Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

Strains of Escherichia coli which lack detectable guanosine 3',5'-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth.

The RpoS sigma factor (also called S or 38 ) is known to regulate at least 50 genes in response to environmental sources of stress or during entry into stationary phase. Regulation of RpoS abundance and activity is complex, with many factors participating at multiple levels. One factor is the nutritional stress signal ppGpp. The absence of ppGpp blocks or delays the induction of rpoS during entry into stationary phase. Artificially inducing ppGpp, without starvation, is known to induce rpoS during the log phase 25-to 50-fold. Induction of ppGpp is found to have only minor effects on rpoS transcript abundance or on RpoS protein stability; instead, the efficiency of rpoS mRNA translation is increased by ppGpp as judged by both RpoS pulse-labeling and promoter-independent effects on lacZ fusions. DksA is found to affect RpoS abundance in a manner related to ppGpp. Deleting dksA blocks rpoS induction by ppGpp. Overproduction of DksA induces rpoS but not ppGpp. Deleting dksA neither alters regulation of ppGpp in response to amino acid starvation nor nullifies the inhibitory effects of ppGpp on stable RNA synthesis. Although this suggests that dksA is epistatic to ppGpp, inducing ppGpp does not induce DksA. A dksA deletion does display a subset of the same multiple-amino-acid requirements found for ppGpp 0 mutants, but overproducing DksA does not satisfy ppGpp 0 requirements. Sequenced spontaneous extragenic suppressors of dksA polyauxotrophy are frequently the same T563P rpoB allele that suppresses a ppGpp 0 phenotype. We propose that DksA functions downstream of ppGpp but indirectly regulates rpoS induction.Eubacteria have developed complex regulatory networks that recognize and respond to a variety of environmental sources of physiological stress. One element common to many such networks in gram-negative bacteria is RpoS (29), a regulator defined by sequence and functional studies as an alternative sigma subunit of RNA polymerase (38). Over the past decade, it has come to be appreciated that RpoS participates in the regulation of at least 50 genes and that RpoS is itself regulated by nearly half as many factors. RpoS has been referred to as "the master regulator of the general stress response in Escherichia coli" (6).Regulation of RpoS itself is arguably the most complicated system in bacteria. Regulation of RpoS involves transcription, mRNA turnover, translation initiation, and proteolysis. Reported transcription regulators include BarA (37), cyclic AMP/ cyclic AMP receptor protein (30), and ppGpp (27). Leader mRNA is a regulatory target affecting efficiency of translation initiation in different ways. With the rpoS transcript originating in nlpD and the initiating AUG at ϩ565 (50), a structure extending from ϩ458 to ϩ565 sequesters the ribosomal binding sequence through a much smaller cis-acting antisense element. One hypothesis is that translation initiation is positively regulated by hfq (7, 36) with HF-1 binding to leader RNA changing antisense element conformation (8). A small RNA, called DsrA, is normally ma...

The spoT gene of Escherichia coli encodes a guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3',5'-bis(diphosphate) synthetase (designated PSII). To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid-borne deletion mutations for their ability to complement chromosomal mutations defective in each activity. We found that a region containing the first 203 amino acids of the 702-amino-acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67-374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein. A ppGppase-defective delta 1-58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild-type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation. We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.