Alternative Roots

Exploring Bacterial Chemotaxis

Quantification Microscopy Chemotaxis on Agar

Naringenin Chemotaxis

An Introduction

We examined how three species of free-living nitrogen-fixing bacteria respond to the presence of the flavonoid naringenin. The three species, Azorhizobium caulinodans (ORS571), Azospirillum brasilense (SP245), and Herbaspirillum seropedicae (Z67), were selected because they all have potential to form different types of interactions with plant roots. A. caulinodans has been shown to fix nitrogen both as a free-living microbe and when in symbiosis with the semi-aquatic leguminous tree Sesbania rostrata [1]. H. seropedicae is a root endophyte and has shown potential to colonise popular crops such as wheat and maize [2].

Bacteria Characterisation

Colony Morphology

Before commencing chemotaxis studies, we needed to understand the growth characteristics of the three free-living nitrogen-fixing bacteria to be used in our project. We first examined the colony morphology of these three species in the absence of any chemoattractants. Familiarisation with the bacteria allows identification of abnormal behaviour and contamination. For colony morphology, the size after a minimum of 24 hours and morphology (shape and pigmentation) was recorded (Table 1, Figs 1-3).

Table 1: Qualitative analysis of Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae colonies grown on solid media.

Species (Strain)	Colony Pigmentation	Colony Morphology	Points of Interest
Azorhizobium caulinodans (ORS571)	White	Regular form, Typically raised, Entire margin	Colonies rarely grow to a measurable size when grown at 30˚c on YEB media after 24 hours
Azospirillum brasilense (SP245)	Orange/Pink	Non-slimy, Regular and round form, Entire margins	Both immature and dead colonies lack the orange/pink pigment, Colonies wrinkle with age
Herbaspirillum seropedicae (Z67)	Cream/Light Green	Circular or Irregular form (occasionally rhizoid), Raised elevation, Shiny	Colonies took on a different morphology depending on how the media was innoculated; stab-innoculation lead to rhizoid form while spreading leads to circular/irregular form

A. caulinodans (Figure 1a): Colonies do not grow to a measurable size within 24 hours at 30 ˚C on Yeast Extract Broth agar. Colonies contain white pigmentation and are raised in elevation with an entire margin – a continuous, uninterrupted border of the colony. Colonies rarely grow larger than 2 mm whilst smaller colonies, which are much more numerous, could not be accurately measured.

A. brasilense (Figure 1b): Colonies are distinguishable by their distinctive orange/pink pigmentation though both immature and dead colonies lack this pigmentation. Older colonies became ingrained into the agar, making them hard to remove without damaging the agar. Older colonies also began to wrinkle with time. The average diameter for a colony of this species after 24 hours incubation at 37 ˚C on LB Agar was 3 mm, making A. brasilense the fastest growing of our nitrogen-fixing bacteria. Young A. brasilense colonies were shiny, round and with entire margins. These young colonies may have some pigmentation near the centre as the colony matures. This is in contrast to older colonies which maintain a different phenotype; losing their shine and gaining the odd wrinkle. Wrinkling often leads to the loss of the round shape.

H. seropedicae (Figure 1c): the colonies take different forms depending on how the plate is inoculated. If the plate is stab-inoculated, the colony takes a rhizoid appearance (Figure 3a). If the culture is spread across the plate, then it typically takes a circular or irregular form (Figure 3b). Colonies possess a green-cream pigmentation and are raised from the surface. Most colonies were shiny and typically 1.5 mm in diameter after 24 hours at 30 ˚C.

Figure 1: Observations of bacterial preservation plates. a) A. caulinodans colonies grown on 1% YEB agar after incubation at 30°c for 56 hours. Plates were inoculated via streaking. b) A. brasilense colonies grown on 1% LB after incubation at 37°c for 16 hours. Plate inoculated via streaking. c) H. seropedicae colonies showing circular growth on 1% LB agar after incubation at 30°c for 24 hours. Plates were inoculated via streaking. d) H. seropedicae colonies showing rhizoid growth on 1% LB agar after incubation at 30°c for 24 hours. Plates were stab-innoculated.

Growth Rates in Liquid Media

From initial iterations of our community modelling, it became apparent that quantitative data on the growth rates of the bacteria were required in order to inform the model. For this, we observed changes in absorbance at 600 nm over 72 hours of the three nitrogen-fixing bacteria and E. coli in liquid culture at 30 °C using a ThermoFisher Scientific Varioskan LUX Microplate Reader.

The data showed that A. brasilense grew at a slow, steady rate before sharply dying off after approximately 60 hours. The slow growth rate is likely to be because its optimal growth temperature is 37 °C rather than 30 °C. H. seropedicae and A. caulinodens showed very similar growth curves when grown at 30 °C: initial growth rate was very fast and then growth became very slow or static after 20 hours. E. coli grew at a medium pace to begin with and steadily slowed down with time.

Effect of Naringenin on Growth Rate in Liquid Culture

Initial research for the Alternative Roots project noted that naringenin possesses antimicrobial properties, particularly towards E. coli [3] [link to the notebook data where you observed this]. As E. coli (DH5α) was to be used as both a control in our chemotaxis assays and as the organism in which our naringenin biosynthesis operon would first be assembled, it was deemed important to characterise the effect of increasing naringenin concentrations on growth rates of both our free-living nitrogen-fixing bacteria, and E. coli in LB medium. This was essential to guide the chemotaxis assays enabling an understanding of naringenin concentrations which would not have detrimental impacts upon the cell. If cell health is impaired, then there is potential for cell death to lead to the appearance of chemorepulsion. This is particularly problematic when applying the response index as a semi-quantitative measure of chemotactic response as the method utilises ratios between colony edges to determine the significance of chemotaxis [4].

Figure 2: Absorbance at 600 nm of four bacterial species (A. brasilense, A. caulinodans, H. seropedicae, and E. coli) after 24 hours of growth when grown in liquid media containing different concentrations of naringenin.

All species successfully grew in the presence of 0-150 μM naringenin (Figure 4). However, it was noted that E. coli showed a reduced growth rate even at lower concentrations of naringenin. When the concentration of naringenin exceeded 100 μM, there exists greater flux in all species suggesting that naringenin begins to have a greater impact on some, but not all, bacteria. As such, naringenin concentrations of <100 μM were used as part of subsequent chemotaxis assays to avoid negatively impacting bacterial growth.

Characterising Chemotactic Behaviour

Quantification Utilising Capillaries

To characterise chemotactic behaviour in response to naringenin, a quantitative approach is desirable. This allows for direct comparison of the strength of the response between different species. Results from a quantitative assay would also be better suited for our community model [link to modelling page] as it allows a ranking of bacterial responses to naringenin.

Table 2: Colony forming units of four bacterial species from capillaries containing 1 µl 100 µM naringenin or motility buffer solution (10 mM potassium phosphate, 0.1 mM EDTA, 10 mM glucose, pH 7.0) after 60 minutes open-end submersion in sterile conditions at room temperature/pressure. Values are mean cfu.μl^-1. Difference between colony counts from capillaries containing naringenin or motility buffer was non-significant for all species (P>0.05).

Species (Strain)	Colony Count (Naringenin)	± Standard Error	Colony Count (Control)	± Standard Error	Significant Difference
A. caulinodans (ORS571)	0	0	0	0	No
A. brasilense (SP245)	0	0	0	0	No
H. seropedicae (Z67)	458	86	376	236	No
E. coli	0	0	0	0	No

After 24 hours incubation at either 30 °C (A. caulinodans and H. seropedicae) or 37 °C (A. brasilense and E. coli), the number of colonies which grew on the LB agar plate was counted (Table 2). The results showed that of the four test bacterial species, only one was able to move into the capillary. This species was H. seropedicae which 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 5). Both methods of agar inoculation (spreading and pipetteing) lead to colony growth.

After counting colonies from the contents of both the control and naringenin capillaries, no significant difference between mean colony count of the two conditions was observed (P>0.05). The results therefore show no evidence for positive chemotaxis using this method. It should be considered, however, that H. seropedicae was the only species that demonstrated growth on agar, and therefore the only one able to enter the capillaries. We concluded that this methodology is not yet sufficiently optimised for our application and may be having a confounding effect upon chemotactic response. Further details of these potential factors can be found here:

Figure 5: a) Growth of H. seropedicae on Typtone and Yeast Extract agar inoculated with contents of a 1 µl capillary containing 100 µM naringenin after 60 minutes open-end submersion in bacterial solution. Plate was incubated for 24 hours at 30 °C. b) Growth of H. seropedicae on 1 % LB agar inoculated with contents of a 1 µl capillary containing motility buffer after 60 minutes open-end submersion in bacterial solution. Plates were innoculated via streaking technique and incubated for 24 hours at 30 °C.

Microscopy Observations

An alternative method of observing chemotactic responses is through the use of microscopy. Brightfield microscopy allows direct observations of bacterial responses. This will allow comparisons of motility and morphology from our experimental data to that of the published literature that was used to underpin our first iteration of the community model [link to modelling]. Using microscopy enables the development of a cell density:optical density index (CD:OD index), a method of converting the two values. This index was also used in the community model to adapt the growth curve data collected during bacterial characterisation in standard laboratory conditions.

Figure 6: Example of haemocytometer square at 40x objective containing H. seropedicae utilised for cell counting, cells along the bottom and/or right lines were not counted to avoid double counting.

The CD:OD index was produced utilising data collected from a haemocytometer. A haemocytometer is a specialised microscopy slide of a known volume, it also contains a grid at the centre. By counting the number of cells in 16 squares at the top right (Figure 6) and performing a series of mathematical calculations ([x], we were able to determine cell density. By utilising a spectrophotometer, we were also able to take a reading of the absorbance (600 nm) and thus link the two together (Table 3).

Table 3: Cell density (cells.ml^-1) of A. brasilense, A. caulinodans, H. seropedicae and E. coli at different optical densities and as a ratio of OD₆₀₀=1

Species (Strain)	Optical Density	Cell Density	Colony Count (Control)
A. caulinodans (ORS571)	0	0	0
A. brasilense (SP245)	0	0	0
H. seropedicae (Z67)	145.33	85.58	109.78
E. coli (DH5α)	0	0	0

Utilising a haemocytometer to count cells also allows observations of cell morphology and behaviour. This was used to an advantage as it allowed us to explore whether our bacteria’s morphology aligns with the literature that was followed to produce the community model. It also enabled observations of motility which was important after theories that the species were no longer motile which is why only H. seropedicae showed movement into the capillary during the attempt to quantify chemotaxis.

Table 4: Microscopy observations of diameter (µm), cell length (µm), and motility in A. brasilense, A. caulinodans, H. seropedicae and on a haemocytometer at 40x objective compared to information in utilised literature.

Species (Strain)	Length in Literature	Mean Diameter	Cell Length in Literature	Mean Cell Length	Motile (Y/N)
A. caulinodans (ORS571)	1.5-2.5[5]	1.9	0.5-0.6 [5]	0.5	No
A. brasilense (SP245)	2.1-3.8 [6]	2.0	1.0 [6]	0.7	Yes
H. seropedicae (Z67)	1.5-5 [7]	2.4	0.7 [7]	0.65	Yes (Highly)

Chemotaxis on Agar

Our third approach to understanding bacterial chemotaxis involved both qualitative and semi-quantitative analysis. Growth of the nitrogen-fixing bacteria on solid media was observed to understand chemotactic behaviour in response to naringenin.

Multiple different variants of agar assays were conducted to optimise the methodology.

Method 1:

The original method utilised 0.75 % LB agar plates with 10μl of 200 μM naringenin applied to one side of the plate and a sterile water control to the other. Bacteria were inoculated into the centre of the plate and it was hypothesised that colony growth would be distorted towards the side that contained naringenin, in the case of positive chemotactic behaviour. This assay was conducted with A. brasilense and E. coli. Neither bacterium showed a growth response favouring either the side with naringenin or the control.

From the results in this iteration, several key elements were identified that were incorporated moving forward. For example, as bacterial growth exhibits inherent variability, a qualitative assay may not be sufficient to identify differences in behaviour in response to different treatments. As such, a more qualitative approach was adopted for future assays. Another issue noted was that there is the potential that the agar percentage was too high resulting in poor diffusion through the medium. This may also have impacted the bacteria’s ability to move and grow towards the naringenin source.

Method 2:

The second iteration of agar assays reduced the agar concentration to 0.5 % and the naringenin concentration to 100μM to align with the findings of the impact of naringenin on growth rate. The plate was also laid out in a more quantifiable manner. This followed concerns of the chemoattractant diffusing onto the side of the control when on the same plate. In this method, the distance of bacterial growth towards the naringenin/control source was measured (Table 5).

Table 5: Mean distance of colony growth towards either naringenin or control source of A. brasilense, A. caulinodans, H. seropedicae and E. coli measured from the point of inoculation after 24 hours incubation. Distance is given in mm

Species (Strain)	Growth Distance Towards Naringenin	Growth Distance Towards Control
A. caulinodans (ORS571)	3.70	4.01
A. brasilense (SP245)	7.32	7.10
H. seropedicae (Z67)	5.87	5.66
E. coli (Z67)	8.33	7.67

Once again, no evidence of chemotaxis towards naringenin was observed in any species. It was noted that the ‘halo’ around H. seropedicae colonies on plates containing naringenin were constricted and more closely situated to the colony margin. Through consulting the literature, it was revealed that high concentrations of naringenin can repress genes involved in chemotactic behaviour in this species [8]. This may provide an explanation as to why chemotaxis was not observed in this species

Method 3:

The third and final iteration of agar assays was based on the gradient plate experiment used by Reyes-Darias et al. (2016) [9]. In this variant, 0.25 % Minimal A Salt agar was utilised and the naringenin concentration was further reduced to 50 μM. The concentration gradients were also left for 16 hours at 4 ˚C in order to form instead of 12 hours at room temperature. Initially, bacterial species were inoculated at different distances from the centre line where the naringenin or control was added; this interval increased by 5 mm until 40 mm. After analysing initial results, the inoculation distance was changed to reflect that which gave the best response index. The control was also altered to 1.5 % (v/v) ethanol as the method of dissolving naringenin was changed to be within the same percentage.

The response index, developed by Pham and Parkinson [10], accounts for a ratio between the edge of the colony nearest the chemoattractant source and the edge furthest from the same source. This ratio is then used to determine if there has been positive chemotaxis (RI >0.52), no effect (RI = 0.48-0.52) or negative chemotaxis (RI <0.48).

Results (Table 6) indicated that both A. brasilense (Figure 7a and 7b) and H. seropedicae (Figure 7c and 7d) experienced positive chemotaxis towards 50 μM between distances of 5-25 mm and 5-10 mm respectively. As such, further investigation utilised the distance that corresponded with the greatest RI value (15mm and 10mm respectively). For H. seropedicae, the colonies nearer the centre line again showed more constricted halos which may indicate that the naringenin concentration may still be too high. The response index of the control for all species at 5 mm was <0.48, suggesting chemorepulsion. This was anticipated as the control contains ethanol which possesses known antimicrobial properties and is commonly used to disinfect lab equipment.

Table 6: Average Response Index and standard error of A. caulinodans, A. brasilense, H. seropedicae and E. coli colonies grown on 0.25% Minimal A Salt agar containing a gradient of either 100µM naringenin or 1.5% ethanol (control). RI = D1/(D1+D2) in which D1 represents distance between colony edge nearest chemical source to site of inoculation whilst D2 represents distance between colony edge furthest from chemical source to site of innoculation [10]. Bacteria were innoculated 15mm or 10mm from naringenin source and incubated at 30

Species (Strain)	Naringenin Response Index	Control Response Index
A. caulinodans (ORS571)	3.70	4.01
A. brasilense (SP245)	7.32	7.10
H. seropedicae (Z67)	5.87	5.66
E. coli (Z67)	8.33	7.67

The response index for E. coli and A. caulinodans indicated no effect of naringenin upon chemotaxis. This may be due to the fact that the species is no longer motile, as observed during microscopy. However, it would be expected that A. caulinodans would still grow towards the source. As the other two species of nitrogen-fixers were demonstrated to show chemoattraction and both of which were motile, then the former may be the explanation. However, it may also be that the set up was not suitable. This will be, however, work for the future.

Conclusions

After successfully characterising how A. brasilense, A. caulinodans, H. seropedicae and E. coli behave in a laboratory environment through means of understanding colonies and growth rates, we began to explore bacterial chemotaxis toward naringenin. While it may be possible to observe this behaviour in a quantitative fashion via microscopy or microfluidic methods. These methods, from the data gathered from these series of experiments, require a higher level of optimisation than semi-quantitative based methods.

Importantly, we were able to successfully demonstrate chemotaxis of A. brasilense and H. seropedicae toward 50 µM naringenin, these results came from semi-quantitative agar-based assays. While no evidence for chemotaxis was demonstrated in A. caulinodans, it may be possible to do so in the future with aforementioned optimisation.

InterLab

REFERENCES

1. Jousset, A., et al. (2009). "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters." The Isme Journal 3: 666

2. Jousset, A., et al. (2009). "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters." The Isme Journal 3: 666

3. Vanitha SC & Umesha S (2011) Pseudomonas fluorescens mediated systemic resistance in tomato is driven through an elevated synthesis of defense enzymes. Biologia Plantarum 55(2):317-322.

4. United Nations, Department of Economic and Social Affairs, Population Division (2017) World Population Prospects: The 2017 Revision, Key Findings and Advance Tables. https://population.un.org/wpp/Publications/Files/WPP2017_KeyFindings.pdf

5. Food and Agriculture Organization of the United Nations (2015) World Fertilizer Trends and Outlook to 2018. http://www.fao.org/3/a-i4324e.pdf

6. 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).

7. Pfromm PH (2017) Towards sustainable agriculture: Fossil-free ammonia. Journal of Renewable and Sustainable Energy 9(3):034702.

8. Bitew YA, M (2017) Impact of Crop Production Inputs on Soil Health: A Review. Asian Journal of Plant Sciences 16(3):109-131.

9. 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.

10. Carmichael WW (2001) Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”. Human and Ecological Risk Assessment: An International Journal 7(5):1393-1407.

11. New Partnership for Africa's Development (2013) Agriculture in Africa - Transformation and Outlook. http://www.un.org/en/africa/osaa/pdf/pubs/2013africanagricultures.pdf

12. Food and Agriculture Organization of the United Nations (2017) World Fertilizer Trends and Outlook to 2020. http://www.fao.org/3/a-i6895e.pdf

13. Bergey, D. H., et al. (1984). Bergey's manual of systematic bacteriology. Baltimore, MD, Williams & Wilkins.

14. Gómez-Lama Cabanás C, Schilirò E, Valverde-Corredor A, & Mercado-Blanco J (2014) The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots. Frontiers in Microbiology 5:427.

15. Gross, H. and J. Loper (2009). Genomics of secondary metabolite production by Pseudomonas spp.

16. Sharma SB, Sayyed RZ, Trivedi MH, & Gobi TA (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587.

17. Ruffner, B., et al. (2013). "Oral insecticidal activity of plant-associated pseudomonads." Environmental Microbiology 15(3): 751-763.

18. Jousset, A., et al. (2009). "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters." The Isme Journal 3: 666

19. Vanitha SC & Umesha S (2011) Pseudomonas fluorescens mediated systemic resistance in tomato is driven through an elevated synthesis of defense enzymes. Biologia Plantarum 55(2):317-322.

20. Maheshwari DK (2012) Bacteria in Agrobiology: Plant Probiotics (Springer Berlin Heidelberg).

21. Despommier D (2011) The vertical farm: Controlled environment agriculture carried out in tall buildings would create greater food safety and security for large urban populations. J fur Verbraucherschutz und Leb 6(2):233–236.

22.World Health Organization. (2018). Q&A: genetically modified food. [online] Available at: http://www.who.int/foodsafety/areas_work/food-technology/faq-genetically-modified-food/en/ [Accessed 13 Sep. 2018].

Team:Newcastle/Results/Chemotaxis