The affinities of a range of penicillins and cephalosporins for ther penicillinbinding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant.

36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 1.0 to 12.4 5.1 ,uM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50%o inhibited by a 3TC 5'-triphosphate concentration of 23.4 + 2.5 ,uM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50%o inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 31, 24.8 10.9, and 43.8 16.4 ,uM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 ,uM for DNA polymerase beta and 15.8 0.8 ,uM for DNA polymerase gamma.

The competition of a number of beta-lactam morphogenic probes for the penicillin-binding proteins (PBPs) of Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella aerogenes, Proteus rettgeri, and Escherichia coli has been studied. The results indicate that the various gram-negative bacteria have similar, but not identical, PBP patterns and that the individual proteins probably perform similar morphogenic functions as in E. coli K-12. Comparison of the 50% binding concentrations of the compounds for the various PBPs of the five strains with their antibacterial activity indicates that the different antibiotics are excluded to a greater or lesser degree by the outer membrane permeability barrier and that the exclusion is most pronounced in P. aeruginosa.

The competition of a new aminothiazolyl cephalosporin, ceftazidime, for the penicillin-binding proteins (PBP's) of Escherichia coli K12, Pseudomonas aeruginosa and Staphylococcus aureus has been studied. Ceftazidime caused filamentation and eventually cell lysis of both E. coli and Ps. aeruginosa at its minimum inhibitory concentration, due to its primary activity against PBP-3. The antibiotic also inhibited PBP's 1 a and 1 bs, the 'essential' cell elongation proteins at higher, therapeutically achievable concentrations and consequently induced rapid lysis of both E. coli and Ps. aeruginosa. In Staph. aureus ceftazidime showed high affinity for PBP-1, -2 and less affinity for PBP-3. The results indicate that in E. coli K12 and Ps. aeruginosa, ceftazidime owes its good antibacterial activity to high affinity for PBP-3, the 'essential' binding protein involved in cell division combined with favourable outer membrane penetration.

Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by recombinant rhinovirus (HRV 14) 3C proteinase. Systematic removal or replacement of individual residues indicated that the minimum sequence required for effective cleavage by the viral cysteine proteinase was P,-Val/Thr-P,-P,-Gln-Gly-Pro.

A series of substituted imidazo[1,5-b]pyridazines have been prepared and tested for inhibitory activity against the reverse transcriptase of HIV-1 (RT) and their ability to inhibit the growth of infected MT-4 cells. Crystal data are reported on two compounds, 15c and 33. From the structure-activity relationships developed within this and other series, it is proposed that key features of the interaction with RT include hydrogen-bond acceptor and aromatic pi-orbital bonding with the imidazopyridazine nucleus and a benzoyl function separated from the heterocycle by a suitable spacer group. Exceptional activity against the reverse transcriptase of HIV-1 (IC50 = 0.65 nM) was obtained with a 2-imidazolyl-substituted derivative, 7-[2-(1H-imidazol-1- yl)-5-methylimidazo-[1,5-b]pyridazin-7-yl]-1-phenyl-1-heptanone (33) which is attributed to additional binding of the imidazole sp2 nitrogen atom. A number of the compounds in this series also inhibit the replication of HIV-1 in vitro in MT-4 and C8166 cells at levels observed with the nucleoside AZT.

SUMMARYThe 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephatomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.

bXenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (⌬xcnKL strain), xenocoumacin and the newly discovered antibiotic F (⌬xcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace's insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ⌬xcnKL strain or ⌬xcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ⌬xcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction. Microorganisms employ various strategies to compete for space, nutrients, and other resources (1). In exploitative competition, limiting nutrients are quickly utilized by specific microorganisms without direct interaction between competitors. Interference competition, on the other hand, makes use of direct, antagonistic interactions. We study a tripartite model system in which the pathogenic bacterium, Xenorhabdus nematophila, establishes a mutualistic partnership with an entomopathogenic nematode, Steinernema carpocapsae, that vectors the bacterium into susceptible insect hosts. This tractable model system provides an excellent opportunity to identify biologically relevant competitors and study the role of X. nematophila antimicrobials in interspecies competition in a host environment.Xenorhabdus nematophila exhibits two distinct phases in its life cycle (2-5). It engages in a species-specific mutualistic relationship with S. carpocapsae, where it resides in a specialized region of the anterior intestine of the infective juvenile (IJ) stage of the nematode (6). The IJs invade s...