Introduction:

The term prokaryote describes a cell type or collectively the organisms in the Domains Bacteria and Archaea. The two prokaryotic domains, Arcahe and Bacteria, are not closely related, although both are prokaryotes. In many ways, archae resemble eukaryotes more closely than they resemble bacteria. 

True multicellularity, in which the activities of individual cells are coordinated and the cells themselves are in contact, occurs only in Eukaryotes and is one of their major characteristics. Bacteria and many single-cell Eukaryoties form coloney aggregates of many cells, but the cells in the aggregates have little differentiation or integration of function. 

Bacterial genomes consist of nucleoids (about 4 million base pairs) and 1-50 plasmids (about 1500 base pairs) per cell. Plasmids are almost always circular and replicate independently. They commonly code for genes which confer antibiotic resistance. 

Structure/Differences with Eukaryotes:

Although all replicating cells share the common characteristics of having ribosomes, cytoplasm, a plasma membrane and DNA, there are important differences between eukaryotic and prokaryotic cells. With few exceptions, prokaryotes are single celled. Prokaryotes lack a membrane bound nucleus; instead they usually have a single circular chromosome made up of DNA and histone like proteins in a nucleoid region of the cytoplasm. Prokaryotic cells also have smaller circular DNA molecules called plasmids, which typically confer some selective advantage but are not essential. Cell division in prokaryoties takes place mainly by binary fission instead of by mitotic or meiotic cell division. Prokaryotes also do not have extensive membrane bound organelles like eukaryotes. Most bacteria and archaea have cell walls that confer shape and rigidity upon the cell and pevent cell lysis due to high osmotic pressures. 

Cell walls: Most, but not all, prokaryotes possess a cell wall and some number of other external structures. A cell wall, if present, is often complex, consisting of many layers. Minimally, it consists of peptidoglycan, a polymer composed of a rigid network of polysaccharide strands cross-linked by peptide side chains. The cell wall maintains the shape of the cell and protects the cell from swelling and rupturing in hypotonic solutions, which are commonly found in the environment. In bacteria, the structure of the cell wall can be used as the basis of differentiating between gram-positive and gram-negative bacteria. 

Arachae do not possess peptidoglycan, but some have a similar structure called pseudomurein. 

–Gram Positive Cell Walls: The thick peptidoglycan layer encassing gram positive bacteria traps crystal violet dye, so the bacteria appear purple in a gram stain. The gram positive cell wall is much simpler than the gram engative cell wall. The gram positive cells wall is compsed of a single, thick layer of peptidoglycan, molecuels of lipoteichoic aid and teichoic acid are embedded in the wall and exposed on the surface of the cell.

–Gran Negative Cells Walls: The gram negative cell wall is composed of multiple layers. The peptidoglycan layer is thinner than in gram positve bacteria and is surrounded by an additional membrane composed of lipopolysaccharide. Porin proteins form aqueous pores in the oter membrane. The space between the outer membrane and the peptidoglycan is called the periplasmic space. 

Capsules and slime layers: In some bacteria, an additional gelatinous layer, the cpasule, surrounds the outer wall layers and can be detected by stiaing. A more loosely organized form of capsule that is harder to detect is called a slime layer. A capsule enables a prokaryotic cell to adhere to surfaces adn to other cells and to evade an immune response by interfering with recognition by phagocytic cells. Thus, a cpasule contribues to teh ability of some bacteria to casue disease. 

Falgella and pili; Some prokaryoties are motile. Many prokaryotic cells possess one falgellum at one end while others have one at each end of the cell. Some prokaryotic cells possess many flagella in clusters or dsitributed around the cell. 

Endospores: Some prokaryotes are able to form endospores, a thick wall around their geneomes and a small amount of cytoplasm when they are exposed to environmental stress. These endospores are highly resistant to environmental stress such as heat and when conditions imporve, they can germinate adn return to normal cell division to form vegetative cells after decades or even centuries of dormancy. The bacteria that cause tetanus, botulism and anthrax are all capable of forming spores. With a puncture wound, tetanus endosores may be driven deep into the skin, where conditions are favorable for them to gemrinate adn casue disease or even death. 

Nucleoid region: Prokaryotic cells do not package their chromosomes in a membrane bounded nucleus. Instead, theri circular DNA chromosomes are condensed to form a visible region of the cell called the nucleoide region. 

Plasmids: Many prokaryotic cells possess plasmids, which are small, extrachromosomal, independently replicating circles of DNA. Plasmids contain only a few genes, and although these genes may confer a selective dvantage, they are not essential for the cell’s survival.

Smaller Ribosomes: Prokaryotic ribosomes are smaller than eukaryotes and differ in protein and RNA content. Antibiotics such as tetracycline and chloramphenicol bind to prokaryotic rebosomes adn block protein synthesis but do not bind to eukaryotic ribosomes.

Lack of internal compartments: While neither bacteria nor archaea have consistent internal compartments found in all cells, they can have both lipid and protein bounded compartments. 

Horizontal Gene Transfer: In sexually reproducing populations, traits can only be trasnferred vertically form parent to child. Prokaryotes do not reproduce sexually; however, they can exchange DNA between different cells of the same species and in many cases, between cells of different species. This horizontal gene transfer occurs when genes move from one cell to another by conjugation requiring cell to cell contact by transduction which requires viruses and even by transformation where genetic material is picked up directly from the environment. 

–Transformation: Natural transformation occurs in some gram engative and gram positive species which are “competent”. Transformation occurs when one bacterial cell has died and ruptured, spilling its fragmented chromosomal DNA or palsmids into the environment. This DNA can be absorbed by anotehr cell and incorporated into its genome. 

–Transduction: is the transfer of DNA between prokaryotes by viruses. 

–Conjugation: Plasmids encode functions that are not necessary to the roganism but that provide a selective advantage in particualr environmetns. For example, antibiotic resistance is not necessary, but is advantageous in the presence of antbiotics. The best known trasmissible plasmid is the E. coli F plasmid (fertility factor). Cells cotnaining F plasmids are termed F+ or donor cells and cells that lack the F plasmid are F-or recipient cells. The F plasmid is an extrachromosomal piece of DNA that uses cellualr machinery for replication. After being tranferred fro a donor to a recipient cell, the F plasmid can integrate into the recipients cell’s genome by recombination. These are regions of homology with the E. coli chromosomes called insertion sequences (IS) adn recombinbation between the insertion sequences adn the chromosome integrates the F plasmid into the chromosome. Some conjugative plasmids can acquire antibiotic resistance genes, becoming resistance plasmids, or R plasmids. 

–CRISPR innate defenses: Prokaryotes use a variety of innate defenses against viral infection including restriction modification systems (the source of restriction endonucleases) and toxin-antitoxin systems. A structure of repeated sequences with “spacer regions” termed CRISPR for “clustered regularly interspaced short palindromic repeats” have also been identified which form a adaptive protection against viral infection. In response to viral challenge, bacteria and archaea will integrate short segments of viral nucleic acid in these CRISPR loci, then sue them to produce an RNA that can be used to guid a complex that degrades viral nucleic acid. 

–Metabolic Diversity: Prokarytoes not only have great metabolic diversity but also exhibit considerable metabolic flexibility which is not seen in any other group of organisms. Carbon is obtained in reduced forms by heterotrophs and in oxidized form (CO2) by autotrophs. Chemotrophs transform energy by oxidizing reduced chemicals obtained form the environment; phototrophs, on the other hand, transform energy by harvesting light. Lithotrophs obtain electrons from reduced inorganic substances in the environment, which organotrophs obtain electrons from reduced carbon sources. Prokaryotes can be classified into 5 different nutritional types and at times can even switch nturitional strategy based on nutritional needs and the avaiability of nutrients. This rarely, if even, occurs with eukaryotes.

One names a prokaryote nutritional strategy by stringing together into one word the terms that describe the approaches used to obtain carbon, electrons and energy. For example, with chemolithoheterotrophs, the source of energy and electrons is a reduced chemical in the environment. However, the chemical is inorganic not organic. “litho” means “stone” and so these organisms are known as “stone-eaters”. Photolithoautotrophs reduce atmospheric CO2 to produce molecules for biosynthesis. The source of elections for this reduction is inorganic.  Some prokarytoes use water as an electron source; however otehrs can use hydrogen and different forms of sulfur as electrons sources. Prokaryotic photosynthesis can differ significantly form green plant photosynthesis. Green plant photosynthesis uses water as a source of electrons and produces NADPH to drive the Calvin cycle. Regardless of the nutritional strategy used, any energy transformation msut ultimately convert one source of chemical potential energy intot he form of chemical potential energy usable by all living organisms: ATP. 

–Prokaryotes have diverse respirations and fermentations:

The transformation of energy in one chemcial potential energy source into usable source for the cell involves either phtosyntehsis, respriation or fermentation. Tehre can be significant differences in how prokaryotes and eukaryotes perform these processes. In eukaryotes, cellular respiration uses an electron transport chain to recycle electron donors into electron acceptors (primarily NADH and FADH2), reduce oxygen to water and produce a proton motive force to drive ATP synthesis. The final electron acceptor is oxygen, so these respirations are aerobic.

Prokaryotic respirations, on the other hand, show rgreat diversity of both eelctron donors and acceptors. Respriations that use a final electron acceptor other than oxygen are anaerobic respriations. Such acceptors include oxidized substances such as nitrate and sulfate. Some bacteria can even use carbon dioxide as an elector acceptor in a respiration, producing methane gas as a product. 

Fermentations may be used in a cell when a teminal electorn acceptor is not available to allow respiration. Fermentation can recycle reduced electron acceptors, such as NADH to their oxidized form, which is needed in oxidation reactions that drive the formation of small amounts of ATP. There is some diversity in the kinds of fermentations seen in eukaryotes: yeasts can ferment sugars to produce ethanol; animal muscle can ferment sugars to produce lactic acid and under certain conditions, plant cells can ferment sugars to produce ethanol. Prokaryotes, however, show much greater diversity of fermentations. A variety of metabolic pathways used by different prokaryotes can produce different alcohols and acids from fermentation of pyruvate, the product of glycolysis. Some of these products have industrial application and contribute to the flavorings of milk and cheese products. The production of chocolate is possible only due to the fermentation of coca. 

An unusal fermentation seen in prokaryotes is performed by teh Clostridium genus of bacteria. This genus includes species that are industrially valuable but also includes dangerous human pathogens.

C. tetani is the causative agent of tetanus and is introduced into the body most commonly through deep puncture wounds to the skin. The environment deep in a wound is anaerobic and C tetani can grow by fermenting the abundant amino acids in tissues. 

C. botulinum is a pathogenic bacteria producing “Botox” toxin used in cosmetic enahncements to reduce wrinkling of the skin. This bacteria can be introduced itno foods during the canning process and if an abudant source of prtoein is avaiable in the anaerobic interio of a can, the bacteria can ferment the constituent amino acids to support grwoth. Ingesting the toxin produced by the apthogen can lead to serius illness and even death. This pathogen is particularly dangeorus to young children with poorly developed gastrointestinal (GI) flora. 

Classification/Evolution:

The eukaryotes developed at least 2.7 billion years ago, following some 1-1.5 billion years of prokaryotic evolution. (Cooper, “The origin and evolution of cells” NCBI Bookshelf, “The cell: a molecular approach, 2nd edition, Sunderland (MA), Sinauer Associates, 2000).

Present day prokaryotes, which include all the various types of bacteria, are divided into two groups –the archabacteria and the eubacteria –which diverged early in evolution. Studies of their DNA sequences indicate that the archaebacteria and eubacteria are as different from each other as either is from present day eukarotes. Thus, a very early event in evolution appears to have been the divergence of 3 lines of descent from a common ancestor, giving rise to present day archaebacteria, eubacteria and eukaryotes. (Cooper, “The origin and evolution of cells” NCBI Bookshelf, “The cell: a molecular approach, 2nd edition, Sunderland (MA), Sinauer Associates, 2000).

Cyanobacteria: are the largest and most complex prokaryotes. Cyanobacteria bacteria are the bacteria in which photosynthesis evolved. (Cooper, “The origin and evolution of cells” NCBI Bookshelf, “The cell: a molecular approach, 2nd edition, Sunderland (MA), Sinauer Associates, 2000). 

Eubacteria: Some archaebacteria live in extreme envionrments, which are unusual today but may have been prevalent in primitive Earth. For example, thermoacidonphiles live in hot sulfur springs with temperatures as high as 80C and pH values as low as 2. The eubacteria include the common forms of present day bacteria –a large group of organisms that live in a wide range of environments, including soil, water, and other organisms (e.g., human pathogens). (Cooper, “The origin and evolution of cells” NCBI Bookshelf, “The cell: a molecular approach, 2nd edition, Sunderland (MA), Sinauer Associates, 2000).

Important Roles of Prokaryote:

Carbon fixation:

The role of photsyntehtic prokaryotes in fixing carbon is critcal in biogeochemical cyclin of carbon. The organic compoudns that plants, algae, and photsynthetic prokaryotes produce fro CO2 pass through food cahins to form the bodies of all the econsystem’s heterotrophs. Some prokaryotes, called emthanogens, can also contribute to reduce carbon in the environment by producing methane.Ancient cyanobacteria are thought o ahve added oxygen to the Earth’s atomsphere as a by produce of their photosynthesis to produce reduced carbon compounds. Modern photosynthetic prokartyoes that oxidize water continue to contribute to teh production of oxygen and are partly responsible for the reduction of atmospheric and soil CO2 levels.

Cycling of Nitrogen:

The ntigoren in the Earth’s atmosphere is in the form of nitrogen gas. The triple covalent bond that linds the two nitrogen atoms is not easy to break. Only a very few species of prokaryotes are able to accomplish this, reducing N2 to ammonia (NH3) which is used to build amino acids and other nitrogen containing biolgocial molecules. When the organisms thatcontain these moelcuesl die, decompasers return ntirogen to the soil as ammonia. This is then converted to nitrate (NO2-) by nitrifying bacterial, making nitrogen available for plants. The nitrate can also be converted back into molecular nitrogen by denitrifiers that retun the ntirogen to teh atomsophere completing the cycle. 

Transcription

Bacteria contain a single type of RNA polymerase. In contrast to eukaryotes, Bacterial RNA polymerase is able to initiate transcription in vitro without the help of additional proteins. The variable subunit of the RNA polymerase is called “sigma”. There are 5 subunits which make up the RNA polymerase which is referred to as the “holoenzyme”. Each type of promoter is recognized by its own sigma subunit.

 In bacteria, there are 2 sequences on the 5′ (upstream) side of the first nucleotide to be transcribed which serve as promoter sites. At -10 on the DNA template there is a  TATAAT sequence (“pribnow box”) and there is another sequence at the -35 region. 

Unlike eukaryotes, there is no  or of the mRNA transcript in prokaryotes.

Translation

In bacteria, the mechanism for selecting a start codon is a little different since bacteria have no . Instead, bacterial mRNA contains a specific ribosome binding site which is rich in A/G, called the Shine-Dalgarno sequence, located a few nucleotides upstream of the AUG start site of each coding region. This sequence base pairs with the 16S rRNA of the small ribosomal subunit to position the intiating AUG codon in the ribosome. This ability of the bacterial ribosome to assemble directly on a start codon AUG so long as a Shine-Dalgarno sequence precedes it means that bacterial mRNAs are often polycistronic in that they encode several different proteins, each of which is translated from the same mRNA molecule. This is not the case for eukaryotes. 

In prokaryotes, the initiation complex includes a special initiator tRNA molecule charged with a chemically modified methionine, N-formylmethionine. The initiator tRNA is known as tRNAfMet. The initiation complex also includes the small ribosomal subunit and the mRNA strand. The small subunit is positioned correclty on the mRNA dur to a conserved sequence in the 5′ end of the mRNA called the ribosome-binding sequence (RBS) that is complementary to the 3′ end of a small subunit rTNA. Once the complex of mRNA, initiator tRNA, and small ribosomal subunit is formed, the large subunit is added, and translation can begin. With the formation of the complete ribosome, the initiator tRNA is bound to the P site with the A site empty. 

Bacteria employ a rather unique trick to insure that incomplete mRNAs are not translated into proteins which could harm the cell. When the bacterial ribosome translates to the end of an incomplete RNA, a special RNA called tmRNA enters the A site of the ribosome and is itself translated into a special 11 amino acid tag to the C terminus of the truncated protein that signals to proteases that this protein should be degraded. Eukaryotes deal with this problem another way by recognizing the 5′ cap and the poly A tail before translation can start. 

Replication

In Procaryotes: the entire DNA replication unit is called a replicon. Procaryotic chromosomes contain 1 replicon whereas eucaryotic chromosomes contain many replicons. The protein assembly that begins DNA replication in procaryotes is referred to as a primosome. 

Each replicon contains an origin of replication where DNA replication starts. In E coli, for example, this origin (“OriC”) is a sequence of 245 bp which contains 3 nearly identical nucleotide sequences which are AT rich as well as 4 binding sequences further upstream for the dnaA protein which initiates the bending and unwinding of the template DNA. 

Since bacteria are circular, bacteria do not have the special problems of  where telomeres need to be added to the chromosome end.

Gene Regulation

Bacteria avoid making enzyme(s) of a pathway when substrate is absent, but are always ready to produce these enzymes if the substrate should appear in the environment. In this way, bacterial cells are able to adapt very quickly to any change in concentration of nutrients in their environment. The primary mechanisms that bacteria have evolved to minimize the energy cost for this type of on-and-off regulation is by grouping genes that encode enyzmes of a particular pathway in a structural unit called an operon. An operon is a group of genes adjacent to one another on the bacterial chromosome which are transcribed from a single promoter as one long mRNA molecule. 

Operons can be under either positive or negative control. Operons (or genes) under negative control are expressed, unless they are switched off by a repressor protein which will bind to a specific DNA sequence called the operator, making it impossible for RNA polymerase to initiate transcription at the promoter. Inversely, genes whose expression is under positive control will not be transcribed unless an active regulator protein is present which binds to a specific DNA sequence and assists the RNA polymerase in the initiation steps.

Lactose (lac) operon:

The lactose (lac) operon responsible for degradation of the sugar lactose in an inducibile operon because it functions only in the presence of an inducer (lactose here). The lac operon is also under negative regulation. In the absence of lactose, the operon is repressed by the binding of the repressor protein to the operator sequence, thus impeding the RNA polymerase function. Addition of lactose will, however, reverse this repression. (the repressor complexed with the inducer does not recognize the operator because of a conformation change in the repressor)

Full expression of the lac operon also requires a protein-mediated positive control mechanisms. In  E. coli a protein called CAP forms a complex with cAMP acquiring the ability to bind to a specific DNA sequence present in the promoter. The CAP-cAMP complex enhances binding of RNA polymerase to the promoter, thus allowing an increase in the frequency of transcription initiation.

This dual combination of positive and negative control allows E. coli to use alternative carbon sources such as lactose when glucose is absent. Falling levels of glucose induce an increase in cyclic AMP which binds to the CAP protein enabling it to bind to its specific DNA sequence near target promoters and thereby turn on the appropriate genes. But it would be wastful for CAP to induce expression of the lac operon if lactose is not present. So as a negative control, bacteria uses the lac repressor above to shut off the lac operon in the absence of lactose. The combination of both this positive and negative control acts as a type of genetic switch to make sure that the lac operon is off when lactose is not available and also that it is off when glucose is available. For the operon to be on, the cell needs -glucose and + lactose. 

Most of the routinely employed expression vectors fely on the lac control in order to overproduce a gene of choice. The lac promoter/operator functions as it does due to the interplay of three main components. First, the wild-type lac 10 region (TATGTT) is very weak. c-AMP activated CAP protein is able to bind to the CAP site just upststream of the –35 region which stimulates binding of RNA polymerase to the weak -10 site. Repression of the lac promer is observed when gluose is the main carbon source because very little c-AMP is present whihc results in low amounts of available c-AMP activate CAP protein. When poor carbon sources such as lactose of glycerol are used, c-AMP levels rise and large amounts of c-AMP activated CAP protein become availalbe. Thus induction of the lac promoter can occur. Second, Lac repressor binds to the lac operator. Lac repressor can be overcome by alloactose which is a natural byproduct of lactose utilizaiton in the cell or by the gratuitous inducter, IPTG. Third, the lac operator can form stable loop structures which prevents the initiation of transcripton due to the interaction of the Lac repressor with the lac operator (O1) and one of two auxiliary operators, O2, which is located downstream of the coding region of the lacZ gene, or O3 which is located just upstream of the CAP binding site. In summary, DNA binding sites include the operators O3 and O1, catabolite gene activator protein (CAP), the -35 site and the -10 site. Important RNA protein sites include the LacI translation stop site (TGA), the +1 lacZ transcription start site, the Shine Delgarno (SD) ribosome binding site for lacZ and the LacZ translation start site (ATG). (Altman (US 2012/0059145)

Tryptophan operon:

The tryptophan operon which contains the structural genes necessary for tryptophan biosynthesis is also under dual transcription control mechanisms. Again, it is under negative control in that an active repressor can bind to the operator blocking any transcription of the trp mRNA by the RNA polymerase. But here tryptophan is a corepressor rather than an inducer in that its presence changes the conformation of an inactive repressor protein which is then able to bind the operator. The trp operon is also under the control of an attenuation-antitermination mechanism (the leader mRNA possesses 4 repeates which can pair differently according to tryptophan availability, leading to an early termination of transcription of the operon or its full transcription.

Genetic Exchange between Bacteria:

4 types of genetic exchange occur among bacteria:

• transformation where bacteria uptake DNA. This does not occur frequently in nature but transformation is commonly used in the laboratory. E. Coli can take up DNA by transformation if they are made “competent” with calcium or magnesium. 

• transduction is the transfer of bacterial DNA from one cell to another by means of a bacteriophageinfection. Bacteriophages are viruses which infect bacteria. Their life cycle can be either lytic where they replicate and cause cell lysis or lysogenic where they integrate into the host chromosome. Transduction can either be  1) specialized where only specific genes adjacent to the site of integration are transferred along with the phage genome or 2) generalized which results from a random and accidental packaging of host DNA into the phage capsid. Thus generalized transducing particles should contain all or nearly all bacterial DNA and little or no phage DNA. 

• conjugation where DNA (usually plasmids) are exchanged between bacterial cells. This is a common mechanisms of genetic exchange between bacteria. This transferred DNA can either be integrated into the recipient chromosome or stably maintained as a plasmid and passed on to daughter bacteria as an autonomously replicating unit. 

• Transposons are segments of DNA which are able to move from one position to another in the genome or from the chromosomal DNA to a plasmid or the reverse. The simplest ones contain inverted repeats at each of their ends. Complex transposons usually code for antibiotic resistance and can contain transposases that excise and integrate the transposon into a new site.