ISOLATION, CHARACTERIZATION AND

MODIFICATION OF CHEMOTACTIC
MOTILITY BEHAVIOR IN BACILLUS
SPECIES OBTAINED FROM CONCRETE
SOIL
Jaikishan Advani, Dr. Arjumanara Surti*, Sophia College, Mumbai
 Introduction
Bacteria has been considered to be simple hardwired organism since
decades which exhibits very little behaviors.

Behavior of bacteria towards the chemicals is immensely studied,

mainly the chemotaxis of the cells towards the gradients of attractants
or away from the repellants.

Whether this behavior can be altered has never been addressed in

bacteria, as behavior modification (learning) is considered as an
phenomenon of higher organisms. Genetic adaptation is the only
phenomenon widely known in bacteria which causes the cell behavior
to alter, e.g: drug resistance, which is far different from behavioral
modification (learning).
 Only a single recent study has been reported so far illustrating the change in the
expression of metabolic pathways in E.coli, for survival in low O2, based on
associative training with high temperature before exposing the cells to O2 drop.
This type of repeated training with high temperature followed by O2 drop led E.coli
to express the metabolic pathways for low O2 just on the exposure to high
temperature alone, and this character was inherited to subsequent generations,
before getting diminished.

Suggesting the predictive learning based on associative conditioning, very different

phenomenon from classical genetic adaptation.

Schematic representation of E.coli Associative Learning

temp= A protein
oxygen= B protein

temp + oxygen = A+B proteins

(conditioning/training)

temp= A+B proteins (behavioral change, i.e,

learning)
 Pavlov`s type of associative conditioning model was designed and
developed for the motile bacterial isolate, obtained from soil

Loaded Electric
Cells Attractant+ Shock
Model for Migrated Cells in Pulses
Capillary
Associative Conditioning
through motility:

Loaded Attractant
(Conditioned (No migration No
Behavioral Modification Cells) of cells in Shock

of cells through motility capillary)

(Learning Assay):
 Aims and Objectives
Aim: Modification of the chemotactic motility behavior of
motile rods through associative conditioning, to determine
the phenomenon of learning in bacterial motility.
Long term objective: to modify the normal chemotactic behavior through
associative conditioning by coupling unconditional attractant with the
aversive electric shock.
Intermediate term objective:. to develop/standardize chemotactic
behavioral assays, to understand whether the movement is directional or
random.
Short term objective: to determine their characters by staining and
biochemical tests.
Immediate term objective: to isolate motile rods (in chains) or
filamentous rods in pure form from soil sample.
 Materials & Methods
Sample Collection
Preservation of Samples

Site of Motile Rods

Isolation of Motile Rods
Biochemical typing: Biochemical characterization of the isolated motile rod
was carried out using Bergey`s manual (gram positive long rods category),
and minimal biochemical tests: catalase test, indole test, voges- proskaeur test,
nitrate reduction test, citrate test, maltose utilization tests were performed.

Preparation of Agar-Slide chemotactic chamber:

Standardization of Chemotaxis:
Control: 100 microlitres of loaded cells of O.D 0.05 /0.03 vs
Chemotaxis buffer
Test: 100 microlitres of loaded cells (of standardized O.D) vs
Lysine (concentrations 5 micromolar/10 micromolar)

Remove the cells from capillary by suspending into 1ml st.saline

Dispense 0.1ml from the saline and perform spread plating on

st.NA

Calculate the cells migrated in the capillary from the cell count
obtained after incubation for 24hrs at RT

Observe the pattern of the colony count in control and test

Plot the graph of number of cells migrated in capillary vs the

concentration of lysine/control
Cell Tracking and Motion Analysis to detect the chemotaxis:

Cells are taken on the cavity slide and 10 micromolar of lysine is added from
the sides of the coverslip in the drop form with capillary, to create the
gradient

Live video is recorded by camera mounted on the microscope

The video is loaded in the Tracker software and the tracks of the cells are
traced, all the motile cells in 20 observed fields are tracked, to avoid any
biasness in tracking the cell response

Similarly the control set is run, with the drop of distilled water

The direction of the motility of the cells in the lysine is observed as compared
to expected random motility in the control set

The orientation and displacement of the cells

Is calculated by using Image processing
Software ImageJ
Development of chemotactic behavioral modification system
through associative shock conditioning:
Fabricating Electric Pulse administering system for electric
shock conditioning of cells to modify the chemotactic motility
behavior:

Fabricated electric pulse administering system which can generate

desired voltage of 300 Volts for desired time duration (250
Administering 4 pulses of 300 volts per
250 milliseconds into the cell suspension
along with 10 micromolar of lysine so that
the cells associate the attractant stimulus
of lysine with the aversive stimulus of shock

Assaying the chemotactic behavioral modification after associative conditioning

(Learning and Memory assay):

The conditioned cells are taken and assayed now with lysine gradient
alone, and the video recorded and tracked, further the directionality
of the cells is checked by determining orientations of the cells and
displacement covered by cells
 Result and Discussion

Pure
culture
of motile
rod

Gram
Positi
ve

Catalase +ve Indole ve VP ve Citrate ve

Nitrate ve Maltose-ve
Chemotactic response towards Lysine:

Control runs with chemotactic buffer (average cell count= 43 cells/0.1 ml)
Test runs with 5 micromolar of lysine (average cell count=81 cells/0.1ml)
Control run with chemotactic
buffer showing
(80 cells/0.1 ml) and Test
run with 10 micromolar
of lysine showing
(more than 300 cells/0.1ml)

Plot of number of cells migrated in capillary to concentration

of lysine
Cell Tracking and Motion analysis of cells for chemotaxis towards lysine:
micromol
ar
Directed motility of cells towards Lysine(Unconditioned)

Modification of directed motility

away from Lysine(Conditioned)

Displacement calculations in control cells and test cells towards
10 micromolar of lysine:

Displacement of the conditioned(learned) cells away

from 10 micromolar of lysine:
 Discussion
Lysine is an attractant for Bacillus at the threshold
concentrations of 10^-6 molar (i.e, Bacillus has
threshold concentration for lysine of 3
micromolar)
Number of cells migrated in capillary was about 3000
in 10 micromolar lysine for possible
B.circulans/macerans, which is less than known,
7,800 in 3 micromolar lysine in B.subtilis
Voltage-time paradigm which effects the motility of
E.coli is 150 V for 250 mS, such affect on Bacillus
motility haven`t been studied, which is found to be 4
pulses of 300V for 250 ms
Associative learning in paremoecium suggests that
paremoecium is able to associate the vibration and
electric shock, and alter its behavior towards
vibration alone, such study haven`t been done in
 Conclusion
The bacteria were able to show the directed motility towards 10
micromolar of lysine within 4 seconds in cell tracking test. 4 pulses of
300V for 250 mS were found to affect the motility of Bacillus and
increased speed of random runs with low tumbling were observed within
a minute of treatment. On the shock conditioning by coupling the 10
micromolar lysine with 4 pulses of 300V for 250 mS the bacteria was
conditioned/trained to associate the attractant stimulus of lysine with the
aversive stimulus of shock, and the alteration/modification of the directed
motility was seen in further trials even without lysine, for atleast 2 mins
showing the memory retention for atleast following 4 mins, plausibly
suggesting that Bacillus species is able of associative learning.

Future Prospects:
Detecting how long the retention of the memory of the conditioned
stimulus is possible.
Detecting whether any possible role of Ca-calmodulin dependent
kinases(CAM kinases) or cAMP in bacterial learning.
Acknowledgements:
Dr. Arjumanara Surti (Guide and HOD of Microbiology Department, Sophia
college), for guiding the research, and providing facilities
ArtScienceBlr, Public Laboratory at Srishti Institute, Bangalore for assisting in
fabricating electrode
Mustafa, TechResource, Mumbai for assisting in electrode development and guiding
in cell tracking
MC Arunan, CUBE lab, HBCSE,TIFR, for organizing Fabrication Workshop and
Pagalapos study which assisted in developing research question on behavioral
modification in bacteria

References:
Pamela Lyon, The cognitive cell: bacterial behavior reconsidered, Front Microbiol. 2015
Ilias Tagkopoulos, Predictive behavior within microbial genetic networks, Science. 2008 Jun
CT Fernando, Molecular circuits for associative learning in single-celled organisms, J R Soc Interface. 2009
Todd M. Hennessey, Classical conditioning in paramecia, Animal Learning & Behavior, 1979 December
G W Ordal and K J Gibson, Chemotaxis toward amino acids by Bacillus subtilis., J Bacteriol. 1977 Jan
Wenyaun Shi, et al, Behavioral responses of Escherichia coli to changes in temperature caused by electric
shock , J Bacteriol, 1993
Weizmann Institute of Science, Science Daily, Scientists Show Bacteria Can 'Learn' And Plan
Ahead, 2009