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.

The outer membrane of imipenem-resistant mutants ofPseudomonas aeruginosa with decreased permeability to imipenem was shown by Western (immuno-) blotting to contain protein Dl and to lack protein D2. Protein D2 was purified and was shown to allow the permeation of imipenem at a rate higher than expected from its molecular weight. Spontaneous imipenem-resistant mutants of P. aeruginosa PAO1 appeared at a frequency of 10-8 in the laboratory and did not synthesize protein D2. Experiments performed with intact cells carrying plasmid pHN4 containing the gene for L-1 ,-lactamase from Pseudomonas maltophiia showed that this channel could also be used by SM-7338, Sch 33755, and Sch 33440 but apparently not by Sch 34343 or Sch 29482.Nutrients and waste products have to go through the outer membrane of gram-negative bacteria to reach the cytoplasm and the outer medium, respectively. This membrane is semipermeable and acts as a molecular sieve allowing the passage of small hydrophilic molecules (21). The overall permeability of the outer membrane usually depends on the number and properties of the pore-forming proteins that are generically called porins (21). Although most of the small hydrophilic molecules utilize this nonspecific pathway, there are some specific channels that allow the passage of compounds that have an insufficient or negligible rate of permeation through the porin channels and of compounds that are found in the external medium at low concentrations (21). Some of these specific channels are synthesized at higher levels when the substrate is present in the medium of growth: for example, the well-known lambda phage receptor of Escherichia coli, protein LamB, which allows the passage of maltose and maltodextrins (16), and the Dl protein of Pseudomonas aeruginosa, which allows the permeation of glucose (26), are induced by maltose and glucose, respectively. Other specific permeation systems, such as the Tsx protein that shows a high specificity for nucleosides in E. coli (18) and the TonB-related iron and cobalamine uptake systems of the outer membrane, function in the uptake of building blocks for the cell rather than of substrates for energy generation. They are derepressed when the substrate is missing from the medium or present at very low concentrations. The TonB-dependent transport systems in E. coli are energy dependent and include the following outer mem-

The cell wall of Mycobacterium smegmatis mc2155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to beta-lactams ranged from 10 x 10(-7) to 0.5 x 10(-7) cms-1. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel-forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage-gated cation-selective channel. The mycobacterial porin channel appeared to be water-filled since the single-channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single-channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point-charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10- to 20-fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.

The cell wall of mycobacteria is an efficient permeability barrier that makes mycobacteria naturally resistant to most antibiotics. Liposome swelling assays and planar bilayer experiments were used to investigate the diffusion process of hydrophilic molecules through the cell wall of Mycobacterium chelonae and identify the main hydrophilic pathway. A 59-kilodalton cell wall protein formed a water-filled channel with a diameter of 2.2 nanometers and an average single-channel conductance equal to 2.7 nanosiemens in 1 M potassium chloride. These results suggest that porins can be found in the cell wall of a Gram-positive bacterium. A better knowledge of the hydrophilic pathways should help in the design of more effective antimycobacterial agents.

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.