BACTEBIAL GENEIICS

No discussion of bacterial genetics is complete without first describing deoxyribonucleic acid (DNA) and

ribonucleic acid (RNA). Historically, DNA was first discovered by Frederick Miescher in 1869. In the 1920s,

Phoebus A.T. Levine discovered that DNA contained phosphates, five-carbon sugars, and nitrogen-containing bases. Later, Rosalind Franklin discovered the helical structure by x-ray crystallography. Most everyone is

familiar with James Watson and Francis Crick who both described the three-dimensional structure of the DNA

molecule in the 1950s

 

Anatomy of a DNA and RNA Molecule

DNA is a double helical chain of nucleotides. The helix is a double strand twisted together, which many scientists

refer to as a “spiral staircase” (resembling th  handrail, sides, and steps of a spiral staircase). Others refer to it as a “zipper with teeth.” A nucleotide is a complex combination of a phosphate group (POJ, a five-carbon (pentose) sugar  (deoxyribose), which makes up the “handrails and sides,” and a nitrogen-containing base, or the “steps,” either a purine or a pyrimidine. A purine consists of a fused ring of nine carbon atoms and nitrogen. There are two purines in the molecule: adenine and guanine. A pyrimidine consists of a single ring of six atoms of carbon and nitrogen. There are two pyrimidines in the molecule: thymine and cytosine. These are the basic building blocks of DNA (Figure 1-13). In the chain of nucleotides, bonds form between the phosphate group of one nucleotide and the sugar of the next nucleotide. The base extends out of the sugar. Adenine of one chain always pairs with thymine of the other chain, and cytosine of one chain pairs with guanine of the other chain. The bases are held together by hydrogen bonds. The information contained in DNA is determined primarily by the sequence of letters along the “staircase” or “zipper.” The sequence of ACGCT represents different information than the sequence AGTCC. This would be like taking the word “stops” and using the same letters to form the word “spots” or “posts,” which have different meanings but all the same letters. The two complementary sugar phosphate strands run in opposite directions, 3′ to 5′ and 5′ to 3′, like one train with its engine going one way alongside a caboose of a train going the opposite direction (Figure 1-14). DNA is also involved in the production of RNA.In RNA, the nitrogenous base thymine is replaced by uracil, another pyrimidine. Unlike DNA, RNA is single stranded and short, not double stranded and long, and contains the sugar ribose not deoxyribose. An interesting aspect is introduced. Human beings are 99.9% identical. In a human genome of three billion “letters,” even one-tenth of 1%translates into 3 million separate lettering differences, an important characteristic useful in forensic science, but with related importance in diagnostic microbiology utilizing the bacterial genome. Bacterial genetics is increasingly important in the diagnostic microbiology laboratory. New diagnostic tests have been developed that are based on identifying unique RNA or DNA sequences present in each bacterial species. The polymerase chain reaction (PCR) technique is a means of amplifying specific DNA sequences and thus detecting very small numbers of bacteria present in a specimen. Genetic tests circumvent the need to culture bacteria, providing a more rapid method of identifying pathogens. An understanding of bacterial genetics is also necessary to understand the development and transfer of antimicrobial resistance by bacteria. The occurrence of mutations can result in a change in the expected phenotypic characteristics of an organism and provides an explanation for atypical results sometimes encountered on diagnostic biochemical tests. This section briefly reviews some of the basic terminology and concepts of bacterial genetics. For a detailed discussion of DNA and molecular diagnostics, see Chapter 11

Terminology

The genotype of a cell is the genetic potential of the DNA of an organism. It includes all the characteristics that are coded for in the DNA of a bacterium and that have the potential to be expressed. Some genes are silent genes, expressed only under certain conditions. Genes that are always expressed are constitutive. Genes that are expressed only under certain conditions are inducible. The phenotype of a cell consists of the genetic characteristics of a cell that actually are expressed and can be observed. The ultimate aim of a cell is to produce the proteins that are responsible for cellular structure and function and to transmit the information for accomplishing this to the next generation of cells.

Information for protein synthesis is encoded in the bacterial DNA and transmitted in the chromosome to each generation. The general flow of information in a bacterial cell is from DNA (which contains the genetic information) to messenger RNA (mRNA) (which acts as a blueprint for protein construction) to the actual protein itself. Replication is the duplication of chromosomal DNA for insertion into a

daughter cell. Transcription is the synthesis of singlestranded RNA (with the aid of the enzyme RNA polymerase) using one strand of the DNA as a template. Translation is the actual synthesis of a specific protein

from the mRNA code. The term protein expression also refers to the synthesis (Le., translation) of a protein. Proteins are polypeptides composed of amino acids. The number and sequence of amino acids in

a polypeptide, and thus the character of that particular protein, is determined by sequence of codons in the mRNA molecule. A codon is a group of three nucleotides in an mRNA molecule that signifies a specific amino acid. During translation, ribosomes containing ribosomal RNA (rRNA) sequentially add amino acids to the growing polypeptide chain. These amino acids are brought to the ribosome by transfer RNA (tRNA) molecules that “translate” the codons. Transfer RNA molecules temporarily attach to the mRNA using their complementary anticodon regions. An anticodon is the triplet of bases on the tRNA that bind the triplet of bases on the mRNA. It identifies which amino acid will be in a specific location in the protein

 

Genetic Elements and Alterations The Bacterial Genome

The bacterial chromosome (also called the genome) consists of a single, closed, circular piece of doublestranded DNA that is supercoiled in order to fit inside the cell. It contains all the information needed for cell growth and replication. Genes are specific DNA sequences that code for the amino acid sequence in one protein (e.g., one gene equals one polypeptide), but this may be sliced up and/or combined with other polypeptides to form more than one protein. In front of each gene on the DNA strand is an untranscribed area containing a promoter region, which the RNA polymerase recognizes for transcription initiation. This area may also contain regulatory regions to which molecules may attach and cause either a decrease or an increase in transcription.

 

Extrachromosomal Elements

In addition to the genetic information encoded in the bacterial chromosome, many bacteria contain extra information on small circular pieces of DNA called plasm ids. Genes that code for antibiotic resistance (and sometimes toxins or other virulence factors) are often located on plasmids. Antibiotic therapy selects for bacterial strains containing plasmids encoding antibiotic resistance genes; this is one reason antibiotics should not be overprescribed. The number of plasmids present in a bacterial cell may vary from one (low copy number) to hundreds (high copy number). Plasm ids are located in the cytoplasm of the cell and can be replicated and passed to daughter cells just like chromosomal DNA. They may also sometimes be passed from one bacterial species to another. This is one way resistance to antibiotics is acquired.

 

Mobile Genetic Elements

Certain pieces of DNAare mobile and may jump from one place in the chromosome to another place. These are sometimes referred to as “jumping genes.” The simplest mobile piece of DNAis an insertion sequence (IS) element. It is about 1000 base pairs long with inverted repeats on each end. Each IS element codes for only one gene, a transposase enzyme that allows the IS element to pop into and out of DNA. Bacterial genomes contain many IS elements. The main effect of IS elements in bacteria is that when an IS element inserts itself into the middle of a gene, it disrupts and inactivates the gene. This can result in loss of an observable characteristic, such as the ability to ferment a particular sugar. Transposons are related mobile elements that contain additional genes. Transposons often carry antibiotic-resistance genes and are usually located in plasmids.

 

Mutations

A gene sequence must be read in the right “frame” for the correct protein to be produced. This is because every set of three bases (known as a codon) specifies a particular amino acid, and when the reading frame is askew, the codons are interpreted incorrectly. Mutations are changes that occur in the DNAcode and often (not always; “silent mutations” do not make a change in the protein) results in a change in the coded protein or in the prevention of its synthesis. A mutation may be the result of a change in one nucleotide base (a point mutation) that leads to a change in a single amino acid within a protein or may be the result of insertions or deletions in the genome that lead to disruption of the gene and/or a frameshift mutation. Incomplete, inactive proteins are often the result. Spontaneous mutations occur in bacteria at a rate of about one in 109 cells. Mutations also occur as the result of error during DNAreplication at a rate of about one in 107 cells. Exposure to certain chemical and physical agents can greatly increase the mutation rate.

 

Genetic Recombination

Genetic recombination is a method by which genes are transferred or exchanged between homologous (similar) regions on two DNAmolecules. This method provides a way for organisms to obtain new combinations of biochemical pathways and copy with changes in their environment.

 

Mechanisms of Gene Transfer

Genetic material may be transferred from one bacterium to another in tree basic ways

*transformation

*transduction

*conjucation

Transformation

Transformation is the uptake and incorporation of naked DNAinto a bacterial cell (Figure 1-15,A). Once the DNAhas been taken up, it can be incorporated into the bacterial genome by recombination. If the DNAis a circular plasmid and the recipient cell is compatible, the plasmid can replicate in the cytoplasm and be transferred to daughter cells. Cells that can take up naked DNAare referred to as being competent. Only a few bacterial species, such as Streptococcus pneumaniae, Neisseria gonorrhoeae, and H. influenzae, do this naturally. Bacteria can be made competent in the laboratory,

and transformation is the main method used to introduce genetically manipulated plasmids into.

bacteria, such as E. coli, during cloning procedures.

 

Transduction

Transduction is the transfer of bacterial genes by a bacteriophage (virus infected bacteria) from one cell to another (see Figure 1-15, B). A bacteriophage consists of a chromosome (DNA or RNA) surrounded by a protein coat. When a phage infects a bacterial cell, it injects its genome into the bacterial cell, leaving

the protein coat outside. The phage may then take a lytic pathway, in which the bacteriophage DNA directs

the bacterial cell to synthesize phage DNA and phage protein and package it into new phage particles. The

bacterial cell then lyses (lytic phase), releasing new phage, which can infect other bacterial cells. In some

instances, the phage DNA instead becomes incorporated into the bacterial genome, where it is replicated along with the bacterial chromosomal DNA; this state is known as lysogeny, and the phage is referred to as

being temperate. During lysogeny, genes present in the phage DNA may be expressed by the bacterial

cell. An example of this in the clinical laboratory is Corynebacterium diphtheriae. Strains of C. diphtheriae

that are lysogenized with a temperate phage carrying the gene for diphtheria toxin cause disease. Strains lacking the phage do not produce the toxin and do not cause disease.

Under certain conditions a temperate phage can be induced, the phage DNA is excised from the bacterial genome, and a lytic state occurs. During this process, adjacent bacterial genes may be excised with the

phage DNA and packaged into the new phage. The bacterial genes may then be transferred when the phage

infects a new bacterium. In the field of biotechnology, phages are often used to insert cloned genes into

bacteria for analysis.

 

Conjugation

Conjugation is the transfer of genetic material from a donor bacterial strain to a recipient strain (see Figure 1-15, C). It requires close contact between the two cells. In the E. coli system, the donor strain (F+) possesses a fertility factor (F factor) on a plasmid that carries the genes for conjugative transfer. The donor strain produces a hollow surface appendage called a sex pilus, which binds to the recipient F- cell and brings the two cells in close contact. Transfer of DNA then occurs. Both plasmids and chromosomal genes can be transferred by this method. When the F factor is integrated into the bacterial chromosome rather than a plasmid, there is a higher frequency of transfer of adjacent bacterial chromosomal genes. These strains are known as high-frequency (Hfr) strains.

 

Restriction Enzymes

Bacteria have evolved a system to restrict the incorporation of foreign DNA into their genomes. Specific

restriction enzymes are produced that cut incoming  foreign DNA at specific DNA sequences. The bacteria methylate their own DNA at these same sequences so that the restriction enzymes do not cut the DNA in their own cell. Many restriction enzymes with a variety of recognition sequences have now been isolated from various microorganisms. The first three letters in the restriction endonuclease name indicate the bacterial source of the enzyme. For instance, the enzyme EcoRI was isolated from E. coli, and the enzyme Hindill was isolated from H. influenzae type d. These enzymes are used in the field of biotechnology to create sites for insertion of new genes. In clinical microbiology, epidemiologists use restriction enzyme fragment analysis to determine whether strains of bacteria have identical restriction sites in their genomic DNA.

SUMMARY

The study of microbes of medical importance includes bacteria, parasites, fungi, and viruses. Bacteria are prokaryotes in nature, meaning they lack a true nucleus and nuclear membrane. Eukaryotes, such as parasites, have a true nucleus and other membranous organelles. Fungi can exist in a unicellular or filamentous form. Viruses are true obligate parasites that consist of DNA or RNA, not both. In the nomenclature of bacteria, the genus (first letter is uppercase) and species (letters are lowercase) are used most often and are italicized or underlined. The genotype of an organism is the actual set of genes in its genome. Phenotype refers to functional and physical traits of an organism (e.g., morphology, structures, and metabolism). Of all the stains used, the Gram stain is the most prevalent in the laboratory. It is the first step in classifying bacteria through microscopic morphology and stain reaction-gram-positive (purple) or gram-negative (pink or red). Bacteria are also classified on their nutritional requirements for growth. Most human inhabitants are heterotrophs that require complex substances for growth, although some are more fastidious than others. Many microbes vary widely in their use of various compounds or substrates, and a variety of biochemical pathways exist for substrate breakdown. Microbiologists use these phenotypic characteristics in the identification of bacteria and other microbes. DNA and RNA differ in the structure of their monomers (nucleotide). Each nucleotide consists of a phosphoric acid, a pentose sugar, either ribose or deoxyribose, and one of five cyclic nitrogenous bases:

adenine, guanine, cytosine, thymine, and uracil. The purines contain double-ring molecules of adenine and guanine; pyrimidines are single rings of uracil, cytosine, and thymine. Strands of DNA are complementary of each other: adenine on one strand of hydrogen bonds with thymine on the other, and guanine hydrogen bonds with cytosine on the other. In RNA, uracil replaces the

thymine.

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