Genomes of bacteria exist on a single double-stranded circular DNA molecule that contains approximately 4000 kb of DNA and are regulated by operons. A mutation is a change in the nucleotide sequence and can create new cellular functionalities or lead to the dysfunction of others. Mutations can occur spontaneously or be caused by exposure to mutation-inducing agents.
While the majority of bacterial genes exist on one circular chromosome, there are other genetic elements within the bacterial genome. Elements like plasmids, transposons, integrons, or gene cassettes are shorter sequences that mainly contribute to recombination events. Bacterial DNA replication and transcription co-occur and utilize the same template DNA. Replication forks proceed bi-directionally with a single origin of replication, OriC.
Bacterial genes with similar functions often share one promoter (RNA polymerase binding site) and are transcribed simultaneously; this system is called an operon. Typical operons consist of several structural genes that code for the enzymes required for the pathway. Regulation occurs through transcription factors binding to a short sequence of DNA between the promoter region and the structural genes called an operator.
A mutation is a change in the nucleotide sequence of a short region of a genome, and phenotypic results may vary on the severity and location of the mutation. Mutations can be the result of errors during DNA replication or induced by exposure to mutagens (like chemicals and radiation). Spontaneous mutations occur at a rate of 1 in 10^5 to 10^8 and contribute to random population variation. Since bacteria are haploid for the majority of their genes and have short generation turnover, phenotypic variation due to point mutations can occur relatively quickly.
Results of mutations can produce changes in structural or colony characteristics or loss in sensitivity to antibiotics. Some potential consequences of mutations are as follows:
Spontaneous mutations occur without mutation induction and are the result of errors during DNA replication. When DNA Pol III is synthesizing a new strand of DNA, occasionally a nucleotide will be mispaired, added, or omitted. Thus a point mutation will occur. For example, when nucleotides are mispaired, it will appear that one nucleotide substitutes for another leading to one mutated granddaughter DNA strand. Two separate malfunctions must happen in the bacteria's DNA replication machinery for this to occur:
DNA bases can exist in many different forms, referred to as tautomers. Nucleotide bases dominantly exist in the keto (C-O) and amino (C-NH2) forms, while the imino (C≡NH) and enol (C-OH) occur rarely. Tautomerization, during DNA replication, will alter nucleotide base pair formation. For example, assume that thymine undergoes keto-enol tautomerization during replication. This enol species will preferentially bind to guanine during the first replication cycle. Due to the semiconservative nature of DNA replication, at the end of the 2nd round of replication, there will be (3) A-T base pairs and (1) G-C in the locus of mutation.
The mechanism is as follows:
T – A --> Tautomerization --> T' – A --> replication 1 --> T' – G and A –T
T – G --> Replication 2 --> T – A and G – C
(enol form of thymine indicated as T')
Errors in DNA replication can result in the addition of erroneous nucleotides or deletion of template nucleotides. For example, loci with a high number of short repeat nucleotides are prone to polymerase slippage. During replication, the DNA Pol III temporarily dissociates from the template strand. Along with its newly synthesized strand, the DNA Polymerase may relocate a few repeats upstream or downstream of its original locus. Slip strand mispairing can result in addition/deletion mutations because some nucleotides are replicated twice while others do not replicate. If the repeats are not in a multiple of three, the mutation can result in a frameshift (A shift in the coding sequence downstream of the mutation). These mutations lead to loss of normal protein functionality. Slip-strand mispairings can increase variation of short tandem repeats (STRs) in a bacterial population and are useful in genetic testing. When an addition or deletion occurs, the potential genomic outcomes are as follows:
The mutation's phenotypical severity depends on the structure of the substituted amino acid's effect on the final protein product. More specifically, non-synonymous amino acid substitutions produce dramatic changes in protein structures because of the chemical dis-similarities of the mutated strand amino acid. However, there are inherent protections against these types of mutations. The redundancy of codon translation mechanisms and occurrence of non-coding regions result in few mutations expressing phenotypically.
Mutagens may be of physical, chemical, or of biological origin. Mostly they act on the DNA directly, causing damage which may result in errors during replication. Although, severely damaged DNA can prevent replication and cause cell death. SOS is an example of a cellular response to DNA damage that results in cell cycle arrest and induction of mutagenesis. Rec A induces SOS response by recognizing single-stranded DNA and activating mutagenic DNA polymerases (II, IV, and V).
The following are several classes of mutagens and their subsequent effects:
Examples of physical mutagens include radiation or UV exposure. UV radiation damages DNA by creating covalent linkages between adjacent pyrimidine bases. This pyrimidine dimer cannot fit well in the double helix structure of DNA and thus inhibiting replication and translation. However, dimer formation usually results in a deletion mutation. Other types of radiation can have a variety of effects (Depending on intensity and wavelength), but mostly insertions/deletions occur. Purine dimers rarely occur.
Chemical mutagens are agents that either directly or indirectly induce mutations. A chemical mutagen can either replace a base in DNA, alter a base's composition and pairing behavior, or damage the base so that it can no longer pair. These include DNA reactive chemicals such as:
Structurally similar enough to nucleotides in that they can incorporate into DNA. For example, 5-bromouracil, an analog of thymine, acts as a substrate during DNA replication and causes point mutations. This mispairing occurs because the base analog forms a tautomer and pairs with guanine instead of adenine.
Reactive oxygen species:
Hydroxyl radicals attack guanine thereby producing 8-hydroxy-deoxyguanosine (8-OhdG) which mispairs with adenine instead of cytosine which resulting in a (G -> T) transversion during replication.
These agents remove amino groups on nucleotide bases. Deaminating agents produce an adenine species that pairs with cytosine and a cytosine species (Uracil) that pairs to adenine. Deamination of guanine results in xanthine which inhibits replication, thereby not creating a mutation.
Flat aromatic compounds:
Acridines like ethidium bromide can intercalate with adjacent pyrimidine base pairs. This interaction slightly unwinds the helix and increases the distance between adjacent base pairs. This intercalation disrupts the reading frame during translation and can cause insertions or deletions.
Agents like ethyl methanesulfonate and dimethyl nitrosoguanidine alter the nucleotide base by adding alkyl groups. The nature and position of the alkylation can vary but usually leads to point mutations through base mispairing. However, alkylation can cause crosslink formation which inhibits replication.
Biological agents of mutation are sources of DNA from elements like transposons and viruses. Transposons are sequences of DNA that can relocate and replicate autonomously. Insertion of a transposon into a DNA sequence can disrupt gene functionality. Transposition is not technically a type of recombination but is mechanistically similar. Transposons often pair with short regions of nucleotide repeats on either side of the transposition sequence. There are three types of transposons:
Antibiotics work through a variety of mechanisms:
When an antibiotic loses the capacity to kill or control bacterial growth, antibiotic resistance occurs. This can occur in two ways:
These circumstances exacerbate under selective pressure (i.e., the use of antibiotics). Antibiotic resistance can spread both vertically and horizontally through a population. Horizontal transfer is considered the primary mediator of antibiotic resistance. Following are non-comprehensive examples of how two of the classes of antibiotics mentioned above encounter resistance mutations.
DNA Synthesis Inhibitor
In gram-negative bacteria, such as Helicobacter pylori, mutation resistance occurs relatively quickly to fluoroquinolones and thereby poses clinical issues for these therapies. Levofloxacin, moxifloxacin, and ciprofloxacin, examples of fluoroquinolones, inhibit DNA synthesis by targeting two homologous enzymes (DNA topoisomerase II and IV). These enzymes are necessary for supercoiling of bacterial DNA.
Gram-negative bacterial resistance to fluoroquinolones includes accumulation of substitution mutations in the coding regions for particular subunits of DNA topoisomerase II. Resistance can be enhanced further by efflux pump modification. Ciprofloxacin targets only the parC subunit while other quinolones target one or more of these subunits. For example, Garenoxacin targets both DNA Topoisomerases II and IV thus is less prone to resistance. Resistance to Garenoxacin requires both proteins to have resistance mutations.
Combination therapy for Helicobacter pylori typically includes clarithromycin (protein synthesis inhibitor), metronidazole (DNA synthesis inhibitor), amoxicillin (Cell wall synthesis inhibitor) or tetracycline (Protein synthesis inhibitor), and a proton pump inhibitor.
Protein Synthesis Inhibitor
Linezolid prevents protein synthesis and is active against resistant Gram-positives. Linezolid inhibits the formation of the 70S ribosomal initiation complex through binding to the 23S portion of the 50S subunit. Infrequent resistance found in strains of staphylococci aureus and coagulase-negative staphylococci have mutations in the central loop of the domain V region of the 23S rRNA gene. More specifically, clinical isolates had a substitution of Thymine for Guanine at the 2576 position.
Intrinsically, resistant bacteria have a characteristic resistance within all members of a species or genus. Such resistance may arise because:
Antibiotic resistance mechanisms can also occur through the incorporation of resistance genes into plasmids, transposons, and integrons. These genes spread through horizontal transfer by conjugation, transformation, or transduction mechanisms. However, mutation is essential for the evolution or assortment of these genes.