MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 3. BACTERIAL GROWTH AND REPRODUCTION

Bacterial Cell Division. The Cell Cycle

The coordinated Synthesis of Cell components by a microorganism drives its growth, reproduction, and ultimately The formation of a microbial culture or colony.

Like All living organisms, microorganisms are characterized by GROWTH AND REPRODUCTION. Cell growth refers to the coordinated increase in all chemical constituents (e.g., protein, DNA, RNA), accompanied by an increase in Cell size and mass. Cell growth is not limitless; upon reaching a certain size, The Cell stops growing and begins to reproduce.

Reproduction is the increase in the number of microbial Cells within a population. The time span from The Emergence of a cell to its division is called the generation time or ontogeny. In nature, this generally follows a predictable pattern (with minor exceptions): the smaller the Organism, the faster a new generation appears. For instance, E. coli cells divide every 20 minutes. If nothing hindered their division, within a day they would produce enough cells to build a pyramid with a base of 1 km² and a height of 1,000 m.

A definite correlation has been established between a cell's growth rate, its average mass, and the state of its chromosome. The initial event leading to Cell Division is the initiation of Chromosome Replication. Under normal conditions, this process is closely tied to cell mass. The rate of bacterial division depends on the availability of nutrients in the growth medium. If any essential substrates are lacking, the rate of replication initiation drops, though the rate of replication itself remains unchanged.

The duration of replication in most Prokaryotic Cells is constant (about 40 min). Once DNA duplication has begun, it proceeds to completion regardless of any Changes in the nutrient medium composition during that time. The time preceding the initiation of METABOLISM/36.html">DNA Replication is variable (ranging from 20 to 220 min). A prolonged cycle occurs when There is a significant delay before initiation starts. In this case, once replication begins, new initiation sites on the chromosome do not appear until all events of the Cell Cycle are completed. The time required for new initiation sites to appear is variable and largely dictates the total duration of the cell cycle. A short cycle is observed when new initiation sites emerge before the replication cycle is fully complete. Thus, the next replication cycle begins even before the current cell division is finished.

Replication is initiated by specific Proteins. If cells are placed in a medium lacking a key essential substrate, Protein Synthesis stops immediately. Although DNA Replication and cell division continue, a new replication cycle does not begin until protein synthesis resumes. Hence, initiators are proteins that interact with DNA and prime it to bind DNA polymerase.

DNA replication begins at the attachment point of the circular chromosome to the cytoplasmic membrane (CM), where the enzymatic machinery responsible for replication is localized. Initiating at the attachment point, replication proceeds in two opposite directions, forming an intermediate Structure characteristic of circular Chromosomes. In the replication zone (the Replication fork), Hydrogen Bonds maintaining the double-stranded Introduction/20.html">DNA Structure are broken over a short segment.

On the single-stranded segments thus prepared, which serve as templates, begins

the synthesis of complementary DNA strands (Fig. 3.1).

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Fig. 3.1. Schematic representation of circular bacterial chromosome replication:

a - parent DNA molecule; b - intermediate replicative forms; c - daughter DNA molecules after replication and Separation:

1 - replication origin (black arrows indicate the Direction of replication)

Following replication, the DNA molecules remain attached to the cytoplasmic membrane, which begins to grow between the DNA attachment points, thereby pushing them apart as if distributing them to different areas of the cell. This leads to the segregation of daughter DNA molecules and the formation of distinct, separated chromosomes (Fig. 3.2). Next, a transverse cell septum is formed.

Fig. 3.2. Schematic representation of The Mechanism of bacterial chromosome segregation:

a - bacterial cell containing a partially replicated chromosome attached to the membrane at the replication site; b - chromosome replication is completed (the bacterial cell contains two chromosomes, each attached to the cytoplasmic membrane; Cell wall synthesis is shown

1 - cytoplasmic membrane); c - further Synthesis of the cytoplasmic membrane and cell wall leads to the separation of daughter chromosomes. The onset of cell division via transverse septum formation is shown: 1 - DNA; 2 - attachment of the chromosome to the cytoplasmic membrane; 3 - cytoplasmic membrane; 4 - cell wall; 5 - synthesized region of the cytoplasmic membrane; 6 - new cell wall material

According to R. Wittenbury and S. Dow, the Formation of the transverse septum and cell wall varies across different groups of microorganisms, depending on the specific type of cell cycle:

- monomorphic, in which only one distinct morphological cell type is formed under normal conditions;

- dimorphic, where division yields two cells that differ in shape, size, or other distinctive features;

- polymorphic, characteristic of Bacteria capable of forming two or more morphological cell types depending on environmental conditions.

Each of these types is characterized by specific (normal) Features of the cell cycle.

The existence of permanent division types in microorganisms indicates that it is supported by the cell's hereditary material.

A monomorphic cell cycle is observed in most bacteria. A typical representative of bacteria with a monomorphic cell cycle is E. coli. During the growth of an E. coli cell, there is a linear, continuous increase in its volume and surface area. Prior to cell division, its growth rate increases abruptly, and this type of growth can be characterized as bilinear.

The mechanism by which daughter cells of Gracilicutes bacteria separate remains a subject of debate. For instance, some authors describe the synchronous growth of all envelope elements, noting that cells divide by constriction without forming a cellular septum. Other authors believe that the septum is formed from the cytoplasmic membrane and a murein layer.

In Gram-positive bacteria, the growth of the intercellular envelope can be traced more or less clearly. However, The regulatory mechanisms of cell division remain insufficiently studied.

The growth pattern and division of firmicute cocci belonging to various groups differ somewhat. Growth always proceeds along the axis of cell Symmetry, while division occurs in a plane perpendicular to this axis. In streptococci, this direction is stable, meaning they divide in a single plane. In staphylococci, division occurs in two mutually perpendicular planes, and the axis of symmetry shifts by 90⁰ after each division. Cell division in three mutually perpendicular planes, accompanied by corresponding shifts in the axis of symmetry, leads to the formation of clusters.

As the cell grows and the area of the peripheral wall increases, the perimeter of the cellular septum decreases. In proportion to this decrease, the septum ingrows toward the interior of the cell, which is accompanied by a reduction in the aperture connecting the daughter cells. The surface area of the septum remains almost unchanged until it completely closes. Along with the increase in cell wall area, the septum and the new portion of the peripheral wall thicken.

Cell division in staphylococci exhibits certain specific features (Fig. 3.3).

Fig. 3.3. Diagram of cell division in Staphylococcus aureus:

a - division geometry: dashed lines indicate the cross-section of the division plane; dark areas represent the portion of the envelope inherited from the mother cell; light areas represent the newly synthesized portion of the envelope; A–D represent points on the cellular septum; b - mechanism of cell division: 1 - initiation of division; 2 - cross-section of the initiation zone (shaded areas of the maternal envelope); 3 - action of the shedding lytic system; 4 - action of the disintegrating lytic system; 5 - action of the Cleavage lytic system. Dashed lines indicate the localization of the cleavage lytic system; arrows point to the locations where The activity of the respective lytic systems is manifested

In staphylococci, a septum is formed first, and its cleavage occurs precisely at the moment of division. The growing cell remains spherical or slightly oval until the daughter cells separate. The septum cleaves rapidly, but not completely. The daughter cells immediately acquire a rounded shape, but because the cleavage is incomplete, the cells remain connected. Despite the planes of successive division being perpendicular to each other, the undivided Regions of the septum may be arranged randomly, resulting in typical grapelike clusters of cells.

Unlike cocci, bacteria of the genus Bacillus exhibit asynchronous growth and division processes. They divide through the ingrowth and cleavage of a septum, with the septum growing faster than it is cleaved. New murein is synthesized and deposited on the inner side of the envelope. Older murein is pushed toward the cell surface, where it is presumably distributed evenly as the cell enlarges. Consequently, only the cellular septa—the cell poles following division and the narrow surface regions above the septa—are constructed exclusively from newly synthesized murein.

A dimorphic cell cycle is a phenomenon observed among Gram-negative bacteria. This type of cell cycle has been best studied in bacteria of the genus Caulobacter. It involves the formation of two cell types: motile (flagellated) and sessile (stalked). Both are capable of division, but their cell cycles differ (Fig. 3.4). Sessile stalked cells are considered maternal, while motile cells possessing a monotrichous flagellum are considered daughter cells.

Fig. 3.4. Stages of the Caulobacter life cycle

The motile cell possesses several polar pili (fimbriae) that function as phage receptors and also ensure adhesion to a solid substrate. Motile cells are incapable of division until they develop a stalk, which forms at the pole opposite the flagellum. The formation of the stalk is accompanied by the loss of the flagellar apparatus and pili.

A variation of binary fission is budding. During this process, a small outgrowth (bud) forms at one of the poles of the mother cell and enlarges as growth continues. Once the bud reaches the size of the mother cell, it detaches. Thus, cells of Nitrobacter agilis and certain photosynthetic bacteria of the genus Rhodopseudomonas divide such that their growth occurs from a single pole. This results in the formation of two unequal cells. The daughter cell possesses a flagellum, but it is lost before division begins.

Reproduction in typical representatives of Mycoplasmas, such as Mycoplasma mycoides, occurs via binary fission through the disintegration of filaments and rings into coccoid cells, as well as through a process resembling budding (Fig. 3.5). This produces small cells ranging in size from 0.125 to 0.150 µm.

Fig. 3.5. Diagram of mycoplasma division in a liquid medium

Some Representatives of the genus Hyphomicrobium exhibit a capacity for a polymorphic cell cycle depending on cultivation conditions. For instance, when grown on a medium containing methanol, they form hyphae and oval buds (Fig. 3.6), whereas on methylamine, they form brush-like cells from which motile daughter individuals bud off. Polymorphism in certain Hyphomicrobium strains is also observed on other substrates.

Fig. 3.6. Stages of the Hyphomicrobium life cycle

In some cyanobacteria, reproduction by multiple fission has been described. It begins with an increase in the size of the vegetative cell, which subsequently undergoes a series of rapid binary divisions occurring within an additional fibrillar layer of the mother cell wall. This is accompanied by The production of small cells known as baeocytes (small cells).

Their number ranges from 4 to 1,000 in different species. The release of baeocytes occurs upon the rupture of the maternal cell wall (Fig. 3.7); thus, multiple fission is fundamentally based on THE PRINCIPLE OF equal binary fission, with the specific feature that daughter cells do not undergo growth before dividing again.

Thus, both monomorphic as well as di- and polymorphic developmental cycles can be identified during bacterial cell division.

Fig. 3.7. Schematic representation of Prokaryotic Cell division modes:

a - division by transverse septum formation; b - division by constriction; c - budding; d - multiple fission: 1 - cell wall; 2 - cytoplasmic membrane; 3 - membranous structure; 4 - nucleoid; 5 - additional fibrillar layer of The cell wall

The mode of bacterial cell division cannot be determined through a superficial examination of a preparation. This requires prolonged observation of living cell behavior. Simple staining Methods also fail to determine the specific type of division with the necessary precision.

In both laboratory and industrial settings, one of the primary objectives is to obtain the maximum yield of microbial metabolic products. In all cases, particularly in the PRODUCTION OF MICROBIAL biomass, the fundamental indicator of the biotechnological process is the rate of cell growth and reproduction. Microbial growth AND reproduction are not constant values; they depend on the Nutritional Value of the medium, the physicochemical cultivation conditions, and the physiological state (age) of the culture. THE CONCEPT OF microbial age is well-defined in terms of the ontogenetic cycle, but quite relative in terms of actual time. The duration of the youthful stage and the Aging process of a microbial population depend on cultivation conditions. The main cause of aging is the accumulation of metabolic products and the depletion of the nutrient medium. With the continuous removal of metabolic products and the supply of nutrients, microbial cells can remain in a youthful state for an extended period. By employing such technological approaches, conditions can be created under which the microbial population remains in a state of continuous active metabolism.

Under ideal conditions, with the continuous removal of metabolic products and supply of nutrients, unending growth and reproduction of the microbial population can be observed. Knowing the generation time of a given microorganism (g) and the initial cell count (_Х0), one can determine their number at any given cultivation time (t):

Х =Х₀ ·2ⁿ,

where Х is the cell count at time t; Х₀ is the initial cell count at time t₀; n is the number of generations:

n = t / g ,

where t is the cultivation time; g is the generation time (cell doubling time).

In other words, the growth of the microbial population is determined by the exponent or the number of divisions (generations) located in the exponent. Therefore, this relationship is exponential.

Assuming a generation time of 20 min for Escherichia coli, a single cell could yield a total of X = 1 · 2⁷² = 4.72 · 10²¹ cells over a 24-hour period.

A different picture is observed in reality, particularly during batch cultivation. A batch system is defined as one in which, following the inoculation of a medium of appropriate composition, neither fresh sterile nutrient medium is added nor any metabolic products are removed.