1,4-Disubstituted imidazoles are potential antibacterial agents functioning as inhibitors of enoyl acyl carrier protein reductase (FabI).

1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.

Inhibitors of bacterial enoyl acyl carrier protein reductase (FabI): 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as potential antibacterial agents.

An SAR study of a screening lead has led to the identification of 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as inhibitors of Staphylococcus aureus enoyl acyl carrier protein reductase (FabI).

Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus.

Triclosan, a widely used antibacterial agent, possesses potent activity against Staphylococcus aureus. This study reports on an investigation of the antibacterial target of triclosan in this pathogen. A strain of S. aureus overexpressing the enoyl-[acyl-carrier-protein] reductase (FabI), demonstrated by Western immunoblotting, gave rise to an increase in the MIC of triclosan, while susceptibilities to a range of unrelated antibacterials were unaffected. There are approximately 12 000 molecules of FabI per cell in mid-log phase growth. This number increased by approximately three- to four-fold in the S. aureus FabI overexpressor. Triclosan selectively inhibited the incorporation of [(14)C]acetate into TCA-precipitable product, an indicator of fatty acid biosynthesis. Furthermore, it inhibited de novo fatty acid biosynthesis in this organism. In vitro, triclosan inhibited recombinant, purified S. aureus FabI with an IC(50) of approximately 1 microM. The combination of these biochemical and genetic data provide further evidence that the mode of action of triclosan in S. aureus is via inhibition of FabI.

A novel gentamicin resistance gene in Enterococcus.

Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 micrograms/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6')-aph(2") did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5 alpha. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2"). The gene is designated aph(2")-Ic, and its observed phosphotransferase activity is provisionally designated APH(2")-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 micrograms/ml for screening for high-level resistance.

Analyses of traA, int-Tn, and xis-Tn mutations in the conjugative transposon Tn916 in Enterococcus faecalis.

The conjugative transposon Tn916 moves intercellularly via an excision/insertion mechanism that involves products of int-Tn and xis-Tn. Tn5-insertion mutations in these genes were found to be complemented in an Enterococcus faecalis host by specific coresident transposons harboring the corresponding wild-type allele. A determinant designated traA, partially overlapping and divergently transcribed from xis-Tn, is thought to encode a key positively acting regulatory protein needed for expression of conjugation functions. This locus was also shown to express a trans-acting product.

A functional origin of transfer (oriT) on the conjugative transposon Tn916.

The origin of transfer (oriT) of the 18-kb conjugative transposon Tn916 has been localized to a 466-bp region which spans nucleotides 15215 to 15681 on the transposon map. The oriT lies within an intercistronic region between open reading frames ORF20 and ORF21 that contains six sets of inverted repeats ranging from 10 to 20 bp in size. The segment contains three sequences showing identity in 9 of 12 bp to the consensus nicking site (nic) of the IncP family of conjugative plasmids found in gram-negative bacteria. Overlapping one of these sequences is a region similar to the nic site of the F plasmid. Functionality was based on the ability of the oriT-containing sequence to provide a cis-acting mobilization of chimeras involving the shuttle vector pWM401 in response to activation in trans by an intact chromosome-borne transposon Tn916 delta E. Cloned segments of 466 or 376 nucleotides resulted in unselected cotransfer of the plasmid at levels of about 40% when selection was for Tn916 delta E, whereas a 110-bp segment resulted in cotransfer at a frequency of about 7%. Mobilization was specific in that gram-positive plasmids, such as pAD1 and pAM beta 1, and the gram-negative plasmids pOX38 (a derivative of F) and RP1 did not mobilize oriT-containing chimeras.

Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons.

Conjugative transposons are highly ubiquitous elements found throughout the bacterial world. Members of the Tn916-Tn1545 family carry the widely disseminated tetracycline-resistance determinant Tet M, as well as additional resistance genes. They have been found naturally in, or been introduced into, over 50 different species and 24 genera of bacteria. Recent investigations have led to insights into the molecular basis of movement of these interesting mobile elements.

Evidence that coupling sequences play a frequency-determining role in conjugative transposition of Tn916 in Enterococcus faecalis.

The conjugative transposon Tn916 (encodes resistance to tetracycline), originally identified in Enterococcus faecalis, moves by an excision-insertion process in which the rate-limiting step is believed to be excision. Individual transposon-containing strains exhibit characteristic mating frequencies which range over several orders of magnitude; the basis of this phenomenon is addressed in the present study. We were able to generate independent single-copy insertions in identical target locations and with similar orientations within a plasmid hemolysin determinant (cylA); however, transposition from this site occurred at very different frequencies (10(-8) to 10(-4) per donor) depending on the individual isolate. DNA sequencing analyses showed that the coupling (junction) sequences differed between isolates and thus appeared to be responsible for differences in excision frequencies. Other experiments showed that inducible transcription into either end of the transposon had no significant effect on transfer.