Marcin Filutowicz

Autorepressor properties of the π-initiation protein encoded by plasmid R6K

A DNA fusion containing the promoter of the pir gene of plasmid R6K that encodes for the pi-initiation protein and the beta-galactosidase gene of Escherichia coli (lacZ) is described. The synthesis of beta-galactosidase promoted by this pir-lac fusion was almost completely inhibited when an R6K sequence containing the pir gene was provided in trans in E. coli. Transcription in vitro from the pir promoter but not the trp promoter of E. coli, was inhibited by purified pi protein indicating that the pi protein alone is responsible for repression of its own gene and that the effect is promoter specific. The DNA-protein interaction sites in the pir regulatory region have been determined for the pi protein and E. coli RNA polymerase using the DNase I protection method. The binding sites for these two proteins overlap for three helical turns. Competition DNA binding experiments show that the pi protein will displace bound RNA polymerase. From these studies we conclude that repression of the pir gene is accomplished by binding of the pi protein and this association blocks access of RNA polymerase to the pir promoter region.

Regulation of Replication of an Iteron-containing DNA Molecule

Publisher Summary This chapter discusses the structure and mechanisms controlling the initiation frequency of plasmid R6K, primarily the γ ori. Because all three origins share many properties, the small γ ori should reveal the most fundamental mechanisms, regulating the replication of the entire 38-kb R6K. Also, the deceptively simple γ ori was chosen for study, because it has all the features needed to elucidate the regulation of replication of iteron-containing DNA molecules in general. Plasmid R6K is a complex replicon, in which the replication forks emanate from any of the three positions called the α, β, and γ origins. All three origins require the R6K-encoded rep protein for their activity. When the α and β sequences are removed, the remaining γ ori can replicate autonomously and the α and β origins require the γ -ori sequence in cis to function. The mechanism, underlying stable inheritance of plasmid R6K, ensures that the plasmid copy-number is maintained at a constant level of 15-20 per chromosomal equivalent. This level can be reduced substantially, by an increase in the intracellular concentration, of two plasmid-encoded elements: the π protein and its binding sites. These two elements have a dual role: they are essential for replication, but can also inhibit origin activity. The negative role of both the elements has also been demonstrated genetically, either by mutating the iterons or by mutating the pir gene.

Regulatory implications of protein assemblies at the gamma origin of plasmid R6K - a review.

Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems. The objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid R6K. The control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors. The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs). The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5). Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs and IRs have been isolated. The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs. These mutants also suggest that pi polypeptides have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization. Additionally, pi35.0 binds to a novel DNA sequence in the A+T-rich segment of gamma-ori. This binding site is at or near the site from which synthesis of the leading strand begins.

Purification of the Escherichia coli integration host factor (IHF) in one chromatographie step

The integration host factor (IHF) participates in a diverse array of DNA transactions such as replication, recombination and gene expression. We describe a fast and very efficient isolation procedure which yields highly purified IHF in one chromatographic step.

Bacterial conjugation-based antimicrobial agents.

A clear imperative exists to generate radically different antibacterial technologies that will reduce the usage of conventional chemical antibiotics. Here we trace one route into this new frontier of drug discovery, a concept that we call the bacterial conjugation-based technologies (BCBT). One of the objectives of the BCBT is to exploit plasmid biology for combating the rising tide of antibiotic-resistant bacteria. Specifically, the concept utilizes conjugationally delivered plasmids as antimicrobial agents, and it builds on the accumulated work of many scientists dating back to the discoveries of conjugation and plasmids themselves. Each of the individual components that comprise the approach has been demonstrated to be feasible. We discuss the properties of bacterial plasmids to be employed in BCBT.

Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is “handcuffing,” in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded π protein with the seven 22-bp iterons of the γ origin of replication. Like other related Rep proteins, π exists as both monomers and dimers. However, the ability of π dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of π protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using π variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (π·P106L∧F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of π protein (π·M36A∧M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type π. The data suggest that π dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the γ ori system. Thus, dimers of π appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of π expression, in cis competition (for origin binding) with monomeric activator π, and handcuffing-mediated inhibition of replication in trans.

BUFFER COMPOSITION MEDIATES A SWITCH BETWEEN COOPERATIVE AND INDEPENDENT BINDING OF AN INITIATOR PROTEIN TO DNA

The regulation of many biological processes, including DNA replication, is frequently achieved by protein-protein interactions, as well as protein-DNA interactions. Multiple protein-binding sites are often involved. For example, the replication of plasmid R6K involves binding of the initiator protein pi to seven 22-bp direct repeats (DR) in the gamma origin of replication (gamma ori). A mutant protein pi S87N has been isolated, that in Tris.borate buffer (TB) binds cooperatively to seven DR, whereas wild-type (wt) pi binds independently [Filutowicz et al., Nucleic Acids Res. 22 (1994) 4211-4215]. Surprisingly, we found that wt pi can also bind cooperatively when Tris.acetate (TA), Tris.succinate or Tris.glutamate buffers are used instead of TB. The cooperative binding of the wt pi protein was also observed in the TB buffer at high concentrations of Na2EDTA. These results suggest that pi may be able to assume two functionally distinct conformations as a result of either mutation or buffer composition. Moreover, we found that the mode of pi binding is determined not by the composition of the buffer in which the reaction was assembled, but by the composition of the electrophoresis buffer. We discuss the general implications of these findings.

A novel antibacterial gene transfer treatment for multidrug-resistant Acinetobacter baumannii-induced burn sepsis.

Sepsis caused by multidrug-resistant bacterial infections in critically injured patients has become a major clinical problem. Recently, Acinetobacter baumannii (AB) wound infections, especially in our critically injured soldiers fighting in Iraq and Afghanistan, is posing a major clinical problem and an economic burden. ConjuGon, Inc., has developed a novel antibacterial therapeutic technology using bacterial conjugation. The donor cells are attenuated Escherichia coli carrying a conjugative plasmid. The expression of bactericidal genes cloned on the plasmid is tightly repressed in the donor cells but becomes de-repressed once mobilized into a pathogen and disrupts protein synthesis. Here, we tested the efficacy of this novel conjugation technology to control and eradicate a drug-resistant clinical isolate of AB wound infection both in vitro and in a murine burn sepsis model. C57Blk/6J mice were divided into burn (B) and burn sepsis (BS) groups. All animals received a 12% TBSA dorsal scald full-thickness burn. The BS group was inoculated with multidrug-resistant AB (1 × 105 colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (103–1010 CFU) of attenuated donor E. coli encoding bactericidal proteins. The survival rate was monitored for 10 days. The ability of donor cells to significantly diminish AB levels in the burn wound 24 hours after injury was determined by quantitative cultures. Donor cells were highly effective in killing AB in vitro. In the burn sepsis model, 90% B group animals survived, and 40% to 50% BS animals survived with no treatment in 5 to 6 days. Treatment with donor cells at 1010 to 106 provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 × 104 CFU to 7.8 ± 4.4 × 109 CFU in 24 hours in the untreated group. Single treatment with donor cells (1010 CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 ± 0.5 × 104 CFU; P < .05). Taken together, these results indicate that this novel technology is an efficient method to control drug-resistant AB burn wound infections and prevent their systemic spread.

Replication of the R6K γ origin in vitro: dependence on wt π and hyperactive πS87N protein variant

Abstract The π protein of plasmid R6K is involved in control of replication. The aim of this study was to use an in vitro replication system dependent on an R6K-derived γ origin of replication (γori) to compare replication characteristics of wt π and a hyperactive variant of π protein (πS87N; Filutowicz et al., 1994b. Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 22, 4211–4215). The characteristics of in vitro replication from γori reported in this investigation are as follows: (i) πS87N is considerably more active in comparison to wt π. (ii) Replication proceeds through Cairns-type intermediates and the initiation site and directionality of the fork movement are similar in the presence of both proteins. (iii) Replication forks emanate unidirectionally in the vicinity of the cluster of seven 22-bp direct repeats within γori. (iv) Replication dependent on wt π, but not πS87N, is stimulated up to 1.5-fold by rifampicin.

Plasmid R6K replication control.

The focus of this minireview is the replication control of the 39.9-kb plasmid R6K and its derivatives. Historically, this plasmid was thought to have a narrow host range but more recent findings indicate that its derivatives can replicate in a variety of enteric and non-enteric bacterial species (Wild et al., 2004). In the four-plus decades since it was first described, R6K has proven to be an excellent model for studies of plasmid DNA replication. In part this is because of its similarities to other systems in which replication is activated and regulated by Rep protein and iteron-containing DNA. However its apparent idiosynchracies have also added to its significance (e.g., independent and co-dependent replication origins, and Rep dimers that stably bind iterons). Here, we survey the current state of knowledge regarding R6K replication and place individual regulatory elements into a proposed homeostatic model with implications for the biological significance of R6K and its multiple origins of replication.