An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Summary

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Abstract

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD₆₀₀ = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Introduction

Chemotaxis is important for colonization of plant growth-promoting rhizobacterial (PGPR) on roots and for understanding plant-microbe interactions¹. A class of low molecular weight compounds (chemoattractants) in plant root exudates induce the chemotactic movement of PGPR to the rhizosphere². Malic acid, citric acid, and other components in the root exudates stimulate chemotaxis of Bacillus strains³. For example, glucose, citric acid, and fumaric acid in maize root exudates recruit bacteria to the root surface⁴. D-galactose, which is derived from root exudates,induces the chemotaxis of Bacillus velezensis SQR9⁵. Organic acids, including fumarate, malic acid, and succinate, influence chemotaxis and colonization of various PGPR in the Cajanus cajan - Zea mays intercropping system⁶. Oleanolic acid in rice root exudates, acts as a chemoattractant for the strain FP35⁷. Other plant exudates (including histidine, arginine, and aspartate) can play a crucial role in the chemotactic response of bacteria⁸. Plant exudates function as a signal to direct the movement of bacteria, which is the first step during rhizosphere colonization. Plant colonization by PGPR is a process of enormous relevance, as PGPR are beneficial for the plant host.

Many methods have been used for analyzing bacterial chemotaxis. The swimming plate method is one of the methods described previously⁹. In this method, plates were made with a semisolid medium. A chemotactic buffer containing agar (1.0%, w/v) was added to the plate. The buffer is heated, and then mixed with the chemoattractant. Then, 8 µL of bacteria suspension was added dropwise to the middle of the plate and the plate was placed in an incubator at 28 °C. The plate was regularly observed and photographed. However, the experimental cycle of the swimming plate method was very long. In the capillary-like method¹⁰, a pipette tip serves as a chamber for holding 100 µL of bacterial suspension. 1 mL syringe needle was used as a capillary. A syringe needle containing chemoattractants with different concentration gradients was inserted into the 100 µL pipette tip. After incubation at room temperature for 3 h, the syringe needle was removed, the content was diluted and plated on the medium. The bacterial accumulation in the syringe was represented by colony-forming units (CFUs) in the plates. However, the experimental error within replicates for the capillary-like method was large. Another method used a microfluidic SlipChip device¹¹. Briefly, bovine serum albumin (BSA) solution was injected into all channels and removed using a vacuum. The solutions containing different chemoattractants (1 mM concentration for qualitative detection only), bacterial cells suspended in phosphate-buffered saline and phosphate buffered saline buffer (negative control) were added to the top, middle, and bottom microwells, respectively. Incubation was then performed in a dark environment at room temperature for 30 min. The bacterial cells were then detected in the microwells. The microfluidic SlipChip device, however, was expensive. Therefore, each of the methods described above had advantages and disadvantages.

We established an improved chemotaxis assay for the rapid identification of rhizobacterial chemoattractants in root exudates using sterile glass slides without complicated steps. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. Therefore, if used to identify a chemoattractant substance, this new method can save 2-3 days and reduce the cost of experimental materials.

Protocol

1. Materials and equipment

NOTE: Bacillus altitudinis LZP02 (CP075052) was isolated from the rhizosphere of rice in northeast China^12,13 for this study.

1. Culture B. altitudinis LZP02 in Luria-Bertani (LB) medium (peptone, 10 g L^-1; NaCl, 8 g L^-1 and yeast extract, 5 g L^-1) for 10 h. Collect cells by centrifugation at 9,569 x g for 2 min at 4 °C. and store with 15% glycerol at -80 °C.
   NOTE: For this experiment, rice seeds (Oryza sativa Longgeng 46) were provided by the Rice Research Institute of Heilongjiang Academy of Agricultural Sciences.

2. Collection of root exudates

1. Randomly distribute rice seeds in a growth chamber.
   NOTE: Rice seeds were sterilized with 30% H₂O₂ for 30 min and soaked in water overnight. The conditions were as follows: the controlled light (16/8 h light/dark cycle), temperature (22 ± 2 °C) and relative humidity (῀70%).
2. Culture rice seeds for a week and add sterile water twice.
3. Select rice seedlings of similar size and plant in 50 mL of Murashige and Skoog (MS) liquid medium. Incubate for 48 h at 22 °C under aseptic conditions.
   NOTE: Rice root exudates will be released into the MS medium^14,15,16.

3. Liquid chromatography-mass spectrometry analysis of root exudates

1. Collect 100 µL of the sample (MS medium containing root exudates) in a 1.5 mL centrifuge tube. Add 20 µL of the extraction solvent (acetonitrile-methanol-water, 2:2:1, including internal standard). Homogenize the sample at 45 Hz for 4 min, followed by ultrasonication on ice for 5 min in a water bath.
2. Repeat the homogenization and ultrasonic cycle three times. Incubate the sample at -20 °C for 1 h, followed by centrifugation at 133,778 x g and 4 °C for 15 min.
3. Transfer the resulting supernatant to LC-MS vials and store at -80 °C until UHPLC-QE analysis. Prepare the quality control (QC) samples by mixing equal portions of the supernatant of all samples.
   NOTE: Each sample volume was 600 µL (six replicates per experiment) in the presented experiment.
4. Perform LC-MS/MS analysis using UHPLC system, UPLC HSS T3 column (2.1 mm x 100 mm, 1.8 µm) and Q Exactive¹².
   1. Use 0.1% formic acid aqueous solution and 5 mmol/L ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B. Use formic acid and ammonium acetate as positive and negative ion modes, respectively.
   2. Set the elution gradient as follows: 0 min, 1% B; 1 min, 1% B; 8 min, 99% B. 10 min, 99% B. 10.1 min, 1% B; 12 min, 1% B. Set the flow rate and injection volume to 0.5 mL/min and 2 µL, respectively.
      NOTE: The macromolecule (>1,000 Daltons) cannot be detected.

4. Chemotaxis assay

1. Prepare the chemoattractant aqueous solution. Ensure that it is sterile. Filter the chemoattractant solution with a 0.22 µm bacteria filter.
   NOTE: The chemoattractant aqueous solution was the single substance obtained from the LC-MS studies dissolved in water. The concentration and volume can be adjusted appropriately according to different studies. Citric acid solution was used as an example. All actions must be performed by the side of a lamp.
2. Mark the middle position of the glass slide at an interval of 1 cm. Ensure that the glass slide is sterilized several times on the flame.
3. Add the 30 µL of chemoattractant solution on the left of the glass slide. Ensure that bacteria were cultured to the logarithmic stage (2 x 10⁸ CFU/mL) in LB medium. Add 30 µL of the bacterial solution on the right of the glass slide.
   NOTE: Prepare a negative control group with an equal volume of sterile water. The saline solution (0.9% NaCI) was used as the positive control in order to eliminate the changes caused by intermolecular force on the experiment.
4. Sterilize an inoculating loop several times on the flame. Use the inoculating loop to connect the chemoattractant aqueous solution to the bacterial solution and keep it at room temperature for 20 min on a clean bench.
   NOTE: The experiment must be carried out in a windless environment. Adjust the time before disconnecting the connection line for strains of different athletic abilities appropriately.
5. After 20 min, disconnect the connecting line with filter paper.
6. Ensure that the 1.5 mL centrifuge tube is sterile. Collect the chemoattractant aqueous solution on the left of the glass slide. Transfer the solution to the 1.5 mL sterile centrifuge tube.
7. Add the appropriate volume of safranin to the centrifuge tube. After 2 min, collect the miscible liquids for counting bacteria and microscopic observation with a blood counting chamber.

5. Results analysis

1. Determine the number of viable microorganisms attracted using the following equation:
   
   where NBC: Total number of bacteria cells; N: Number of bacteria in 80 grids.
   NOTE: Statistical analysis software was used for data analysis. The error was based on three different replicated experimental values and was calculated using one-way ANOVA followed by Turkey's post-hoc analysis. P ≤ 0.05 was considered significant.

Results

A total of 584 and 937 known metabolites were detected in the positive and negative ion indices, respectively. Previous studies have shown that chemoattractants are typically organic acids, amino acids, and carbohydrates^17,18.

In this study, 16 kinds of chemoattractants from the LC-MS studies in the rice rhizosphere exudates were selected for subsequent experiments (Table 1). Using the swimming plate method, we screene...

Discussion

Increasing research indicates that plant-bacteria interactions mainly occur in the rhizosphere and are influenced by root exudates^{20,21,22,23,24}. Plant root exudates include a diverse array of primary metabolites, including phenolic acids, organic acids and amino acids as well as more complex secondary compounds^25,

Materials

Name	Company	Catalog Number	Comments
2,5-dihydroxybenzoic acid	Beijing InnoChem Science & Technology C.,Ltd.	490-79-9
Acetonitrile	CNW Technologies	75-05-8
Ammonium acetate	CNW Technologies	631-61-8
Caffeic acid	Beijing InnoChem Science & Technology C.,Ltd.	331-39-5
Centrifuge	Thermo Fisher Scientific	Heraeus Fresco17
Citric acid	Beijing InnoChem Science & Technology C.,Ltd.	77-92-9
Clean bench	Shanghai Boxun Industrial Co., Ltd.	BJ-CD
Ferulic acid	Beijing InnoChem Science & Technology C.,Ltd.	1135-24-6
Formic acid	CNW Technologies	64-18-6
Fructose	Beijing InnoChem Science & Technology C.,Ltd.	57-48-7
Galactose	Beijing InnoChem Science & Technology C.,Ltd.	59-23-4
Glycine	Beijing InnoChem Science & Technology C.,Ltd.	56-40-6
Grinding Mill	Shanghai Jingxin Industrial Development Co., Ltd.	JXFSTPRP-24
Histidine	Beijing InnoChem Science & Technology C.,Ltd.	71-00-1
Internal standard: 2-Chloro-L-phenylalanine	Shanghai Hengbai Biotech C.,Ltd.	103616-89-3
Leucine	Beijing InnoChem Science & Technology C.,Ltd.	61-90-5
Malic acid	Beijing InnoChem Science & Technology C.,Ltd.	6915-15-7
Mannose	Beijing InnoChem Science & Technology C.,Ltd.	3458-28-4
Mass Spectrometer	Thermo Fisher Scientific	Q Exactive Focus
Methanol	CNW Technologies	67-56-1
Optical Microscope	Olympus	BX43
Phenylalanine	Beijing InnoChem Science & Technology C.,Ltd.	63-91-2
Proline	Beijing InnoChem Science & Technology C.,Ltd.	147-85-3
Scales	Sartorius	BSA124S-CW
Serine	Beijing InnoChem Science & Technology C.,Ltd.	56-45-1
Threonine	Beijing InnoChem Science & Technology C.,Ltd.	72-19-5
UHPLC	Agilent	1290 UHPLC
Ultrasound Instrument	Shenzhen Leidebang Electronics Co., Ltd.	PS-60AL
Valine	Beijing InnoChem Science & Technology C.,Ltd.	7004-03-7