Unlike eukaryotes, bacteria do not have a formed nucleus, but their DNA is not scattered throughout the cell, but is concentrated in a compact structure called a nucleoid. In functional terms, it is a functional analogue of a nuclear apparatus.
What is a nucleoid
A bacterial nucleoid is a region in their cells that contains structured genetic material. Unlike the eukaryotic nucleus, it is not separated by a membrane from the rest of the cellular contents and does not have a permanent shape. Despite this, the genetic apparatus of bacteria is clearly separated from the cytoplasm.
The term itself means "nucleus-like" or "nuclear region". This structure was first discovered in 1890 by the zoologist Otto Buchli, but its differences from the genetic apparatus of eukaryotes were identified already in the early 1950s thanks to electron microscopy technology. The name "nucleoid" corresponds to the concept of "bacterial chromosome", if the latter is contained in a cell in a single copy.
Nucleoid does not include plasmids thatare extrachromosomal elements of the bacterial genome.
Features of bacterial nucleoid
Usually, the nucleoid occupies the central part of the bacterial cell and is oriented along its axis. The volume of this compact formation does not exceed 0.5 microns3, and the molecular weight varies from 1×109 to 3×10 9 d alton. At certain points, the nucleoid is bound to the cell membrane.
The bacterial nucleoid contains three components:
- DNA.
- Structural and regulatory proteins.
- RNA.
DNA has a chromosomal organization that is different from eukaryotic. Most often, the bacterial nucleoid contains one chromosome or several copies of it (with active growth, their number reaches 8 or more). This indicator varies depending on the type and stage of the life cycle of the microorganism. Some bacteria have multiple chromosomes with different sets of genes.
In the center of the nucleoid DNA is packed quite tightly. This zone is inaccessible to ribosomes, replication and transcription enzymes. On the contrary, the deoxyribonucleic loops of the peripheral region of the nucleoid are in direct contact with the cytoplasm and represent active regions of the bacterial genome.
The amount of the protein component in the bacterial nucleoid does not exceed 10%, which is about 5 times less than in eukaryotic chromatin. Most proteins are associated with DNA and participate in its structuring. RNA is a producttranscription of bacterial genes, which is carried out on the periphery of the nucleoid.
The genetic apparatus of bacteria is a dynamic formation capable of changing its shape and structural conformation. It lacks the nucleoli and mitotic apparatus characteristic of the nucleus of a eukaryotic cell.
Bacterial chromosome
In most cases, bacterial nucleoid chromosomes have a closed ring shape. Linear chromosomes are much less common. In any case, these structures consist of a single DNA molecule, which contains a set of genes necessary for the survival of bacteria.
Chromosomal DNA is completed in the form of supercoiled loops. The number of loops per chromosome varies from 12 to 80. Each chromosome is a full-fledged replicon, since when doubling the DNA is copied entirely. This process always starts from the origin of replication (OriC), which is attached to the plasma membrane.
The total length of a DNA molecule in a chromosome is several orders of magnitude greater than the size of a bacterium, so it becomes necessary to package it, but while maintaining functional activity.
In eukaryotic chromatin, these tasks are performed by the main proteins - histones. The bacterial nucleoid contains DNA-binding proteins that are responsible for the structural organization of the genetic material, and also affect gene expression and DNA replication.
Nucleoid-associated proteins include:
- histone-like proteins HU, H-NS, FIS and IHF;
- topoisomerases;
- proteins of the SMC family.
The last 2 groups have the greatest influence on the supercoiling of the genetic material.
Neutralization of the negative charges of chromosomal DNA is carried out by polyamines and magnesium ions.
The biological role of the nucleoid
First of all, the nucleoid is necessary for bacteria in order to store and transmit hereditary information, as well as to implement it at the level of cellular synthesis. In other words, the biological role of this formation is the same as that of DNA.
Other bacterial nucleoid functions include:
- localization and compaction of genetic material;
- functional DNA packaging;
- regulation of metabolism.
DNA structuring not only allows the molecule to fit in a microscopic cell, but also creates conditions for the normal flow of replication and transcription processes.
Features of the molecular organization of the nucleoid create conditions for the control of cellular metabolism by changing the DNA conformation. Regulation occurs by looping out certain sections of the chromosome into the cytoplasm, which makes them available for transcription enzymes, or vice versa, by pulling them in.
Detection methods
There are 3 ways to visually detect a nucleoid in bacteria:
- light microscopy;
- phase contrast microscopy;
- electron microscopy.
Depending on the methodthe preparation of the preparation and the method of research, the nucleoid may look different.
Light microscopy
To detect a nucleoid using a light microscope, bacteria are preliminarily stained so that the nucleoid has a color different from the rest of the cellular contents, otherwise this structure will not be visible. It is also obligatory to fix bacteria on a glass slide (in this case, microorganisms die).
Through the lens of a light microscope, the nucleoid looks like a bean-shaped formation with clear boundaries, which occupies the central part of the cell.
Coloring methods
In most cases, the following staining methods for bacteria are used to visualize the nucleoid by light microscopy:
- according to Romanovsky-Giemsa;
- Felgen method.
When staining according to Romanovsky-Giemsa, bacteria are pre-fixed on a glass slide with methyl alcohol, and then for 10-20 minutes they are impregnated with a dye from an equal mixture of azure, eonine and methylene blue, dissolved in methanol. As a result, the nucleoid becomes purple and the cytoplasm becomes pale pink. Before microscopy, the stain is drained and the slide is washed with distillate and dried.
The Feulgen method uses weak acid hydrolysis. As a result, the released deoxyribose passes into the aldehyde form and interacts with the fuchsine-sulphurous acid of the Schiff reagent. As a result, the nucleoid becomes red, and the cytoplasm becomes blue.
Phase contrast microscopy
Phase contrast microscopy hashigher resolution than light. This method does not require fixation and staining of the preparation - the observation takes place for living bacteria. The nucleoid in such cells looks like a light oval area against the background of dark cytoplasm. A more effective method can be made by applying fluorescent dyes.
Nucleoid detection with an electron microscope
There are 2 ways to prepare a preparation for examination of a nucleoid under an electron microscope:
- ultra-thin cut;
- Cut frozen bacteria.
In electron micrographs of an ultrathin section of a bacterium, the nucleoid has the appearance of a dense network structure consisting of thin filaments, which looks lighter than the surrounding cytoplasm.
On a section of a frozen bacterium after immunostaining, the nucleoid looks like a coral-like structure with a dense core and thin protrusions penetrating into the cytoplasm.
In electronic photographs, the nucleoid of bacteria most often occupies the central part of the cell and has a smaller volume than in a living cell. This is due to exposure to the chemicals used to fix the preparation.
