High-affinity binding of hemimethylated
            <i>oriC</i>
            by
            <i>Escherichia coli</i>
            membranes is mediated by a multiprotein system that includes SeqA and a newly identified factor, SeqB

Shakibai, Nader; Ishidate, Kohei; Reshetnyak, Elena; Gunji, Shigemichi; Kohiyama, Masamichi; Rothfield, Lawrence

doi:10.1073/pnas.95.19.11117

Cited by 42 publications

(34 citation statements)

References 17 publications

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“…In E. coli, DnaA is thought to bind directly to membranes (18,54), although membrane association of oriC is mediated primarily via SeqA and SeqB (60). In contrast, membrane association in B. subtilis is dependent on DnaB (23,66,74), and there is no evidence for a direct association between DnaA and the membrane.…”

Section: Membrane Attachmentmentioning

confidence: 77%

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

2012

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bMuch of our knowledge of the initiation of DNA replication comes from studies in the Gram-negative model organism Escherichia coli. However, the location and structure of the origin of replication within the E. coli genome and the identification and study of the proteins which constitute the E. coli initiation complex suggest that it might not be as universal as once thought. The archetypal low-G؉C-content Gram-positive Firmicutes initiate DNA replication via a unique primosomal machinery, quite distinct from that seen in E. coli, and an examination of oriC in the Firmicutes species Bacillus subtilis indicates that it might provide a better model for the ancestral bacterial origin of replication. Therefore, the study of replication initiation in organisms other than E. coli, such as B. subtilis, will greatly advance our knowledge and understanding of these processes as a whole. In this minireview, we highlight the structure-function relationships of the Firmicutes primosomal proteins, discuss the significance of their oriC architecture, and present a model for replication initiation at oriC. The Firmicutes are Gram-positive bacteria encompassing three major classes, Bacilli, Clostridia, and Mollicutes. They have a relatively low GϩC content in their genomes and are morphologically and physiologically diverse. Rod-shaped bacilli, spherical cocci, and aerobic, anaerobic spore-forming, and non-sporeforming bacteria are found in this group. They likely represent the most ancestral phylum of prokaryotes, with high-GϩC-content Gram-positive and Gram-negative bacteria having diverged from the Firmicutes at a later stage in evolution (13,70). Bacillus subtilis is the best studied of the Firmicutes and is widely considered the Gram-positive model bacterium, but other bacilli, streptococci, staphylococci, and clostridia have been extensively studied because of their medical and industrial importance.In addition to the universally conserved replication initiation protein DnaA (32, 65) and the replication restart protein PriA (17, 39), the low-GϩC-content Firmicutes generally have two unique essential genes, dnaD and dnaB, coding for the replication initiation proteins DnaD and DnaB, respectively ( Table 1) (note that DnaB in B. subtilis is unrelated to DnaB in Escherichia coli). In some cases-for example, in several Mollicutes-there are no distinct dnaD and dnaB genes, but there is, instead, a single gene annotated as dnaD-like which may combine both functions. No homologous proteins are found outside the Firmicutes, suggesting a replication initiation machinery distinctly different from those of other bacteria (31). In addition, there are a number of regulatory proteins which are not found in the Gram-negative model organism Escherichia coli, including YabA, Soj, SirA, and Spo0A, while other regulatory proteins are found in E. coli but not B. subtilis (these regulatory proteins have been subject to a recent review [29]). DnaA, DnaD, and DnaB, together with the helicase loader DnaI (called DnaC in E. coli), the replicativ...

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Section: Membrane Attachmentmentioning

confidence: 77%

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

2012

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“…Although these results show the predominant role of the SeqA protein in oriC sequestration, the auxiliary role played by the membrane in the process should not be disregarded. Shakibai et al (23) observed that the hemimethylated oriC binding activity of SeqA is stimulated by addition of a membrane protein preparation designated SeqB. It has also been found that the membrane obtained from a seqA mutant stimulates the protective activity of a His-tagged SeqA protein against DNase I attack of hemimethylated oriC DNA (7).…”

mentioning

confidence: 96%

Isolation of a New Hemimethylated DNA Binding Protein Which Regulates dnaA Gene Expression

et al. 2003

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In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.The minimum replication origin of Escherichia coli (oriC) has an elevated number of DNA adenine methylation (Dam) sites (18) that become hemimethylated immediately after initiation of replication and stay in this state for one-quarter of the generation time; these sites differ from most Dam sites located elsewhere on the chromosome (6). The finding that the hemimethylated oriC interacts with an outer membrane preparation of E. coli (20) prompted a search for hemimethylated DNA binding proteins in the membrane fraction. The existence of such a category of proteins in the membrane has been reported previously; in fact, Southwestern blot analysis of the membrane preparation demonstrated the presence of 25-and 16-kDa peptides reacting with a hemimethylated oriC probe (9). These proteins might participate in the hemimethylated oriC membrane sequestration that prevents initiation of chromosome replication in E. coli (16). Lu et al. (17) isolated the seqA gene that codes for a 21-kDa peptide and has an affinity for the hemimethylated oriC DNA (24). In a seqA mutant, the duration of the hemimethylation period of oriC is shortened, and the reinitiation of replication occurs at oriC repeatedly in a single replication cycle (17). These phenotypes may be explained by a lack of hemimethylated oriC sequestration by either SeqA or a SeqA membrane complex; the membrane obtained from the seqA mutant failed to bind the hemimethylated oriC DNA in vitro (2, 24). Although these results show the predominant role of the SeqA protein in oriC sequestration, the auxiliary role played by the membrane in the process should not be disregarded. Shakibai et al. (23) observed that the hemimethylated oriC binding activity of SeqA is stimulated by addition of a membrane protein preparation designated SeqB. It has also been found that the membrane obtained from a seqA mutant stimulates the protective activity of a His-tagged SeqA protein against DNase I attack of hemimethylated oriC DNA (7).In order to isolate auxiliary factors of SeqA, we adopted a strategy of searching for factors among the dnaA46 suppressor gene products. The rationale behind this approach was based on the observation that the seqA mutation partially suppresses the temperature sensitivity of dnaA46 (17). Likewise, mutation of the auxiliary factor gene should also result in suppression of dnaA46. Previously, this type of work has been undertaken by Katayama's group, who used a system involving random insertion of Tn10-Tet r into the chromosome. In this way, they found one new suppressor mutation for dnaA46, hslU (12).Isolation of dnaA46 suppressors by mini-Tn10 insertion. Briefly, random insertion of mini-Tn10 into the chromosome containing dnaA46 (KA413⌬H) ( Table 1) was accomplished by infection of the mutant with a lambda phage (1098) carrying mini-Tn10 (27), followed by plat...

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“…The in vivo origin of this membrane species is not known, and it is possible that membranes from several distinct cellular locations could have this density. This fraction is likely to include adhesion zones (19,25), the membrane domain that binds hemimethylated oriC DNA (5) primarily by SeqA (28,29), envelope proteins such as TolQ, -R, -A, and -B involved in translocations across the periplasm (13) and the group D protein thioredoxin (25) in addition to EntB/G, -E, and -F. For group D proteins, of course, the vast majority of each is found in soluble fractions. We also show here that the envelope protein TonB (24), when monitored on gels in which a constant amount of protein was present in each lane, also was enriched in the intermediate-density fraction under these growth conditions.…”

Section: Vol 182 2000 Notes 1769mentioning

confidence: 99%

Membrane Association of the Escherichia coli Enterobactin Synthase Proteins EntB/G, EntE, and EntF

Hantash

Earhart

2000

J Bacteriol

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The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.

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High-affinity binding of hemimethylated oriC by Escherichia coli membranes is mediated by a multiprotein system that includes SeqA and a newly identified factor, SeqB

Cited by 42 publications

References 17 publications

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

Isolation of a New Hemimethylated DNA Binding Protein Which Regulates dnaA Gene Expression

Membrane Association of the Escherichia coli Enterobactin Synthase Proteins EntB/G, EntE, and EntF

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