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Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Acinetobacter baumannii is an important nosocomial bacterial pathogen. Multidrug-resistant isolates of A. baumannii are reported worldwide. Some A. baumannii isolates display resistance to nearly all antibiotics, making treatment of infections very challenging. As the need for new and effective antibiotics against A. baumannii becomes increasingly urgent, there is a need to understand the mechanisms of antibiotic resistance and virulence in this organism. In this work, comparative genomics was used to understand the mechanisms of antibiotic resistance and virulence in AB030, an extremely drug-resistant and hypervirulent strain of A. baumannii that is a representative of a recently emerged lineage of A. baumannii International Clone V. In order to characterize AB030, we carried out a genomic and phenotypic comparison with LAC-4, a previously described hyper-resistant and hypervirulent isolate. AB030 contains a number of antibiotic resistance- and virulence-associated genes that are not present in LAC-4. A number of these genes are present on mobile elements. This work shows the importance of characterizing the members of new lineages of A. baumannii in order to determine the development of antibiotic resistance and virulence in this organism.