The Vibrio harveyi master quorum-sensing regulator, LuxR, a TetR-type protein is both an activator and a repressor: DNA recognition and binding specificity at target promoters

Quorum sensing is the process of cell-to-cell communication by which bacteria communicate via secreted signal molecules called autoinducers. As cell population density increases, the accumulation of autoinducers leads to co-ordinated changes in gene expression across the bacterial community. The marine bacterium, Vibrio harveyi, uses three autoinducers to achieve intra-species, intra-genera and inter-species cell-cell communication. The detection of these autoinducers ultimately leads to the production of LuxR, the quorum-sensing master regulator that controls expression of the genes in the quorum-sensing regulon. LuxR is a member of the TetR protein superfamily; however, unlike other TetR repressors that typically repress their own gene expression and that of an adjacent operon, LuxR is capable of activating and repressing a large number of genes. Here, we used protein binding microarrays and a two-layered bioinformatics approach to show that LuxR binds a 21 bp consensus operator with dyad symmetry. In vitro and in vivo analyses of two promoters directly regulated by LuxR allowed us to identify those bases that are critical for LuxR binding. Together, the in silico and biochemical results enabled us to scan the genome and identify novel targets of LuxR in V. harveyi and thus expand the understanding of the quorum-sensing regulon.     

Therapeutic attenuation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no current therapy preventing cumulative neuronal loss. There is substantial evidence that mitochondrial dysfunction, oxidative stress, and associated caspase activity underlie the neurodegeneration observed. One potential drug therapy is the potent free radical scavenger and antioxidant cystamine, which has demonstrated significant clinical potential in models of neurodegenerative disorders and human neurological disease. This study examined the oral efficacy of cystamine in the MPTP and 6-hydroxydopamine neurotoxin models of PD. The neuroprotective effects of cystamine treatment significantly ameliorated nigral neuronal loss, preserved striatal dopaminergic projections, and improved striatal dopamine and metabolite levels, as compared to MPTP alone. Cystamine normalized striatal 8-hydroxy-2'-deoxyguanosine levels and ATP concentrations, consistent with reduced oxidative stress and improved mitochondrial function. Cystamine also protected against MPTP-induced mitochondrial loss, as identified by mitochondrial heat shock protein 70 and superoxide dismutase 2, with concomitant reductions in cytochrome c and caspase-3 activities. The neuroprotective value of cystamine was confirmed in the 6-hydroxydopamine model. Together these findings show cystamine's therapeutic benefit to reduce neuronal loss through attenuation of oxidative stress and mitochondrial dysfunction, providing the rationale for human clinical trials in PD patients.     

Costs of parental care on hunting behaviour of Helobdella papillornata (Euhirudinea: Glossiphoniidae)

Helobdella papillornata, an Australian freshwater leech, feeds primarily on snails and has a high level of parental care involving brooding eggs and young, with direct feeding of young. Parental costs and offspring benefits from these behaviours are poorly understood. A potential cost of parental care may be a change in the time taken to hunt prey. To test this hypothesis, the hunting behaviour of adults without progeny, parents with eggs, and parents with young were compared. We found that parents brooding eggs had a significantly (P = 0.029) longer lag time to begin hunting than parents brooding young, and spent significantly (P = 0.018) less time actively hunting than non-brooding adults. These costs, which may represent lost potential for the parent’s future reproductive success, should be outweighed by the fitness benefits of improved growth and survival of offspring, if parental care is favoured by selection. The hunting costs of care in Helobdella and other benthic, dorsoventrally flattened leeches in the family Glossiphoniidae may be smaller than the costs of brood tending that would be imposed on other freshwater leeches, and this difference may help explain the restriction of care to a single clade of the Euhirudinea.     

Cyst bank life-history model for a fairy shrimp from ephemeral ponds

1. Ephemeral wetland habitats provide a useful model system for studying how life‐history patterns enable populations to persist despite high environmental variation. One important life‐history trait of both plants and crustaceans in such habitats involves hatching/germination of only some of the eggs/seeds at any time. This bet‐hedging leads to the development of a bank composed of dormant propagules of many ages. 2. The San Diego fairy shrimp, Branchinecta sandiegonensis (Crustacea: Anostraca), a dominant faunal element of ephemeral ponds in San Diego, California, is a suitable organism for studying the consequences of highly fluctuating environmental conditions. As a result of large‐scale habitat loss, the species is also endangered, and this motivated our specific study towards understanding the hatching dynamics of its egg bank for planning conservation efforts such as pool restoration and re‐creation. 3. We formulated a matrix population model using egg age within the bank to study the relationship between adult survival and reproduction, and survival in and hatching from the egg bank. As vital rates for fairy shrimp are only poorly known, we generated 48 matrices with parameters encompassing ranges of likely values for the vital rates of B. sandiegonensis. We calculated population growth rates and eigenvalue elasticities both for a static model and a model with periodic reproductive failure. 4. The model shows that in good filling events, population growth rate is very high and the egg bank is increased dramatically. While population growth rate is insensitive to long‐term survival in the egg bank in our static deterministic model, it becomes sensitive to survival in the egg bank when a regime of periodically failed reproductive events is imposed. 5. Under favourable conditions, it was best for shrimp to hatch from eggs as soon as possible. However, under a regime where failed reproductive events were common, it was best to hatch after several pool fillings. Because conditions change from favourable to unfavourable unpredictably, variation in age within the egg bank appears to be critical for the persistence of the population. This attribute needs to be carefully considered when restoring or creating new pools for conservation purposes.     

A nonalkaline method for isolating sequencing-ready plasmids

We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damage to the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the presence of urea or N,N-dimethylformamide.