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7.20: Culturing Prokaryotes - Biology

7.20: Culturing Prokaryotes - Biology


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Microbiologists typically grow prokaryotes in the laboratory using an appropriate culture medium containing all the nutrients needed by the target organism. After an incubation time at the right temperature, there should be evidence of microbial growth (Figure 1).

The process of culturing bacteria is complex and is one of the greatest discoveries of modern science. German physician Robert Koch is credited with discovering the techniques for pure culture, including staining and using growth media. His assistant Julius Petri invented the Petri dish whose use persists in today’s laboratories. Koch worked primarily with the Mycobacterium tuberculosis bacterium that causes tuberculosis and developed postulates to identify disease-causing organisms that continue to be widely used in the medical community. Koch’s postulates include that an organism can be identified as the cause of disease when it is present in all infected samples and absent in all healthy samples, and it is able to reproduce the infection after being cultured multiple times. Today, cultures remain a primary diagnostic tool in medicine and other areas of molecular biology.

Some prokaryotes, however, cannot grow in a laboratory setting. In fact, over 99 percent of bacteria and archaea are unculturable. For the most part, this is due to a lack of knowledge as to what to feed these organisms and how to grow them; they have special requirements for growth that remain unknown to scientists, such as needing specific micronutrients, pH, temperature, pressure, co-factors, or co-metabolites. Some bacteria cannot be cultured because they are obligate intracellular parasites and cannot be grown outside a host cell.

In other cases, culturable organisms become unculturable under stressful conditions, even though the same organism could be cultured previously. Those organisms that cannot be cultured but are not dead are in a viable-but-non-culturable (VBNC) state. The VBNC state occurs when prokaryotes respond to environmental stressors by entering a dormant state that allows their survival. The criteria for entering into the VBNC state are not completely understood. In a process called resuscitation, the prokaryote can go back to “normal” life when environmental conditions improve.

Is the VBNC state an unusual way of living for prokaryotes? In fact, most of the prokaryotes living in the soil or in oceanic waters are non-culturable. It has been said that only a small fraction, perhaps one percent, of prokaryotes can be cultured under laboratory conditions. If these organisms are non-culturable, then how is it known whether they are present and alive? Microbiologists use molecular techniques, such as the polymerase chain reaction (PCR), to amplify selected portions of DNA of prokaryotes, demonstrating their existence. Recall that PCR can make billions of copies of a DNA segment in a process called amplification.


Microbiological culture

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagnostic methods used as a research tool in molecular biology.

Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. For example, a throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. [1] Furthermore, the term culture is more generally used informally to refer to "selectively growing" a specific kind of microorganism in the lab.

It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another. For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. A cheap substitute for agar is guar gum, which can be used for the isolation and maintenance of thermophiles.


Needs of Prokaryotes

The diverse environments and ecosystems on Earth have a wide range of conditions in terms of temperature, available nutrients, acidity, salinity, and energy sources. Prokaryotes are very well equipped to make their living out of a vast array of nutrients and conditions. To live, prokaryotes need a source of energy, a source of carbon, and some additional nutrients.

Macronutrients

Cells are essentially a well-organized assemblage of macromolecules and water. Recall that macromolecules are produced by the polymerization of smaller units called monomers. For cells to build all of the molecules required to sustain life, they need certain substances, collectively called nutrients. When prokaryotes grow in nature, they obtain their nutrients from the environment. Nutrients that are required in large amounts are called macronutrients, whereas those required in smaller or trace amounts are called micronutrients. Just a handful of elements are considered macronutrients—carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur. (A mnemonic for remembering these elements is the acronym CHONPS.)

Why are these macronutrients needed in large amounts? They are the components of organic compounds in cells, including water. Carbon is the major element in all macromolecules: carbohydrates, proteins, nucleic acids, lipids, and many other compounds. Carbon accounts for about 50 percent of the composition of the cell. Nitrogen represents 12 percent of the total dry weight of a typical cell and is a component of proteins, nucleic acids, and other cell constituents. Most of the nitrogen available in nature is either atmospheric nitrogen (N2) or another inorganic form. Diatomic (N2) nitrogen, however, can be converted into an organic form only by certain organisms, called nitrogen-fixing organisms. Both hydrogen and oxygen are part of many organic compounds and of water. Phosphorus is required by all organisms for the synthesis of nucleotides and phospholipids. Sulfur is part of the structure of some amino acids such as cysteine and methionine, and is also present in several vitamins and coenzymes. Other important macronutrients are potassium (K), magnesium (Mg), calcium (Ca), and sodium (Na). Although these elements are required in smaller amounts, they are very important for the structure and function of the prokaryotic cell.

Micronutrients

In addition to these macronutrients, prokaryotes require various metallic elements in small amounts. These are referred to as micronutrients or trace elements. For example, iron is necessary for the function of the cytochromes involved in electron-transport reactions. Some prokaryotes require other elements—such as boron (B), chromium (Cr), and manganese (Mn)—primarily as enzyme cofactors.

Practice Question

The substances needed to sustain life are _____.


Regulation of Gene Expression in Prokaryotes | Gene Regulation

Gene transcription is regulated in bacteria through a complex of genes termed operon. These are transcriptional units in which several genes, with related functions, are regulated together. Other genes also occur in operons which encode regulatory proteins that control gene expression. Operons are classified as inducible or repressible.

Inducible and Repressible System:

The β galactosidase in E. coli is responsible for hydro­lysis of lactose into glucose and galactose.

If lactose is not supplied to E. coli cells, the presence of β galactosidase is hardly detectable. But as soon as lactose is added, the production of β galactosidase enzyme increases. The enzyme falls as quickly as the substrate (lactose) is removed.

Such enzymes whose synthesis can be induced by adding the substrate are known as inducible enzymes and the genetic system responsible for the synthesis of such an enzyme is called inducible system. The substrate whose addition induces the synthesis of an enzyme is inducer.

In some other cases, the situation is reverse. For instance, when no amino acids are supplied from outside, the E. coli cells can synthesize all the enzymes needed for the synthesis of different amino acids. However, if a particular amino acid, for instance, histidine, is added, the production of histidine synthesizing enzyme falls.

In such a system, the addition of the end product of biosyn­thesis checks the synthesis of the enzymes needed for the biosynthesis. Such enzymes whose synthesis can be checked by the addition of the end product are repressible enzymes and the genetic system is known as repressible sys­tem. The end product, the addition of which check the synthesis of the enzyme is co-repressor.

A class of molecules called repressors are found in cells and these repressors check the activity of genes. An active repressor can be made inactive by adding inducer, while an inac­tive repressor can be made active by adding a co-repressor.

Operon Model:

A hypothesis to explain the induction and repression of enzyme synthesis was first pro­posed by Jacob and Monod. The scheme pro­posed by them is called Operon Model.

This consists of the components:

These are directly con­cerned with the synthesis of cellular proteins. They produce the mRNAs through transcription and determine the sequence of amino acids in the synthesized proteins. All the structural genes under an operon may form one long poiycistronic or polygenic mRNA molecule.

This is located adjacent to the structural gene. It determines whether the structural genes are to be repressed by the repre­ssor protein, a product of regulator gene. The operator gene is the site of binding of the repre­ssor protein, the latter binds to the operator form­ing an operator-repressor complex. When the repressor binds to the operator, transcription of the structural genes cannot occur.

These genes synthesize repressor. Repressor may be either an active repressor or an inactive repressor. Repressor pro­tein has one active site for operator recognition and other active site for inducer. In absence of an inducer protein, the repressor binds to the ope­rator gene and blocks the path of RNA poly­merase. Thus the structural genes are unable to transcribe mRNA and consequently protein syn­thesis does not occur.

In presence of an inducer, the repressor protein binds to the inducer to form an inducer-repressor complex. The repressor when binds with inducer undergoes a change and becomes ineffective and as a result it cannot bind to the operator gene and the protein syn­thesis is possible.

The actual site of transcrip­tion initiation is known as promoter gene which lies to the left of the operator gene. It is believed that RNA polymerase binds to and moves from the promoter site.

Effector is a small molecule (sugar or amino acid) that can be linked to a regulator protein and will determine whether repressor will bind the operator or not. In the inducible operon, these effector molecules are called inducer. In repressible operon, these effector molecules are called co-repressor.

Inducible Operon:

The best known operon is the lac operon. The lac operon exercises both positive and nega­tive control. Negative control is in the sense that the operon is normally “on” but is kept “off” by the regulator gene, i.e., the genes are not allowed to express unless required.

The lac repressor exercises negative control. Positive control is that in which the regulator gene will stimulate the production of the enzyme. Catabolite activator protein (CAP) facilitates transcription, so it exer­cises positive control. Two unique proteins are thus involved in the regulation of the lac operon which are lac repressor and CAP.

Lactose is a disaccharide molecule. In order to utilize lactose as a carbon and energy source, the lactose molecules must be transported from the extracellular environment into the ceil, and then undergo hydrolysis into glucose and galac­tose. These reactions are catalysed by three enzymes. The lac operon consists of three struc­tural genes (lac Z, Y, A) which code for these three enzymes (Fig. 17.2).

lac Z gene — codes for enzyme β galactosidase which breaks lactose into galactose and glucose

lac Y gene — codes for permease which transports lactose into the cell

lac A gene — codes for transacetylase which transfer the acetyl group from acetyl CoA to galactose.

Negative Control of lac Operon:

lac repres­sor is synthesized through the activity of the lac I gene called the regulator gene. This repressor is an allosteric protein

(i) That can bind the lac DNA at the operator site, or

(ii) That can bind to inducer.

In the absence of inducer, DNA binding site of repressor is functional. The repressor protein binds to the DNA at the operator site of the lac locus and blocks the transcription of the lac genes by RNA polymerase. Thus lac enzyme syn­thesis is inhibited (Fig. 17.3A).

Lactose is not the real inducer of the lac operon. It binds to repressor to increase its affi­nity for operator. On the other hand, the bound protein of the inactive repressor is the allolactose. While β galactosidase breaks lactose into glucose and galactose, a side reaction changes galactose to allolactose and galactobiose.

This allolactose prevents the anti-inducing lac I lac lac effect of lactose. When the allolactose (inducer) binds to the repressor, it changes the form of DNA binding site making the repressor inactive and release from- the operator site. Thus trans­cription of lac genes are possible.

Positive Control of lac Operon:

It is an additional regulatory mechanism which allows the lac operon to sense the presence of glucose, an alternative and preferred energy source to lactose. If glucose and lactose are both present, cells will use up the glucose first and will not uti­lize energy splitting lactose into its component sugars.

The presence of glucose in the cell switches off the lac operon by a mechanism called catabolite repression which involves a regulatory protein called the catabolite activator protein (CAP). CAP binds to a DNA sequence upstream of the lac promoter and enhances bind­ing of the RNA polymerase and transcription of the operon is enhanced (Fig. 17.3B).

CAP only binds in the presence of a deri­vative of ATP called cyclic adenosine monophos­phate (cAMP) whose levels are influenced by glucose. The enzyme adenylate cyclase cata­lyzes the formation of cAMP and is inhibited by glucose. When glucose is available to the cell, adenylate cyclase is inhibited and cAMP levels are low.

Under these conditions CAP does not bind upstream of the promoter and the lac ope­ron is transcribed at a very low level. Conversely, when glucose is low, adenylate cyclase is not inhibited, cAMP is higher and CAP binds increasing the level of transcription from the operon.

If glucose and lactose are present together, the lac operon will only be transcribed at a low level. However when the glucose is used up, catabolite repression will end and trans­cription from the lac operon increases allowing the available lactose to be used up.

Repressible Operon:

The trp operon consists of the following components:

(i) Structural genes (trp E, D, C, B and A):

This operon contains five structural genes encoding enzymes involved in biosynthe­sis of the amino acid tryptophan. The genes are expressed as a single mRNA transcribed from an upstream promoter.

(ii) Promoter gene (trp P):

It is the promoter region which is the binding site for RNA polymerase.

(iii) Operator gene (trp O):

It is the operator region which binds with the repressor.

It is the leader region which is made of 162 nucleotides prior to the first structural gene trp E. It has four regions, region 1 has the codon for tryp­tophan, region 2, 3 and 4 regulate the mRNA synthesis of the structural genes.

Expression of the operon is regulated by the level of tryptophan in the cell (Fig. 17.4). A regu­latory gene upstream of the trp operon encodes a protein called the trp repressor. This protein binds a DNA sequence called the trp operator which lies just downstream of the trp promoter partly overlapping it.

When tryptophan is present in the cell it binds to the trp repressor protein enabling it to bind the trp operator sequence, obstructing binding of the RNA polymerase to the trp promoter and preventing transcription of the operon.

In the absence of tryptophan, the trp repressor is incapable of binding the trp operator and transcription of the operon proceeds. Tryptophan, the end product of the enzymes encoded by the trp operon, thus acts as a co-repressor with the trp repressor protein and inhibits its own synthesis by end product inhibition.

Attenuation is an alternative regulatory mechanism that allows fine adjust­ment of expression of the trp operon and other operons (phe, his, leu, thr operon). The trans­cribed mRNA sequence between the trp promo­ter and the first trp gene are capable of forming either a large stem-loop structure that does not influence transcription or a smaller stem loop which acts as transcription terminator (Fig. 17.5).

The relative position of the sequences does not allow the formation of both stem-loops at a time. Attenuation depends on the fact that transcrip­tion and translation are linked, i.e., ribosomes attach to mRNAs as they are being transcribed and begin translating them into protein.

Binding of ribosomes to the trp mRNA influences which of the two stem-loops can form and so deter­mines whether termination occurs or not (Fig. 17.5).

A short coding region upstream of the stem-loop region contains tryptophan codons which is translated before the structural genes. When tryptophan levels are adequate, RNA polymerase transcribes the leader region closely followed by a ribosome which prevents forma­tion of the larger stem-loop, allowing the termi­nator loop to form ending transcription.

If trypto­phan is lacking, transcription is initiated, but not subsequently terminated because the ribosome is stalled, the RNA polymerase moves ahead and the large stem-loop forms. Formation of the ter­minator loop is blocked and transcription of the operon proceeds. When tryptophan present at intermediate levels, some transcripts will termi­nate and others not.

Attenuation thus allows the cell to synthesize tryptophan according to its exact requirements. Overall, the trp repressor determines whether the operon is switched on or off and attenuation determines how efficiently it is transcribed.

The sequence of the mRNA suggests that ribosome stalling influences termination at the attenuator. The ability of the ribosome to pro­ceed through the leader region may control transition between these structures. The structure determines whether the mRNA can provide the features needed for termination or not.

When tryptophan is present, ribosomes are able to synthesize the leader peptide. They will continue along the leader section of the mRNA to the UGA codon, which lies between regions 1 and 2. By progressing to this point, the ribosomes extend over region 2 and prevent it from base pairing.

The result is that region 3 is available to base pair with region 4, generating the termi­nator hairpin. Under these conditions, therefore, RNA polymerase terminates at the attenuator.

However, when there is no tryptophan, ribo­somes initiate translation of the leader peptide but stall at the trp codons which is at the region 1. Thus the region 1 cannot base pair with region 2. If this happens, even while the mRNA itself is being synthesized, region 2 and 3 will be base- paired before region 4 has been transcribed.

This compels region 4 to remain in a single stranded form. In the absence of the terminator hairpin, RNA polymerase continues transcription past the attenuator.

The ara (arabinose) operon of F. coli con­tains:

(i) Three structural genes (ara A, ara B and ara D) – which encode three different enzymes (isomerase, kinase, epimerase) for metabolism of arabinose three sructuretural genes are co-transcribed on a single mRNA.

(ii) Promoter gene(PBAD)- which initiates transcription.

(iii) Regular gene (ara C)- the regulatory protein of this gene ara C.

(iv) Promoter gene (Pc)- This initiates transcription of are C.

Two promoters PBAD and Pc are situated 100 nucleotide pairs away in the same inducer region and they initiate transcription in opposite direc­tions.

The induction of ara operon depends on the positive regulatory effects of two proteins, the ara C protein and CAP (the cAMP binding catabolite activator protein), the binding sites of these two proteins are located in a region called ara I which is situated in between the three structural genes (ara B, ara A and ara D) and the regulator gene (ara C) (Fig. 17.6A).

The ara C protein acts as a negative regulator (a repressor) of transcription of the ara B, ara A and ara D structural genes from the PBAD promoter in absence of arabinose and cyclic AMP (cAMP). But it acts as a positive regulator (an activator) of tran­scription of these genes from the PBAD promoter when arabinose and cAMP are present.

Depending on the presence or absence of effector molecule like arabinose and cAMP, the ara C regulatory gene product may exert either a positive or negative effect on transcription of the ara B, ara A and ara D structural genes (Fig. 17.6B).

Post-Transcriptional Regulation of Gene Expression in Prokaryotes:

Gene regulation may also occur in prokaryotes at the time of translation.

Autogenous Regulation of Translation:

There are number of examples where a protein or RNA regulates its own production. Several proteins work as repressors, bind to the ribosome binding site (or SD-Shine-Dalgarno sequence) or initiation codon of mRNA. In these cases mRNA remains intact but cannot be translated. There are some other systems where mRNA may be degraded by the binding of protein on the short specific sequences of mRNA.

Regulation by Anti-sense RNA:

Translational control of protein synthesis can be exercised by using RNA which is complementary to mRNA, these complementary RNA will form RNA- mRNA hybrids and prevent mRNA from being translated. These kind of RNAs are called anti- sense RNA or micRNA (mic = mRNA interfering complementary RNA).

Repression of Translation:

Repression of translation occurs by the following ways:

(a) A repressor-effector molecule may recog­nise and bind to a specific sequence or to a specific secondary structure (involving SD region and AUG codon), thus blocking initiation of translation through blocking of the ribosomal bind­ing region.

(b) A repressor-effector molecule may bind to an operator (not involving SD region and AUG codon) thus stabilizing an inhibitory mRNA secondary structure.

(c) An effector molecule (an endonuclease) can inhibit initiation of translation by endonucleolytic cleavage of SD region.

Activation of Translation:

Some positive effectors or activators cause activation of trans­lation by destabilizing the inhibitory secondary structures in mRNA either through simple bind­ing or by endonucleolytic cleavage. Translation of certain genes may be influenced by certain other genes – the phenomenon is called trans­lational coupling.

In some cases, the end product of a particular biosynthetic pathway gets accumulated and this accumulation may stop further synthesis of this substance. The end product acts through allosteric transformation of the first enzyme of biosynthetic pathway (Fig. 17.7).


Gene Regulation in Prokaryotes | Genetics

Gene regulation refers to the control of the rate or manner in which a gene is expressed. In other words, gene regulation is the process by which the cell determines (through interactions among DNA, RNA, proteins, and other substances) when and where genes will be activated and how much gene product will be produced.

Thus, the gene expression is controlled by a complex of numerous regulatory genes and regulatory proteins. The gene regulation has been studied in both prokaryotes and eukaryotes.In prokaryotes, the operon model of gene regulation is widely accepted.

This model of gene regulation was proposed by Jacob and Monod in 1961 for which they were awarded Nobel Prize in 1965. The operon refers to a group of closely linked, genes which together code for various enzymes of a particular biochemical pathway.

In other words, operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in-DNA recognized by regulator gene product(s). Thus operon is a model which explains the on-off mechanism of protein synthesis in a systematic manner. The main points of operon model of gene regulation are presented below.

(i) Developed By:

In prokaryotes, the operon model of gene regulation was developed by Jacob and Monod in 1961 for which they were awarded Nobel prize in 1965. Now this model of gene regulation is widely accepted.

(ii) Organism Used:

The operon model was developed working with lactose region [lac region] of human intestine bacteria E. coli. The gene regulation was studied for degradation of the sugar lactose.

(iii) Genes Involved:

In the operon model of gene regulation, four types of genes viz:

(iv) Regulator gene are involved.

In addition, repressor, co-repressor, and inducer molecules are also involved.

(iv) Enzymes Involved:

Four types of enzymes are involved in gene regulation of prokaryotes. These are beta-galactosidase, galactosidase permease, transacetylase and RNA polymerase. The beta-galactosidase catalyses the breakdown of lactose into glucose and galactose.

The galactosidase permease permits entry of lactose from the medium into the bacterial cell. The enzyme transacetylase transfers an acetyl group from acetyl co-enzyme A to beta galactosidase. The enzyme mRNA polymerase controls on-off of the transcription.

Types of Operon in Gene Regulation:

In prokaryotes, operons are of two types, viz., inducible and repressible. The example of an inducible operon is the lactose operon, which contains genes that encode enzymes responsible for lactose metabolism. An example of repressible operon is the Trp operon, which encodes enzymes responsible for the synthesis of the amino acid tryptophan (trp for short).

A. Inducible Operon:

An enzyme whose production is enhanced by adding the substrate in the culture medium is called inducible enzyme, and such system is called inducible system. The example of an inducible operon is the lactose operon, which contains genes that encode enzymes responsible for lactose metabolism.

In bacteria, operon refers to a group of closely linked genes which act together and code for the various enzymes of a particular biochemical pathway.

The model of lac operon of E. coli looks like this:

There are three structural genes of the lac operon i.e. lac Z, lac Y and lac A. The main function of structural genes is to control of protein synthesis through messenger RNA. Function of these genes is as follows.

It encodes the enzyme beta-galactosidase, which catalyses the breakdown of lactose into glucose and galactose.

It encodes the enzyme galactosidase permease, which permits entry of lactose from the medium into the bacterial cell.

It encodes the enzyme transacetylase, which transfers an acetyl group from acetyl co-enzyme A to beta galactosidase.

The above three structural genes genes are under the control of the promoter gene [designated P]. In the promoter, RNA polymerase binds to the DNA and prepares to initiate transcription. The main function of promoter gene is to initiate mRNS transcription.

The other regulatory element in an operon is the operator (designated O). This is the element that determines whether or not the genes of the operon are transcribed. The main function of operator gene is to control function of structural genes.

This is designated as I. It is expressed all the time, or constitutively and plays an important role in operon function. This is the lac I gene, which encodes a protein called the lac repressor. The lac repressor has two functional domains or regions: one that binds to the DNA of the operator region, and one that binds to lactose.

When the repressor binds to the operator, it prevents RNA polymerase advancing along the operon, and transcription does not occur. The regulation of the operon depends on regulating whether or not the repressor binds to the operator. The function of regulator gene is to direct synthesis of repressor, a protein molecule. Its function differs in the presence and absence of lactose as discussed below.

When Lactose is absent:

When the lactose is absent in the environment, events take place in this way. The lac I gene is transcribed [constitutively i.e. continuously] and the mRNA is translated, producing the lac repressor. The repressor binds to the operator, and blocks RNA polymerase.

When RNA polymerase is blocked, there is no transcription. Thus the enzymes for lactose metabolism are not synthesized, because there is no lactose to metabolize. Thus when lactose is absent, lactose-metabolizing enzymes are not produced.

When Lactose is Present:

When the lactose is present in the environment, the events occur in a different way. A small amount of the lactose enters into the cell and affects regulation of the operon. The lac repressor is still synthesized. The repressor can bind to lactose.

After binding to lactose, the repressor undergoes a conformational change (change of shape). Molecules that change shape when they bind to another molecule are called allosteric molecules. With this change, the lac repressor is unable to bind to the operator region. Hence RNA polymerase is not blocked, and is able to transcribe the genes of the operon.

The enzymes encoded by those genes are produced. The lac permease transports more lactose into the cell and beta-galactosidase cleaves the lactose into glucose and galactose. This can be further metabolized by other enzymes, producing energy for the cell.

Lactose, therefore, is able to induce the synthesis of the enzymes necessary for its metabolism (by preventing the action of the repressor). As such, lactose is the inducer of the lac operon. Thus when lactose is absent, lactose-metabolizing enzymes are not produced, and when lactose is present, those enzymes are produced.

Mutations of the Lac Operon:

Mutations can affect the regulation of the lac operon in different ways as given below:

(i) Mutation of the lac I gene in such a way that the repressor encoded no longer binds to lactose. In this case, the repressor would bind to the operator regardless of the presence or absence of lactose, and the operon would never be transcribed at high levels.

(ii) Mutation of the lac I gene in such a way that the repressor no longer binds to the operator. In this case, the operon would never be repressed, and transcription would be carried out continuously. This is known as constitutive transcription.

(iii) Mutation in the operator region in such a way that the wild-type repressor does not recognize it (the repressor recognizes the specific DNA sequence of the operator legion): In this case, there will be no binding of the repressor to the operator, and transcription will go on continuously.

Catabolite Repression:

Expression of the lac operon can also be regulated in another way. Glucose is preferable to lactose as an energy source. Hence if glucose is present in the environment, the transcription is reduced or lac operon is down-regulated.

Transcription of the lac operon requires another protein, called catabolite activator protein (CAP for short). This CAP protein binds to the lac promoter and enhances transcription. But it occurs only after CAP binds to a small molecule called cyclic AMP (cAMP).

Without cAMP, CAP will not bind to the promoter, and no transcription will occur. In the previous examples involving the lac operon, we can assume that cAMP was present, and the CAP-cAMP complex was bound to the promoter.

The cAMP is produced by an enzyme called adenyl-cyclase. In the presence of glucose in the environment, adenyl-cyclase is inhibited, and cAMP production drops. Thus there is no cAMP to bind to CAP. In this situation, the CAP will not bind to the lac promoter, and no lac transcription takes place.

In this way, the bacterium does not produce enzymes for lactose metabolism when they are not necessary because of the presence of glucose. Beta-galactosidase breaks lactose in to glucose and galactose. When enough lactose has been metabolized, glucose (one of the products) accumulates and causes repression of the lac operon.

Merits of Operon Model in Gene Regulation:

1. It is a very simple yet informative model of gene regulation in prokaryotes.

2. It is a very well understood model of gene regulation in prokaryotes.

3. This model is based on empirical results and has been studied on different prokaryotes.

4. This model is of two types, viz:

B. Repressible Operon:

A protein molecule which prevents transcription is called repressor and the process of inhibition of transcription is called repression. Repressible operons are regulated by the end product of the metabolic pathway and not by a reactant in the metabolic pathway (such as lactose in lac operon).

An example of repressible operon is the Trp operon. This encodes enzymes which are responsible for the synthesis of the amino acid tryptophan (trp for short). The trp operon is regulated by trp, which is the product of the metabolic pathway.

In trp operon, the trp repressor only binds to the operator when trp is present, (opposite to the lac repressor). The repressor binds to trp, and undergoes a conformational change [change of shape]. This change in shape allows it to bind to the operator, blocking transcription. Because trp is needed for repression, it is referred to as a co-repressor in this system (as opposed to lactose being an inducer).

When trp is absent, the repressor will not bind to the operator, and transcription occurs. Thus, if there is plenty of trp around [and no more is needed], the transcription is blocked. If there is no trp around [it needs to be synthesized], transcription occurs. In other words, it allows production of the enzymes for trp synthesis.

Repressible operons are organized in much the same way as inducible operons: there are structural genes under the control of a promoter and operator, and there is a gene encoding a repressor.

The mutation will affect the gene regulation as follows:

(i) mutation in the repressor gene in such a way that it no longer binds trp When repressor does not bind trp, there will be no change in its structure and it will not bind with operator and transcription will occur.

(ii) mutation in the repressor gene in such a way that it no longer binds the repressor: In such situation transcription will take place.

(iii) mutation in the operator in such a way that it no longer binds to the repressor: In such situation also transcription will occur.

Mechanism of Gene Regulation:

The mechanism of gene regulation is of two types, viz:

(1) Negative regulation, and

The mechanism of gene regulation in E. coli operon and tryptophan operon are discussed below:

1. Negative Control:

The first switch in the lac operon of E. coli, is the repressor protein. In negative control, the transcription is controlled by repressor protein, which is an allosteric protein. The repressor protein binds to operator region and prevents transcription. It prevents transcription by blocking RNA polymerase. Thus, when repressor is bound to operator, the transcription is switched off.

Thus the on-off switch of protein synthesis is governed by free or occupied position of the operator gene. When the operator is free, transcription will take place and when the operator gene is blocked, the transcription is prevented. If an isomer of lactose [allolaptose] is present, it will bind to repressor protein and change its shape. The changed repressor does not bind to operator and thus allows transcription.

2. Positive Control:

The second switch in the lac operon of E. coli, is the catabolite activator protein [CAP].The CAP is an allosteric protein. The CAP binds to DNA and small molecule called cyclic adenosine mono phosphate [cAMP], The CAP only binds to promoter region and stimulates transcription when cAMP binds to allosteric site.

The concentration of cAMP is controlled by ATP concentrations. The low ATP leads to high cAMP and high ATP leads to low cAMP. If E. coli is growing on glucose, there will be high [ATP] & low [cAMP], If no glucose is present, there will be a low [ATP] & high [cAMP]

In the absence of glucose, [cAMP] is high, binds to CAP which binds to promoter region and stimulates transcription. If glucose is present, [cAMP] is low. doesn’t bind to CAP which cannot bind to promoter and doesn’t allow transcription.

Tryptophan Operon:

The tryptophan operon [in short trp operon] is regulated by trp, which is the product of .the metabolic pathway. The trp operon contains genes that make 5 enzymes in the biosynthetic pathway for the production of amino acid tryptophan.

In trp operon, the negative control is associated with a repressor protein. However, the repressor protein only binds with operator gene when an allosteric effector is bound to it. The tryptophan is an allosteric effector, which is called a co-repressor in trp operon also, the transcription is controlled by the free or occupied position of repressor.

If the repressor protein doesn’t bind with operator gene, transcription will take place. If tryptophan is present, there is no need to synthesize enzymes. In such situation tryptophan binds to repressor protein and both these [trp and repressor] bind to operator gene preventing transcription. When trp is absent, the repressor will not bind to the operator, and transcription will take place.

In the negative control, repressor protein binds DNA and stops transcription. In positive control, activator protein binds DNA and stimulates transcription. In the inducible system, allosteric effector binds and releases repressor protein from DNA resulting in transcription. In the repressible system, allosteric effector binds and causes repressor protein to bind to DNA preventing transcription.


Biology 171


In the recent past, scientists grouped living things into five kingdoms—animals, plants, fungi, protists, and prokaryotes—based on several criteria, such as the absence or presence of a nucleus and other membrane-bound organelles, the absence or presence of cell walls, multicellularity, and so on. In the late 20 th century, the pioneering work of Carl Woese and others compared sequences of small-subunit ribosomal RNA (SSU rRNA), which resulted in a more fundamental way to group organisms on Earth. Based on differences in the structure of cell membranes and in rRNA, Woese and his colleagues proposed that all life on Earth evolved along three lineages, called domains. The domain Bacteria comprises all organisms in the kingdom Bacteria, the domain Archaea comprises the rest of the prokaryotes, and the domain Eukarya comprises all eukaryotes—including organisms in the kingdoms Animalia, Plantae, Fungi, and Protista.

Two of the three domains—Bacteria and Archaea—are prokaryotic. Prokaryotes were the first inhabitants on Earth, appearing 3.5 to 3.8 billion years ago. These organisms are abundant and ubiquitous that is, they are present everywhere. In addition to inhabiting moderate environments, they are found in extreme conditions: from boiling springs to permanently frozen environments in Antarctica from salty environments like the Dead Sea to environments under tremendous pressure, such as the depths of the ocean and from areas without oxygen, such as a waste management plant, to radioactively contaminated regions, such as Chernobyl. Prokaryotes reside in the human digestive system and on the skin, are responsible for certain illnesses, and serve an important role in the preparation of many foods.

Learning Objectives

By the end of this section, you will be able to do the following:

  • Describe the evolutionary history of prokaryotes
  • Discuss the distinguishing features of extremophiles
  • Explain why it is difficult to culture prokaryotes

Prokaryotes are ubiquitous. They cover every imaginable surface where there is sufficient moisture, and they also live on and inside virtually all other living things. In the typical human body, prokaryotic cells outnumber human body cells by about ten to one. They comprise the majority of living things in all ecosystems. Some prokaryotes thrive in environments that are inhospitable for most living things. Prokaryotes recycle nutrients —essential substances (such as carbon and nitrogen)—and they drive the evolution of new ecosystems, some of which are natural and others man-made. Prokaryotes have been on Earth since long before multicellular life appeared. Indeed, eukaryotic cells are thought to be the descendants of ancient prokaryotic communities.

Prokaryotes, the First Inhabitants of Earth

When and where did cellular life begin? What were the conditions on Earth when life began? We now know that prokaryotes were likely the first forms of cellular life on Earth, and they existed for billions of years before plants and animals appeared. The Earth and its moon are dated at about 4.54 billion years in age. This estimate is based on evidence from radiometric dating of meteorite material together with other substrate material from Earth and the moon. Early Earth had a very different atmosphere (contained less molecular oxygen) than it does today and was subjected to strong solar radiation thus, the first organisms probably would have flourished where they were more protected, such as in the deep ocean or far beneath the surface of the Earth. Strong volcanic activity was common on Earth at this time, so it is likely that these first organisms—the first prokaryotes—were adapted to very high temperatures. Because early Earth was prone to geological upheaval and volcanic eruption, and was subject to bombardment by mutagenic radiation from the sun, the first organisms were prokaryotes that must have withstood these harsh conditions.

Microbial Mats

Microbial mats or large biofilms may represent the earliest forms of prokaryotic life on Earth there is fossil evidence of their presence starting about 3.5 billion years ago. It is remarkable that cellular life appeared on Earth only a billion years after the Earth itself formed, suggesting that pre-cellular “life” that could replicate itself had evolved much earlier. A microbial mat is a multi-layered sheet of prokaryotes ((Figure)) that includes mostly bacteria, but also archaeans. Microbial mats are only a few centimeters thick, and they typically grow where different types of materials interface, mostly on moist surfaces. The various types of prokaryotes that comprise them carry out different metabolic pathways, and that is the reason for their various colors. Prokaryotes in a microbial mat are held together by a glue-like sticky substance that they secrete called extracellular matrix.

The first microbial mats likely obtained their energy from chemicals found near hydrothermal vents. A hydrothermal vent is a breakage or fissure in the Earth’s surface that releases geothermally heated water. With the evolution of photosynthesis about three billion years ago, some prokaryotes in microbial mats came to use a more widely available energy source—sunlight—whereas others were still dependent on chemicals from hydrothermal vents for energy and food.


Stromatolites

Fossilized microbial mats represent the earliest record of life on Earth. A stromatolite is a sedimentary structure formed when minerals are precipitated out of water by prokaryotes in a microbial mat ((Figure)). Stromatolites form layered rocks made of carbonate or silicate. Although most stromatolites are artifacts from the past, there are places on Earth where stromatolites are still forming. For example, growing stromatolites have been found in the Anza-Borrego Desert State Park in San Diego County, California.


The Ancient Atmosphere

Evidence indicates that during the first two billion years of Earth’s existence, the atmosphere was anoxic , meaning that there was no molecular oxygen. Therefore, only those organisms that can grow without oxygen—anaerobic organisms—were able to live. Autotrophic organisms that convert solar energy into chemical energy are called phototrophs , and they appeared within one billion years of the formation of Earth. Then, cyanobacteria , also known as “blue-green algae,” evolved from these simple phototrophs at least one billion years later. It was the ancestral cyanobacteria ((Figure)) that began the “oxygenation” of the atmosphere: Increased atmospheric oxygen allowed the evolution of more efficient O2-utilizing catabolic pathways. It also opened up the land to increased colonization, because some O2 is converted into O3 (ozone) and ozone effectively absorbs the ultraviolet light that could have otherwise caused lethal mutations in DNA. The current evidence suggests that the increase in O2 concentrations allowed the evolution of other life forms.


Microbes Are Adaptable: Life in Moderate and Extreme Environments

Some organisms have developed strategies that allow them to survive harsh conditions. Almost all prokaryotes have a cell wall, a protective structure that allows them to survive in both hypertonic and hypotonic aqueous conditions. Some soil bacteria are able to form endospores that resist heat and drought, thereby allowing the organism to survive until favorable conditions recur. These adaptations, along with others, allow bacteria to remain the most abundant life form in all terrestrial and aquatic ecosystems.

Prokaryotes thrive in a vast array of environments: Some grow in conditions that would seem very normal to us, whereas others are able to thrive and grow under conditions that would kill a plant or an animal. Bacteria and archaea that are adapted to grow under extreme conditions are called extremophiles , meaning “lovers of extremes.” Extremophiles have been found in all kinds of environments: the depths of the oceans, hot springs, the Arctic and the Antarctic, in very dry places, deep inside Earth, in harsh chemical environments, and in high radiation environments ((Figure)), just to mention a few. Because they have specialized adaptations that allow them to live in extreme conditions, many extremophiles cannot survive in moderate environments. There are many different groups of extremophiles: They are identified based on the conditions in which they grow best, and several habitats are extreme in multiple ways. For example, a soda lake is both salty and alkaline, so organisms that live in a soda lake must be both alkaliphiles and halophiles ((Figure)). Other extremophiles, like radioresistant organisms, do not prefer an extreme environment (in this case, one with high levels of radiation), but have adapted to survive in it ((Figure)). Organisms like these give us a better understanding of prokaryotic diversity and open up the possibility of finding new prokaryotic species that may lead to the discovery of new therapeutic drugs or have industrial applications.

Extremophiles and Their Preferred Conditions
Extremophile Conditions for Optimal Growth
Acidophiles pH 3 or below
Alkaliphiles pH 9 or above
Thermophiles Temperature 60–80 °C (140–176 °F)
Hyperthermophiles Temperature 80–122 °C (176–250 °F)
Psychrophiles Temperature of -15-10 °C (5-50 °F) or lower
Halophiles Salt concentration of at least 0.2 M
Osmophiles High sugar concentration


Prokaryotes in the Dead Sea

One example of a very harsh environment is the Dead Sea, a hypersaline basin that is located between Jordan and Israel. Hypersaline environments are essentially concentrated seawater. In the Dead Sea, the sodium concentration is 10 times higher than that of seawater, and the water contains high levels of magnesium (about 40 times higher than in seawater) that would be toxic to most living things. Iron, calcium, and magnesium, elements that form divalent ions (Fe 2+ , Ca 2+ , and Mg 2+ ), produce what is commonly referred to as “hard” water. Taken together, the high concentration of divalent cations, the acidic pH (6.0), and the intense solar radiation flux make the Dead Sea a unique, and uniquely hostile, ecosystem 1 ((Figure)).

What sort of prokaryotes do we find in the Dead Sea? The extremely salt-tolerant bacterial mats include Halobacterium, Haloferax volcanii (which is found in other locations, not only the Dead Sea), Halorubrum sodomense, and Halobaculum gomorrense, and the archaean Haloarcula marismortui, among others.


Unculturable Prokaryotes and the Viable-but-Non-Culturable State

The process of culturing bacteria is complex and is one of the greatest discoveries of modern science. German physician Robert Koch is credited with discovering the techniques for pure culture, including staining and using growth media. Microbiologists typically grow prokaryotes in the laboratory using an appropriate culture medium containing all the nutrients needed by the target organism. The medium can be liquid, broth, or solid. After an incubation time at the right temperature, there should be evidence of microbial growth ((Figure)). Koch’s assistant Julius Petri invented the Petri dish, whose use persists in today’s laboratories. Koch worked primarily with the Mycobacterium tuberculosis bacterium that causes tuberculosis and developed guidelines, called Koch’s postulates , to identify the organisms responsible for specific diseases. Koch’s postulates continue to be widely used in the medical community. Koch’s postulates include that an organism can be identified as the cause of disease when it is present in all infected samples and absent in all healthy samples, and it is able to reproduce the infection after being cultured multiple times. Today, cultures remain a primary diagnostic tool in medicine and other areas of molecular biology.


Koch’s postulates can be fully applied only to organisms that can be isolated and cultured. Some prokaryotes, however, cannot grow in a laboratory setting. In fact, over 99 percent of bacteria and archaea are unculturable. For the most part, this is due to a lack of knowledge as to what to feed these organisms and how to grow them they may have special requirements for growth that remain unknown to scientists, such as needing specific micronutrients, pH, temperature, pressure, co-factors, or co-metabolites. Some bacteria cannot be cultured because they are obligate intracellular parasites and cannot be grown outside a host cell.

In other cases, culturable organisms become unculturable under stressful conditions, even though the same organism could be cultured previously. Those organisms that cannot be cultured but are not dead are in a viable-but-non-culturable (VBNC) state. The VBNC state occurs when prokaryotes respond to environmental stressors by entering a dormant state that allows their survival. The criteria for entering into the VBNC state are not completely understood. In a process called resuscitation , the prokaryote can go back to “normal” life when environmental conditions improve.

Is the VBNC state an unusual way of living for prokaryotes? In fact, most of the prokaryotes living in the soil or in oceanic waters are non-culturable. It has been said that only a small fraction, perhaps one percent, of prokaryotes can be cultured under laboratory conditions. If these organisms are non-culturable, then how is it known whether they are present and alive? Microbiologists use molecular techniques, such as the polymerase chain reaction (PCR), to amplify selected portions of DNA of prokaryotes, e.g., 16S rRNA genes, demonstrating their existence. (Recall that PCR can make billions of copies of a DNA segment in a process called amplification.)

The Ecology of Biofilms

Some prokaryotes may be unculturable because they require the presence of other prokaryotic species. Until a couple of decades ago, microbiologists used to think of prokaryotes as isolated entities living apart. This model, however, does not reflect the true ecology of prokaryotes, most of which prefer to live in communities where they can interact. As we have seen, a biofilm is a microbial community ((Figure)) held together in a gummy-textured matrix that consists primarily of polysaccharides secreted by the organisms, together with some proteins and nucleic acids. Biofilms typically grow attached to surfaces. Some of the best-studied biofilms are composed of prokaryotes, although fungal biofilms have also been described, as well as some composed of a mixture of fungi and bacteria.

Biofilms are present almost everywhere: they can cause the clogging of pipes and readily colonize surfaces in industrial settings. In recent, large-scale outbreaks of bacterial contamination of food, biofilms have played a major role. They also colonize household surfaces, such as kitchen counters, cutting boards, sinks, and toilets, as well as places on the human body, such as the surfaces of our teeth.

Interactions among the organisms that populate a biofilm, together with their protective exopolysaccharidic (EPS) environment, make these communities more robust than free-living, or planktonic, prokaryotes. The sticky substance that holds bacteria together also excludes most antibiotics and disinfectants, making biofilm bacteria hardier than their planktonic counterparts. Overall, biofilms are very difficult to destroy because they are resistant to many common forms of sterilization.


Compared to free-floating bacteria, bacteria in biofilms often show increased resistance to antibiotics and detergents. Why do you think this might be the case?

Section Summary

Prokaryotes existed for billions of years before plants and animals appeared. Hot springs and hydrothermal vents may have been the environments in which life began. Microbial mats are thought to represent the earliest forms of life on Earth. A microbial mat is a multi-layered sheet of prokaryotes that grows at interfaces between different types of material, mostly on moist surfaces. Fossilized microbial mats are called stromatolites and consist of laminated organo-sedimentary structures formed by precipitation of minerals by prokaryotes. They represent the earliest fossil record of life on Earth.

During the first two billion years, the atmosphere was anoxic and only anaerobic organisms were able to live. Cyanobacteria evolved from early phototrophs and began the oxygenation o the atmosphere. The increase in oxygen concentration allowed the evolution of other life forms.

Bacteria and archaea grow in virtually every environment. Those that survive under extreme conditions are called extremophiles (extreme lovers). Some prokaryotes cannot grow in a laboratory setting, but they are not dead. They are in the viable-but-non-culturable (VBNC) state. The VBNC state occurs when prokaryotes enter a dormant state in response to environmental stressors. Most prokaryotes are colonial and prefer to live in communities where interactions take place. A biofilm is a microbial community held together in a gummy-textured matrix.

Art Connections

(Figure) Compared to free-floating bacteria, bacteria in biofilms often show increased resistance to antibiotics and detergents. Why do you think this might be the case?

(Figure) The extracellular matrix and outer layer of cells protects the inner bacteria. The close proximity of cells also facilitates lateral gene transfer, a process by which genes such as antibiotic-resistance genes are transferred from one bacterium to another. And even if lateral gene transfer does not occur, one bacterium that produces an exo-enzyme that destroys antibiotic may save neighboring bacteria.

Free Response

Describe briefly how you would detect the presence of a non-culturable prokaryote in an environmental sample.

As the organisms are non-culturable, the presence could be detected through molecular techniques, such as PCR.

Why do scientists believe that the first organisms on Earth were extremophiles?

Because the environmental conditions on Earth were extreme: high temperatures, lack of oxygen, high radiation, and the like.

A new bacterial species is discovered and classified as an endolith, an extremophile that lives inside rock. If the bacteria were discovered in the permafrost of Antarctica, describe two extremophile features the bacteria must possess.

Footnotes

    Bodaker, I, Itai, S, Suzuki, MT, Feingersch, R, Rosenberg, M, Maguire, ME, Shimshon, B, and others. Comparative community genomics in the Dead Sea: An increasingly extreme environment. The ISME Journal 4 (2010): 399–407, doi:10.1038/ismej.2009.141. published online 24 December 2009.

Glossary


Frontiers in mammalian cell culture

For the past 60 years, fundamental discoveries in eukaryotic biology using mammalian cell cultures have been significant but modest relative to the enormous potential. Combined with advances in technologies of cell and molecular biology, mammalian cell culture technology is becoming a major, if not essential tool, for fundamental discovery in eukaryotic biology. Reconstruction of the milieu for cells has progressed from simple salt solutions supporting brief survival of tissues outside the body to synthesis of the complete set of structurally defined nutrients, hormones and elements of the extracellular matrix needed to reconstruct complex tissues from cells. The isolation of specific cell types in completely defined environments reveals the true complexity of the mammalian cell and its environment as a dynamic interactive physiological unit. Cell cultures provide the tool for detection and dissection of the mechanism of action of cellular regulators and the genes that determine individual aspects of cell behavior. The technology underpins advances in virology, somatic cell genetics, endocrinology, carcinogenesis, toxicology, pharmacology, hematopoiesis and immunology, and is becoming a major tool in developmental biology, complex tissue physiology and production of unique mammalian cell-derived biologicals in industry.


Microbes Are Adaptable: Life in Moderate and Extreme Environments

Some organisms have developed strategies that allow them to survive harsh conditions. Almost all prokaryotes have a cell wall, a protective structure that allows them to survive in both hypertonic and hypotonic aqueous conditions. Some soil bacteria are able to form endospores that resist heat and drought, thereby allowing the organism to survive until favorable conditions recur. These adaptations, along with others, allow bacteria to remain the most abundant life form in all terrestrial and aquatic ecosystems.

Prokaryotes thrive in a vast array of environments: Some grow in conditions that would seem very normal to us, whereas others are able to thrive and grow under conditions that would kill a plant or an animal. Bacteria and archaea that are adapted to grow under extreme conditions are called extremophiles , meaning “lovers of extremes.” Extremophiles have been found in all kinds of environments: the depths of the oceans, hot springs, the Arctic and the Antarctic, in very dry places, deep inside Earth, in harsh chemical environments, and in high radiation environments (Figure 4), just to mention a few. Because they have specialized adaptations that allow them to live in extreme conditions, many extremophiles cannot survive in moderate environments. There are many different groups of extremophiles: They are identified based on the conditions in which they grow best, and several habitats are extreme in multiple ways. For example, a soda lake is both salty and alkaline, so organisms that live in a soda lake must be both alkaliphiles and halophiles (Table 1). Other extremophiles, like radioresistant organisms, do not prefer an extreme environment (in this case, one with high levels of radiation), but have adapted to survive in it (Figure 4). Organisms like these give us a better understanding of prokaryotic diversity and open up the possibility of finding new prokaryotic species that may lead to the discovery of new therapeutic drugs or have industrial applications.

Table 1: Extremophiles and Their Preferred Conditions
Extremophile Conditions for Optimal Growth
Acidophiles pH 3 or below
Alkaliphiles pH 9 or above
Thermophiles Temperature 60–80 °C (140–176 °F)
Hyperthermophiles Temperature 80–122 °C (176–250 °F)
Psychrophiles Temperature of -15-10 °C (5-50 °F) or lower
Halophiles Salt concentration of at least 0.2 M
Osmophiles High sugar concentration

Figure 4: Radiation-tolerant prokaryotes. Deinococcus radiodurans, visualized in this false color transmission electron micrograph, is a prokaryote that can tolerate very high doses of ionizing radiation. It has developed DNA repair mechanisms that allow it to reconstruct its chromosome even if it has been broken into hundreds of pieces by radiation or heat. (credit: modification of work by Michael Daly scale-bar data from Matt Russell)

Prokaryotes in the Dead Sea

One example of a very harsh environment is the Dead Sea, a hypersaline basin that is located between Jordan and Israel. Hypersaline environments are essentially concentrated seawater. In the Dead Sea, the sodium concentration is 10 times higher than that of seawater, and the water contains high levels of magnesium (about 40 times higher than in seawater) that would be toxic to most living things. Iron, calcium, and magnesium, elements that form divalent ions (Fe 2+ , Ca 2+ , and Mg 2+ ), produce what is commonly referred to as “hard” water. Taken together, the high concentration of divalent cations, the acidic pH (6.0), and the intense solar radiation flux make the Dead Sea a unique, and uniquely hostile, ecosystem 1 (Figure 5).

What sort of prokaryotes do we find in the Dead Sea? The extremely salt-tolerant bacterial mats include Halobacterium, Haloferax volcanii (which is found in other locations, not only the Dead Sea), Halorubrum sodomense, and Halobaculum gomorrense, and the archaean Haloarcula marismortui, among others.

Figure 5: Halophilic prokaryotes. (a) The Dead Sea is hypersaline. Nevertheless, salt-tolerant bacteria thrive in this sea. (b) These halobacteria cells can form salt-tolerant bacterial mats. (credit a: Julien Menichini credit b: NASA scale-bar data from Matt Russell)

Unculturable Prokaryotes and the Viable-but-Non-Culturable State

The process of culturing bacteria is complex and is one of the greatest discoveries of modern science. German physician Robert Koch is credited with discovering the techniques for pure culture, including staining and using growth media. Microbiologists typically grow prokaryotes in the laboratory using an appropriate culture medium containing all the nutrients needed by the target organism. The medium can be liquid, broth, or solid. After an incubation time at the right temperature, there should be evidence of microbial growth (Figure 6). Koch’s assistant Julius Petri invented the Petri dish, whose use persists in today’s laboratories. Koch worked primarily with the Mycobacterium tuberculosis bacterium that causes tuberculosis and developed guidelines, called Koch’s postulates , to identify the organisms responsible for specific diseases. Koch’s postulates continue to be widely used in the medical community. Koch’s postulates include that an organism can be identified as the cause of disease when it is present in all infected samples and absent in all healthy samples, and it is able to reproduce the infection after being cultured multiple times. Today, cultures remain a primary diagnostic tool in medicine and other areas of molecular biology.

Figure 6: Bacteria growing on blood agar plates. In these agar plates, the growth medium is supplemented with red blood cells. Blood agar becomes transparent in the presence of hemolytic Streptococcus, which destroys red blood cells and is used to diagnose Streptococcus infections. The plate on the left is inoculated with non-hemolytic Staphylococcus (large white colonies), and the plate on the right is inoculated with hemolytic Streptococcus (tiny clear colonies). If you look closely at the right plate, you can see that the agar surrounding the bacteria has turned clear. (credit: Bill Branson, NCI)

Koch’s postulates can be fully applied only to organisms that can be isolated and cultured. Some prokaryotes, however, cannot grow in a laboratory setting. In fact, over 99 percent of bacteria and archaea are unculturable. For the most part, this is due to a lack of knowledge as to what to feed these organisms and how to grow them they may have special requirements for growth that remain unknown to scientists, such as needing specific micronutrients, pH, temperature, pressure, co-factors, or co-metabolites. Some bacteria cannot be cultured because they are obligate intracellular parasites and cannot be grown outside a host cell.

In other cases, culturable organisms become unculturable under stressful conditions, even though the same organism could be cultured previously. Those organisms that cannot be cultured but are not dead are in a viable-but-non-culturable (VBNC) state. The VBNC state occurs when prokaryotes respond to environmental stressors by entering a dormant state that allows their survival. The criteria for entering into the VBNC state are not completely understood. In a process called resuscitation , the prokaryote can go back to “normal” life when environmental conditions improve.

Is the VBNC state an unusual way of living for prokaryotes? In fact, most of the prokaryotes living in the soil or in oceanic waters are non-culturable. It has been said that only a small fraction, perhaps one percent, of prokaryotes can be cultured under laboratory conditions. If these organisms are non-culturable, then how is it known whether they are present and alive? Microbiologists use molecular techniques, such as the polymerase chain reaction (PCR), to amplify selected portions of DNA of prokaryotes, e.g., 16S rRNA genes, demonstrating their existence. (Recall that PCR can make billions of copies of a DNA segment in a process called amplification.)


Carl Woese

In 1977, Carl Woese overturned one of the major dogmas of biology. Until that time, biologists had taken for granted that all life on Earth belonged to one of two primary lineages, the eukaryotes (which include animals, plants, fungi and certain unicellular organisms such as paramecium) and the prokaryotes (all remaining microscopic organisms). Woese discovered that there were actually three primary lineages. Within what had previously been called prokaryotes, there exist two distinct groups of organisms no more related to one another than they were to eukaryotes. Because of Woese’s work, it is now widely agreed that there are three primary divisions of living systems – the Eukarya, Bacteria, and Archaea, a classification scheme that Woese proposed in 1990.

The new group of organisms – the Archaea – was initially thought to exist only in extreme environments, niches devoid of oxygen and whose temperatures can be near or above the normal boiling point of water. Microbiologists later realized that Archaea are a large and diverse group of organisms that are widely distributed in nature and are common in much less extreme habitats, such as soils and oceans. As such, they are significant contributors to the global carbon and nitrogen cycles.

The method Woese used to identify this “third form of life,” which involved comparing the sequences of a particular molecule central to cellular function, called ribosomal RNA, has become the standard approach used to identify and classify all organisms. These techniques have also revolutionized ecology, because it is now possible to survey an ecosystem by collecting ribosomal DNA from the environment, thus sidestepping the often impossible task of culturing the organisms that are there. These microorganisms and the revolutionary methods that Woese introduced into science can offer insights into the nature and evolution of cells.

In 1996, Woese and colleagues (University of Illinois professor Gary Olsen and researchers from the Institute for Genomic Research) published in the journal Science the first complete genome structure of an archaeon, Methanococcus jannaschii. Based on this work, they concluded that the Archaea are more closely related to humans than to bacteria. “The Archaea are related to us, to the eukaryotes they are descendants of the microorganisms that gave rise to the eukaryotic cell billions of years ago,” Woese said at the time.

Woese’s experimental discoveries were made in the context of his search for a deep understanding of the process of evolution. As early as the 1970’s Woese was thinking about what sort of theory of evolution one would need in the era before genes as we know them had emerged. At such a time, the standard population genetics theory of evolution would not be applicable. Woese articulated early clear proposals about the nature of what has come to be known as the last universal common ancestor, concluding for a variety of reasons that the universal ancestor was not a single organism, but rather groupings of loosely structured cells that existed together during a time when genetic mutation rates were high and the transfer of genes between cells occurred more frequently than in the present day. The most detailed version of these proposals was put forward on the basis of Woese’s work here at IGB (with University of Illinois professor Nigel Goldenfeld). These groups of primitive cells, called progenotes, evolved together and eventually formed the three ancestral lineages.

“Carl's work, in my view, ranks along with the theory of superconductivity as the most important scientific work ever done on this campus – or indeed anywhere else,” says Dr. Nigel Goldenfeld, leader of the IGB Biocomplexity research theme and long-time colleague of Dr. Woese. “It remains one of the 20th century's landmark achievements in biology, and a rock solid foundation for our growing understanding of the evolution of life.”

Woese passed away in December of 2012 at the age of 84.

Publications and Resources

Read the groundbreaking 1977 publication “Phylogenetic structure of the prokaryotic domain: The primary kingdoms,” by Carl R. Woese and George E. Fox, in which Archaea, the third domain of life, is identified.

Commentaries on the 1977 publication include “Woese and Fox: Life, rearranged,” by Prashant Nair, and “Phylogeny and beyond: Scientific, historical, and conceptual significance of the first tree of life,” by Norman R. Pace, Jan Sapp, and Nigel Goldenfeld.

The 30 th anniversary of the first report of the discovery of Archaea was celebrated in 2007 at the IGB, with a symposium covering the historical aspects of the discovery and how this knowledge has transformed microbial ecology. The program, including videos of the presentations, is available at archaea.igb.uiuc.edu.

Carl R. Woese Memorial

On January 26, 2013, a memorial was held with a number of speakers sharing their remembrances of their interaction with Carl.

The speakers included IGB Director Gene Robinson, President Robert Easter, University of Illinois (at the 4:20 mark), Professor Larry Gold, University of Colorado (at the 9:15 mark), Professor Nigel Goldenfeld (at the 16:20 mark), Professor Richard Herman (at the 24:10 mark), Professor Gary Olsen (at the 31:15 mark), Professor Norman Pace, University of Colorado (at the 36:10 mark), Professor Emeritus Karl Stetter, Universität Regensburg (at the 37:46 mark), LAS Dean Ruth Watkins (at the 41:08 mark), Chancellor Phyllis Wise (at the 47:05 mark), and Professor Emeritus Ralph Wolfe (at the 49:32 mark). The open mike comments begin at the 54:14 mark.

Memories shared via the online guest book can also be viewed here.

Carl R. Woese Research Fund

Donations may be made to the Carl R. Woese Research Fund. Dr. Woese approved this fund to support research on evolution, systems biology and ecosystem dynamics at the Carl R. Woese Institute for Genomic Biology. Gifts may be sent to the “University of Illinois Foundation” in care of the Carl R. Woese Institute for Genomic Biology, 1206 W. Gregory Drive, Urbana, IL 61801 or via the secure website https://www.uif.uillinois.edu/Gifts/StartGiving.aspx. At the bottom of the page is a section "I would like my donation allocated to the following specific fund(s):" Specify your donation amount, and in the field "Other - Indicate where to direct donation here" please type “Carl R. Woese Research Fund” in the box. Click the continue button on the bottom of the page, and you will be directed to a secure page for contact and credit card information. You will have the chance to review this information before submission.

About Dr. Woese

Carl Woese was a professor of microbiology at the University of Illinois at Urbana-Champaign and a faculty member of the Carl R. Woese Institute for Genomic Biology. He was awarded the John D. and Catherine T. MacArthur Foundation “genius” award in 1984, and the National Academy of Sciences elected him to membership in 1988. In 1992 the Dutch Royal Academy of Science gave him the highest honor bestowed upon any microbiologist, the Leeuwenhoek Medal, awarded only once every 10 years. He was given the National Medal of Science in 2000 “for his brilliant and original insights, through molecular studies of RNA sequences, to explore the history of life on Earth.” In 2003 the Royal Swedish Academy of Sciences awarded Woese the Crafoord Prize in Biosciences for his discovery of the third domain of life. The Crafoord award honors scientists whose work does not fall into any of the categories covered by Nobel Prizes. The Royal Society, the world’s oldest continuously active scientific organization, elected Woese as a foreign member in 2006. He held the Stanley O. Ikenberry Endowed Chair and served as Center for Advanced Study Professor of Microbiology.


Biology 171

By the end of this section, you will be able to do the following:

  • Explain the need for nitrogen fixation and how it is accomplished
  • Describe the beneficial effects of bacteria that colonize our skin and digestive tracts
  • Identify prokaryotes used during the processing of food
  • Describe the use of prokaryotes in bioremediation

Fortunately, only a few species of prokaryotes are pathogenic! Prokaryotes also interact with humans and other organisms in a number of ways that are beneficial. For example, prokaryotes are major participants in the carbon and nitrogen cycles. They produce or process nutrients in the digestive tracts of humans and other animals. Prokaryotes are used in the production of some human foods, and also have been recruited for the degradation of hazardous materials. In fact, our life would not be possible without prokaryotes!

Cooperation between Bacteria and Eukaryotes: Nitrogen Fixation

Nitrogen is a very important element to living things, because it is part of nucleotides and amino acids that are the building blocks of nucleic acids and proteins, respectively. Nitrogen is usually the most limiting element in terrestrial ecosystems, with atmospheric nitrogen, N2, providing the largest pool of available nitrogen. However, eukaryotes cannot use atmospheric, gaseous nitrogen to synthesize macromolecules. Fortunately, nitrogen can be “fixed,” meaning it is converted into a more accessible form—ammonia (NH3)—either biologically or abiotically.

Abiotic nitrogen fixation occurs as a result of physical processes such as lightning or by industrial processes. Biological nitrogen fixation (BNF) is exclusively carried out by prokaryotes: soil bacteria, cyanobacteria, and Frankia spp. (filamentous bacteria interacting with actinorhizal plants such as alder, bayberry, and sweet fern). After photosynthesis, BNF is the most important biological process on Earth. The overall nitrogen fixation equation below represents a series of redox reactions (Pi stands for inorganic phosphate).

The total fixed nitrogen through BNF is about 100 to 180 million metric tons per year, which contributes about 65 percent of the nitrogen used in agriculture.

Cyanobacteria are the most important nitrogen fixers in aquatic environments. In soil, members of the genera Clostridium and Azotobacter are examples of free-living, nitrogen-fixing bacteria. Other bacteria live symbiotically with legume plants, providing the most important source of fixed nitrogen. Symbionts may fix more nitrogen in soils than free-living organisms by a factor of 10. Soil bacteria, collectively called rhizobia, are able to symbiotically interact with legumes to form nodules , specialized structures where nitrogen fixation occurs ((Figure)). Nitrogenase, the enzyme that fixes nitrogen, is inactivated by oxygen, so the nodule provides an oxygen-free area for nitrogen fixation to take place. The oxygen is sequestered by a form of plant hemoglobin called leghemoglobin, which protects the nitrogenase, but releases enough oxygen to support respiratory activity.

Symbiotic nitrogen fixation provides a natural and inexpensive plant fertilizer: It reduces atmospheric nitrogen to ammonia, which is easily usable by plants. The use of legumes is an excellent alternative to chemical fertilization and is of special interest to sustainable agriculture, which seeks to minimize the use of chemicals and conserve natural resources. Through symbiotic nitrogen fixation, the plant benefits from using an endless source of nitrogen: the atmosphere. The bacteria benefit from using photosynthates (carbohydrates produced during photosynthesis) from the plant and having a protected niche. In addition, the soil benefits from being naturally fertilized. Therefore, the use of rhizobia as biofertilizers is a sustainable practice.

Why are legumes so important? Some, like soybeans, are key sources of agricultural protein. Some of the most important legumes consumed by humans are soybeans, peanuts, peas, chickpeas, and beans. Other legumes, such as alfalfa, are used to feed cattle.

The commensal bacteria that inhabit our skin and gastrointestinal tract do a host of good things for us. They protect us from pathogens, help us digest our food, and produce some of our vitamins and other nutrients. These activities have been known for a long time. More recently, scientists have gathered evidence that these bacteria may also help regulate our moods, influence our activity levels, and even help control weight by affecting our food choices and absorption patterns. The Human Microbiome Project has begun the process of cataloging our normal bacteria (and archaea) so we can better understand these functions.

A particularly fascinating example of our normal flora relates to our digestive systems. People who take high doses of antibiotics tend to lose many of their normal gut bacteria, allowing a naturally antibiotic-resistant species called Clostridium difficile to overgrow and cause severe gastric problems, especially chronic diarrhea ((Figure)). Obviously, trying to treat this problem with antibiotics only makes it worse. However, it has been successfully treated by giving the patients fecal transplants from healthy donors to reestablish the normal intestinal microbial community. Clinical trials are underway to ensure the safety and effectiveness of this technique.

Scientists are also discovering that the absence of certain key microbes from our intestinal tract may set us up for a variety of problems. This seems to be particularly true regarding the appropriate functioning of the immune system. There are intriguing findings that suggest that the absence of these microbes is an important contributor to the development of allergies and some autoimmune disorders. Research is currently underway to test whether adding certain microbes to our internal ecosystem may help in the treatment of these problems, as well as in treating some forms of autism.

Early Biotechnology: Cheese, Bread, Wine, Beer, and Yogurt

According to the United Nations Convention on Biological Diversity, biotechnology is “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.” 1 The concept of “specific use” involves some sort of commercial application. Genetic engineering, artificial selection, antibiotic production, and cell culture are current topics of study in biotechnology and will be described in later chapters. However, humans were using prokaryotes before the term biotechnology was even coined. Some of the products of this early biotechnology are as familiar as cheese, bread, wine, beer, and yogurt, which employ both bacteria and other microbes, such as yeast, a fungus ((Figure)).

Cheese production began around 4,000 to 7,000 years ago when humans began to breed animals and process their milk. Fermentation in this case preserves nutrients: Milk will spoil relatively quickly, but when processed as cheese, it is more stable. As for beer, the oldest records of brewing are about 6,000 years old and were an integral part of the Sumerian culture. Evidence indicates that the Sumerians discovered fermentation by chance. Wine has been produced for about 4,500 years, and evidence suggests that cultured milk products, like yogurt, have existed for at least 4,000 years.

Using Prokaryotes to Clean up Our Planet: Bioremediation

Microbial bioremediation is the use of prokaryotes (or microbial metabolism) to remove pollutants. Bioremediation has been used to remove agricultural chemicals (e.g., pesticides, fertilizers) that leach from soil into groundwater and the subsurface. Certain toxic metals and oxides, such as selenium and arsenic compounds, can also be removed from water by bioremediation. The reduction of SeO4 -2 to SeO3 -2 and to Se 0 (metallic selenium) is a method used to remove selenium ions from water. Mercury (Hg) is an example of a toxic metal that can be removed from an environment by bioremediation. As an active ingredient of some pesticides, mercury is used in industry and is also a by-product of certain processes, such as battery production. Methyl mercury is usually present in very low concentrations in natural environments, but it is highly toxic because it accumulates in living tissues. Several species of bacteria can carry out the biotransformation of toxic mercury into nontoxic forms. These bacteria, such as Pseudomonas aeruginosa, can convert Hg +2 into Hg 0 , which is nontoxic to humans.

One of the most useful and interesting examples of the use of prokaryotes for bioremediation purposes is the cleanup of oil spills. The significance of prokaryotes to petroleum bioremediation has been demonstrated in several oil spills in recent years, such as the Exxon Valdez spill in Alaska (1989) ((Figure)), the Prestige oil spill in Spain (2002), the spill into the Mediterranean from a Lebanon power plant (2006), and more recently, the BP oil spill in the Gulf of Mexico (2010). In the case of oil spills in the ocean, ongoing natural bioremediation tends to occur, since there are oil-consuming bacteria in the ocean prior to the spill. In addition to these naturally occurring oil-degrading bacteria, humans select and engineer bacteria that possess the same capability with increased efficacy and spectrum of hydrocarbon compounds that can be processed. Bioremediation is enhanced by the addition of inorganic nutrients that help bacteria to grow.

Some hydrocarbon-degrading bacteria feed on hydrocarbons in the oil droplet, breaking down the hydrocarbons into smaller subunits. Some species, such as Alcanivorax borkumensis, produce surfactants that solubilize the oil (making it soluble in water), whereas other bacteria degrade the oil into carbon dioxide. Under ideal conditions, it has been reported that up to 80 percent of the nonvolatile components in oil can be degraded within one year of the spill. Other oil fractions containing aromatic and highly branched hydrocarbon chains are more difficult to remove and remain in the environment for longer periods of time.

Section Summary

Pathogens are only a small percentage of all prokaryotes. In fact, prokaryotes provide essential services to humans and other organisms. Nitrogen, which is not usable by eukaryotes in its plentiful atmospheric form, can be “fixed,” or converted into ammonia (NH3) either biologically or abiotically. Biological nitrogen fixation (BNF) is exclusively carried out by prokaryotes, and constitutes the second most important biological process on Earth. Although some terrestrial nitrogen is fixed by free-living bacteria, most BNF comes from the symbiotic interaction between soil rhizobia and the roots of legume plants.

Human life is only possible due to the action of microbes, both those in the environment and those species that call us home. Internally, they help us digest our food, produce vital nutrients for us, protect us from pathogenic microbes, and help train our immune systems to function properly.

Microbial bioremediation is the use of microbial metabolism to remove pollutants. Bioremediation has been used to remove agricultural chemicals that leach from soil into groundwater and the subsurface. Toxic metals and oxides, such as selenium and arsenic compounds, can also be removed by bioremediation. Probably one of the most useful and interesting examples of the use of prokaryotes for bioremediation purposes is the cleanup of oil spills.

Free Response

Your friend believes that prokaryotes are always detrimental and pathogenic. How would you explain to them that they are wrong?

Remind them of the important roles prokaryotes play in decomposition and freeing up nutrients in biogeochemical cycles remind them of the many prokaryotes that are not human pathogens and that fill very specialized niches. Furthermore, our normal bacterial symbionts are crucial for our digestion and in protecting us from pathogens.

Many people use antimicrobial soap to kill bacteria on their hands. However, overuse may actually increase the risk of infection. How could this occur?

Soap indiscriminately kills bacteria on skin. This kills harmful bacteria, but can also eliminate “good” bacteria from the skin. When the non-pathogenic bacteria are eliminated, pathogenic bacteria can colonize the empty surface.

Footnotes

Glossary


Watch the video: Biology - What is Binary fission? #5 (July 2022).


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