Microorganisms as Freight Haulage Systems. Colin Ingham Wageningen University, NL

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1 Microorganisms as Freight Haulage Systems Colin Ingham Wageningen University, NL

2 Swarming bacteria Surface, collective, flagellar-driven, motility common in soil bacteria. Often involves surfactants/lubricants to assist migration. What happens when moving microbial communities encounter other microorganisms? Approached this question with Paenibacillus vortex.

3 Paenibacillus vortex 1 Gram + curved, flagellated rod from soil. Over a centimetre an hour continuous swarming on rich media (inhibited PNPG). Vigorous circular vortex movement of cells and colonies A: DIC microscopy of swarming colony (0.5 mm diameter) B: SEM stressed microcolony (100 µm diameter) C: Colonization of 14 cm plate (colour from tetrazolium dye). D: Patterning on low nutrient media agar (pseudocoloured).

4 Paenibacillus vortex 2 Genome sequenced (very high proportion of 2 component systems, membrane transporters, antimicrobials/resistance markers common Rhizosphere package ). Limited genetic tractability. Spore former. 6,437 open reading frames (ORFs) and 73 non-coding RNA genes A: 16S derived phylogenetic tree B: highly represented categories of genes

5 Aspergillus fumigatus Filamentous fungus Pathogenic and allergenic. Spores (Asexual, i.e. conidia) spread long distances, primarily through the air. Conidia are c. 3 µm diameter then swell due to water uptake early in germination. Germinating conidia swell (take up water) after a few hours with outgrowth after 6 to 8 h (approximately) with formation of hyphae. A sophisticated and versatile microorganism but not motile. Conidia on 14 cm RMHA plates 12 hours 5 days 9 days

6 The approach Day 1: Day 2: Culture P. vortex from freezer stocks on RMHA plate. Harvest actively swarming plate, combine with A. fumigatus conidia. Inoculate in centre of a large Petri dish. [Minimal culture. Initiation of swarming within 20 min. Almost no swarm lag ] Controls include: Conidia + P. vortex with PNPG RMHA = (reduced strength Mueller Hinton) media.

7 The result fungal dispersal Swarming P. vortex facilitates dispersal of A. fumigatus (control non-swarming P. vortex does not e.g. inhibited by PNPG) A: Plate after 72 h (fungal colonies visible, bacteria ubiquitous but not visualized). B: Microcolony imaging after 24 h fungi always initiate growth within P. vortex.

8 P. Vortex transports conidia movie 1 Time lapse movies coupled with conidial recovery by toothpick confirms that conidia are directly transported. A and B: Transport of aggregates of conidia. C: Control P. vortex with no cargo.

9 P. Vortex transports conidia movie 2 Time lapse movie 2 conidial loading onto rotating colony on 1.5% w/v agar

10 P. Vortex transports conidia movie 3 Resolution at level of individual conidia

11 But we know microbes can move stuff. Example 1: MEMS Motile bacteria drive a wheel. Di Leonardo R. et al (2010) Bacterial ratchet motors. Proc Natl Acad Sci USA 107:9541 Example 2: Microoxen Motile algae move a covalently coupled bead. Weibel D. et al. (2005) Microoxen: microorganisms to move microscale loads. Proc Natl Acad Sci USA 102:

12 But we know microbes can move stuff 2 Eukaryotic microorganisms can transport bacteria Fungal conidia can be carried by insects, mammals or even white blood cells But never have swarming bacteria been shown to cooperatively move a larger/eukaryotic organism. This is the first time this type of transport of conidia by bacteria has been shown in a way which could occur in nature. Transport of A. fumigatus up to 30 cm by P. vortex.

13 Tracking bacteria motion Stills from movie with trajectory of real conidia (red) compared with virtual trajectory (white). Modelling vs movie comparisons suggests conidia co-migrate with nearby P. vortex.

14 Conidia associate with the bacteria Scanning electron microscopy of conidia during transport by P. vortex. C = conidia V = P. vortex

15 Conidial transport Conidia swell and germinate as rapidly during transport as directly on RMHA (from toothpick recovery and microscopy) Therefore conidia are metabolically active and sense their environment during transport. P. vortex does not inhibit germination or the early stages of outgrowth. Outgrowth inhibits transport (deduced from loading pregerminated conidia and direct deposition of different sized microcolonies). Conidia can reach the edge of a 14 cm diameter RMHA plate in 7 h. This is over 30 times faster than growth or growth-conidia-airborne routes that fungus without bacteria would use to reach the plate edge.

16 Rescue of conidia Swarming P. vortex can pick up conidia from a location they cannot grow in, and deposit them in a more favourable environment. A: Co-inoculation of P. vortex, conidia and voriconazole (antifungal). B: P P. vortex. X A. fumigatus conidia + voriconazole.

17 Antibiotic refractory swarmers Swarming P. vortex can cross agar containing otherwise effective conc. of antibiotics. This is a temporary state of what appears to be a slower growing subpopulation. How does the subpopulation organise/emerge.? Pioneering bacteria crossing dangerous territory in search of a better life? If you are going through hell keep going. - Churchill

18 Conidia can exploit this Transport of conidia can occur into habitats where P. vortex cannot thrive.

19 Mutual benefit in the soil? Mycelia cross air gaps, most bacteria struggle Soil On an agar plate - simulated air gap Fungal Highways Warmink JA, van Elsas JD (2009) Migratory response of soil bacteria to Lyophyllum sp. strain Karsten in soil microcosms. Appl Environ Microbiol 75:

20 Mutual benefit (related to soil) 2? Swarming P. vortex can cross simulated air gaps when assisted by mycelial growth. 5 day experiment starting at position Y. 0.5 mm gap. Process inhibited by PNPG. Also works from position X conidia transported to Y, germinate, bridge gap, facilitate bacteria reaching target. Set up 0.5 mm air gap 0.8 mm gap Target reached..! On mycelia Its therefore possible to have a situation where each microorganism facilitates the dispersal of the other.

21 A. fum A. fum P. cit P. cit P. exp P. exp P. cam P. cam C. glo C. glo Is transport cargo specific? Conidia transport Species Not all conidia transported equally (White close to start point Black further away) Changes to A. fumigatus conidia surface inhibitory

22 Can any swarmer transport? Apparently not (so far) attempts to transport conidia of 6 fungal species, beads and non-motile bacteria with Proteus mirabilis and Paenibacillus polymyxa have failed.

23 So, what about P. vortex spores? RMHA does not support sporulation in P. vortex. Exogenously added spores (from liquid culture) are transported (SEM, dispersal of heat resistant CFU, surface stained spores). Given the propensity of P. vortex to swarm into dangerous areas having prefabricated spores may make sense.

24 Summary P. vortex can transport spores over tens of centimetres under conditions which suggest this is possible in the natural environment (soil). For Aspergillus conidia this is a new method of dispersal and the first time a smaller microorganism has been shown to cooperatively transport a larger Eukaryote. It may permit targeting niches that are poorly reached by airborne dispersal and possibly allow nutrient rich or other good environments to be reached. Aspergillus can in turn aid P. vortex dispersal (though relationship between these two organisms is complex its not always cooperative). P. vortex can also transport its own spores with potential in stress situations this would seem to fit with the pioneering nature of this microorganism. Not all cargo organisms are beneficial. Not all swarming microorganisms transport (?). The surface/size of the cargo matters.

25 Swarming and meeting strangers Swarms transporting other microorganisms: a moving ecosystem? Could extended motile extended colonies act as logistics systems? Territorial excludes competitors of the same species (Dienes/ids system) Gibbs et al. J. Bact :3286. Maybe also not inclusive other species (not easily transported)?

26 THE END and Thanks Collaboration: Eshel ben Jacob group University Tel Aviv Thanks to Joëlle Dupont, Rolf Geisen, Peter Schneeberger and Amir Sharon for strains of fungi and advice on fungal culture and Adriaan van Aelst for help with electron microscopy. Episode 23