Alternative Roots

Utilising Capillaries to Quantify Chemotaxis

Principle

To truly characterise chemotactic behaviour in response to naringenin, a quantitative approach would be best suited. This allows for direct comparison of the strength of the response between different species through statistical analysis. Results from a quantitative assay would also be better suited for our community model as it allows a hierarchy of which bacteria would be more likely to respond first.

The quantitative assessment of chemotaxis used was a variant of Pfeffer’s capillary method [1]. This method is based on the premise that a capillary containing a chemoattractant will contain more bacteria than one containing a control after incubation in which the open end is submerged in bacterial solution of known and equal cell density. This is as the bacteria are attracted to the chemoattractant and thus move into the capillary at a higher rate than the control – which only demonstrates random movement. In order to observe differences in the bacterial density between the test solutions, the contents of the capillaries were spread over solid media and the number of colonies was counted.

Results

After 24 hours incubation at either 30°C (Azorhizobium caulinodans and Herbaspirillum seropedicae) or 37°C (Azospirillum brasilense and Escherichia coli), the number of colonies was manually counted for each segment (Table 1). The results showed that of the 4 test bacterial species, only 1 was able to swim into the capillary - H. seropedicae.

H. seropedicae was able to move successfully into capillaries containing either the control (buffer solution) or the chemoattractant. This was demonstrated by the growth of colonies on LB agar from the contents of each capillary (Figure 1). Both methods of agar inoculation (spread and pipetteing) lead to colony growth.

After counting colonies from the contents of both the control and naringenin capillaries, statistical analysis was conducted in order to observe if there was a difference between said counts. Analysis was completed using MiniTab 17. A 2-sample t-test revealed that there was no significant difference between mean colony count of the two conditions (P>0.05).

This result did not align with the hypothesis that more colonies of the nitrogen fixing bacteria would grow on agar inoculated with the contents of the naringenin-containing capillary. This is as it was predicted that more bacteria would be present in the capillary due to the chemoattraction of H. seropedicae to naringenin. While the results may indicate that there was no significant evidence for positive chemotaxis in this scenario, it must be considered that H. seropedicae was the only species out of 4 that showed growth on agar. As such, it may be suggested that an aspect of the protocol was having a confounding effect upon chemotactic response.

Reflecting upon this fact, multiple potential flaws with the protocol used were identified. The first potential flaw with the protocol is the use of a chemotaxis buffer. This buffer (10mM potassium phosphate, 0.1mM EDTA, 10mM glucose, pH 7.0) has been utilised to maintain motility in E. coli strains [1]; however, it may be possible that this buffer was not suitable for our nitrogen-fixing bacteria. Another potential flaw was that the assay requires a very low OD600 of between 0.04-0.05. This could mean that the bacterial cell density was too low and thus our OD readings were not accurate. This theory was overruled after observing growth from LB agar plates that were spread with 60µl of each bacterial suspension that was placed in the wells of the microtitre plate.

The third potential flaw was made apparent when considering the ecology of the bacteria. As the bacteria are able to colonise the rhizoplane [2-4], it may also be possible that the cells adhered to the inner surface of the capillary. This may explain why no bacteria were able to grow on the plate as a single wash with distilled water may not have been substantial enough to sheer the cells off the surface. The low cell density may also play a role here as this a higher percentage of cells would be able to adhere to the surface. However, it was very unlikely that all cells adhered to the surface and thus it is hard to explain no colonies grew.

The final potential flaw of this protocol and potential reason behind the results is that the naringenin concentration was too high. This could mean that when the capillary end was submerged in the bacterial solution, there was an exchange of chemicals and thus naringenin entered the well at a concentration that saturated both areas. As such, there was no concentration gradient for the bacteria to move along thus their behaviour was unchanged. This principle, however, does not explain why there was no random movement into the capillary tubes.

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Results overview

From our results, we can conclude that the quantitative approach utilised to understanding chemotaxis was likely an inappropriate choice for our bacteria. Results indicated that only H. seropedicae was able to move into the capillary tube; however, the nature of the bacteria means that H. seropedicae was the only one to also leave the capillary after entering. No growth from the E. coli capillaries may be explained due to the fact that the bacteria has no natural chemotactic response to the flavonoid, as well as the fact that naringenin is toxic to the species [5]. Other species may not have demonstrated evidence of chemotaxis via this method due to flaws in the protocol; however, the factor that had this effect is hard to define without future studying.

REFERENCES & Attributions

1. Yang X-e, Wu X, Hao H-l, & He Z-l (2008) Mechanisms and Assessment of Water Eutrophication. Journal of Zhejiang University. Science. B 9(3):197-209.

2. Usman MN, MG; Musa, I (2015) Effect of Three Levels of NPK Fertilizer on Growth Parameters and Yield of Maize-Soybean Intercrop. International Journal of Scientific and Research Publications 5(9).

3. Liu W, et al. (2017) Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems. Frontiers in Microbiology 8:1327..

4. Pedrosa FO, et al. (2011) Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses. PLoS Genetics 7(5):e1002064.

Attributions:

Primary Author: Connor Trotter

Stylisation: Connor Trotter, Umar Farooq

Troubleshooting: Umar Farooq