..Recently, an increasing body of experimental and theoretical research investigates macroscopic movement in different types of bacterial colonies.

Experimentally, it has been shown that the macroscopic colony of bacteria can expand and move in 2D and 3D geometric space in search of food and can cross empty gaps, but how?Most people think that bacteria use pili (sometimes called flagella or pseudolegs) to "walk" (in horizontal orientation) or crawl (in vertical orientation) [1,2].Reference [1] stated that Myxococcus xanthus is a motile bacterium that does not produce flagella but glides slowly on solid surfaces.On the other hand, the experimental study shows that the flat or surface macroscopic growth of a bacterial colony in the case of two concentric food rings [3,4] follows two directions,i-its own electric field in the radial direction i.e. along its pseudo-legs and also,ii - along its own magnetic field in concentric circles, i.e. perpendicular to its pseudo-legs.In short, the current theory that bacteria move or search for food via their pseudo-legs as the main driving force is not valid.This obviously leads us to an alternative hypothesis that nature itself equips each bacterial cell with one or more free electrons as a "walking" mechanism to be able to survive and search for food which is the subject of this article.

Surface interface physiology is particularly important for single-celled organisms with respect to the maintenance of optimal cellular function. Bacterial cell surfaces possess a net negative electrostatic charge due to ionized phosphoryl and carboxylate substituents on outer cell envelope macromolecules that are exposed to the extracellular environment [1,2,5]. The degree of peripheral electronegativity influences the overall polarity of the cell surface and can be assessed based on the zeta potential which is most often determined by estimating the electrophoretic mobility of cells in an electric field. The purpose of this investigation is to help bacteriologists better apply instrumentation and fundamental principles regarding estimation of zeta potential as it relates to bacterial surface physiology.

The bacterial cell carries a negative charge Q= N e on its surface, (where N is the number of free electrons and e is the electronic charge equal to -1.6 E- 19 Coulomb).

This may be the consequence of evolution by natural selection.

However, we will not discuss in detail the origin or the mechanism of generation of negative charges in different types of bacterial cells in order to focus on the mechanism of its transport movement.

To this end, we propose the following hypotheses:

i- The so-called legs are actually negatively charged appendages that extend radially and act like strings or ropes pulling the bacterial cells in a direction anti-parallel to its own intrinsic E and at the same time they influence the bacteria rich in iron via magnetic force, i.e. in circular motion normal to E.

ii- The higher the number of free electrons in a particular bacterial species,

the greater its negative charge on its surface. This may be a function of the number of evolutionary stages it has passed through and therefore inhibits its ability to survive and adapt to food scarcity conditions over time in 3D geometric space.

Colonies of bacterial cells are the main source of disease and food. Bacterial microbes are unicellular microorganisms lacking a nuclear membrane, metabolically active and when they grow, beyond a certain limit, under appropriate conditions of food, water and oxygen, they divide by binary fission.

In other words, bacterial growth is the proliferation of bacteria into two daughter cells, in a process called binary fission. Provided no event occurs, the resulting daughter cells are genetically identical to the original cell. Therefore, exponential bacterial growth occurs.

The progression of bacterial colonies over time goes through three main phases, namely the lag phase, the logarithmic or exponential growth phase and finally the exponential death or decay phase [1].

The physical interactions of growing bacterial cells with each other and with their growth environment, expressed mathematically via Diririclet boundary conditions, significantly affect the structure and movement dynamics of biofilms.

The currently burgeoning field of theoretical and experimental studies, especially of the movement or walking of bacterial cells, focuses on the role of their legs and falagellae Fig1.

Fig.1 Bacteria cell anatomy

The "walking mechanism" of bacterial cells and the role of their legs and falagellae in their transfer or walking is the subject of this article.

Katsumi Imada et al 2018 [2] states that the bacterial flagellum is a mobile organelle composed of thousands of protein subunits. The filamentous part that extends from the cell membrane is called the axial structure and consists of three major parts, the filament, hook, and rod, and other minor components. Each of the three main parts shares a similar self-assembly mechanism and a common basic architecture of subunit arrangement while showing quite distinct mechanical properties to achieve its specific function. Structural and molecular mechanisms produce these different mechanical properties of the axial structure, such as the so-called filament. He adds that Gram-negative bacteria, such as Escherichia coli and Salmonella, swim by rotating helical filamentous organelles called the flagellum. The flagellum rotates at a relatively high speed and is driven by a reversible rotary motor embedded in the cell membrane at the base of the filament.

Sandra Postel et al 2016 [3] stated that Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Considering that the flagellin filaments on which the FliD oligomers are attached vary in number of protofilaments between bacteria, their results suggest that the stoichiometries of the FliD oligomers vary from one bacterium to another to complete their filament assemblies.

He adds: “Many bacteria, including several that cause disease in humans, have long, whip-like appendages called flagella that extend well beyond their cell walls. Flagella can spin and propel bacteria through liquids, such as water or blood, and they are mostly made up of thousands of copies of a single protein called flagellin. During the construction of flagella, the flagellin proteins are placed in their appropriate positions by another protein called FliD, several copies of which form a cap at the end of the flagella. Without FliD, bacteria cannot properly assemble flagella and therefore cannot swim; it also hinders their ability to cause disease

Marko Nedeljkovic, 2021 [4] stated that the bacterial flagellum is one of the most complex and dynamic biological nanomachines known and has attracted attention since its discovery in the late 19th century. The history of flagellar research has gradually moved from biological, purely morphological studies, to biochemical and biophysical studies at the atomic level, enabling current understanding to the point of being able to modify it for various purposes. The bacterial flagellum is a complex and dynamic nanomachine that propels bacteria through liquids. It consists of a basal body, a hook and a long filament.

The flagellar filament is composed of thousands of copies of the flagellin protein (FliC) arranged in a helix and ending in a filamentary cap composed of an oligomer of the FliD protein. The overall structure of the filament core is preserved across bacterial species, while the outer domains show great variability and in some cases are even completely absent.

Flagellar assembly is a complex and energetically costly process triggered by environmental stimuli and, therefore, highly regulated at the transcriptional, translational, and post-translational levels. Besides its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction, and pathogenicity, such as adhesion to surfaces, secretion of virulence factors, and formation. of biofilms. Additionally, due to their ability to elicit potent immune responses, flagellins play a role as adjuvants in vaccine development.

W W Wilson et al 2007[5] stated that

Never the less, up to our knowledge the preceding work never described the effect of boundary geometry on the macroscopic colonial growth especially in food empty zones.

It is perhaps the first comprehensive theoretical and experimental study to fuse experimental microbiology with the foundations of theoretical mathematical physics to further reveal the growth properties of bacteria in two-dimensional geometric space.

Here, an agent-based 3D model is formulated to describe the establishment of single expanding bacterial colonies by the physical force of their growth. With a single set of parameters, the model captures key dynamic characteristics of colony growth by nonmotile, non-EPS-producing E. coli cells on hard agar. The model, supported by experiments on colony growth in different nutrient types and concentrations, suggests that the radial expansion of colonies is not nutrient-limited as commonly believed, but by mechanical forces. Rather, nutrient penetration governs the vertical growth of colonies, through thin layers of vertically oriented cells lifting their ancestors from below. Overall, the model provides a versatile platform to study the influences of metabolic and environmental factors on bacterial colony growth and morphology.

Bacteria have legs.? But in a sense, it's true: The movies show that the Pseudomonas aeruginosa bacteria wiggle themselves up into a vertical position and move leglike projections known as Type IV pili to wander around a surface surfaces

Xiaowei Zhao finds that [6]2014,the flagellum is one of the most sophisticated self-assembled molecular machines in bacteria. Propelled by the proton motive force, the flagellum rapidly rotates clockwise or counterclockwise, which ultimately controls the motility and behavior of bacteria. Escherichia coli and Salmonella enterica have served as important model systems for in-depth genetic, biochemical, and structural analysis of the flagellum, providing unprecedented insight into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part due to the limited structural information available regarding the intact rotor-stator complex and secretory apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Since the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional information on the in situ structure of the flagellar motor and other cellular components.

Wolfram Siede 2018 [7] performed a groundbreaking experiment to study the microscopic and macroscopic growth of bacterial colony in a multi-hole perforated plate Fig 2 and stated that,

based on cutting holes in standard medium agar plates where mature mycelium is grown. In terms of ease, time required and spore yield, we have found this method to be superior to others.

Fig 2. Macroscopic growth of bacteria colony containing macroscopic holes in the agar

He added,To start, an existing microconidia sample (or any source of spores/cells) is used for plating or streaking at a density that leads to a dense mycelium cover of a petri plate within a few days. After two days incubation at 30 °C, plates are typically sealed with Parafilm to prevent excessive drying and as a means to avoid spreading of spores of this BSL2 organism. After about 8 days of incubation in total (in the dark), holes of ∼6 mm diameter are cut into the now mycelium-covered agar, preferably by using a flamed cork borer or a similar cutting tool (Fig. 1A–C). The circular agar pieces are removed and discarded. The resulting holes should be not placed less than 10 mm apart. After continued incubation, additional growth may become evident around the holes (Fig. 1A). In roughly the same area, developing red pigmentation should be visible when the plate is viewed from the bottom (Fig. 1B). This appears to accompany formation of spores which are mostly microconidia in our case (Fig. 1D). In another Trichphyton species, carotenoid production has been associated with arthrospore formation.

.

However, it is clear from Figure 2-A that bacteria cannot travel or walk from the outside of the hole to the inside as if the barrier cannot be traversed.

In the previous work mentioned above, the authors have shown experimentally that bacterial cells have many nanometer-fine legs and one or more micrometer-wide flagella and that they play an important role in orienting bacterial populations towards food/agar.

However, they provide a conclusion that the hole or barrier (Fig 2-A) cannot be traversed, which is not entirely true.

In fact, the same bacteria can travel or walk from the inside of the circular food ring outwards as if there were no barrier Fig 3a&3b[8,9].

Fig. 3b

The obvious reason is that the electric field E is zero in the first case ( Fig. 3and exists in sufficient quantity in the second case( Fig. 3b).

The "walking dynamics" of bacterial cells and the role of their own thermal diffusion field or electric and magnetic field constructed by the geometric boundary conditions of Dirichlet in their transfer or walking is the core subject of this article.

In other words, how the food-free barrier can be crossed by bacterial cells where they are born and transported by E continuously for days, as explained in the following section II.

Ref

1- I.M. Abbas et al ,ijisrt, Does the growth of bacteria follow the solution of the heat diffusion equation with boundary conditions ,Oct 21.

2- 2-Katsumi Imada

Biophys Rev. 2018 Apr; 10(2): 559–570.

Published online 2017 Dec 12. doi: 10.1007/s12551-017-0378-z

Bacterial flagellar axial structure and its construction

3-Sandra Postel et al 2016

4-Marko Nedeljkovic,2021

5-W W Wilson et al, Spaceflight alters bacterial gene expression and virulence and reveals the role of the global regulator Hfq

J.W Wilson, C.M. Ott, K. Höner zu Bentrup, +37, and C.A. Nickerson [email protected] Author information and affiliations

Edited by Arnold Demaine, Drew University, Madison, NJ, and approved August 27, 2007

6-Xiaowei Zhao finds that [6]2014,

7-Wolfram Sied,[7]

Heliyon,

Volume 4, Number 7, July 2018, e00676

A "Hole Punched Plate" method to easily generate and harvest microconidia in the dermatophyte Trichophyton rubrum

8-I.M. Abbas et al, IJISRT Journal, How Colonial Pattern Formation in Macroscopic Bacterial Growth Follow its Own Electric and Magnetic Fields

9- I.M. Abbas, Researchgate, Formation of colonial patterns in the macroscopic growth of bacteria under its own electric and magnetic fields-II,Dec 21..