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We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1͞3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (؉) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (؉) supercoils in front of the chromosomal fork.topo IV ͉ coumarins ͉ positive supercoiling T opoisomerases are needed in all three phases of bacterial DNA replication: initiation at the origin of replication, elongation with progressive outward movement of the forks until they meet halfway around the chromosome, and termination, including the final disentanglement of the catenated daughter chromosomes. Bacterial cells have two indispensable topoisomerases: gyrase and topoisomerase IV (topo IV) (1). (Ϫ) supercoiling by gyrase is involved in all three phases of replication: (Ϫ) supercoiling facilitates unwinding in initiation, reduces links in front of the fork during elongation, termed the swivel function (2), and compacts DNA in termination.It was widely believed that gyrase was the only topoisomerase needed during elongation (3, 4). This was inferred from the observation that replication stops in the absence of gyrase (5), whereas inhibition of topo IV has only a small effect on DNA synthesis (6). However, the interpretation of past results is complicated by the multiple roles of gyrase in replication and the failure until quite recently to recognize that drugs used to inhibit gyrase also inhibit topo IV (6, 7).Here we demonstrate that topo IV can indeed act as a replication swivel. Our demonstration depended on a use of a whole genome Escherichia coli microarray and on the observation that topo IV is a secondary target for coumarin antibiotics such as novobiocin. The evidence for the role of topo IV in DNA elongation is supported by our findings that topo IV and gyrase have overlapping roles in replication fork movement and that topo IV is sufficient to remove (ϩ) supercoils in vivo. Materials and MethodsBacterial Strains and Plasmids. The bacterial strains used in this study are listed in supplemental Table 1 (see www.pnas.org). Plasmids pBR322 and pGP509 have been described in refs. 8 and 9, respectively. Enzymes, Reactions, and Chemicals. Norf loxacin, novobiocin, coumermycin A1, hydroxyurea, and pancreatic DNaseI were obtained from Sigma. DNA supercoils were removed by nicking substrates with pancreatic DNase I in the presence of 300 g͞ml ethidium bromide. Assays of plasmid topology and DNA replication were done as previously described (6).Use of Genomic Microarrays to Study DNA Replication. Four thousand one hundred fifteen of all 4,290 annotated E. coli ORFs (10) were successfu...

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Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by ␤-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2-to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 Å of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.In eubacteria and their endosymbiotic descendants (the plastids of plants and apicomplexan parasites) fatty acids are produced by what is known as the type II fatty acid biosynthetic pathway (1-3). The steps in this pathway are catalyzed by a universal set of enzymes, each encoded by a separate gene, that have been most closely studied in the model organism Escherichia coli (4, 5). The growing acyl chain is shuttled between the pathway enzymes attached to the 4Ј-phosphopantetheine prosthetic group of a dedicated carrier protein, ACP.1 This system contrasts with the type I fatty acid biosynthesis system that exists in metazoans, where multifunctional polypeptide chains (6) encode all activities in chain initiation and elongation. The intermediates in the type I system are shuffled from one catalytic site to another without being released from the complex. In light of the profound differences in these two systems, enzymes of the type II pathway have emerged as attractive targets for the development of novel antimicrobial or antiparasitic agents (2,7,8). The core feature of the type II pathway is the fatty acid elongation cycle, which extends the fatty acid chain by two carbons in each round. There are four steps in the cycle, and the proteins involved in E. coli are 1) condensation of malonyl-ACP with acyl-ACP, catalyzed by the FabB-and FabF-condensing enzymes, 2) reduction of the ␤-keto moiety by the NADPH-dependent FabG re...