Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system.

The Escherichia coli activator and repressor proteins AraC and LexA bind DNA as homodimers. Here we show that their heterodimerization through fused cognate dimerization domains results in repression of AraC-dependent gene activation by LexA. Repression also requires a LexA operator half-site located several helical turns downstream of the AraC operator. This requirement for a specific spatial organization of the operators suggests the formation of a DNA loop between operator-bound Ara/LexA heterodimers, and we propose that heterodimerization with the AraC hybrid provides co-operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA looping, repression increases when the E. coli DNA looping and transcriptional effector protein IHF binds between the AraC and LexA operators. Thus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA looping, in which the activator protein is an essential part of the repressor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA. The requirement for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli-based two-hybrid system.

High-pressure liquid chromatographic analysis of aztreonam in sera and urine.

Aztreonam (SQ 26,776) is a new synthetic monocyclic beta-lactam antibiotic which is specifically active against aerobic gram-negative bacteria. High-pressure liquid chromatographic (HPLC) systems were developed for the quantitative analysis of aztreonam in human, monkey, rat, mouse, and rabbit sera and urine. The HPLC conditions employed for these analyses were a muBondapak C18 column, a mobile phase made up of 0.005 M tetrabutylammonium hydrogen sulfate at pH 3.0 and acetonitrile or methanol, UV detection at 293 nm, and a flow rate of 2.0 ml/min. For human sera and urine, the mobile phase was 80% 0.005 M tetrabutylammonium hydrogen sulfate-0.005M (NH4)2SO4 and 20% acetonitrile (vol/vol). For the range of sera and urine, HPLC analyses were shown to have excellent detector linearity of aztreonam over a concentration range of 1.0 mg/ml to 0.5 microgram/ml. Correlation coefficients for plots of aztreonam peak area versus its concentration were greater than or equal to 0.990. The detection limit of aztreonam was 1.0 micrograms/ml in sera and 5.0 micrograms/ml in urine. HPLC and microbiological assays of aztreonam in human sera and urine were in good agreement.

Diastereomeric 7-ureidoacetyl cephalosporins. III. Contribution of D- and L-isomers to the growth inhibiting activities of 7alpha-H and 7alpha-OCH3 derivatives for gram-positive and gram-negative bacteria.

A series of 7beta-ureidoacetyl, 7alpha-H and 7alpha-OCH3 cephalosporin antibiotics have shown broad-spectrum antibacterial activity in vitro. In the 7alpha-H but not in the 7alpha-OCH3 series, contrary to experience in the antibiotic field, the L-isomers were substantially more active than the D-isomers both in vitro and in vivo particularly, but not exclusively, against Enterobacteriaceae that produce potent chromosomal cephalosporinases. Enhanced resistance to and inhibition of beta-lactamase (s) appeared to be responsible for this effect. Studies in vitro specifically with 7beta-thienylureidoacetyl derivatives showed that D-isomers interacted with L-isomers in the 7alpha-OCH3 series in a synergistic manner against "cephalosporinase-type" enzyme producers while isomers in the 7alpha-H series did not. Examples were presented in which this favorable event resulted in improved efficacy of the racemic mixture over the pure D- or L-isomer alone in appropriate experimental infections.