Amplified fragment length polymorphism (AFLP) analysis, a genetic fingerprinting technique based on polymerase chain reaction (PCR), can present financial and logistical challenges, as the use of radioactive isotopes and sequencing gels can be dangerous and expensive. Our goal was to optimize an AFLP protocol that did not require these materials and could distinguish among clone lines of pale anemones (Exaiptasia pallida). Anemone DNA was digested with EcoRI and MseI endonucleases. Adapters were ligated to cut sites, and fragments were amplified via nested PCR using increasingly selective EcoRI- and MseI-specific primers. In the final amplification step, EcoRI primers were labeled with 5(6)-carboxyfluorescein (FAM). Amplified fragments and a FAM-labeled ladder were electrophoresed through pre-cast polyacrylamide mini-gels. Gels were photographed and bands were marked manually. Fragment lengths were determined by comparison with the FAM-labeled ladder. Estimates of genetic similarity were then calculated for each pair of fingerprints using the Jaccard index of similarity, S_J. Samples were clustered on a dendrogram into five significantly different groups using similarity profile (SIMPROF) analysis. Comparisons of sample replicates yielded a mean S_J of 0.74, and mean S_J values within groups ranged from 0.51 to 0.72. Our safe and cost-effective genetic fingerprinting method was used to distinguish among E. pallida clone lines without the use of radioactive isotopes or sequencing gels. As E. pallida is increasingly used as a model for studying the establishment, maintenance, and breakdown of the symbiotic relationship in cnidarians, our protocol facilitates genetic fingerprinting of this vulnerable lineage of marine organisms.

In recent years, Acinetobacter baumannii has emerged as a major threat to human health. Yet studies to uncover the molecular mechanisms used by A. baumannii to mediate human infections have only recently begun. For example, the methods used by A. baumannii to resist antibacterial treatment are not fully understood. Using a candidate approach, we sought to identify proteins found in the A. baumannii cell envelope that are important for resistance to antimicrobial compounds. We screened selected mutants in genes needed for assembly of the bacterial cell envelope, biofilm formation, or capsule production for sensitivity to different antibiotics and detergents. Compared to wild-type, these mutants displayed an increased sensitivity to bacitracin yet not to vancomycin, both antibiotics that target the cell wall. Only the ompA and lnt mutants had significantly increased sensitivity to the DNA gyrase inhibitor novobiocin. Similarly, there was also a spectrum of sensitivity to bile salts, with ompA and lnt mutants being most sensitive while pgaA, pgaB, and capA mutants showed a modest growth defect. While we do not fully understand the specificity of these sensitivity profiles, our findings suggest a potential use for these antimicrobial compounds in genetic selections for mutants that disrupt cell envelope biogenesis. This approach could potentially inform future efforts to combat multi-drug resistant Acinetobacter infections.

Adaptation to low temperature presents many challenges. On a cellular level, structural integrity and metabolism are profoundly affected in cold temperatures, and at freezing temperatures, ice crystal formation must be prevented or controlled for an organism to survive. Antifreeze proteins (AFP) and glycoproteins (AFGP) are protective molecules expressed by organisms to effectively cope with these challenges. These proteins are extremely effective at protecting tissues from chilling and freezing damage, and are active at low concentrations compared to other cryoprotectants, such as glycerol. Many organisms can prevent ice crystal formation in their tissues by supercooling, where the freezing point of cellular fluid is decreased below the normal freezing point of water. Those that can tolerate freezing use ice nucleating proteins (INP) and AFPs to restrict the growth and coalescence of ice crystals. Studies on the evolution of AFPs and AFGPs in polar species reveal that the genes arose from a variety of mechanisms - including lateral gene transfer, gene duplication and convergent evolution - in response to climate change. AFPs directly bind to ice crystal faces, slowing or preventing crystal growth and coalescence. They also act to reduce membrane permeability by interacting with bilayer lipids, thereby preventing leakage during chilling. Practical applications of these molecules include tissue preservation, cold tolerance in crops and food animals, frozen food storage, and freeze-proof surface coatings.

Agriculture can be hindered by low soil nutrient availability, leading to the need for nutrient amendments to maintain crop productivity. Casuarina equisetifolia, commonly known as Australian pine, is a highly invasive species that represses native plant growth, but some studies have suggested that C. equisetifolia litter could be used as an agricultural amendment. Our objective was to investigate the effects of C. equisetifolia litter on cauliflower productivity. In a greenhouse experiment, individual cauliflower seeds were grown in pots with no litter addition, non-composted litter, or composted litter. We hypothesized that cauliflower in the composted litter treatment would have the highest carbon assimilation, biomass, and number of leaves. We found no effect of treatment on carbon assimilation, which suggests that C. equisetifolia litter does not increase nitrogen (N) availability. Aboveground biomass of cauliflower and the number of leaves per plant were lower in the composted litter treatment than in the non-composted litter or control treatments. This suggests that composted C. equisetifolia litter had allelopathic effects on cauliflower or facilitated an increase in microbial biomass, which increased competition and reduced the amount of N available for cauliflower. This study indicates that C. equisetifolia litter is not an ideal soil amendment for cauliflower production due to its potential allelopathic and inhibitory effects when composted and its inability to increase productivity when not composted.