Unlike eukaryotes, bacteria do not have a 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 an area in their cells that contains structured genetic material. Unlike the nucleus of eukaryotes, it is not separated by a membrane from the rest of the cellular contents and does not have a constant shape. Despite this, the genetic apparatus of bacteria is clearly demarcated from the cytoplasm.
The term itself means “nucleus-like” or “nuclear region”. For the first time this structure was discovered in 1890 by zoologist Otto Buchli, but its differences from the genetic apparatus of eukaryotes were already identified 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.
The nucleoid does not include plasmids, which are extrachromosomal elements of the bacterial genome.
Features of the bacterial nucleoid
Typically, the nucleoid occupies the central portion of the bacterial cell and is oriented along its axis. The volume of this compact formation does not exceed 0.5 μm 3 , and the molecular weight varies from 1 × 10 9 to 3 × 10 9 daltons. 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 other than 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 several chromosomes with a different set of genes.
In the center of the nucleoid, the DNA is equipped with a fairly tight. This zone is not accessible for ribosomes, replication and transcription enzymes. In contrast, the deoxyribonucleic loops of the peripheral region of the nucleoid are in direct contact with the cytoplasm and represent the active sites 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 the chromatin of eukaryotes. Most proteins are associated with DNA and are involved in its structuring. RNA is a product of the transcription of bacterial genes that occurs at the periphery of a nucleoid.
The genetic apparatus of bacteria is a dynamic formation capable of changing its shape and structural conformation. Nucleoli and mitotic apparatus, characteristic of the nucleus of a eukaryotic cell, are absent in it.
Bacterial chromosome
In most cases, the chromosomes of the bacterial nucleoid have a closed circular shape. Linear chromosomes are much less common. In any case, these structures consist of a single DNA molecule that contains a set of genes necessary for the survival of the bacteria.
Chromosomal DNA is equipped with supercoiled loops. The number of loops per chromosome varies from 12 to 80. Each chromosome is a full-fledged replicon, since when duplication of DNA is copied as a whole. This process always starts from the point of origin of replication (OriC), which is attached to the plasma membrane.
The total length of the DNA molecule in the chromosome is several orders of magnitude larger than the size of the bacterium, so there is a need for its packaging, but while maintaining functional activity.
In eukaryotic chromatin, these tasks are performed by the main proteins - histones. The bacterial nucleoid incorporates DNA-binding proteins that are responsible for the structural organization of genetic material, and also affect gene expression and DNA replication.
Nucleoid-associated proteins include:
- histone-like proteins HU, H-NS, FIS and IHF;
- topoisomerase;
- proteins of the SMC family.
The last 2 groups have the greatest impact on the supercoiling of genetic material.
The neutralization of negative charges of chromosomal DNA is carried out due to 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 realize 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.
Structuring of DNA not only allows the molecule to fit in a microscopic cell, but also creates the conditions for the normal course of replication and transcription.
Features of the molecular organization of the nucleoid create the conditions for controlling cellular metabolism by changing the conformation of DNA. Regulation occurs due to the weaving of certain sections of the chromosome into the cytoplasm, which makes them available for transcription enzymes, or vice versa, retraction inward.
Detection methods
There are 3 ways to visually detect a nucleoid in bacteria:
- light microscopy;
- phase contrast microscopy;
- electron microscopy.
Depending on the preparation method of the preparation and the research method, the nucleoid may look different.
Light microscopy
To detect a nucleoid using a light microscope, the bacteria are pre-stained so that the nucleoid has a color different from the rest of the cell contents, otherwise this structure will not be visible. Also required is the fixation of bacteria on a glass slide (while microorganisms die).
Through the lens of a light microscope, the nucleoid looks like a bean-like formation with clear boundaries that occupies the central part of the cell.
Painting methods
In most cases, the following methods of staining bacteria are used to visualize the nucleoid by light microscopy:
- according to Romanovsky-Giemsa;
- Felgen's method.
When stained according to Romanowski-Giemsa, the 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 azur, eonin and methylene blue, dissolved in methanol. As a result, the nucleoid becomes violet, and the cytoplasm becomes pale pink. Before microscopy, the paint is poured, and the drug is washed with distillate and dried.
The Felgen method uses slightly acid hydrolysis. As a result, the released deoxyribose transforms into the aldehyde form and interacts with the fuchsulfuric acid of the Schiff reagent. As a result, the nucleoid becomes red, and the cytoplasm becomes blue.
Phase contrast microscopy
Phase contrast microscopy has a higher resolution than light. This method does not require fixation and staining of the drug; observation is made of live bacteria. The nucleoid in such cells looks like a bright oval area against a dark cytoplasm. A more effective method can be made by applying fluorescent dyes.
Nucleoid Detection Using an Electron Microscope
There are 2 ways to prepare the drug for the study of the nucleoid under an electron microscope:
- ultra-thin cut;
- slice of frozen bacteria.
In electron micrographs of an ultrathin section of a bacterium, the nucleoid has the form of a dense mesh structure consisting of thin filaments that looks lighter than the surrounding cytoplasm.
In a section of a frozen bacterium after immunostaining, the nucleoid looks like a coral-like structure with a dense core and thin protrusions penetrating the cytoplasm.
In electronic photographs, the bacterial nucleoid 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 chemicals used to fix the drug.