CYTO L5

BACTERIAL GENETICS

-   The study of mechanisms of heritable information in bacteria and their chromosomes, plasmids, transposons and phages.

-   Originally developed from a refined E. Coli as a consequence wherein elegant plasmid as well as the cloning expression and mutagenesis systems were being developed over the years of science.

-   A good number of them are commercially available in the recent market 

GENETIC CHANGE IN BACTERIA 

• Organisms adapt to changing environments 

• Natural selection favors those with greater fitness

• Bacteria adjust to new circumstances:

  • Regulation of gene expression
  • Genetic change
  • Bacteria excellent system for genetic studies
  • Rapid growth, large numbers
  • More known about E. coli genetics than any other

BACTERIAL GENOMES

• A dynamic structure influenced by several events may include: gene acquisition, duplication or loss, or maybe in genome reduction or rearrangement 
• Basic properties of genomes of a bacteria:
• Genome that consists of a single DNA molecule (i.e., one chromosome) that is several million base pairs in size and is “circular”. It doesn’t have ends like chromosomes or like the eukaryotic organisms. 
• May have one or more circular DNA molecules called plasmids,  containing non-essential genes.
• IIn general a single bacterium will be replicating its DNA whenever possible 
  • (Ex. If food chemicals are present in an aqueous environment in the right temperature range. Once the genome is completely replicated, the two circular DNA separate and the cell divides. This process is a lot simpler than mitosis and meiosis because bacteria don’t have multiple chromosomes that have to be sorted out correctly to the daughter cells. Thus bacteria are able to grow and divide much faster than eukaryotic cells. 

Genome sequences show that parasitic bacteria have 500-1200 genes, free-living bacteria have 1500-7500 genes, and archaea have 1500-2700 genes.

REVERSE TRANSCRIPTASE

• Another process in this pathway is reverse transcription, which involves copying RNA information into DNA using reverse transcriptase.

  

So, basically what organisms use reverse transcriptase?

• Reverse transcriptases have been identified in many organisms, which include: 

  • the viruses,
  • bacteria, 
  • animals,
  • as well as in plants. 

• And, in these organisms, the general role of reverse transcriptase is being able to convert RNA sequences to complementary DNA sequences that are capable of inserting into different areas of the genome. 

• And, reverse transcriptase is an enzyme wherein this is found in retroviruses that converts the RNA genome carried into the retroviruses particle into double stranded DNA.

• So basically, reverse transcriptase reverse transcribes a complementary strand of DNA to make an RNA on DNA hybrid. 

  

• Reverse transcriptase also, recently, has been defined and may expand the central dogma.

Ex. Retroviruses. They use the enzyme reverse transcriptase to transcribe the DNA from rRNA template. With this, the viral DNA then integrates into the nucleus of the host cell in which it is transcribed and further translated into protein. In other words, the product is “protein”. 

\n This biological process effectively adds into the Central Dogma of molecular biology. Going back to our previous lesson, we have tackled the Central Dogma of Biology. 

Now, once again, the function or the importance or significance of  Reverse transcriptase is to identify or to convert the RNA sequences to complementary DNA sequences that are capable of inserting into different areas of the genome.

Ex. RT-PCR. The ability of the Reverse transcriptase to synthesize DNA from RNA has been used inthe laboratory like the RT-PCR wherein it is commonly used to quantify the amount of mRNA transcribe from a gene.

DNA REPLICATION IN BACTERIA

Schematic diagram for the DNA replication in bacteria

• So what really happens in bacterial replication?

1. First things first, initiation of bacteria.

• Replications of bacterial chromosomes are initiated at a single region which proceeds from both directions. It will also terminate at a terminal region.
• During slow growth, replication is initiated once per cycle. When bacterial chromosomes replicate, both strands are being duplicated and each strands function as a template
• During replication as well, enzymes known as polymerase transports the nucleotides from a cytoplasm that are complimentary to the template and fit them into place. This will result to two strands:

1. One (1) parental, and

2. One (1) new strand.

• This (replication) process is said to be semi-conservative because of the parental strand is conserved and it remains the same.

WHY IS DNA REPLICATION IMPORTANT IN BACTERIA?

• This is the accurate copying of genetic information in the double helix of the DNA which is very essential for the inheritance for the traits that define the phenotype of cells and as well as to the other organisms.

 The core machineries that copy DNA are conserved in all three domains of life:

1. bacteria,

2. archaea, and

3. Eukaryotes.

\n STEPS IN DNA REPLICATION

1. DNA unwinds with enzymes.

   1.  A replication fork can be seen

2. Complimentary bases added to the template/ parent strand using the enzyme.

3. Replication Fork moves down the strand and newly replicated DNA rewinds. This process is called the “semi-conservative replication.” It is copied from the 5’ to 3’ direction.

4. DNA polymerase can only add nucleotides to 3’ ends.

In prokaryotes, replication begins in specific sites in chromosomes called “the origin of replication.”

Replication of DNA also happens in a specific site in the DNA template wherein it is termed as the “origin” and also proceeds in both directions from the origin until nuclear division and cytokinesis will take place.

PROTEIN SYNTHESIS IN BACTERIA

• Protein synthesis is carried out in the cytoplasm.
• It begins in DNA Replication by mRNA through Transcription.
• From mRNA, it migrates to the ribosomes where the tRNA transfer the information from mRNA to rRNA. This is processed through translation
• Transcription is the synthesis of RNA that involves the assembly of basic nucleotides by an enzyme called “RNA polymerase.”
• This RNA polymerase binds to the DNA at the promoter side near the gene to be transcribed.
• Afterwards, the enzyme-RNA polymerase will travel to the length of the DNA using its template to duplicate.
• This RNA polymerase will continue to duplicate until it reaches the termination site at which the Transcription process is complete.

Summary:

Enzyme used is RNA polymerase

1. It will bind with the DNA at the promoter’s side

2. It will travel to the length of the DNA until its template duplicates

3. It will continue until it reaches the termination site and this signifies the completion of the transcription process

 (Video)

Protein synthesis in bacteria involves three stages:

1. Initiation

2. Elongation

3. Termination

PROTEIN SYNTHESIS

• a three-step process including initiation, elongation, and termination.

In bacteria such as E coli initiation requires three small proteins called initiation factors IF1,IF2, and IF3. As well as the first tRNA, the mRNA, and the small ribosomal subunit also called the 30s subunit.

INITIATION

• IF3 - readily binds to this small ribosomal subunit and its presence blocks the large and small subunits from prematurely associating. IF3 facilitates the binding of the mRNA to the small subunit of the ribosome. The binding occurs just 4 to 8 bases upstream of the AUG start where a consensus sequence called the shine-dalgarno sequence in the mRNA anneals near the end of the 16s ribosomal RNA in the small ribosomal subunit.
•  IF1- binds to the small subunit at a location called the A site where incoming tRNA's normally bind. The first tRNA called N- formylmethionyl Trna. Thus enters another site called the P site note that the initiator T RNA has been escorted to the P site by IF2 which is bound to GTP (a high-energy molecule similar to ATP). The anticodon of the tRNA is complementary to the AUG start codon. Once the initiator tRNA is in place, IF3 is released with the loss of IF3 the 50s subunit can dock on the 30s subunit. The docking of the 50s subunit triggers the hydrolysis of GTP on IF2 and the subsequent release of the initiation factors. The ribosome is now ready for elongation.

ELONGATION

• elongation involves the repetition of three steps.

• 1st step an elongation factor called EF-Tu associated with GTP binds to free charged aminoacyl tRNAs. This complex enters the A or acceptor site correct selection of the T RNA complex depends mainly on codon, anticodon pairing. In this example the anti-codon CCC pairs with a GGG codon the tRNA carries the amino acid glycine.

• In the 2nd step the ribosomes peptidyl transferase activity catalyzes the formation of a peptide bond between the new amino acid in the A site and the previous amino acid in the P site. Simultaneously GTP is hydrolyzed and the resulting in EF-Tu GDP is expelled.

• The 3rd step is called translocation and elongation factor called EFG associated with GTP binds to the ribosome. The GTP is hydrolyzed providing the energy to ratchet the 50s and 30s subunits ahead one codon. This maneuver opens up the A site and slides the uncharged tRNA into the last site called the E or exit site.

• The next aminoacyl T RNA that enters the A site creates a conformational change in the ribosome that telegraphs through to E sites and ejects the uncharged tRNA. These elongation steps repeat along the mRNA. The ribosomes of the coli can speed through these elongation steps linking together 16 amino acids per second.

TERMINATION

• Eventually the ribosome arrives at the end of the coding region marked by one of three stop codons UAA. This stage of translation is called termination no corresponding tRNA's exist/stop codons instead a protein called a release factor either RF1 or RF2 which has the general shape of a tRNA mimics a tRNA and enters the A site. \n The release factor activates the peptidyl transferase function of the ribosome which cuts the bond tethering the completed peptide to tRNA in the P site, another factor called RF3 then triggers RF1 or RF2 to depart the ribosome.
• Finally, another factor called ribosome recycling factor or RRF along with EFG bind to the A site and the accompanying GTP hydrolysis undocks the two ribosomal subunits. IF3 then reassociates with the 30s subunit preventing the 50s and 30s subunits from coming together again.
• The liberated ribosomal subunits are now free to diffuse through the cell ready to bind yet another MRI and begin the translation sequence anew.

The protein synthesis involved in bacteria in which it has three stages:

• Initiation
• Elongation 
• Termination

INITIATION 

• The beginning of protein synthesis starts with the methionine which is the start codon. 
• This start codon is known as “Formylmethionine” or f-met. 
• It  is coded as AUG (start codon)

ELONGATION

• In the elongation stage, it begins by a complex with f-met, amino acid attach to form a chain (amino acids joined repeatedly to form proteins). TERMINATION

  \n

• In the last stage, it ends when the synthesis comes to a termination codon. 

• There are three stop codons or termination codon and is coded as UAA, UAG, UGA

Again the stages of protein synthesis in bacteria is the initiation, elongation, and termination.

LAC OPERON

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• A group of genes with a single promoter is  transcribed as a single mRNA wherein the specific genes in the operon encode the proteins that will allow the bacteria to use lactose as an energy source.

As you can see in the diagram, we have the lacZ, lacY, and lacA these are the three lactose metabolism genes that are being organize together in a cluster called the “lac operon”. The coordinated transcription and translation of the lac operon structural genes is supported by a shared promoter, operator, and the terminator. So, the  lac regulator gene with its promoter as being found just outside on the lac operon. 

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• *

Summary: The mentioned above are the structure of lac operon and the three lactose metabolism genes the lacZ, lacY, and lacA and is being supported with a structural genes or supported by a promoter, operator, and terminator. 

CHANGES IN THE DNA MOLECULES CAN CAUSE MUTATIONS

1. One base pair is exchanged for another in the DNA molecule.
2. One or more base pairs are inserted in the DNA molecule.
3. One or more base pairs are deleted in the DNA molecule.
4. There is rearrangement of sections in the molecule.
5. There is an exchange of DNA region with another DNA molecule (Recombination). 

The enumerated above is just a brief description of the specific types of mutation and, give at least one example.

• Some mutations are considered harmful, some beneficial, and some neutral. 

There are also specific examples of this type of mutation and Miss wanted us to explore and discover more by reading about different types of mutation and its example.

THREE METHODS OF GENETIC RECOMBINATION IN BACTERIA

• Transformation 
• Transduction
• Conjugation

GENETIC TRANSFER AND RECOMBINATION 

• Recombination: exchange of homologous genes on a chromosome

• Transformation: genes transferred from one bacterium to another. After cell death, some bacteria are lysed and release cellular contents into the surrounding environment. The recipient cell is in a physiological state that will allow it to take up DNA

TAKE NOTE: Transformation occurs naturally among a few organisms.

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GENETIC CHANGE IN BACTERIA

• Mutation and horizontal gene transfer

• Horizontal gene transfer: newly acquired DNA which is being incorporated into the genome of the recipient cell through either recombination process or insertion.

• Recombination is basically essential in the regrouping of genes that native and foreign to the DNA segment are homologous and are being edited and combined.

  

HORIZONTAL GENE TRANSFER AS A MECHANISM OF GENETIC CHANGE

• Could either be through plasmid transfer or through chromosome transfer
• Genes are naturally transferred by three mechanisms

a. Transformation: naked DNA uptake by bacteria

b. Transduction: bacterial DNA transfer by viruses

c. Conjugation: DNA transfer during cell-to-cell contact

• Horizontal Gene Transfer is also known as the lateral gene transfer. It refers to the movement of genetic information across its normal mating barriers and between more or less distantly related organisms. Thus, this stands in distinction to the standardization of vertical transmission of genes from parent to offspring.
• Horizontal gene transfer also enables the bacteria to respond and adapt to the environment, acquiring large DNA sequences.
• DNA is replicated only through replicon.

®     Replicon is an entire region of the DNA that is independent, replicated to a single origin of replication. This bacterial chromosome contains a single origin and therefore, the whole bacterial chromosome is a replicon.  \n How does Horizontal Gene Transfer contribute to genetic variation? 

• HGT spreads genetic diversity by moving genes across species’ boundaries. 

• By rapidly introducing newly evolved genes into existing genomes. 

• HGT circumvents the slow step of initial gene creation and accelerates genome innovation. 

  \n BACTERIAL TRANSFORMATION

• is the process wherein a horizontal gene transfer by which some bacteria take up from foreign genetic material as 

• mentioned earlier that the feature of (???) is taking DNA from the environment so its was reported through the use of bacteria streptococcus pneumoniae.

  

1. Removing the plasmid from bacterial cell 
2. Isolate the gene of interest 
3. Using the enzyme dna ligase. So you need to cut open plasmid with restriction enzymes and this will leave sticky ends 
4. You will insert the gene of interest and the insertion of the plasmid with the recombinant DNA into a new bacterium

Furthermore bacterial transformation is important because this process is the uptake of genetic material by bacteria from its surroundings in which it will be  utilized in genetic engineering to introduce foreign genes into the bacterium. 

\n TRANSDUCTION

• other type of transfer mechanism 
• transfer of genes by bacteriophages

A = is the formation of transducing particles

B = is the process of transduction 

PRODUCTION OF TRANSDUCING PARTICLES

** **

1. A bacteriophage attaches to a specific receptor on a host cell

2. The phage DNA enters the cell. The empty phage coat remains on the outside of the bacterium

3. Enzymes encoded by the phage genome cut the bacterial DNA into small pieces 

4. Phage nucleic acid is replicated and coat proteins synthesized 

5. During construction of viral particles, bacterial DNA  can mistakenly enter a protein coat. This creates a transducing particle that carries bacterial DNA instead of phage DNA

   \n Once you have the transducing particle then you will be using this one in the process of transduction.

TRANSDUCTION PROCESS

• *

1. A transducing particle attaches to a specific receptor on a host cell

2. The bacterial DNA is injected into a cell

3. The injected bacterial DNA integrates into the chromosome by homologous recombination 

4. Bacteria multiply with new genetic material. replaced host DNA is degraded

   \n In addition to the transduction process there are types of transduction, we have the generalized transduction and the specialized transduction.

SPECIALIZED TRANSDUCTION

• specialized meaning there are only specific genes 
• only a few restrictive bacteria are being transferred from the donor to the recipient bacteria 
• this is carried out by a temperate bacteriophage which undergoes a cycle 

GENERALIZED TRANSDUCTION

• this occurs in any gene of the donor cell
• the type where the bacteriophage first infects the donor cell and begins the lytic cycle
• example: the virus develops its components using the host cell machinery and then the host cell DNA is being hydrolyzed into small fragments by the viral enzymes 

CONJUGATION

• Another form of transfer of DNA from one cell to another is called the conjugation in which this requires a cell-to-cell contact which transfers plasmid through the F-factor pili

• Is the DNA transfer between bacterial cells 

• requires contact between donor and recipient cells 

  LEDEBERG AND TATUM:

• Another description of conjugation was first described by Lederberg and Tatum in the year 1946 as a phenomenon involving the exchange of markers between closely related strains of e-coli (Escherichia coli)

• In which an agent responsible for this process was later found to be a site of a chromosome called the F factor or the F (fertility) factor 

• Why is bacterial conjugation important? 

  • Bacterial conjugation transfer mechanism is very important not only for bacterial evolution but also for human health since it represents the most sophisticated form of HGT (horizontal gene transfer) in bacteria.
  • it also provides, for instance, a platform for spread and persistence of antibiotic resistance genes
  • And this F cell and F factor cells that contain the plasmid called the fertility factor that allows the cells to initiate conjugation in which when an F cell undergoes conjugation, with the F cell, the plasmid is being transferred without without the presence of bacterial genes and this F cells are the same that lock this fertility factor, plasmin.

STAGES OF BACTERIAL CONJUGATION

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• As you can see in the figure, we have here the steps or the stages for conjugation (bacterial conjugation)

• From (1)making contact to (2)initiating transfer, then (3)transferring of dna and the last stage is (4)transfer complete –

  • –in which at the end of a transfer process, both the donor and the recipient cells are F+ and synthesize the F pilus 

  

• Chromosomal DNA transfer 

  • Less common
  • This involves the Hfr cells. They are a high frequency of recombination 
  • Then, the F plasmid is being integrated into the chromosome by homologous recombination in which through the process is reversible. So, the F plasmid is resulted when small pieces of chromosomes are being removed with F plasmid DNA. 
  • And then this Hfr cell produces the F pilus and this transfer begins with genes on one side of the other or in the side of a region of transfer of plasmin.
  • Part of chromosome transfer to recipient cells in which the chromosome usually breaks before complete transfer and the recipient cell remains the F- (F minus) since incomplete F plasmids are being transferred 

WHAT ARE PLASMIDS?

So, what are really plasmids?

Plasmids

• Plasmids are extrachromosomal structures in the cells of bacteria which have the ability to self-replicate.
• They do not combine with the genetic material of the host cell and stay independently.
• They are genetically modified, and are used in the recombinant DNA technology.

Example: RNA plasmids 

• Very rare and poorly characterized
• Examples are known from plants, fungi, animals
• Some strains of yeast (Scientific Name: Saccharomyces cerevisiae) may contain Linear RNA plasmids which are similar to the RNA found in the mitochondria of some varieties of maize plants/corn plants.

Role of plasmids in bacteria: Plasmids transfer information from one cell to another.

Examples: Transfer of important genes,  confer resistance of particular antibiotics for their bacterial cells, and this enables them to metabolize a nutrient which a bacteria is normally unable to.

\n PROKARYOTES VS. EUKARYOTES

Prokaryotes: Have no true nucleus Eukaryotes: Have a true nucleus

• All cells (prokaryotic or eukaryotic) share the following organelles:

• DNA

• Plasma membrane

• Cytoplasm

• Ribosomes

TRANSCRIPTION AND TRANSLATION

• In Prokaryotic cells, transcription and translation are coupled. Meaning, translation begins during mRNA synthesis.
• In Eukaryotic cells, transcription and translation are not coupled; transcription occurs in the nucleus, producing the mRNA.

(TAKE NOTE: Take note of the differences between the two in terms of transcription and translation processes.) 

• In the Eukaryotic cells, the mRNA exits the nucleus in the transcription and translation, and the translation occurs in the cell’s cytoplasm. Take note for that.
• Differences include: 
• Structural Variation - Whether a nucleus is present or absent, and whether the cell has membrane bound organelles.
• Molecular Variation - Whether the DNA is in a circular and linear form. 

So you can see here, (in the image) that there's a presence of pili in the Prokaryotic cell. And on the other hand, for the Eukaryotic cell, you cannot see a pili as well as a plasmid. So prokaryotes have a capsule, cell wall, nucleoid, or the presence of DNA, niya ribosome, flagellum, and cytoplasm. 

THE MOBILE GENE POOL

Table of the summary of the gene pool.

What is a mobile gene pool?

A gene pool refers to the:

• combination of the genes
• may include the allele; 
• present in a reproducing population or species

Basically, a large gene pool has extensive genomic diversity wherein it is able to withstand environmental challenges.

1. Transposons 

• Insertion Sequences (ISs) 

  • Composition: Transposase gene flanked by short repeat sequences

• Property: Move to different locations in DNA in same cell

• Composite transposons 

  • Composition: Recognizable gene flanked by insertion sequences

• Property: Same as insertion sequences, but encode additional information

• Plasmids 

• Composition: Circular double-stranded DNA replicon; smaller than chromosomes (These are  structures of the plasmids)

• Property: Generally a code only for non-essential genetic information

• Genomic Islands

• Composition: Large fragment of DNA in a chromosome or plasmid

• Property: Code for genes that allow cell to occupy specific environmental locations

• Phage DNA

• Composition: Phage genome

• Property: May encode proteins important to bacteria

This is the mobile gene pool wherein this is important for the variation of gene pool of single species. 

SOME PLASMID-CODED TRAITS

• Plasmids are found in many bacteria and archaea–some eukarya, and this usually is the origin of replication, and then you can see here (the table) the organisms in which trait is found in some plasma-coded traits. So for antibiotic resistance, there are many–including E.coli, Salmonella, Neisseria, Staphylococcus, and Shigella, and so on and so forth.* 

R PLASMID

R plasmid 

• also called as the resistance plasmid 
• are responsible for the resistance of the drugs 

Sex factor plasmids

• cells which possess this plasmid are called the F positive (F+) or the male or donor cells 
• cells that do not possess this plasmid are called the F negative (F-) or the recipient cell

So what is a R plasmid and F plasmid? Take note of the differences between the two.

R plasmid has:

• antibiotic resistance gene 
• resistance factor

F plasmid has: 

• F or fertility factor necessary for conjugation \n

  TRANSPOSON

  

  • These are able to direct synthesis of copies of themselves and become incorporated into the chromosomes

  Another term for transposon is jumping genes (ability to insert themselves into a chromosome or change their location)