Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms

Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans . Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro , suggesting that 5MPCA modification by protein amine groups occurs in vivo . The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79382/1/j.1365-2958.2010.07414.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79382/2/MMI_7414_sm_Figures_Table.pd

The Iron Assimilatory Protein, FEA1, from Chlamydomonas reinhardtii Facilitates Iron-Specific Metal Uptake in Yeast and Plants

We demonstrate that the unique green algal iron assimilatory protein, FEA1, is able to complement the Arabidopsis iron-transporter mutant, irt1, as well as enhance iron accumulation in FEA1 expressing wild-type plants. Expression of the FEA1 protein reduced iron-deficient growth phenotypes when plants were grown under iron limiting conditions and enhanced iron accumulation up to fivefold relative to wild-type plants when grown in iron sufficient media. Using yeast iron-uptake mutants, we demonstrate that the FEA1 protein specifically facilitates the uptake of the ferrous form of iron. Significantly, the FEA1 protein does not increase sensitivity to toxic concentrations of competing, non-ferrous metals nor facilitate their (cadmium) accumulation. These results indicate that the FEA1 protein is iron specific consistent with the observation the FEA1 protein is overexpressed in cadmium stressed algae presumably to facilitate iron uptake. We propose that the FEA1 iron assimilatory protein has ideal characteristics for the iron biofortification of crops and/or for facilitated iron uptake in plants when they are grown in low iron, high pH soils, or soils that may be contaminated with heavy metals

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Chlamydomonas reinhardtii Secretes Compounds That Mimic Bacterial Signals and Interfere with Quorum Sensing Regulation in Bacteria

The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-l-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene expression. More than a dozen chemically separable but unidentified substances capable of specifically stimulating the LasR or CepR but not the LuxR, AhyR, or CviR AHL bacterial quorum sensing reporter strains were detected in ethyl acetate extracts of C. reinhardtii culture filtrates. Colonies of C. reinhardtii and Chlorella spp. stimulated quorum sensing-dependent luminescence in Vibrio harveyi, indicating that these algae may produce compounds that affect the AI-2 furanosyl borate diester-mediated quorum sensing system of Vibrio spp. Treatment of the soil bacterium Sinorhizobium meliloti with a partially purified LasR mimic from C. reinhardtii affected the accumulation of 16 of the 25 proteins that were altered in response to the bacterium's own AHL signals, providing evidence that the algal mimic affected quorum sensing-regulated functions in this wild-type bacterium. Peptide mass fingerprinting identified 32 proteins affected by the bacterium's AHLs or the purified algal mimic, including GroEL chaperonins, the nitrogen regulatory protein PII, and a GTP-binding protein. The algal mimic was able to cancel the stimulatory effects of bacterial AHLs on the accumulation of seven of these proteins, providing evidence that the secretion of AHL mimics by the alga could be effective in disruption of quorum sensing in naturally encountered bacteria

Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein

Sinorhizobium meliloti, the nitrogen-fixing bacterial symbiont of Medicago spp. and other legumes, secretes a considerable amount of riboflavin. This precursor of the cofactors flavin mononucleotide and flavin adenine dinucleotide is a bioactive molecule that has a beneficial effect on plant growth. The ribBA gene of S. meliloti codes for a putative bifunctional enzyme with dihydroxybutanone phosphate synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps of the riboflavin biosynthesis pathway. We show here that an in-frame deletion of ribBA does not cause riboflavin auxotrophy or affect the ability of S. meliloti to establish an effective symbiosis with the host plant but does affect the ability of the bacteria to secrete flavins, colonize host-plant roots, and compete for nodulation. A strain missing the RibBA protein retains considerable GTP cyclohydrolase II activity. Based on these results, we hypothesize that S. meliloti has two partly interchangeable modules for biosynthesis of riboflavin, one fulfilling the internal need for flavins in bacterial metabolism and the other producing riboflavin for secretion. Our data also indicate that bacteria-derived flavins play a role in communication between rhizobia and the legume host and that the RibBA protein is important in this communication process even though it is not essential for riboflavin biosynthesis and symbiosis