Bacteria and Their Structure
The old kingdom Monera contained organisms that have now been classified into the domains Bacteria and Archaea. Organisms in these domains are unicellular (one-celled) and prokaryotic (lacking a membrane-bound nucleus). Bacteria are among the simplest, smallest, and most ancient of organisms, found in nearly every environment on earth. While some bacteria are autotrophic (capable of making their own food), most are heterotrophic (forced to draw nutrients from their environment or from other organisms). For most of human history, the existence of bacteria was unknown. It was not until the late 1800s that bacteria were first identified. Their role in nature is that of decomposers: they break down organic molecules into their component parts. Along with fungi, they are the major recyclers in nature. They are also capable of changing atmospheric nitrogen to a form that is usable by plants and animals.
It has long been known that some bacteria are pathogens, or causers of disease. Scientists have expended tremendous effort to describe the role such bacteria play in disease and to create agents that can kill them. Other bacteria, such as Escherichia coli, may be part of a mutualistic relationship with another organism, such as humans. Bacteria have been used extensively in genetics research because of their small size and because they reproduce rapidly; some bacteria produce a new generation every twenty minutes. Because they have been so thoroughly studied, a great deal is known about their structure and genetics.
Most bacteria are less than one micron (one-millionth of a meter) in length. They do not contain mitochondria (organelles that produce the energy molecule adenosine triphosphate, or ATP), chloroplasts (plant organelles in which the reactions of photosynthesis take place), lysosomes (organelles that contain digestive enzymes), or interior membrane systems such as the endoplasmic reticulum or Golgi bodies. They do, however, contain RNA, ribosomes (organelles that serve as the sites of protein synthesis), and DNA, which is organized as part of a single, circular chromosome. The circular chromosome is centrally located within the cell in a region called the nucleoid region and is capable of supercoiling. Bacteria often have additional genes carried on small, circular DNA molecules called plasmids, which have been used extensively in genetic research. Some plasmids carry genes that impart antibiotic resistance to the cells that contain them.
Bacteria have three basic morphologies, or cell shapes. Bacteria that are spherical are called cocci. Some coccus bacteria form clusters (staphylococcus), while others may form chains (streptococcus). Bacteria that have a rodlike appearance are called bacilli. Spiral or helical bacteria are called spirilla (sometimes called spirochetes).
Classification of Bacteria
Bacteria fall into three basic types: those that lack cell walls, those with thin cell walls, and those with thick cell walls. Mycoplasmas lack cell walls entirely. The bacteria that cause tuberculosis,
Mycobacterium tuberculosis
, do have cell walls, and unlike Archaea, their cell walls are composed of peptidoglycan, a complex organic molecule made of two unusual sugars held together by short polypeptides (short chains of amino acids). In 1884, Hans Christian Gram, a Danish physician, found that certain bacterial cells absorb a stain called crystal violet, while others do not. Those cells that absorb the stain are called gram-positive, and those that do not are called gram-negative. It has since been found that gram-positive bacteria have thick walls of peptidoglycan, while gram-negative bacteria have thin peptidoglycan walls covered by a thick outer membrane. It is this thick outer membrane that prevents crystal violet from entering the bacterial cell. Distinguishing between gram-positive and gram-negative bacteria is an important step in the treatment of disease, as some antibiotics are more effective against one class than the other.
In contrast to bacteria, members of the domain Archaea have cell walls that do not contain peptidoglycan. Archaea are usually found in extreme environments, such as hot springs, extremely saline environments, and hydrothermal vents. Methanogens are the most common and are strict anaerobes, which means that they are killed by oxygen. They live in oxygen-free environments, such as sewers and swamps, and produce methane gas as a waste product of their metabolism. Halobacteria live in only those environments that have a high concentration of salt, such as salt ponds. Thermoacidophiles
grow in very hot or very acidic environments.
Bacteria can be further differentiated by the presence or absence of certain surface structures. Some strains produce an outer slime layer called a “capsule.” The capsule permits the bacterium to adhere to surfaces (such as human teeth, for example, where the buildup of such bacteria causes dental plaque) and provides some protection against other microorganisms. Some strains display pili, which are fine, hairlike appendages that also allow the bacterium to adhere to surfaces. Some pili, such as F pili in E. coli, are involved in the exchange of genetic material from one bacterium to another in a process called conjugation. Some bacterial strains have one or more flagella, which allow them to be motile (capable of movement). Any bacterium may have one or more of these surface structures.
Research in molecular genetics is continuing to expand insight into bacterial classification and gene function. Many researchers have been actively sequencing the genomes
of bacteria from a broad spectrum. The number of species that have been sequenced is now in the hundreds and includes many human pathogens, such as those that cause tuberculosis, bacterial pneumonia, ulcers, bacterial influenza, leprosy, and Lyme disease. The genomes of a wide range of nonpathogenic bacteria have also been sequenced. Comparisons of the genomes that have been sequenced are beginning to show extensive evidence that bacteria of different species have transferred genes back and forth many times in the past, thus making it difficult to trace their evolutionary lineages.
Bacterial Reproduction
Bacteria reproduce in nature by means of binary fission,
wherein one cell divides to produce two daughter cells that are genetically identical. As bacteria reproduce, they form clustered associations of cells called colonies. All members of a colony are genetically identical to one another, unless a mutagen (any substance that can cause a mutation) has changed the DNA sequence in one of the bacteria. Changes in the DNA sequence of the chromosome often lead to changes in the physical appearance or nutritional requirements of the colony. While a bacterium is microscopic, bacterial colonies can be seen with the naked eye, and changes in the colonies are relatively easy to perceive. This is one of the reasons bacteria have been favored organisms for genetic research.
For the most part, there is very little genetic variation between one bacterial generation and the next. Unlike higher organisms, bacteria do not engage in sexual reproduction, which is the major source of genetic variation within a population. In laboratory settings, however, bacteria can be induced to engage in a unidirectional (one-way) exchange of genetic material via conjugation, first observed in 1946 by biochemists Joshua Lederberg
and Edward Tatum. The unidirectional nature of the gene transfer was discovered by William Hayes in 1953. He found that one bacterial cell was a donor cell while the other was the recipient. In the 1950s, molecular biologists François Jacob
and Elie Wollman used conjugation and a technique called “interrupted mating” to map genes onto the bacterial chromosome. By breaking apart the conjugation pairs at intervals and analyzing the times at which donor genes entered the recipient cells, they were able to determine a correlation between time and the distance between genes on a chromosome. The use of this technique led to a complete map of the sequence of genes contained in the chromosome. It also led to a surprise: it was the use of interrupted mating with E. coli that first demonstrated the circularity of the bacterial chromosome. The circular structure of the chromosome was in striking contrast to eukaryotic chromosomes, which are linear.
Transformation and Transduction
The bacterium
Streptococcus pneumoniae
was used in one of the early studies that eventually led to the identification of DNA as the master chemical of heredity. Two strains of S. pneumoniae were used in a study conducted by microbiologist Frederick Griffith in 1928. One strain, S, produces a smooth colony that is virulent (infectious) and causes pneumonia. The other strain, R, produces a rough colony that is avirulent (noninfectious). When Griffith injected mice with living type-R bacteria, the mice survived, and no bacteria were recovered from their blood. When he injected mice with living type S, the mice died, and type-S bacteria were recovered from their blood. However, if type S was heat-killed before the mice were injected, the mice did not die, and no bacteria were recovered from their blood. This confirmed what Griffith already knew: only living type-S S. pneumoniae caused lethal infections. Something interesting happened when Griffith mixed living type R with heat-killed type S, however: mice injected with this mixture died, and virulent type-S bacteria were recovered from their blood. An unknown agent apparently transformed avirulent type R into virulent type S. Griffith called the agent the “transforming principle.” It was his belief that the transforming principle was a protein.
Sixteen years later, in 1944, bacteriologists Oswald Avery, Colin MacLeod, and Maclyn McCarty designed an experiment that showed conclusively that the transforming principle was DNA rather than protein. They showed that R bacteria could be transformed into S bacteria in a test tube. They then progressively purified their extract until only proteins and the two nucleic acids, RNA and DNA, remained. They placed some of the mixture onto agar plates (glass dishes containing a gelatin growth medium). At this point, transformation still occurred; therefore, it was clear that one of these three molecules was the transforming agent. They treated their extract with protein-degrading enzymes, which denatured (destroyed) all the proteins in the extract. Despite the denaturing of the proteins, transformation still occurred when some of the extract was plated; had protein been the transforming agent, no transformation could have occurred. Protein was eliminated as the transforming agent. The next step was to determine which of two nucleic acids was responsible for the transformation of the R strain into the S strain. They introduced RNase, an enzyme that degrades RNA, to the extract. The RNA was destroyed, yet transformation took place. RNA was thus eliminated. At this point, it was fairly obvious that DNA was the transforming agent. To conclusively confirm this, they introduced DNase to the extract. When the DNA was degraded by the enzyme, transformation did not take place, showing that DNA was indeed the transforming agent.
Another way that genetic material can be exchanged between bacteria is by transduction. Transduction requires the presence of a bacteriophage
(a virus that infects bacteria). A virus
is a simple structure consisting of a protein coat called a capsid that contains either RNA or DNA. Viruses are acellular, nonliving, and extremely small. To reproduce, they must infect living cells and use the host cell’s internal structures to replicate their genetic material and manufacture viral proteins. Bacteriophages, or phages, infect bacteria by attaching themselves to a bacterium and injecting their genetic material into the cell. Sometimes, during the assembly of new viral particles, a piece of the host cell’s DNA may be enclosed in the viral capsid. When the virus leaves the host cell and infects a second cell, that piece of bacterial DNA enters the second cell, thus changing its genetic makeup. Generalized transduction (the transfer of a gene from one bacterium to another) was discovered by Joshua and Esther Lederberg and Norton Zinder in 1952. Using E. coli and a bacteriophage called P1, the Lederbergs and Zinder were able to show that transduction could be used to map genes to the bacterial chromosome.
Hershey-Chase Bacteriophage Experiments
The use of bacteriophages has been instrumental in confirming DNA as the genetic material of living cells. In 1953, Alfred Hershey and Martha Chase
devised a series of experiments using E. coli and the bacteriophage T2 that conclusively established DNA as genetic material. Bacteria are capable of manufacturing all essential macromolecules by utilizing material from their environment. Hershey and Chase grew cultures of E. coli in a growth medium enriched with a radioactive isotope of phosphorus, phosphorus-32. DNA contains phosphorus; as the succeeding generations of bacteria pulled phosphorus from the growth medium to manufacture DNA, each DNA strand also carried a radioactive label. T2 phages were used to infect the cultures of E. coli. When the new T2 viruses were assembled in the bacterial cells, they too carried the radioactive label phosphorus-32 on their DNA. A second culture of E. coli was grown in a medium enriched with radioactive sulfur-35. Proteins contain sulfur but no phosphorus. T2 viruses were used to infect this culture. New viruses contained the sulfur-35 label on their protein coats.
Since the T2 phage consists of only protein and DNA, one of these two molecules had to be the genetic material. Hershey and Chase infected unlabeled E. coli with both types of radioactive T2 phages. Analysis has shown that the phosphorus-32 label passed into the bacterial cells, while the sulfur-35 label was found only in the protein coats that did not enter the cells. Since the protein coat did not enter the bacterial cell, it could not influence protein synthesis. Therefore, protein could not be the genetic material. The Hershey-Chase experiment confirmed DNA as the genetic material.
Restriction Enzymes and Gene Expression
Using the aforementioned methods, it has been possible to construct a complete genetic map showing the order in which genes occur on the chromosome of E. coli and other bacteria. Certain genes are common to all bacteria. There are also several genes that are shared by bacteria and higher life-forms, including humans. Further research showed that genes can be either inserted into or deleted from bacterial DNA. In nature, only bacteria contain specialized enzymes called restriction enzymes. Restriction enzymes are capable of cutting DNA at specific sites called restriction sites. The function of restriction enzymes in bacteria is to protect against invading viruses. Bacterial restriction enzymes are designed to destroy viral DNA without harming the host DNA. Hundreds of different restriction enzymes have been isolated from bacteria, and each is named for the bacterium from which it comes. The discovery and isolation of restriction enzymes led to a new field of biological endeavor: genetic engineering.
Use of these enzymes has made gene cloning possible. Cloning is important to researchers because it permits the detailed study of individual genes. Restriction enzymes have also been used in the formation of genomic libraries
(a collection of clones that contains at least one copy of every DNA sequence in the genome). Genomic libraries are valuable because they can be searched to identify a single DNA recombinant molecule that contains a particular gene or DNA sequence.
Bacterial studies have been instrumental in understanding the regulation of gene expression, or the translation of a DNA sequence first to a molecule of messenger RNA (mRNA) and then to a protein. Bacteria live in environments that change rapidly. To survive, they have evolved systems of gene regulation that can either “turn on” or “turn off” a gene in response to environmental conditions. François Jacob and Jacques Monod discovered the
lac operon, a regulatory system that permits E. coli to respond rapidly to changes in the availability of lactose, a simple sugar. Other operons, such as the tryptophan operon, were soon discovered as well. An operon is a cluster of genes whose expression is regulated together and involves the interaction of regions of DNA with regulatory proteins. The discovery of operons in bacteria led to searches for them in eukaryotic cells. While none have been found, using a strict definition of the term, some eukaryotes have very similar methods of regulating the expression of genes.
Impact and Applications
Diabetes mellitus is a disease caused by the inability of the pancreas to produce insulin, a protein hormone that is part of the critical system that controls the body’s metabolism of sugar. Prior to 1982, people who suffered from diabetes controlled their disease with injections of insulin that had been isolated from other animals, such as cows. In 1982, human insulin became the first human gene product to be manufactured using recombinant DNA. The technique is based on the knowledge that genes can be inserted into the bacterial chromosome; that once inserted, the gene product, or protein, will be produced; and that once produced, the protein can be purified from bacterial extracts. Human proteins are usually produced by inserting a human gene into a plasmid
vector, which is then inserted into a bacterial cell. The bacterial cell is cloned until large quantities of transformed bacteria are produced. From these populations, human proteins, such as insulin, can be recovered.
Many proteins used against disease are manufactured in this manner. Some examples of recombinant DNA pharmaceutical products that are already available or in clinical testing include atrial natriuretic factor, which is used to combat heart failure and high blood pressure; epidermal growth factor, which is used in burns and skin transplantation; factor VIII, which is used to treat hemophilia; human growth hormone, which is used to treat dwarfism; and several types of interferons and interleukins, which are proteins that have anticancer properties.
Bacterial hosts produce what are called the “first generation” of recombinant DNA products. There are limits to what can be produced in and recovered from bacterial cells. Since bacterial cells are different from eukaryotic cells in a number of ways, they cannot process or modify most eukaryotic proteins. Nor can they add sugar groups or phosphate groups, additions that are often required if the protein is to be biologically active. In some cases, human proteins produced in prokaryotic cells do not fold into the proper three-dimensional shape; since shape determines function in proteins, these proteins are nonfunctional. For this reason, it may never be possible to use bacteria to manufacture all human proteins. Other organisms are used to produce what are called the second generation of recombinant DNA products.
The impact of the study of bacterial structures and genetics and the use of bacteria in biotechnology cannot be underestimated. Bacterial research has led to the development of an entirely new branch of science, that of molecular biology. Much of what is currently known about molecular genetics, the expression of genes, and recombination comes from research involving the use of bacteria. Moreover, bacteria have had and will continue to have applications in the production of pharmaceuticals and the treatment of disease. The recombinant DNA technologies developed with bacteria are now being used with other organisms to produce medicines and vaccines.
Key Terms
cloning
:
the generation of many copies of DNA by replication in a suitable host
eukaryote
:
an organism made up of cells having a membrane-bound nucleus that contains chromosomes
mutation
:
the process by which a DNA base-pair change or a change in a chromosome is produced; the term is also used to describe the change itself
prokaryote
:
an organism lacking a membrane-bound nucleus
recombinant DNA
:
a DNA sequence that has been constructed or engineered from two or more distinct DNA sequences
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