Proteome Analyses of Soil Bacteria Grown in the Presence of Potato Suberin, a Recalcitrant Biopolymer.

Suberin is a complex lipidic plant polymer found in various tissues including the potato periderm. The biological degradation of suberin is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as the carbon source, and a metaproteomic approach was used to identify bacteria that developed in the presence of suberin over a 60-d incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community markedly decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species with the ability to develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria on the last days of the assay. Nevertheless, two putative lipases from Rhodanobacter thiooxydans (I4WGM2) and Myxococcus xanthus (Q1CWS1) were detected in the culture supernatants, suggesting that at least some bacterial species degrade suberin. When grown in suberin-containing medium, R. thiooxydans strain LCS2 and M. xanthus strain DK 1622 both produced three lipases, including I4WGM2 and Q1CWS1. These strains also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and ß-oxidation enzymes, suggesting that they participate in the degradation of suberin. However, only the R. thiooxydans strain appeared to retrieve sufficient carbon and energy from this recalcitrant polymer in order to maintain its population over an extended period of time.

Feeding behaviour of Caenorhabditis elegans is an indicator of Pseudomonas aeruginosa PAO1 virulence.

Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. The standard virulence assays rely on the slow and fast killing or paralysis of nematodes but here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. We monitored the food preferences of nematodes fed the wild type PAO1 and mutants in the type III secretion (T3S) system, which is a conserved mechanism to inject secreted effectors into the host cell cytosol. A ΔexsEΔpscD mutant defective for type III secretion served as a preferred food source, while an ΔexsE mutant that overexpresses the T3S effectors was avoided. Both food sources were ingested and observed in the gastrointestinal tract. Using the slow killing assay, we showed that the ΔexsEΔpscD had reduced virulence and thus confirmed that preferred food sources are less virulent than the wild type. Next we developed a high throughput feeding behaviour assay with 48 possible food colonies in order to screen a transposon mutant library and identify potential virulence genes. C. elegans identified and consumed preferred food colonies from a grid of 48 choices. The mutants identified as preferred food sources included known virulence genes, as well as novel genes not identified in previous C. elegans infection studies. Slow killing assays were performed and confirmed that several preferred food sources also showed reduced virulence. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for P. aeruginosa PAO1.