Alternative Roots

Exploring Bacterial Chemotaxis

Introduction Colony Morphology

Growth in LB Naringenin & Growth

Quantification Microscopy

Chemotaxis on Agar Conclusions

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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 strain ORS571, Azospirillum brasilense strain SP245, and Herbaspirillum seropedicae strain 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) were recorded (Table 1, Figure 1).

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 Yeast Extract Broth agar 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 inoculated; stab-inoculation 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 1a). If the culture is spread across the plate, then it typically takes a circular or irregular form (Figure 1b). 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 % Yeast Extract Broth 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-inoculated.

Growth Rates in Liquid Media

From initial iterations of our community model, 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 (Figure 2).

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. caulinodans 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.

Figure 2: Growth curves showing changes in absorbance at 600 nm of E. coli, A. caulinodans, H. seropedicae, and A. brasilense in LB at 30 °C for 72 hours. n=4 replicates, error bars indicate standard error of the mean.

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]. 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 that cell death may lead to results similar to 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 3: Optical density at 600 nm wavelength of 4 bacterial species (A. brasilense, A. caulinodans, H. seropedicae, and E. coli) after 24 hours of growth when grown in liquid media (LB) containing different concentrations of naringenin. n=4 replicates, error bars indicate standard error of the mean.

All species successfully grew in the presence of 0-150 μM naringenin (Figure 3). However, it was noted that when the concentration of naringenin exceeded 100 μM, the amount of error also increases. This suggests that naringenin begins to have a greater impact on some, but not all, bacteria in the solution. 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 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 (DH5α)	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 3). 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. 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.

Figure 3: 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 inoculated 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. 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.

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 and performing a series of mathematical calculations, 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).

A. brasilense was unable to be counted accurately. This was as the bacteria was difficult to view under the microscope due to human limitations, in addition to highly variable optical density readings but consistently low cell densities despite more than sufficient incubation times. This happened throughout all replicates and as such results for this species have been omitted. Exploration of why this occurred shall be work for the future with the potential alternative approach of utilising flow cytometry.

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

Species (Strain)	Optical Density	Cell Density
A. caulinodans (ORS571)	1.0	3.14x10⁵
H. seropedicae (Z67)	0.05	1.05x10⁶
E. coli (DH5α)	1	7.4x10⁸

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)

While completing microscopy evaluations, we utilised the ibidi µ-Slide III 3-in-1 Chemotaxis Microscopy Slide provided by our sponsors at ibidi. This specialised microscopy slide was designed to allow real time observations of chemotactic behaviour in response to an adjustable chemical gradient. Through working with experts at ibidi, we were able to successfully seed all 4 species of our bacteria onto the uncoated, hydrophobic variant of the slide. This would allow us to begin quantifying naringenin chemotaxis in a modern and easily repeatable manner. Continued work with ibidi in the future demonstrates the exciting potential to quantify naringenin to further improve the design of our community model in the future. More information on ibidi and the µ-Slide III 3-in-1 Chemotaxis Microscopy Slide can be found here.

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 10 μl 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 100 μM 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 (DH5α)	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 [4], 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 and H. seropedicae 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 (15 mm and 10 mm 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 100 µM 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 inoculation [4]. Bacteria were inoculated 15 mm (A. brasilense and E. coli) or 10 mm (A. caulinodans and H. seropedicae) from naringenin source and incubated at 30 ˚C.

Species (Strain)	Naringenin Response Index	Control Response Index	Chemotactic Response
A. caulinodans (ORS571)	0.478	0.473	Negative
A. brasilense (SP245)	0.552	0.515	Positive
H. seropedicae (Z67)	0.625	0.5	Positive
E. coli (DH5α)	0.472	0.493	Negative

The response index for E. coli and A. caulinodans indicated negative chemotaxis in response to naringenin. This may be due to the fact that the naringenin was dissolved in 1.5 % ethanol which is commonly used to to sterilise due to ethanol's antimicrobial properties. As the other two species of nitrogen-fixers were demonstrated to show chemoattraction and both of which were motile, unlike A. caulinodans, it may be possible that the loss of motility combined with the antimicrobial properties of ethanol are triggering this result. This would mean the experimental set-up was not appropriate and thus requires further work. This will be 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.

Chemotaxis

REFERENCES & ATTRIBUTIONS

1. 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.

2. Pedrosa FO, et al. (2011) Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses. PLoS Genetics 7(5):e1002064. 261.

3. Lee K-A, Moon SH, Kim K-T, Mendonca AF, & Paik H-D (2010) Antimicrobial Effects of Various Flavonoids on Escherichia coli O157:H7 Cell Growth and Lipopolysaccharide Production. Food Science and Biotechnology 19(1):257.

4. Pham HT & Parkinson JS (2011) Phenol Sensing by Escherichia coli Chemoreceptors: a Nonclassical Mechanism. Journal of Bacteriology 193(23):6597-6604.

5. Dreyfus BG, JL; Gillis, M (1988) Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a Stem-Nodulating Nitrogen-Fixing Bacterium Isolated from Sesbania rostrata. International Journal of Systematic Bacteriology 38:89-98.

6. Tarrand JJ, Kried NR, Doebereiner J (1978) A Taxonomic Study of the Spirillum lipoferum Group, with Descriptions of a New Genus, Azospirillum gen. nov. and Two Species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Canadian Journal of Microbiology 24: 967-980.

7. Baldani JI, Baldani VLD, Seldin L, Doebereiner J (1986) Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a Root-Associated Nitrogen-Fixing Bacterium International Journal of Systematic and Evolutionary Microbiology 36: 86-93, doi: 10.1099/00207713-36-1-86.

8. Tadra-Sfeir MZ, et al. (2015) Genome Wide Transcriptional Profiling of Herbaspirillum seropedicae SmR1 Grown in the Presence of Naringenin. Frontiers in Microbiology 6:491.

9. Reyes-Darias JA, García V, Rico M, Corral-Lugo A, & Krell T (2016) Identification and Characterization of Bacterial Chemoreceptors Using Quantitative Capillary and Gradient Plate Chemotaxis Assays. Bio-protocol 6(8):e1789.

Attributions:

Authors: Connor Trotter and Sadiya Quazi

Troubleshooting: Umar Farooq and Patrycja Ubysz

Proofreading: Dr Alice Banks

Team:Newcastle/Results/Chemotaxis