Periplasm: Definition, Discovery, Functions and Periplasmic Space

It is the compartment in which the cytoplasm is surrounded in some prokaryotic cells.

The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in gram-negative bacteria.

Using cryoelectron microscopy, it has been found that a much smaller periplasmic space is also present in gram-positive bacteria.

The periplasm can constitute up to 40% of the total cell volume of gram-negative bacteria, but it is a much lower percentage in gram-positive bacteria.

Although bacteria are conventionally divided into two main groups:

Gram-positive and Gram-negative: according to its property of retention of Gram spots, this classification system is ambiguous, since it can refer to three different aspects (result of staining, organization of the cell envelope, taxonomic group).

They do not necessarily bind for some bacterial species. However, although the Gram stain response of bacteria is an empirical criterion, it is based on the marked differences in the ultrastructure and chemical composition of the two main types of bacteria.

These bacteria are distinguished from each other based on the presence or absence of an outer lipid membrane, which is a more reliable and fundamental characteristic of bacterial cells.

All gram-positive bacteria are limited by a single lipid membrane unit; they generally contain a thick layer (20-80 nm) of peptidoglycan responsible for retaining the Gram stain.

A number of other bacteria that are bound by a single membrane but stain gram-negatively due to the lack of the peptidoglycan layer (i.e., mycoplasmas) or their inability to retain the Gram stain due to their cell wall composition .

They are also closely related to gram-positive bacteria.

For bacterial cells ( prokaryotes ) that are joined by a single cell membrane, the term “monodermal bacteria” or “monodermal prokaryotes” has been proposed.

Unlike gram-positive bacteria, all archetypal gram-negative bacteria are bound together by a cytoplasmic membrane and an outer cell membrane; they contain only a thin layer of peptidoglycan (2–3 nm) between these membranes.

The presence of internal and external cell membranes forms and defines the periplasmic space or periplasmic compartment.

These bacterial cells with two membranes have been designated as ddermal bacteria. The distinction between monoderm and diderm prokaryotes is supported by conserved signature indels in several important proteins (namely, DnaK, GroEL).

In ddermic bacteria, the periplasm contains a thin cell wall composed of peptidoglycan.

In addition, it includes solutes such as ions and proteins, which participate in a wide variety of functions ranging from:

  • Nutrient binding, transport, folding, degradation, substrate hydrolysis, peptidoglycan synthesis, electron transport, and alteration of substances toxic to the cell (xenobiotic metabolism).

Importantly, the periplasm lacks adenosine triphosphate (ATP).

Discovery of the periplasm

Once considered an empty space, the periplasm is now recognized as a specialized region of great importance.

The existence of a region between the membranes of gram-negative bacteria became apparent when electron microscopy technology was developed to the point where samples could be chemically preserved, mounted on a resin, and very finely cut.

The so-called thin sections allowed electrons to pass through the sample when placed in the electron microscope.

Areas that contained more material provided more contrast and therefore appeared darker in the electronic image.

The region between the outer and inner membranes was white in appearance. For a time, this was interpreted as indicative of a void. From this visual appearance arose the idea that space lacked function.

In fact, the region was first described as the periplasmic space.

Techniques were developed that allowed the outer membrane to become extremely permeable or completely removed, while preserving the integrity of the underlying membrane and another stress-supporting structure called peptidoglycan.

This made it possible to extract and examine the contents of the periplasmic space.

The periplasm, as it is now called, proved to be a true cell compartment. It is not an empty space, but is filled with a periplasmic fluid that has a gel consistency.

The periplasm contains a number of proteins that perform various functions. Some proteins bind to molecules such as sugars, amino acids, vitamins, and ions.

By associating with other proteins bound to the cytoplasmic membrane, these proteins can release the bound compounds, which can then be transported to the cytoplasm of the bacterium.

Proteins, known as chaperones, are released into the periplasm and bind to another incoming molecule. Other proteins break down large molecules, such as nucleic acid and large proteins, to a size that is easier to transport.

These periplasmic proteins include proteases, nucleases, and phosphatases.

Additional periplasmic proteins, including beta-lactamase, protect the bacteria by degrading incoming antibiotics before they can penetrate the cytoplasm and its site of lethal action.

The periplasm represents a buffer between the external environment and the interior of the bacterium. Gram-positive bacteria, which do not have a periplasm, excrete degrading enzymes that act beyond the cell to digest compounds in forms that can be absorbed by the cell.

Periplasmic space

Between the inner and outer membranes is the periplasm, an aqueous environment that contains a high concentration of proteins and peptidoglycan, which probably forms a hydrated gel.

In contrast to the reducing environment of the cytoplasm, the periplasm is an oxidizing environment, and therefore cysteine ​​residues of periplasmic proteins are frequently involved in disulfide bonds.

A variety of functional categories of proteins are found in the periplasm, including.

  • Disulfide oxidoreductases, peptidyl-prolyl isomerases, chaperones, and proteases involved in protein folding and degradation.
  • Solute-binding proteins that carry sugars, amino acids, ions, and vitamins through space.
  • Lipoprotein classification proteins; detoxifying enzymes; and enzymes involved in peptidoglycan, lipopolysaccharide (LPS), and capsule biogenesis.

The gram-negative bacterial periplasm: size matters

Gram-negative bacteria are surrounded by two layers of membrane separated by a space called the periplasm.

The periplasm is a multipurpose compartment separate from the cytoplasm whose distinctive reducing environment allows for more efficient and diverse mechanisms of protein oxidation, folding, and quality control.

The periplasm also contains important structural elements and environmental sensing modules, and allows complex nanomachines to span the cell envelope.

Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer.

This intermembrane distance from the periplasm is critical for detecting outer membrane damage and dictates the length of the flagellar periplasmic rotor, which controls motility.

These exciting results resolve long-standing debates about whether periplasmic distance has a biological role and increase the possibility that mechanisms for the maintenance of periplasmic size can be harnessed for the development of antibiotics.

Gram-negative bacteria, such as the energy organelles of plants and animals (the chloroplast and mitochondria), have two layers of membrane called the outer and inner membranes.

The space between these two membranes is called the periplasm.

Long before single-celled eukaryotes, the periplasm evolved as the first extracytoplasmic compartment to provide an important competitive adaptation to gram-negative bacteria.

Early knowledge and discovery of the periplasm developed even before its morphological visualization.

In the 1960s, scientists were trying to understand how toxic enzymes involved in the breakdown of important biological molecules, such as ribonucleases and phosphatases produced by the gram-negative bacterium Escherichia coli, were not toxic to the cell.

Biochemical extraction methods suggested a separate compartment, since such extraction preserves the cytoplasm attached to the inner membrane, and these spheroplasts could grow back and synthesize more enzymes.

The development of electron microscopy led to the visualization of the two membrane bilayers separated by the periplasm.

The additional membrane allows the creation of the periplasm as a separate cell compartment whose novel functions probably provided a significant and perhaps even more important selective advantage than toxin exclusion.

These novel functions include protein transport, folding, oxidation, and quality control similar to the endoplasmic reticulum of eukaryotic cells.

The periplasm also allows the sequestration of enzymes that can be toxic in the cytoplasm, important functions of signaling and regulation of cell division.

In addition, it contributes to the cell’s ability to withstand turgor pressure by providing structural systems that work in conjunction with the outer membrane.

Such as peptidoglycans and lipoproteins, specific solutes and multidrug exit systems that contribute to a Donnan or ionic potential across the outer membrane.

The periplasm also contains the assembly platforms involved in the secretion of single-frame beta-barrel proteins, lipoproteins, and glycerol phospholipids to the outer membrane.

The outer membrane is a single organelle connected to other parts of the cell envelope through the periplasm. Gram-positive bacteria lack an outer membrane but have a more extensive peptidoglycan polymer that protects their surface.

In contrast to the bacterial inner membrane, which is a glycerol phospholipid bilayer similar to that of most mammalian membranes and which has a specific flux characterized by lateral diffusion, the outer membrane has a restricted flux.

It is a unique bilayer, with the inner leaflet having a typical glycerolipid content of phosphotidylethanolamine, phosphatidylglycerol and cardiolipin and the outer leaflet largely composed of a single glycolipid, lipopolysaccharide (LPS).

Lipopolysaccharide phosphates confer a negative charge on the surface, and a specific Donnan potential is created across the outer membrane into the periplasm.

The outer membrane functions as a selective barrier that allows the transport of valuable nutrients while providing a barrier against toxic compounds, such as cationic antimicrobial compounds produced by all organisms, including many gram-positive bacteria.

Another component of this barrier is outer membrane proteins with a unique beta barrel structure that insert into the outer membrane through a specific periplasmic chaperone system.

These proteins assemble on the outer membrane as specific points, indicating that the outer membrane likely assembles into specific discrete patches containing proteins and the unique asymmetric lipid bilayer.

These outer membrane proteins include porins, which can act as selective channels that allow hydrophilic substrates of a specific size to enter the periplasm.

Fortunately for humans, these porins carry hydrophilic beta-lactam antibiotics, allowing their penetration into the periplasm, where they target the synthesis of the important structural element of the cell wall: polymeric peptidoglycan.

The outer membrane in some bacteria is anchored to the peptidoglycan polymer through abundant lipoproteins, which insert into the inner leaflet of the outer membrane through specific secretion systems.

A variety of important protein complexes function as nanomachines, using the hydrolysis of adenosine triphosphate to secrete macromolecules or convert a motile organelle called flagella.

Therefore, the outer membrane and the inner membrane are also connected through the periplasm by membrane-spanning protein complexes.

Therefore, the outer membrane is made up of clearly assembled patches comprising a complex organelle that can be attached to the peptidoglycan layer and the inner membrane through covalent and non-covalent protein bonds.

The assembly of the outer membrane and its bond with the peptidoglycan and the cytoplasm creates a space between the inner membrane and the outer membrane, which is the periplasm.

Despite the important functions contained within the periplasmic space, for many years there has been a debate about the intermembrane distance or size of this compartment and whether there is a uniform separation between the inner and outer membranes throughout the cell.

There was concern that many of the visualizations of this space as being of a specific size were fixation artifacts for electron microscopy imaging and that, in fact, the space was actually just a potential space.

Bayer’s first electron microscopy studies demonstrated adhesions between the outer and inner membrane that destroyed part of these spaces.

He suggested that the adhesion points were areas in which the main outer lipid, lipopolysaccharide, was delivered to the outer membrane from its synthesis site on the inner membrane.

However, his work was later discredited as stemming from the observation of possible fixation artifacts.

Although many experts today believe that there may be actual protein-based adhesions between membranes because some outflow and transport systems do not contain components of sufficient dimensions to span the visualized space.

The presence of specific areas where the membranes are close together would explain how some of these flow pumps and outflow pumps from the adenosine triphosphate binding cassette might work.

These systems have periplasmic protein components that are essential for efflux, lipopolysaccharides, or other glycolipid transport, but lack an intrinsic size or polymeric nature large enough to reach the outer membrane and thus provide a mechanism. to promote transportation.

In addition, the periplasm contains many other components that require at least some volume for the periplasmic space, primarily the polymeric peptidoglycan layer that surrounds the cell.

At present, it is not clear how these transporters avoid this polymer and the width of the periplasm to come into contact with the membrane.

Although recent work demonstrating that outer membrane lipoproteins can coordinate peptidoglycan synthesis through direct contact indicates that at least some proteins can pass through pores in peptidoglycan to serve important functions.

Periplasm functions

The periplasm of gram-negative bacteria provides a unique and challenging environment for protein folding and stabilization, as it is devoid of adenosine triphosphate and is highly exposed to fluctuations in the external environment.

The lack of an energy source provides a particular barrier to processes that require energy input, for example, biosynthesis in the outer membrane.

To overcome these two problems, organisms like E. coli employ a series of general and specialized chaperones that help with normal function of the periplasm and also alleviate the effects of environmental stress.

Periplasmic chaperones are classified into two categories according to their functional properties:

Folding chaperones, which perform a function analogous to classical cytoplasmic heat shock proteins such as GroEL / GroES and DnaK, and carrier chaperones, which are involved in the stabilization and transport of specific substrates.

However, it becomes apparent when trying to assign chaperones such as SurA or PapD that there is substantial overlap between the two classes.

In fact, perhaps only small dimeric chaperones like FkpA and DsbC are totally limited to one of the two activities.

The normal functioning of the periplasm relies on both carrier chaperones and collapsible chaperones.

For proper export of most integral outer membrane proteins, which by necessity expose large patches of lipophilic surface area, there is an absolute requirement for at least one of the two Skp or SurA chaperones.

These two chaperones are capable of picking up outer membrane proteins as they emerge in the unfolded state of the Sec translocon and carry them to the BAM complex in the outer membrane, which then catalyzes insertion of the membrane.

In this case, the two chaperones would essentially function as carrier proteins for the outer membane protein.

It should be noted that there are also reports that some outer membrane proteins cross the periplasm in a relatively folded state and this could represent an alternative route for outer membrane biogenesis.

In fact, the pore-forming secretins of many bacterial secretion systems are not reported to use the standard Skp / SurA pathway and are instead believed to localize to the outer membrane with the help of specific ‘pilotines’, so they self-assemble into their final multimeric structures.

Lipoproteins also have a specific chaperone requirement, as their hydrophobic lipid anchors cannot traverse the periplasm without the help of LolA.

The LolCDE complex in the inner membrane consumes adenosine triphosphate to load lipoproteins into LolA, which transports them across the periplasm and delivers them to LolB for membrane insertion.

Chaperones of the chaperone / usher pathway play a similar role in pilus biogenesis, taking pilus subunits and transporting them to the pilus assembly site, the usher.

However, their involvement in the correct folding of the pilus subunits further blurs the distinction between folding and transport.

One of the key characteristics of the carrier chaperones identified so far is the ability to compensate for the lack of adenosine triphosphate in the periplasm.

These energy functions, required only by the specific environment in which their substrates reside, can be divided into two categories:

Energy transduction, in which energy is stored in the energy assembly steps in the outer membrane, and kinetic trap mechanisms, where secreted proteins are folded into conformations from which they cannot unfold once the chaperone.

These two distinct functions are helpful in understanding the functions of the periplasmic chaperones; however, they are by no means exclusive:

The chaperone / usher route uses both mechanisms to ensure fast and efficient production of pili that are highly stable once displayed on the cell surface.

Energy transduction systems in the periplasm include the insertion pathways for membrane proteins and the chaperone / usher pilus biogenesis pathway.

For example, SurA and Skp are believed to store the folding energy of outer membrane proteins, assisting with their insertion into the outer membrane.

Similarly, PapD only partially folds its cognate pilus subunits, storing the remainder of the folding energy to drive the DSE reaction.

And the lipoprotein loading in LolA makes use of the hydrolysis of adenosine triphosphate in the inner membrane, using this energy to drive its substate to LolB.

Bacterial secretion systems also require energy transduction systems to drive the export of their substrates across the outer membrane.

However, most of these are large complexes that span the entire cell envelope and are believed to use the energy of adenosine triphosphate directly.

The exception to this are type V secretion systems (autotransporters and two-partner secretion systems) that use a pore-forming translocator domain to secrete a functional passenger domain.

The detailed secretion processes of these systems have not yet been established, but in many cases the final step of export is thought to be due to the folding of the passenger domain on the extracellular side of the outer membrane.

This would imply a mechanism by which premature folding on the periplasmic side of the membrane is prevented, perhaps involving extrinsic periplasmic chaperones or a domain with IMC activity.

The kinetic trap mechanism of periplasmic chaperones, utilized by protease propeptides, chaperone / usher pathways, and possibly lipase folding factors, makes use of the ability of some chaperones to catalyze folding directly.

By reducing the energy barrier to folding or assembly, they allow non-covalent reactions to occur on a time scale that is physiologically relevant.

However, once their target proteins have been secreted or transferred to the cell surface, the chaperones are no longer present and therefore the reverse reaction is blocked by an effective kinetic barrier.

This function of the chaperones explains the high stability of the pili involved in bacterial adhesion and of some secreted proteases.

Although the best characterized chaperones in the periplasm have specific substrates and carrier chaperone functionality, a variety of chaperones and more general folding catalysts have been identified in the periplasm, including DegP, FkpA, DsbC, DsbG, and PpiD.

These could potentially be involved in the normal folding of any other type of protein, including membrane-bound, secreted, or soluble proteins.

However, apart from the disulfide-bonding activity of Dsb enzymes and the proteolytic function of DegP, there is little evidence for its direct requirement under normal physiological conditions.

Instead, a likely role for this group of proteins is protection against protein unfolding caused by extrinsic factors such as heat shock.

Such environmental changes can cause native proteins to unfold, reducing their levels within the periplasm and exposing normally buried hydrophobic surfaces to the external environment.

In these circumstances, chaperones with an otherwise poorly understood function could bind to these hydrophobic surfaces, preventing aggregation and allowing partially unfolded proteins to regain their proper folded state.

It has been shown that very few soluble periplasmic proteins require the presence of chaperones for their correct folding or stabilization under normal conditions.

In fact, most of the studies that identified chaperone activity in the aforementioned chaperones have used model cytoplasmic substrates, rather than the periplasmic proteins that they would find in vivo.

It has been suggested that this lack of established chaperone targets in the periplasm could be explained by an inherent resistance of all periplasmic proteins to aggregation.

And while the detailed conclusions of the previous article are questionable, it is nonetheless plausible that periplasmic proteins have evolved in a propensity to fold properly without external assistance and remain stable under a variety of different conditions.

However, as illustrated in the specific example of the acid shock response, there is a definite requirement for protection of proteins against folding stress, and it seems likely that chaperones conserved within the periplasm are responsible for this protection.

It would be interesting to further understand the various functions of the general chaperone in protein folding and stabilization, and how their interaction keeps bacterial cells alive under varying conditions.

Understanding these processes could not only lead to improved recombinant expression systems in bacteria, but also provide targets for antibacterial agents that act only under specific conditions.