Question

what experiments could be done to learn more about quorum sensing in myxoccus xanthus??

(microbiology)

Answer 1

The myxobacterium Myxococcus xanthus is a predatory member of the soil microfauna, able to consume bacteria (Gram-negative, Gram-positive), archaea, and fungi. Many potential prey of M. xanthus communicate amongst themselves using acyl homoserine lactones (AHLs) as quorum signals.  Four AHLs of different side chain length were tested and all found to delay sporulation of M. xanthus vegetative cells, and to stimulate germination of myxospores, increasing the proportion of predatory vegetative cells in the population.

The predatory activity and expansion rates of M. xanthus colonies were also found to be stimulated by AHLs. Thermally inactivated AHLs had no effect on M. xanthus cells, and the response to AHLs depended (non-linearly) on the length of AHL side chain, suggesting that the effect of AHLs was mediated by specific signaling within M. xanthus, rather than being a consequence of the chemical or physical properties of AHLs. Therefore, it seems that the presence of xenic quorum signaling. molecules enhances the predatory activity of M. xanthus. AHLs increase the proportion of the population capable of predation, and stimulate the motility and predatory activity of vegetative cells. We therefore propose that in the wild, M. xanthus uses AHLs as markers of nearby prey, potentially eavesdropping on the conversations between prey organisms.

The addition of AHLs affects several behaviors exhibited by M. xanthus. Several lines of evidence suggest this is a consequence of signaling rather than due to the chemical and/or physical properties of the added AHLs.Loss of activity upon heating [presumably due to enhanced pH-dependent ring-opening lactonolys  also indicates an effect due to specific signaling rather than non-biological properties.

. The demonstration of a predator responding to signals secreted by prey, suggests that predator-prey interactions can be much more sophisticated than mere chance encounters between predator and prey, and phenomena such as QS, which exhibits a clear fitness advantage in the laboratory, may well have profound downsides in the wild.

Bacterial Strains, Media, and Cultivation

Myxococcus xanthus wild-type strain DK1622 (Kaiser, 1979) was used throughout, maintained on the rich medium DCY as described previously (Whitworth et al., 2008), with incubation at 30°C. Low nutrient DCY/10 medium is DCY diluted to 10% v/v. Media were solidified with the addition of 1.5% agar. TM was used as a nutrient free medium for developmental assays (Whitworth et al., 2008). E. coli strain TOP10 (Invitrogen) and B. megaterium (strain NCIMB 4821) were cultivated on LB (Sambrook et al., 1989), solidified with 1.5% agar as required. Cells were harvested from liquid cultures by centrifugation (4000 × g for 10 min). OMVs and soluble culture supernatant were purified from late-exponential cultures of M. xanthus by differential centrifugation as described previously (Evans et al., 2012).

Signaling Compounds

N-Acyl homoserine lactones (5 mg of >95% purity determined by high field proton NMR spectroscopy) were acquired from the University of Nottingham, dissolved in ethylacetate and aliquots lyophilised overnight before storage at 5°C. AHLs were dissolved in TM to a stock concentration of 2 mM immediately before being used at a working concentration of 6 μM. Control experiments used TM added to a lyophilised aliquot of ethylacetate without AHL. Heat-inactivation of AHLs was performed in a liquid state, on aliquots that had been resuspended in TM.

Physiological Assays

To monitor developmental sporulation, cells from late-exponential phase cultures were sedimented, washed with TM and spotted onto TM plates. Harvested spots were scraped from plates and spores enumerated as described previously (Higgs and Merlie, 2007). To assess motility rate, concentrated cells were spotted onto solid media as described by Higgs and Merlie (2007) and the increase in colony size recorded. To mark the original extent of the colony, Indian Ink was added to the cell suspension to a concentration of 0.1% v/v prior to spotting. To assess germination, spores were produced as before, enumerated, and added to DCY liquid medium and incubated at 30°C. As spores germinate they lose refractility, which was monitored as a decrease in turbidity at 560 nm as described by Otani et al. (1995). Killing of E. coli by OMVs and culture supernatant was assessed by killing plate assays as described by Evans et al. (2012), in which a known number of E. coli cells are mixed and incubated with a sample, and then plated to enumerate viable survivors.

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