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.

Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. While the standard virulence assays rely on the slow and fast killing or paralysis of nematodes, here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. The type III secretion system is a well-conserved virulence trait that is not required for slow or fast killing of C. elegans. However, ΔexsE mutants that are competent for hypersecretion of ExoS, ExoT and ExoY effectors were avoided as food sources in binary assays. Conversely, mutants lacking the secretion machinery or type III effectors were preferred food sources for PAO1. In binary feeding assays, both food sources were ingested and observed in the gastrointestinal tract, but non-preferred food sources were ultimately avoided. Next we developed a high throughput feeding behaviour assay to test a library of 2370 transposon mutants in order to identify preferred food sources. After primary and secondary screens, 37 mutants were identified as preferred food sources, which included mutations in many known virulence genes and that showed reduced virulence in the slow killing assay. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for bacterial strains that have moderate worm killing activity.

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 β-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.

Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in 2Pseudomonas aeruginosa. While the standard virulence assays rely on the slow and fast killing or 3 paralysis of nematodes, here we developed a behaviour assay to monitor the preferred bacterial 4 food sources of C. elegans. The type III secretion system is a well-conserved virulence trait that 5is not required for slow or fast killing of C. elegans. However, ΔexsE mutants that are competent 6 for hypersecretion of ExoS, ExoT and ExoY effectors were avoided as food sources in binary 7 assays. Conversely, mutants lacking the secretion machinery or type III effectors were preferred 8 food sources for PAO1. In binary feeding assays, both food sources were ingested and observed 9 in the gastrointestinal tract, but non-preferred food sources were ultimately avoided. Next we 10 developed a high throughput feeding behaviour assay to test a library of 2370 transposon mutants 11 in order to identify preferred food sources. After primary and secondary screens, 37 mutants were 12 identified as preferred food sources, which included mutations in many known virulence genes 13 and that showed reduced virulence in the slow killing assay. We propose that C. elegans feeding 14 behaviour can be used as a sensitive indicator of virulence for bacterial strains that have moderate 15 worm killing activity. C. elegans is an important model organism for developmental biology and infectious 22 diseases research. The nematode has been used for numerous studies as an infection host for 23Pseudomonas aeruginosa and many other bacteria (Sifri, Begun & Ausubel, 2005). C. elegans is 24 a bacteriovore that forages for bacteria in rotting plants and soil. When C. elegans is fed a lawn 25 of P. aeruginosa PA14, the gut is colonized and numerous virulence factors contribute to worm 26 death over a period of days, also known as slow killing (Tan, Mahajan-Miklos & Ausubel, 1999; 27 Feinbaum et al., 2012). Defects in virulence of transposon mutants fed to synchronized worms 28 are determined by decreased nematode killing kinetics relative to the wild type strain. After 29 ingestion, P. aeruginosa caused gut distension, the production of biofilm-like extracellular matrix 30 in the lumen, penetrated the intestinal barrier and invaded epithelial cells to some extent 31 (Irazoqui et al., 2010). P. aeruginosa PA14 uses a wide range of virulence phenotypes in the slow 32 killing assay (Feinbaum et al., 2012), but when PA14 is grown on rich, high osmolarity medium, 33 the fast killing pathway does not require live bacteria and is due to the production of toxic 34 phenazine compounds (Cezairliyan et al., 2013). P. aeruginosa strains display a range of 35 virulence phenotypes and PAO1 is among the strains with moderate slow killing activity (Lee et 36 al., 2006) . However, PAO1 was shown to induce a rapid, paralytic killing mechanism dependent 37 on hydrogen cyanide production (Gallagher & Manoil, 2001 known that C. elegans does have a feeding preference for cer...