Although macroscopically, ecosystems on Earth appear to be composed of plants and animals, there are some key players that exist beyond what we can see. Bacteria are tiny, single celled microbes that help all life exist. They have a very simple structure with no nucleus, a structure that protects the DNA in more complex cells. Bacteria range from helpful organisms that break down dead material and provide food for ecosystems to pathogenic bacteria that can cause disease.
When most people think of bacteria, they think of disease causing organisms, like the Streptococcus bacteria growing in culture in this picture, which were isolated from a man with strep throat. While pathogenic bacteria are notorious for such diseases as cholera, tuberculosis, and gonorrhea, such disease causing species are a comparatively tiny fraction of the bacteria as a whole.
Bacteria grow in a wide variety of habitats and conditions:
Bacteria are so widespread that it is possible only to make the most general statements about their life history and ecology. They may be found on the tops of mountains, the bottom of the deepest oceans, in the guts of animals, and even in the frozen rocks and ice of Antarctica. One feature that has enabled them to spread so far, and last so long is their ability to go dormant for an extended period.
Bacteria have a wide range of environmental and nutritive requirements:
Most bacteria may be placed into one of three groups based on their response to gaseous oxygen.
- Aerobic Bacteria Aerobic bacteria thrive in the presence of oxygen and require it for their continued growth and existence.
- Anaerobic Bacteria Anaerobic, and cannot tolerate gaseous oxygen, such as those bacteria which live in deep underwater sediments, or those which cause bacterial food poisoning.
- Facultative anaerobes The bacteria which prefer growing in the presence of oxygen, but can continue to grow without it.
Bacteria may also be classified on the basis of mode by which they obtain their energy:
Classifying according to the source of their energy there are two categories of Bacteria:
- Heterotrophs: Heterotrophs derive energy from breaking down complex organic compounds that they must take in from the environment. This includes saprobic bacteria found in decaying material, as well as those that rely on fermentation or respiration.
- Autotrophs: The other group, the autotrophs, fix carbon dioxide to make their own food source; this may be fueled by light energy (photoautotrophic), or by oxidation of nitrogen, sulfur, or other elements (chemoautotrophic). While chemoautotrophs are uncommon, photoautotrophs are common and quite diverse. They include the cyanobacteria, green sulfur bacteria, purple sulfur bacteria, and purple nonsulfur bacteria. The sulfur bacteria are particularly interesting, since they use hydrogen sulfide as hydrogen donor, instead of water like most other photosynthetic organisms, including cyanobacteria.
Application of Bacteria in Molecular Biology A variety of microorganisms are used by molecular biologists to study gene structure and function. Some scientists study microorganisms because they are pathogenic to plants, humans or other animals, and through learning more about these pathogenic organisms, eﬀective drugs and strategies for infection control can be developed. Microorganisms, particularly bacteria and yeasts, are also used by many scientists as a tool for molecular biology research. Bacteria cultures grow very quickly, and most strains used in molecular research divide in less than 45min. Thus, a single bacterial cell can, in 16h, produce suﬃcient numbers of cells for isolating deoxyribonucleic acid (DNA) and many proteins. By contrast, a mammalian cell requires 18h to complete cell division, and several days of growth are needed to produce comparable cell numbers. Media contain essential components needed for cell growth, including a carbon source, a nitrogen source and essential vitamins and cofactors. Additionally, media often contain antibiotics or deﬁned components that allow for selective growth of cells, especially cells containing recombinant DNA. An essential skill for culturing microorganisms is aseptic technique. Media prepared in the laboratory must be kept sterile, and cultures must be free of contamination by other microorganisms present in the laboratory.
Bacteria: Prokaryotic Unicellular Organism Organisms are generally classiﬁed as prokaryotic (no nucleus or other organelles) or eukaryotic (containing organelles). Bacteria are unicellular prokaryotes: the organism is one cell. Some bacteria have very simple growth requirements. This, in combination with their rapid division time, makes them ideal research organisms. By far the most frequently used bacterial species in molecular biology is Escherichia coli. E. coli is a Gram-negative bacteria found in the intestines of many mammals and can often be pathogenic. However, the commonly used laboratory strains do not carry toxins and therefore are not considered pathogenic. E. coli is regularly used for cloning genes and growing plasmid DNA (discussed later) from many diﬀerent organisms. Since the genetic code is conserved among living organisms, DNA from any source can be replicated in E. coli. See also: Escherichia coli as an Experimental Organism; Gene Expression in Escherichia coli Not all bacteria are, however, easy to culture. For example, the bacterium that causes tuberculosis grows very slowly in the laboratory, and thus is diﬃcult to culture. Other bacteria are naturally found in extreme or unique environments and thus require specialized conditions for culture in the laboratory. Some examples include bacteria that grow in deep oceanic thermal vents, in anaerobic environments or in acid lakes. Although these bacteria are not routinely cultured in the laboratory, they contain enzymes which have been cloned and are now essential to molecular biology research. Probably the most widely used are the DNA polymerases isolated from thermophilic (heat loving) bacteria, used in high-throughput sequencing and ampliﬁcation of DNA by a technique called polymerase chain reaction (PCR). Molecular biologists also use restriction enzymes to clone and analyse DNA. Restriction enzymes, isolated from many diﬀerent species of bacteria, act as a natural host defence mechanism to prevent bacteriophage infection. These enzymes cut DNA molecules at speciﬁc sequences and many are routinely used in research and forensic DNA analysis.
Bacterial Genes and Genome
The bacterial genome consists of one circular chromosome, attached to the bacterial cell wall. This chromosome contains all the genes the bacterium needs to replicate DNA and make proteins needed for cell growth. Each time a bacterium divides, the entire chromosome is replicated once and a copy goes with the new cell. This is regarded as asexual reproduction, and the new cell is an exact copy, or clone, of the original cell.
Bacterial Genetics Bacterial cells reproduce asexually, always producing an exact clone. For evolutionary survival, bacteria also have several mechanisms by which they ‘trade’ or share their DNA with other individual bacterial cells, resulting in variability in genomic content. Genes that are critical for survival in speciﬁc situations, such as genes for antibiotic resistance, are exchanged between individuals, including cross-species exchange. There are three main mechanisms for exchanging DNA between individual bacterial cells.
Transformation: Transformation is the process by which bacteria pick up DNA from their immediate surroundings. In the natural environment, the source can be DNA fragments released from dead bacterial cells. In the laboratory, this is the most common method used in molecular biology to clone and replicate genes in bacteria.
Transduction: Transduction is the exchange of genetic information mediated by bacteriophages. Bacteriophages are also commonly used tools for cloning and manipulating DNA.
Conjugation: Conjugation is the process by which bacteria exchange DNA through specialized protein structures called sex pili.
Each of these mechanisms of DNA transfer is employed in a variety of molecular biology techniques to manipulate DNA.
Plasmids as Vectors for Amplifying Foreign DNA In addition to the circular chromosome, bacteria may carry smaller extrachromosomal circular DNA, called plasmids, which are replicated in the cell but not necessarily carried along in cell division. Often these plasmids carry nonessential genes that allow bacteria to grow in special environmental conditions, such as genes to degrade unique nutrients in the environment (e.g. oil) or genes to break down toxins in the environment (e.g. antibiotics). Over the past few decades, bacterial geneticists have isolated and altered these plasmids, and now hundreds of diﬀerent specialized plasmids exist. Plasmids are used by molecular biologists in all ﬁelds of research as vectors for cloning and amplifying DNA from many diﬀerent organisms. The structure and composition of DNA is universal among all living organisms. Thus, DNA from any other ‘foreign’ organism, if inserted or cloned into a bacterial vector, can be ampliﬁed and then isolated from a bacterial cell. This process allows scientists to produce large quantities of a speciﬁc DNA sequence for experimental study.
- A multiple cloning site containing several unique restriction enzyme sites, allowing foreign DNA to be easily inserted into the plasmid.
- A selectable marker to select the bacteria which received the vector DNA during a bacterial transformation. The selectable marker is often an antibiotic resistance gene such as b lactamase, an enzyme that degrades ampicillins. 3. An origin of replication so that the bacterial DNA polymerase will replicate the plasmid, including the inserted foreign DNA. Origins of replication vary so that some vectors will be present in only a few copies, while other vectors may have many copies within an individual cell.
Circular vector DNA can be cut, or linearized, at one or two of the restriction sites in the multiple cloning site. Foreign DNA is then combined with the linearized vector and an enzyme (ligase or topoisomerase) joins the ends together, forming a circular plasmid containing foreign DNA (2), and restriction enzymes. A plasmid can be put into a bacterial cell by transformation, as outlined in Figure 2. In the laboratory this is easily accomplished by placing freshly grown cells into calcium chloride solution, adding the plasmid DNA and allowing the DNA to adsorb to the bacterial membrane.
Following a brief heat shock, some of the bacterial cells will take up the DNA. The cells are then plated and allowed to grow over night to form bacterial colonies. Electroporation is another common transformation process, in which a quick electrical pulse is delivered to cells. During the temporary disruption of the cellular membrane, DNA enters the cell. See also: Electroporation The transformation process is ineﬃcient, so that only a small fraction of the bacterial cells actually take up the plasmid DNA. The selectable marker on the plasmid allows for selection of these cells by plating the cells on media containing antibiotic. For example, after transformation of vector DNA carrying the b-lactamase gene, the bacterial cells are plated on to media containing ampicillin. Bacterial cells that have obtained the plasmid begin producing the enzyme that degrades ampicillin and are able to begin cell division, forming colonies after about 16h. The ampicillin inhibits growth of all bacterial cells lacking the plasmid. A single colony, or clone, containing the plasmid can then be transferred into a large volume of liquid media, and allowed to grow overnight. This culture will yield large quantities of the puriﬁed plasmid for further study and analysis. Bacterial vectors can eﬃciently replicate inserts of DNA of about 10000bp in length. While this is adequate for some genes and sequences, often scientists need to work with longer fragments of DNA. Specialized vectors called BACs, for bacterial artiﬁcial chromosomes, have been developed which can allow cloning of inserts 100–300000bp in length. The vectors are essential for cloning very large genes and transforming these genes into other cell types, such as insect, plant or mammalian cells. BACs are essential to the sequencing of large genomes, as they allow for sequencing of much larger continuous pieces of DNA, signiﬁcantly reducing the time-consuming task of piecing together many small segments of DNA.
Bacteriophages as Vectors for Amplifying Foreign DNA Bacteriophages are viruses of the bacterial world. Their entire life cycle, shown in Figure3, exists within the conﬁnes of a bacterial cell. During this replication process, bacterial DNA may be packaged into the phage particles with the phage DNA. This DNA is then ultimately transferred to another bacterial cell in a process called transduction. Foreign DNA can be inserted into the phage genome so that it is packaged into the phage particles. The bacteriophage genome is much larger than plasmids, and can be used as a tool to clone fragments of DNA up to 24000bp.When bacteria are infected with the recombinant phage, the foreign DNA is then ampliﬁed along with the phage DNA. As the phages replicate and lyse the bacterial cells, phage particles are released into the culture supernatant. These phage particles can then be isolated and the cloned DNA recovered in large quantities for use in other experiments.
The Ecological Role of Bacteria
Bacteria as Producers Producers are organisms that make their own food, which we usually think of as green plants. The truth is that bacteria are the producers in many ecosystems as well. Producers make food for the entire ecosystem, supporting animals that eat plants, or herbivores, which in turn support carnivores. Without producers, there would be no life on Earth. There are bacteria that do photosynthesis using carbon dioxide and sunlight, like plants, and bacteria that do chemosynthesis, where they use chemicals to make food.