Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA

Chakraborti, Anuradha; Gunji, Shigemichi; Shakibai, Nader; Cubeddu, J; Rothfield, Lawrence

doi:10.1128/jb.174.22.7202-7206.1992

Cited by 25 publications

(25 citation statements)

References 14 publications

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“…The DnaA protein is located at the cell membrane (19,26) and is activated by anionic phospholipids (32,41), and the origin of replication (oriC) has been found to bind to a sub-inner membrane domain whose density is the same as that of the one which binds oriV (3).…”

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confidence: 99%

Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

et al. 2003

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The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. Carrying resistance genes to three antibiotics (ampicillin, kanamycin, and tetracycline), it has been isolated from a number of different medical environments (4). Despite the diversity of host cells, only two plasmid-carried loci are necessary to initiate unidirectional replication, the cis-acting origin of replication (oriV) and the trans-acting initiator gene trfA, which codes for two proteins of 44 and 33 kDa, the latter of which is expressed from an internal translational start within trfA (35). Recently, specific binding of TrfA and host initiation protein DnaA of Escherichia coli to oriV was found to be required for initiation of plasmid replication, consistent with the presence of four DnaA boxes within the oriV sequence (15). It appears that the DnaA protein cannot by itself form an open complex in oriV but rather enhances formation of the complex by TrfA. Nevertheless, numerous experiments have failed to demonstrate a direct physical interaction between TrfA and the DnaA protein although a physical association between TrfA and the ClpX chaperone protein has been observed (17). The latter interaction is important in converting the dimer form of TrfA, which is inactive in initiating DNA replication, to the active monomer form (16,38).RK2 replication is associated with the inner membranes of a number of gram-negative hosts (1). The TrfA initiation proteins were detected in both inner and outer membrane fractions of the hosts (1, 25), but further studies of E. coli revealed that oriV, the TrfA initiator proteins, and replication inhibited by specific anti-TrfA antibody were enriched in a specific subdomain found in the inner membrane fraction, representing less than 10% of the total membrane (13, 23)....

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confidence: 99%

Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

et al. 2003

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“…3b) was similar to that of oriC in the in vitro binding studies of Chakraborti et al (2), it was of interest to determine whether oriV binding in vitro would also exhibit this binding pattern (despite the lack of hemimethylation). In addition, such results would suggest that the in vivo association of plasmid DNA was due, in fact, to the specific interaction of the origin region.…”

Section: Resultsmentioning

confidence: 99%

“…This submembrane domain (fraction B in the SG2 gradient, which is one of two subfractions derived from fraction II in the SG1 gradient) may be a general site for membrane-DNA interaction, since oriC from E. coli is also bound to this or a related subfraction (2). However, with RK2, not only does oriV bind to this submembrane domain, but the plasmid-encoded initiation proteins bind as well.…”

Section: Discussionmentioning

confidence: 99%

“…This resolution seemed ideal to further localize the site of plasmid DNA-membrane interactions. Previous studies by Chakraborti et al (2) with this technique demonstrated that two-thirds of the membrane-associated hemimethylated oriCbinding action of E. coli occurred with a small submembrane domain (fraction II), whereas one-third remained with the outer membrane fraction. This latter result with the outer membrane had represented the only membrane-oriC inter-action detected (6,12,17) prior to the study of Ishidate et al (7).…”

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confidence: 99%

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Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli

1995

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It has been possible to locate a submembrane domain representing less than 10% of the total membrane that appears to be responsible for sequestering some essential components required for plasmid RK2 DNA replication. This subfraction, whose cellular location in the membrane prior to extraction is still unknown, is derived from the inner membrane fraction, since it possesses enzyme marker activity (NADH oxidase) exclusively associated with the inner membrane. The site of DNA replication in the prokaryotic cell has long been investigated because of its importance in controlling such replication. Concentration and sequestration of the vital components of the replisome, as well as the transient or permanent expression of control factors at this site, determine when DNA replication will ensue, how it progresses, and when it will terminate. This site has been linked to the cell membrane by a variety of experimental approaches (for a review, see reference 3), but few studies (except our own [10,11,16]) have attempted to localize the site to a specific submembrane domain. Such studies are critical in attempting to understand the DNAmembrane interaction with respect to possible receptor and anchoring sites for both DNA and the replisome.One model system that we have used extensively to investigate these problems has consisted of miniplasmid derivatives of the broad-host-range plasmid RK2. It was demonstrated (i) that plasmid DNA-membrane complexes could be extracted from both the outer and inner membrane fractions of Escherichia coli (11), (ii) that both fractions contained supercoiled plasmid DNA and the essential plasmid-specific initiation (TrfA) proteins (16), (iii) that TrfA-dependent initiation of DNA replication was associated primarily with the inner membrane fraction (16), and (iv) that a host initiation protein (DnaA) required by RK2 for initiation (4) was also present and active in the complex (11).The fact that both membrane fractions contained plasmid DNA and the TrfA proteins whereas only the inner membrane fraction displayed plasmid DNA replication suggested that other factors not present in the outer membrane fraction were required for plasmid replication. In addition, the actual membrane domain involved in such replication could be distributed in both major fractions. That membrane domains other than the inner and outer membrane fractions exist in gram-negative bacteria has been known for some time (7,15). Ishidate et al. (7) have made an extensive study of the heterogeneity of the cell membranes of E. coli and Salmonella typhimurium. By a combination of sedimentation and flotation density gradient centrifugation, several additional submembrane domains were revealed (7). These included a junction region containing inner and outer membrane material and a relatively small intermediate fraction derived from the inner membrane that could be further delineated into a higher-density fraction composed of flagellum fragments and a lower-density fraction. Finally, another small peak derived from the inner membran...

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“…In this context, it is important to recognize that numerous studies, including our own, have shown that, in vivo, DNA replication is membrane associated (for reviews, see references 4, 5, and 22). Thus, both Hda and the DnaA initiator protein are membrane localized (8,16), anionic phospholipids activate the initiation protein (26), and oriC itself binds to a subfraction of the inner membrane (2). Therefore, it is not surprising that when a protein such as Hda is overexpresssed in its membrane environment, profound physiological changes can result.…”

Section: Induction Of the Sos Response By Overexpression Of The Hda Pmentioning

confidence: 99%

Overexpression of the Hda DnaA-Related Protein in Escherichia coli Inhibits Multiplication, Affects Membrane Permeability, and Induces the SOS Response

et al. 2005

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The Hda protein, a recently identified DnaA-related protein from Escherichia coli, is part of the AAA ϩ ATPase family known to be involved with various aspects of initiation of DNA replication in prokaryotes. We report here that overexpression of this membrane-associated protein inhibits multiplication, affects membrane permeability, and is also an unexpected initiator of the bacterial SOS response, which may represent a major new pathway for inducing DNA damage repair mechanismsWe recently identified a small membrane-associated protein (28.4 kDa) in Escherichia coli that is related to the DnaA host initiation protein and that affected the initiation of the broadhost-range plasmid RK2 (8). By interacting physically with the plasmid-encoded initiation protein (TrfA), it acted as a steric inhibitor of either or both of TrfA's two functions: cooperating with the DnaA protein (which is also required by RK2) to open the replication bubble and guiding the DnaB-DnaC complex into the open site (8, 9, 10). The protein is identical to the Hda protein ("homologous to DnaA" protein) that is responsible for controlling overinitiation in E. coli by accelerating the ability of the ␤-clamp subunit of DNA polymerase III to convert the active form of DnaA (ATP-bound DnaA) to its inactive form (ADP-bound DnaA) (1, 7, 11). Hda has a high sequence homology to the domain III ATPase region of DnaA (1,7,8,13) and is important as an accessory component for initiation and, subsequently, replication in prokaryotes.In further assessing the role of Hda protein in RK2 metabolism, we previously constructed a compatible plasmid that placed hda under the control of an inducible promoter and monitored the effects of increasing levels of Hda induction in vivo. Profound inhibitory effects on both maintenance and replication of RK2 were observed (8). Of additional interest, Hda overexpression also inhibited cell multiplication, with only a limited effect on optical density profiles. In this study, we investigated the basis for these inhibitory effects and determined that they involve induction of the SOS response system that may be actuated by perturbation of membrane integrity and/or permeability.Effects of Hda on growth and viability of E. coli. BL21(DE3)/ pLysS, which contains an IPTG [isopropyl-(3-D-thiogalactoside)]-inducible T7 RNA polymerase gene (21), was transformed with plasmid pPK101 or the pET17B vector, as described by Hanahan (6), resulting in strain 1921 or 1110, respectively. pPK101 is a pET17B derivative (Novogen) that expresses a functional N-terminal T7 epitope-tagged Hda protein that was constructed previously (8). These two strains were grown to mid-logarithmic phase, induced with different IPTG concentrations, and viable cells were quantified. Whereas IPTG induction had a significant inhibitory effect on the viability of strain 1921 at all concentrations utilized (Fig. 1a), there was only a modest effect on the viability of the control 1110 strain (Fig. 1b). Of note, there was a threshold level of inhibition of cell viability ...

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Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA

Cited by 25 publications

References 14 publications

Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli

Overexpression of the Hda DnaA-Related Protein in Escherichia coli Inhibits Multiplication, Affects Membrane Permeability, and Induces the SOS Response

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