microbial genetics- prokaryotes

genome

entire genetic complement of an organism, includes is genes and nucleotide sequences

chromosomes

main portion of DNA with associated proteins and RNA, haploid (single chromosome copy), chromosome is a circular molecule of DNA in the nucleoid

plasmids

small molecules of DNA that replicate independently, not essential for metabolism, growth, or reproduction, can confer survival advantages

• fertility plasmids, resistance plasmids, bacteriocin plasmids, virulence plasmids

type of nucleic acids

circular or linear dsDNA

location of DNA

in nucleoid of cytoplasm and in plasmids

polymers of nucleotides

• direction 5’ to 3’

• each nucleotide is made of phosphate, pentose sugar, nitrogenous base

• length of DNA expressed in base pairs

transcription

information in DNA is copied as RNA, occurs in nucleoid in prokaryotes

translation

polypeptides synthesized from RNA

central dogma of genetics

• DNA transcribed to RNA

• RNA translated to form polypeptides

splicing

rare and usually occurs in non-coding RNAs like tRNAs

transcription- initiation

• RNA polymerase binds to a specific region on the DNA (promoter)

• promoter contains special sequence which help RNA polymerase recognize where to start

• protein (sigma factor) helps RNA polymerase attach to the promoter correctly

• RNA polymerase is bound, it unwinds a small part of DNA called “open complex” where transcription can begin

transcription- elongation

• After initiation, sigma factor is released and RNA polymerase moves along the DNA

• as RNA moves, it reads the DNA sequence and adds complementary RNA nucleotides (A, U, C, G)

• only one strand of DNA (template strand) is copied onto RNA

• new RNA strand grows in 5’-3’ direction

transcription- termination

transcription ends when RNA polymerase reaches a termination sequence

Rho-independent termination: GC- rich terminator

RNA forms a hairpin loop followed by a series of U’s making RNA polymerase detach

Rho-dependent termination

Rho protein binds to the RNA and helps it pull away from RNA polymerase

translation

process in which ribosomes use genetic information of nucleotide sequences to synthesize polypeptides

• use messenger RNA, transfer RNA, ribosomes and ribosomal RNA

prokaryotic operons- regulation of gene expression

consists of a promoter and a series of genes, controlled by a regulatory called an operator

inducible operons

must be activated by inducers, example: Lactose operon- regulates lactose catabolism

repressible operons

transcribed continually until deactivated by repressors, example- Tryptophan operon- regulates tryptophan synthesis

lactose operon

includes genes involved in the transport and catabolism of lactose

• activated by positive regulation by a protein called CAP

  • repressor is produced

  • binds to the operator: RNA polymerase cannot bind

• activation of deactivation of repressor molecule

  • repressor is produced

Tryptophan operon

includes gene for synthesis of the amino acid tryptophan, usually active but can be repressed when tryptophan is available in the environment

• trp operon induced- repressor is produced but not active, transcription occurs (trp production)

• trp operon- repressor binds with tryptophan (co-repressor), binds to the operator: RNA polymerase cannot bind

regulatory RNAs

can regulate translation of polypeptides, microRNAs, small interfering RNA, riboswitch

microRNAs (miRNAs)- RNA interference

• bind complementary mRNA and inhibit its translation

• RNA-induced silencing complex (RISC complex)

  • miRNA-mRNA complex

• blocks translocation (imperfect pairing—>unstable mRNA)

small-interfering RNA (siRNA)- RNA interference

• binds a portion of mRNA or DNA that binds and renders the target inactive

  • siRNA-mRNA complex (RISC)

• cleaves the mRNA

riboswitch

RNA molecules that changes shape to help regulate translation—> genetic switch

recombination

exchange of nucleotide sequences often occurs between homologous sequences→ gain of genetic material

recombinants

cells with DNA molecules that contain new nucleotide sequences

horizontal gene transfer- recombination

donor cell contributes part of genome to recipient cell

vertical gene transfer

mutation

fertility plasmids- f-factor/sex pili

• F-factor allows bacteria to transfer genes through conjugation

• bacteria with F-plasmid are called F+ cells (donors) and those without are F- cells (recipients)

• F+ cells build a sex pilus (bridge) to attach to an F- cell and transfer the plasmid

• spreads useful genes

different types of plasmids

resistance plasmids, virulence plasmids, degradative & metabolic plasmids

DNA replication key enzymes

DNA Polymerase (I-V), Topoisomerase, DNA ligase, DNA helicase

transcription key enzymes

RNA polymerases, sigma factor, topoisomerases

translation key enzymes

mRNA, tRNA, ribosomes, and ribosomal RNA

gyrases and topoisomerases

remove supercoils in DNA

DNA replication- definition

replication is semi-conservative- new DNA is composed of one original and one daughter strand, key to replication is the complementary structure of the two strands, anabolic polymerization processes require monomers and energy- triphosphate deoxyribonucleotides serve both functions

DNA replication-process

bacterial DNA replication begins at the origin (OR), DNA polymerases replicates DNA only 5’ to 3’, strands are antiparallel so new strands are synthesized differently- leading strand: synthesized continuously, lagging strand: synthesized discontinuously

formation of replicating fork

DNA helicase separates DNA strands, parent DNA helix splits into two strands, subsequently copied by DNA polymerases

characteristics of bacterial DNA replication

• bidirectional, gyrases and topoisomerases removes supercoils, DNA is methylated (control of genetic expression, initiation of DNA replication, protection against viral infections, repair of DNA)

genetic code and how it works

3 nucleotide (codon)→ amino acid, start codon=initiates the translation (AUG), stop codon= signals the end protein (peptide synthesis)

lag phase- bacterial growth curve

 It’s the temporary period of non-replication where bacteria adjust to a new environment. This is where synthesizing enzymes occur and N-N₀. 

log phase (exponential)- bacterial growth curve

Bacteria will grow logarithmically and divide at their maximum rate because nutrients are plentiful, waste products are low, and the bacteria have adapted to their environments. The generation time is at its shortest with early, mid, and late phase (rapid bacterial growth-high cell division). The late log phase is the deceleration phase which is the number of viable cells being reduced. 

stationary phase- bacterial growth curve

 exponential growth cannot continue forever in a closed system because of the exhaustion of available nutrients, accumulation of inhibitory metabolites, and exhaustion of space. The cells are still active but the division has slowed down and secondary metabolites might be produced.

death phase- bacterial growth curve

The number of viable cells declines where the death rate is also exponential but slower than the growth rate in the log phase. Some cells will survive and adapt by living off nutrients that were released from dead cells which then creates a modified organism that is better adapted and more fit.

diauxic growth

It is a two phase growth pattern where the bacteria have two different carbon sources with one being the preferred source and the other being the one that will be used later. Usually glucose being the preferred one and lactose the one being used later. It results in two different exponential growth phases with a lag phase in between the switch from one carbon source to another. Bacteria will use the preferred source first because it is easier to metabolize and at this phase, the bacteria will grow at a rapid rate = normal log phase growth. At this stage, the genes for the alternative sugar metabolism will be repressed. After the glucose is exhausted, there will be a lag phase where the bacterial growth will be paused, and at this phase, the bacteria will turn on the genes needed to use the second source and enzymes required to metabolize lactose will start getting produced. Once the bacteria have adapted, it will start using the second sugar causing a second exponential growth phase to occur but the growth will be slower.

spontaneous mutation

genetic changes from natural processes that occur randomly at infrequent by characteristic rates. Mutations passed to progeny is a vertical gene transfer. It alters the genotype which can alter the phenotype. The environment selects cells that grow under its conditions, it cannot cause a mutation but can favor the growth of a mutation. 

induced mutation

genetic changes that occur due to an influence outside of the cell. It can be caused by substitution, disruption of DNA/RNA replication, inserts into DNA molecules, and nonsense mutations. 

point mutation

one base pair is affected, more base pairs are affected= gross mutation, can cause substitutions and frameshift mutations

substitution

mismatching of nucleotides or replacement of one base pair by another. It can cause a silent mutation where it is as if the amino acid sequence in the polypeptide is not changed. It can also cause a missense mutation which results in a different amino acid, the effect is dependent on the location of the different amino acid in the polypeptide. It can also cause a nonsense mutation if the codon for an amino acid is changed to a stop codon.

frameshift mutation: insertion

the addition of one or a few nucleotide pairs that creates a new sequence of codons which can result in missense or nonsense mutations.

frameshift mutation: deletion

the removal of one or a few nucleotide pairs creating a new sequence of codons which can result in missense or nonsense mutations

inhibition of cell wall synthesis

The most common agents prevent the cross-linkage of NAM subunits with Beta-lactams being the most prominent. The functional group are the beta-lactam rings which bind to the enzymes that cross link NAM subunits. This causes the bacteria to have weakened cell walls and eventually lyse (die). There are also semisynthetic derivatives of beta-lactams which are more stable in acidic conditions, more readily absorbed, and more active against more types of bacteria. An example would be Penicillin G

inhibition of protein synthesis

It works through interference with prokaryotic ribosomes (30s and 50s) because the drugs can selectively target translation.  Aminoglycoside changes conformation of 30s subunit which causes a misreading. For example, Streptomycin

translation

The Shine-Dalgarno sequence (AGGAGG) on the mRNA pairs with 16s rRNA in the 30s ribosomal subunit- ensures that the ribosome starts at the correct AUG codon. Initiator tRNA binds to the start codon with the help of GTP. Once the initiator tRNA is in place, the GTP is hydrolyzed and the 50s subunit joins which completes the 70s ribosome. Ribosomes form a peptide bond between the amino acids which causes the ribosome to shift forward moving the tRNA to the next site. Process then repeats to add more amino acids to the protein. Ribosomes will reach a stop codon -UAA, UAG, UGA- and the tRNA doesn’t match these codons making a release factor bind to the codons instead. The newly made protein is released and the ribosome will disassemble.

transformation

The reuptake of “naked” DNA by bacterial cells, the recipient cell must be competent meaning in a specific physiological state that allows the cell to take up the DNA; results from alterations in the cell wall and cytoplasmic membrane that allows the DNA to enter. It can occur naturally by incorporating fragments of DNA or can be engineered by making the cells competent by inserting plasmids, genetic recombination, etc.

bacterial conjugation

It is the transfer of genetic material that needs physical contact between donor and recipient cell and the donor cells stays alive. Donor bacterium has to have F plasmid that has the genes needed to form a sex pilus thus making it F+. The recipient is F- because it does not have the F plasmid. The F+ donor bacterium extends a sex pilus which is a thin, hair-like appendage to the F- recipient, and together they form a bridge for DNA transfer. A single strand of the F plasmid DNA is transferred from F+ to F-, resulting in 2 F+ cells. Now that it has received the F plasmid, the recipient cells can form its own pilus and transfer DNA to other bacteria with the F factor directing their own transfer. Though when conjugation is occurring, the F plasmid integrates the bacterial chromosome resulting in an Hfr cell (high frequency recombination). So during conjugation, part of the chromosomal DNA as well as part of the F plasmid is transferred to the recipient cell, and the recipient cells don't always become F+ because the entire F plasmid did not transfer all the way.