DNA exchange and insertional inactivation in spirochetes

Spirochetes have complex life cycles and are associated with a number of diseases in humans and animals. Despite their significance as pathogens, spirochete genetics are in their early stages. However, gene inactivation has been achieved in Borrelia burgdorferi, Brachyspira hyodysenteriae, and Treponema denticola. Here, we review methods that have been used in spirochetes for gene inactivation and DNA exchange, with a primary focus on B. burgdorferi. We also describe factors influencing electrotransformation in B. burgdorferi. In summary, optimal transformation frequencies are obtained with log phase bacteria, large amounts of DNA (up to 50 microg per transformation), and high field strength (12.5-37.5 kV/cm). Infectious B. burgdorferi isolates transform with frequencies 100-fold lower than those found for high passage, non-infectious strains. Surface characteristics of the bacteria, which often correlate with infectivity, are among the obstacles to effective transformation by electroporation.     

DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.     

Successful reintroduction of species: improving on windows of opportunity for biodiversity repair

To overcome resistance of degraded ecological communities to restorative interventions, we need to understand windows of opportunity—limited time frames when species reintroduction attempts are still successful. More specifically, we need to understand what makes these windows close, as this may enable us to stretch or reopen them. We investigated this using models of simple food web modules. We show how joint changes of bottom–up and top–down control may be applied to change windows of opportunity and increase reintroduction success. Which reintroduction densities were most effective seemed system-specific. A more general result is that reintroduction success was strongly enhanced by low to intermediate carrying capacities of basal species (e.g. periphyton and other algae in streams). This can be seen as equivalent to low to intermediate nutrient levels. When these were too high, almost any combination of restorative measures was rendered ineffective. Interestingly, reintroducing primarily and secondarily lost species at the same time was more effective than sequential reintroductions that first attempted to fix secondary extinctions. We could further enhance the success of species reintroductions by reducing the carrying capacities of basal species before the reintroduction of primarily and secondarily lost species. We discuss our results in the light of empirical work on macro-invertebrates in streams. This serves to exemplify how our results can be applied in the practice of ecological restoration.     

Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid. Coumermycin A1 resistance can be transferred to naive B. burgdorferi by transformation with borrelial plasmid DNA from the initial transformants. This work represents the first example of a directed mutation in B. burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.     

Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae

Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β’-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ’-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ’-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ’-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β’-NAC or α-NAC subunits as well as αβ’-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.     

Linear plasmids and chromosomes in bacteria

Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.     

Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase

Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.