Multiple Dictyostelid Species Destroy Biofilms of Klebsiella oxytoca and Other Gram Negative Species.

Dictyostelids are free-living phagocytes that feed on bacteria in diverse habitats. When bacterial prey is in short supply or depleted, they undergo multicellular development culminating in the formation of dormant spores. In this work, we tested isolates representing four dictyostelid species from two genera (Dictyostelium and Polysphondylium) for the potential to feed on biofilms preformed on glass and polycarbonate surfaces. The abilities of dictyostelids were monitored for three hallmarks of activity: 1) spore germination on biofilms, 2) predation on biofilm enmeshed bacteria by phagocytic cells and 3) characteristic stages of multicellular development (streaming and fructification). We found that all dictyostelid isolates tested could feed on biofilm enmeshed bacteria produced by human and plant pathogens: Klebsiella oxytoca, Pseudomonas aeruginosa, Pseudomonas syringae, Erwinia amylovora 1189 (biofilm former) and E. amylovora 1189 Δams (biofilm deficient mutant). However, when dictyostelids were fed planktonic E. amylovora Δams the bacterial cells exhibited an increased susceptibility to predation by one of the two dictyostelid strains they were tested against. Taken together, the qualitative and quantitative data presented here suggest that dictyostelids have preferences in bacterial prey which affects their efficiency of feeding on bacterial biofilms.

A Mouse Model of Multi-Drug Resistant Staphylococcus aureus-induced Ocular Disease.

Staphylococcus aureus infection of the cornea is a significant threat to vision. The percentage of bacterial isolates resistant to antibiotics is increasing as is the percentage of infections caused by methicillin resistant isolates. There is a critical need for additional therapeutic approaches and their development will require the use of animal models to test efficacy. Two mouse models of S. aureus keratitis have been described but only quantified stromal keratitis (corneal clouding and perforation). We have extended these models using the methicillin resistant S. aureus USA300 LAC strain and show that eyelid inflammation and swelling (blepharitis) and corneal neovascularization can be quantified. This expanded model should prove useful in assessing additional effects of antibacterial therapies and additional pathological mechanisms involved in bacterial ocular infection.

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.

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.

Cooperative binding mode of the inhibitors of R6K replication, pi dimers.

The replication initiator protein, pi, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of pi bind to iterons in the gamma origin of plasmid R6K, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. Consequently, pi monomers activate replication, while pi dimers inhibit replication. Recently, it was shown that the monomeric form of pi binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers outcompete dimers for replication initiation when plasmid copy number and pi supply are low. Here, we examine cooperative binding of pi dimers and explore the role that these interactions may have in the inactivation of gamma origin. To examine pi dimer/iteron interactions in the absence of competing pi monomer/iteron interactions using wild-type pi, constructs were made with key base changes to each iteron that eliminate pi monomer binding yet have no impact on pi dimer binding. Our results indicate that, in the absence of pi monomers, pi dimers bind with greater cooperativity to alternate iterons than to adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by pi dimers.

Structure-based functional analysis of the replication protein of plasmid R6K: key amino acids at the pi/DNA interface.

In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of pi protein monomers and dimers. Here we investigate the following six amino acids of pi, encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.

Mechanism of origin activation by monomers of R6K-encoded pi protein.

One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.

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 x 10(5) colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (10(3) - 19(10) 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 10(10) to 10(6) provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 x 10(4) CFU to 7.8 +/- 4.4 x 10(9) CFU in 24 hours in the untreated group. Single treatment with donor cells (10(10) CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 +/- 0.5 x 10(4) 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.

Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity.

The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.

Role of pi dimers in coupling ("handcuffing") of plasmid R6K's gamma 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 pi protein with the seven 22-bp iterons of the gamma origin of replication. Like other related Rep proteins, pi exists as both monomers and dimers. However, the ability of pi 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 pi protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using pi variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (pi.P106L(wedge)F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of pi protein (pi.M36A(wedge)M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type pi. The data suggest that pi dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the gamma ori system. Thus, dimers of pi appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of pi expression, in cis competition (for origin binding) with monomeric activator pi, and handcuffing-mediated inhibition of replication in trans.

Bacterial expression system with tightly regulated gene expression and plasmid copy number.

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K.

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.

Tightly regulated vectors for the cloning and expression of toxic genes.

A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems.

Isomerization and apparent DNA bending by pi, the replication protein of plasmid R6K.

Plasmid R6K-encoded pi protein has multiple regulatory functions in replication and transcription. These functions rely, in part, on a complex set of interactions between monomers and dimers of the protein and distinct DNA targets, the direct and inverted repeats (DRs, IRs). In the work described here, we examine the isomerization and DNA bending properties of pi using electrophoretic mobility shift assays and circular permutation assays. Our data suggest that pi dimers can bend IRs, and dimer subunits seem to readily associate in head-to-head and head-to-tail fashion. The ability of pi to bend DRs is also reexamined using techniques that allow us to discriminate between bending induced by its different isomeric forms. We find that both monomers and dimers bend a single DR to similar degrees while results with 2DRs are more complex. The significance of the bending data in regard to a possible mechanism for replication initiation by pi protein is discussed.

pi protein- and ATP-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid R6K.

R6K-encoded pi protein can bind to the seven, 22 bp tandem iterons of the gamma origin. In this work, we use a variant of pi, His-pi.F107S, that is hyperactive in replication. In vitro, His-pi.F107S-dependent local DNA melting (open complex formation) occurs in the absence of host proteins (IHF/HU or DnaA) and it is positioned in the A + T-rich region adjacent to iterons. Experiments described here examine the effects of ATP, Mg2+ and temperature on the opening reaction. We show that the opening of the gamma origin can occur in the presence of ATP as well as AMP-PCP (a non-hydrolyzable ATP analog). This suggests that, for gamma origin, ATP hydrolysis may be unnecessary for open complex formation facilitated by His-pi.F107S. In the absence of ATP or Mg2+, His-pi.F107S yielded data suggestive of distortions in the iteron attributable to DNA bending rather than DNA melting. Our findings also demonstrate that ATP and pi stimulate open complex formation over a wide range of temperatures, but not at 0 degrees C. These and other results indicate that ATP and/or Mg2+ are not needed for His-pi.F107S binding to iterons and that ATP effects an allosteric change in the protein bound to gamma origin.

Characterization of His-tagged, R6K-encoded pi protein variants.

The pi protein of plasmid R6K is a multifunctional replication (Rep) protein, its different activities attributable, in part, to different oligomeric states: monomers and dimers. We have previously shown that His-tagged variants of the protein can exhibit alterations in dimer stability. Herein, we examined the functional properties of selected His-tagged derivatives of pi (His-pi x wt and three hyperactive replication variants) to determine if the functionality of these proteins in replication, DNA binding, and oligomerization is altered. Our results indicate that these tagged proteins retain the characteristics previously demonstrated for their non-tagged counterparts making them suitable for ongoing studies of pi protein structure and functions in replication and transcription.

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination.

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.