicinal chemistry allows for the design of inhibitor libraries targeting metalloenzymes and the efficient optimization of leads identified. This approach has been successfully applied to the discovery of in vivo active antibacterial agents that are inhibitors of bacterial peptide deformylase and UDP-3-O-(R-3hydroxymyristoyl)-N-acetylglucosamine deacetylase. Other bacterial metalloenzymes are open to the same approach.
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