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 x 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.
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.
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 o-factors, .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. a' is encoded by the promoter-distal member of the three-cistron sporulation operon spoIIA (3-7). The a.F_ encoding cistron is designated spoIIAC and the two upstream cistrons are called spoIIAA and spoIIAB. Although the product of spoIIAC (SpoIIAC or o0F) has been characterized biochemically (7), little is known about the function of the spoIIAA gene product (SpoIIAA) and the spoIIAB gene product (SpoIIAB). SpoIIAA and QF (SpoIIAC) evidently play similar roles in sporulation, since spoIIAA and spoIIAC mutations have indistinguishable phenotypic effects (8-10).It has been uncertain whether SpoIIAB is required for sporulation, because no lesions in spoIIAB were found in the extensive collection of traditionally isolated mutations in the spoIfA operon. Recently, a spoIIAB mutation of a special kind was obtained by a scre...
The Bacilus subtilis divIVBI 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 divIVBI 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 (rodBI) 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 involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.Cells of the gram-positive soil bacterium Bacillus subtilis are capable of entering an alternative developmental pathway that is characterized by the formation of a transverse septum. During vegetative growth, the formation of a septum at the center of the cell partitions the bacterium into identical daughter cells which separate and undergo further cycles of binary fission. The hallmark of the process of sporulation, in contrast, is the formation of a septum that is sited near one pole of the cell. This asymmetrically positioned septum partitions the bacterium into unequal-sized cellular compartments, of which one, the forespore, undergoes metamorphosis into a spore and the other, the mother cell, participates in the formation of the spore but is eventually discarded by lysis. The binary fission septum and the sporulation septum are produced by similar processes (17), involving in both cases the action of B. subtilis homologs to the Escherichia coli septation genes ftsA and ftsZ (2, 3). However, little is known about the mechanisms that govern the alternative placement of the septa at medial or polar positions within the cell.In the non-spore-forming bacterium E. coli, placement of the septum is governed by genes at the minB locus. Cells of E. coli grow by binary fission and are normally capable of producing only medially sited septa. However, certain mutations at the minB locus allow septa to form at a polar position, thereby generating small, anucleate cells with intact cell walls and membranes which except for their lack of DNA appear to be metabolically normal (1). Minicell production occurs as an alternative to normal division, and consequently, the sister cell is filamentous and carries two or more copies of the chromosome. Because the minicell division process appears to be identical to that of the wild type, the defect is apparently with site selection and not with septum formation. As with sporulation in B....
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 arabinosedependent 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 show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S. aureus ⌬fmt strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, the defB gene could be disrupted in an fmt mutant.
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