Micro 3

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**Operon**

**A group of functionally related genes which are transcribed together into one mRNA and whose products are translated from the same mRNA. Bacterial genes are often arranged in an operon. Genes in an operon are coordinated together by means of an operator, a promoter, as well as other cis-acting sequences required for expression and regulation of the genes of the operon. Operons with many structural genes are **polycistronic**. For example, E. coli lac operon comprises all the genes required for lactose metabolism, as well as the control sequences to regulate the expression of the operon in the presence or absence of glucose and/or lactose.**

*EXTRACHROMOSOMAL ELEMENTS*

*Plasmids* *Bacteriophages*

*transformation*

- is the uptake of naked DNA by bacteria from the environment. Both Gram positive and Gram negative bacteria can uptake and stably maintain foreign DNA. Certain species of bacteria are naturally competent to take up exogenous DNA. However, most bacteria are not naturally competent. • Transformation is a three step process. 1) The naked DNA must bind to the bacterial cell surface. 2) The bound DNA is taken up through the cell membrane. 3) All or some of the DNA is integrated into the host chromosome or replicates autonomously as a plasmid.

*The SOS response*

4) The SOS response: Upon encountering damage to their genomes due to physical/chemical mutagens, bacteria such as E. coli respond by activating the SOS network, consisting of about forty genes whose task is to repair/bypass the DNA damage, in order to enable DNA replication. The SOS genetic network deploys a variety of specific functions such as detecting damage, repairing it correctly by nucleotide excision or by recombination, and if these functions do not succeed, *bypassing* damage by mutagenesis. The activation of all these functions requires a high degree of coordination and regulation.

*Frameshift mutation*

A SMALL deletion or insertion that is not in multiples of three (a codon). It can result in a change in the reading frame, usually leading to a useless peptide and premature truncation of the protein.

*Genetic Mutations*

A mutation is any change in the base pair sequence of DNA. Mutations can occur either spontaneously as a result of errors in DNA replication or due to direct changes in the DNA by a variety of chemical agents (mutagens). Mutations occur at different rates in different organisms. In bacteria spontaneous mutation rate is ~ 1 in 107 to 108 cell divisions. A few examples of mutations are: *base substitutions* *deletions* *insertions* *rearrangements*

*Repressor*

A protein that negatively regulates gene expression by preventing the polymerase from binding to the promoter.

*Activator*

A protein that positively regulates gene expression by binding upstream of the promoter at an *upstream activator site (UAS)*, where it can help the RNA polymerase bind to the promoter.

*Null mutation*

An EXTENSIVE insertion, deletion, or gross rearrangement of the chromosome structure which completely destroys gene function.

*MECHANISMS OF GENE TRANSFER IN BACTERIA*

Bacteria are quite capable of taking up foreign DNA or exchanging DNA with other bacteria. The exchange of DNA between different bacteria allows the cells to acquire new genetic material, thus producing new strains of bacteria. This exchange of new material can sometimes be advantageous for the recipient especially if it provides the recipient with a growth advantage (e.g. acquisition of antibiotic resistance). The new DNA can be integrated into the host genome or be stably maintained as an extrachromosomal element *(plasmid)* or a virus *(bacteriophage)* and transferred to daughter bacteria as an autonomously replicating unit. The exchange or transfer of genetic material between bacterial cells can take place by three basic mechanisms *transformation* *transduction* *conjugation*

Bacteriophages:

Bacteriophages: Viruses that infect bacteria are called bacteriophages or phages. Phages are similar to other virus in many of their properties. They contain either DNA or RNA enclosed in a proteinaceous coat (capsid). Phages can survive outside the cell due to the presence of the protein coat. Some phages (e.g. phage T4) also carry a tail-like structure which helps them in attaching to and infecting their bacterial host. Phages infect by attaching to a specific receptor on the surface of the bacterium and the nucleic acid enters the cell. • Virulent/lytic phages • Temperate phages, such as lambda () phage,

*Generalized transduction*

In this case, phage DNA is injected into the host cell and through a series of steps produces its own DNA concatamers (a linear sequence of covalently attached phage genomes), digests the concatamers into single pieces of phage DNA using DNase and then packages the DNA into proteinaceous phage heads. When mature phages are ready the bacterial cells are lysed and phages are released. DNase that digests the phage concatamers can also cut other DNA in the host cell (genomic or plasmid). Some of these DNA pieces are just the right size to be erroneously packaged in the phage head in place of the phage DNA. • Phages that carry the bacterial DNA are known as *transducing phages*.

*Operator*

Sequence close to, or even overlapping the promoter, to which a repressor protein binds.

*GENETIC MUTATIONS AND DNA REPAIR*

Since bacteria are haploid organisms (carrying only one copy of the chromosome), there is no genetic exchange by meiosis and zygote formation. One of two processes is responsible for changes in bacterial genome, mutation or recombination, resulting in progeny DNA with phenotypic properties different from the parent. This can be significant in terms of bacterial virulence and drug resistance. *GENETIC MUTATIONS* *DNA REPAIR*

*DNA Repair*

Since bacteria carry only one chromosome, mutations can be extremely dangerous to their survival. Thus, bacteria have developed several repair mechanisms to combat both spontaneously occurring and induced mutational damage. These repair mechanisms include the following: *1) Direct DNA repair * *2) Excision repair* *3) Recombination or Post-replication repair* *4) The SOS response* *5) Error-prone repair * • DNA damage and repair mechanisms exist in both prokaryotes and eukaryotes with many of the proteins involved being highly conserved throughout evolution. The investigation of bacterial DNA repair has provided a model for understanding similar, more complex processes in humans including issues of cancer and aging. For example, several human disorders are now known to be DNA-repair related. These include diseases like *xeroderma pigmentosum*, characterized by extreme sensitivity to the sun, with great risk for development of a variety of skin cancers such as basal cell carcinoma, squamous cell carcinoma and melanoma.

*Effectors*

Small molecules which bind to regulatory proteins and affect their activity. If the effector induces transcription of an operon, for example, by binding to a repressor and changing it so that the repressor no longer binds to the DNA, the effector molecule is called an *inducer*. If, by binding to a repressor, it causes the operon to be turned off, it is called a *corepressor*.

*Specialized transduction*

Temperate phages (e.g. phage lambda) can either undergo a _lytic cycle resulting in generalized transduction of bacterial DNA_ or a _lysogenic replication cycle in which they are integrated at specialized attachment sites_ in the host genome after injection (prophages). When a prophage is induced, its DNA is excised from the bacterial chromosome, the phage replicates, and the host cell lyses, releasing mature phage particles. On rare occasion, the excision of the integrated prophage DNA is not precise taking some of the adjacent host DNA with it. • Transduction with these phages usually transfers only a restricted number of bacterial genes (usually those adjacent to their integration sites in the genome) to recipient bacterial cell. This is called *specialized transduction*. Plasmid DNA cannot be transferred by this process. • If the host DNA replaces essential phage genes, the resulting phage will be a *defective phage*, which cannot mature and replicate in the absence of a normal phage.

*Promoter*

The sequence to which the RNA polymerase binds.

*GENETIC MATERIAL*

Total genetic material in bacteria includes both the genes it carries on its chromosome as well as any pla8smids *(extrachromosomal genetic elements) it may carry. The bacterial chromosome differs from the human chromosomes in a number of ways

*transducing phages*

When a transducing phage injects chromosomal DNA into another bacterial cell, the incoming DNA may be incorporated into the recipient bacterial DNA by homologous recombination and the recipient is stably transduced. The requirement is that the incoming DNA must be homologous to a region in the host DNA and the host must have a functional rec system. Similar to transformation, plasmid DNA maybe transduced and expressed in a recipient without recombination. • Generalized phages are valuable in genetic mapping of bacterial chromosome. The closer two genes are within the bacterial chromosome; the more likely it is that they will be co-transduced in the same fragment of DNA.

*Bacteriocins*

a class of antibacterial agent that are active only against similar or closely related bacterial strains.

Non-composite transposons

also carry DNA segments (e.g. antibiotic resistance genes), which are flanked by _inverted repeat sequences but not IS elements_. An example is Tn3 encodes B-lactam antibiotic resistance.

*MOBILE GENETIC ELEMENTS*

can be defined as any piece of DNA that can be translocated from one part of a genome to another or between genomes. These elements are not capable of independent replication and thus cannot be translocated to a new host unless associated with a plasmid or bacterial chromosome. They are found in both prokaryotes and eukaryotes. Examples of mobile genetic elements include: *insertion sequences (ISs)* *transposons* *integrons* *pathogenicity islands*

Composite transposon

carry genes flanked by _identical IS elements on either end_. These IS elements function in concert and move together along with the intervening DNA (carrying one or more antibiotic-resistance genes such as kanamycin resistance in Tn5). IS elements at the ends of composite transposons may be either in the same or inverted orientation (i.e. direct or inverted repeats).

*nonsense mutation*

changes a codon for an amino acid to a stop codon, causing premature termination and production of a truncated protein.

*Direct DNA repair*

constitutes either reversal or enzymatic removal of damage, such as abnormally linked pyrimidine bases in DNA (pyrimidine dimers) and alkylated bases. Ultraviolet radiation causes pyrimidine dimer formation in DNA strands. A photoreactivating enzyme directly reverses these dimers restoring the phosphodiester bonds between complementary bases. This repair process is also known as *photoreactivation*.

*Gene Transfer and Recombination*

genetic material is transferred from one bacterium to another resulting in new genotypes. There are two possible consequences of these events. Either, the incoming DNA can recombine with the recipient genome (recombination), or it can be on a plasmid capable of replication in the recipient without recombination. *Homologous recombination* *Site specific recombination*

*R-factors*

if a plasmid contains one or more *antibiotic resistance genes*, the bacteria will be able to survive in the presence of those antibiotics during an infection. Such plasmids are called *R-factors*. Gram negative bacteria carry plasmids that confer resistance to antibiotics such as neomycin, kanamycin, streptomycin, chloramphenicol, tetracyclines, penicillins, and sulfonamides.

*Base substitutions (point mutations)*

in which a single nucleotide is changed into a different nucleotide, arise due to replication errors that cause mispairing between complementary bases. Base substitutions could be *transitions* where one purine is replaced by another purine or one pyrimidine is replaced by another pyrimidine or *transversion* in which case a purine is replaced by a pyrimidine and vice versa. Base substitutions may result in *silent mutation* *missense mutation* *nonsense mutation* • More dramatic changes in the DNA occur when numerous bases are involved. These can result in deletion, replacement, insertion or inversion of several bases.

*Virulence determinants*

include adhesins that are produced by the plasmids of Yersinia enterocolitica and Yersinia pestis. Also in this group are genes that may provide bacteria with unique advantage in metabolizing certain substrates for example genes for degradation of camphor, toluene, octane and salicylic acid produced

*Post-transcriptional regulation*

includes mRNA stability/degradation; translational regulation; post-translational regulation (protein stability, protein modification, and protein degradation) (no further discussion).

*Recombination or Post-replication repair*

involves retrieval of missing information by genetic recombination when both DNA strands are damaged.

*transduction*

is a genetic transfer that is mediated by bacteriophages, which pick up fragments of bacterial DNA, package them into bacteriophage particles and transfer them into another bacterial cell where it is integrated into the chromosome. This transfer takes place in both Gram positive and Gram negative bacteria and does not require cell-to-cell contact (DNase resistant). There are many types of bacteriophages and not all of them take part in transduction. Only double stranded DNA phages are capable of transduction. Bacteriophages are responsible for two types of transduction mechanisms: *Generalized transduction* *Specialized transduction*

*Conjugation (plasmid mediated transfer)*

is a mechanism by which DNA is transferred from one cell to the other by direct contact during the mating of the bacteria. • Conjugation results in a uni-directional transfer of DNA from the donor (male; F+) cell to the recipient (female; F-) through direct contact. • The mating type or sex of the cell depends on the presence or absence of a conjugative plasmid or episome such as the *F plasmid* of E. coli. • Conjugation can occur with most if not all eubacteria. It usually takes place between members of the same or related species but has been observed between prokaryotes and cells from plants, animals, and fungi. • Conjugative plasmids are large, carry genes that code for the production of *sex pilus (tra genes)*, enzymes necessary for conjugation, and genes necessary to initiate DNA synthesis at the *origin of transfer (OriT)*. They may also carry antibiotic resistance genes as well as virulence factors. • The F plasmid transfers itself, converting recipients into F+ male cells. • If a fragment of chromosomal DNA is incorporated into the plasmid, it is termed an F prime (F') plasmid. When such plasmid is transferred into the recipient cell, it carries that fragment with it and converts the recipient into an F' male. • If the F plasmid sequence gets integrated into the bacterial chromosome, the cell is designated an *Hfr (high frequency recombination)* cell. In such cases, transfer of a part of the plasmid and some portion of the bacterial chromosomal DNA can take place. However, conjugation with Hfr donor cells does not result in complete transfer of the integrated plasmid. Thus, the recipient cell does not become Hfr and is incapable of serving as a conjugation donor

*Excision repair*

is exemplified by removal of the damaged DNA base or bases by housekeeping enzymes like DNA glycosylases and endonucleases. This is followed by synthesis of a new DNA strand by DNA polymerase which fills the gap using the intact complementary DNA strand as a template. There are three types of excision repair mechanisms each of which utilizes a specific set of enzymes. These are *base excision repair, nucleotide excision repair, and mismatch repair.*

*Error-prone repair*

is the ONLY remaining option for bacteria before they die. It is used to fill in gaps with a random sequence when the template DNA is not available, resulting in a lot of errors.

*Transcriptional Regulation*

occurs by controlling the amount of mRNA that is made from the genes, making sure that proteins are only produced when and if they are needed. Transcriptional initiation Transcriptional termination

*Site specific recombination*

occurs when one DNA molecule integrates into another DNA molecule with no homology other than a small site on each DNA called an: *attachment* *integration* or *insertion site.* It is a mechanism used to combine circular pieces of DNA like plasmids, temperate phages and transposons, for e.g. integration of F-plasmid into the bacterial chromosome to make Hfr cell; integration of temperate phage into the bacterial genome to create a prophage; movement and insertion of transposons. This type of recombination requires restriction endonucleases and restriction endonuclease sites on each DNA.

*Toxins*

some plasmids carry toxin genes such as enterotoxins from E. coli or Vibrio cholerae.

*Temperate phages*

such as lambda () phage, have the ability to enter a non-lytic *prophage* state in which they integrate into the bacterial chromosome and replication of their nucleic acid is linked to replication of host cell DNA. In this state, almost all the phage genes are completely repressed, as is replication of the nucleic acid, so no phage particles are produced *(latent infection)*. Bacteria carrying prophages are designated *lysogenic* because a physiologic signal can trigger a lytic cycle resulting in excision of the viral DNA from the host chromosome, activation of replication and assembly, cell lysis and viral release. Due to the presence of prophages, some lysogenized bacteria express new characteristics such as an increase in bacterial virulence *(lysogenic conversion)*. For example, Vibrio cholerae produce the cholera toxin only when infected by CTX.

*Virulent/lytic phages*

take over the host replication and protein synthesizing machinery to produce new virus DNA and protein. Some of the phage genes (the early genes coding for proteins required for replication) are expressed almost immediately, using host enzymes. A number of copies of the phage nucleic acid are then made, and expression of the late genes (needed for production of phage particle) starts. Many new virus particles (virions) are then assembled and released into the environment as the bacterial cell ruptures (lyses). This is called the *lytic cycle*. T-even phages of E. coli (e.g. T2, T4) are the most thoroughly studied lytic phages.

*missense mutation*

where a different amino acid is inserted in the translated protein and may or may not alter protein stability or functional properties.

*Silent mutation*

where there is no change in the amino acid sequence of the protein encoded by the gene, because the altered codon also specifies for the Same amino acid

*E. coli F plasmids* are called *episomes*,

which means, that they can insert into the bacterial chromosome, where they become a permanent part of the bacterial genome.

*BACTERIAL GENE REGULATION*

• Each bacterial genome carries many genes (sequences of nucleotides that have a biological function). Majority of these are protein-coding genes *(cistrons)*. Other set of genes are transcribed to produce ribosomal RNA species that provide framework to assemble ribosomal subunits. • Bacterial genomes also carry sequences that are recognition and binding sites (cis-sites for example *promoters and operators*) for regulatory trans-acting elements (*transcription factors - activators and repressors*). • Bacteria can adapt very readily to changing environment by regulating their gene expression. Bacterial gene regulation can occur at two main levels 1) *Transcriptional regulation* 2) *Post-transcriptional regulation*

**Homologous recombination**

• Homologous recombination is the mechanism by which newly introduced genes are stabilized in the recipient cell. It results in an "exchange" of DNA between the linear piece of DNA (exogenote) and a homologous region on the bacterial chromosome. Homologous recombination requires o a region of homology between the DNA strands and o a number of recombination enzymes/factors encoded by recombination genes **recA**, recB, recC and recD (recA being absolutely required).

*integrons*

• Integrons are a diverse group of genetic elements that encode a site-specific recombination system that can capture gene cassettes (most often antibiotic resistance gene cassettes) in tandem arrays. • Even though they lack terminal repeat sequences and enzymes required for transposition, integrons are often found associated with larger mobile genetic elements such as transposons or plasmids. These elements allow horizontal gene transfer of integrons between different bacterial genera. Integrons have also been found on bacterial chromosomes. • The key components of an integron are the integrase (int1) gene, which encodes for the recombinase responsible for the insertion and assortment of gene cassettes and an associated attI site for integration of cassettes and recognition of the integrase.

*insertion sequences (ISs)*

• Mobile genetic elements which have the ability to insert at multiple sites in a target molecule such as the chromosome or plasmids. • They have two distinguishing characteristics. First, they are relatively small in size (700-2500 bp in length) as compared to other transposable elements. Second, they only encode proteins required for insertion function such as the transposase enzyme and a regulatory protein that can either stimulate or inhibit transposition activity. • The coding region of an IS element is usually flanked by short *inverted repeats* (15 to 40 base pairs), which are important for locating and inserting into a DNA target. • As a result of their translocation, IS elements can disrupt coding or regulatory sequences of genes, alter expression of nearby genes by the action of the IS element promoter, cause deletions and inversions in DNA, and also serve as a site for cross-over between duplicated IS elements.

*transposons*

• Mobile genetic elements, similar to IS elements, that can transfer (transpose) DNA within a cell from one position to another in the chromosome or from an extrachromosomal genetic element to the chromosome and vice versa. • In contrast to IS elements, transposons are >2 kb in size and carry genes (like antibiotic resistance genes) in addition to those that encode enzymes required for transposition (the transposase and resolvase genes). The most extensively studied transposable elements are the ones found in E. coli and other Gram negative bacteria. Transposons can be of two types. 1) *Composite transposon* carry genes flanked by identical IS elements on either end. These IS elements function in concert and move together along with the intervening DNA (carrying one or more antibiotic-resistance genes such as kanamycin resistance in Tn5). IS elements at the ends of composite transposons may be either in the same or inverted orientation (i.e. direct or inverted repeats). 2) *Non-composite transposons* also carry DNA segments (e.g. antibiotic resistance genes), which are flanked by inverted repeat sequences but not IS elements. An example is Tn3 encodes -lactam antibiotic resistance. • Transposons utilize two general mechanisms to translocate between different segments of DNA. During *replicative transposition* there is duplication and the transposon leaves a copy of itself at the original location. Thus, the number of transposons doubles during each replicative transposition event. A *non-replicative transposon*, on the other hand, utilizes a "cut and paste" mechanism where it does not leave a copy of itself in the original location. • Since transposons can move into or out of DNA sequences so easily, transposition onto a broad-host range conjugative plasmid can lead to rapid dissemination of resistance among different bacteria. • Transposons can sometimes insert into a gene thereby inactivating it. If the insertion and inactivation takes place in an essential gene, it can be lethal.

*Plasmids*

• Plasmids are small, autonomous, self-replicating DNA molecules that are mostly circular (some bacteria like Borrelia carry linear plasmids). • Some plasmids such as the *E. coli F plasmids* are called *episomes*, which means, that they can insert into the bacterial chromosome, where they become a permanent part of the bacterial genome. • Even though plasmids may not carry genes essential for cell survival, they often carry information that gives bacteria a selective advantage. For e.g. o *R-factors* - if a plasmid contains one or more *antibiotic resistance genes*, the bacteria will be able to survive in the presence of those antibiotics during an infection. Such plasmids are called *R-factors*. Gram negative bacteria carry plasmids that confer resistance to antibiotics such as neomycin, kanamycin, streptomycin, chloramphenicol, tetracyclines, penicillins, and sulfonamides. o *Toxins* - some plasmids carry toxin genes such as enterotoxins from E. coli or Vibrio cholerae. o *Bacteriocins* - a class of antibacterial agent that are active only against similar or closely related bacterial strains. o *Virulence determinants* - include adhesins that are produced by the plasmids of Yersinia enterocolitica and Yersinia pestis. Also in this group are genes that may provide bacteria with unique advantage in metabolizing certain substrates for example genes for degradation of camphor, toluene, octane and salicylic acid produced by Pseudomonas sp.

*pathogenicity islands*

• Special class of genomic islands acquired through horizontal gene transfer. • They carry groups of coordinately regulated virulence genes surrounded by direct or inverted repeat IS elements. The genes carried by pathogenicity islands include adhesins, toxins, iron uptake systems, invasins etc.


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