Syam P. Anand

GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.

RepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTP‐dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPγS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPγS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl‐terminal region and exhibits GTP‐dependent, non‐specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.

Bacillus anthracis and Bacillus cereus PcrA Helicases Can Support DNA Unwinding and In Vitro Rolling-Circle Replication of Plasmid pT181 of Staphylococcus aureus

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange

PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

The Tubulin-Like RepX Protein Encoded by the pXO1 Plasmid Forms Polymers In Vivo in Bacillus anthracis

Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.

Bacterial cell division protein FtsZ is a specific substrate for the AAA family protease FtsH

The role of AAA (ATPases Associated to a variety of cellular Activities) family protease FtsH in bacterial cell division is not known, although mutations in ftsH were found to inhibit cell growth and division (1, 6, 13). Overexpression of heterologous FtsH in Escherichia coli results in the formation of multinucleate ®lamentous cells due to the abolition of cell septation (8). Further, independent studies on FtsH (15) and FtsZ (2), which is the key regulator of bacterial cell division, have shown that FtsH protease and FtsZ protein are localized to the mid-cell site during septation. FtsZ protein is the prokaryotic homologue of tubulin (5, 10, 12), possessing GTP-dependent polymerization activity (4, 11). igni®cantly, the AAA family ATPase member katanin disassembles tubulin polymers in an ATP-dependent manner (7). Based on these observations, we reasoned that an interaction similar to that between katanin and tubulin might hold true for FtsH and FtsZ in prokaryotes as well. To verify this hypothesis, we examined whether the FtsH protease of Escherichia coli

Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time

(FtsH_{Ec})

Plasmid Segregation: Birds of a Feather Try Not To Flock Together

could degrade FtsZ of E. coli

Structure-specific DNA binding and bipolar helicase activities of PcrA

(FtsH_{Ec})

Biochemical Characterization of the Staphylococcus aureus PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

in vitro.

PcrA-mediated disruption of RecA nucleoprotein filaments—essential role of the ATPase activity of RecA

Many questions in molecular and cellular biology can be reduced to questions about ‘who talks to whom, when and how frequently’. Here, we review approaches we have used with single-pair fluorescence resonance energy transfer (spFRET) to follow the motions between two well-placed fluorescent probes to ask similar questions. We describe two systems. We have used a nucleosomal system in which the naked DNA molecule has the acceptor and donor dyes too far apart for FRET to occur whereas the dyes are close enough in the reconstituted nucleosome for FRET. As these individual nucleosomes were tethered on a surface, we could follow dynamics in the repositioning of these two dyes, inferring that nucleosomes stochastically and reversibly open and close. These results imply that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix. In the case of following the binding of recombination protein RecA to double-stranded DNA (dsDNA) and the RecA filament displacement by DNA helicase motor PcrA, the dsDNA template is prepared with the two dyes close enough to each other to generate high FRET. Binding of the RecA molecules to form a filament lengthens the dsDNA molecule 1.5-fold and reduces the FRET accordingly. Once added, DNA motor protein helicase PcrA can displace the RecA filament with concomitant return of the DNA molecule to its original B-form and high FRET state. Thus, appropriately placed fluorescent dyes can be used to monitor conformational changes occurring in DNA and or proteins and provide increased sensitivity for investigating dynamic DNA–protein interactions in real time.