Chapter 28: Bacterial Genetics

Describe the three mechanisms of bacterial gene transfer

• Conjugation: Process in which genes are transferred from one bacterium to another by cell-to-cell contact

• Transformation: Process in which genes are transferred into a cell as free molecules of DNA

• Transduction: Process in which genes are transferred from one bacterium to another via virus particles

Identify the three possible fates of incoming DNA fragments during gene transfer

• It may survive as an independent DNA molecule

• It may be completely degraded

• part may survive by integration or recombination with the host chromosome before the rest is degraded

Describe Oswald Avery’s experiment to prove that DNA is the genetic material

• Avery exploited the difference between S. pneumoniae variants to prove that DNA from one strain could “transform” or change the other strain. Avery used DNA extracted from virulent strains of S. pneumonia. He purified the DNA and added it to harmless strains of the same bacterial species. Some of the harmless bacteria took up the DNA and were transformed into virulent strains (read 3.1)

Define “competent” and describe competence pheromones

• Competent is a cell that is capable of taking up DNA from the surrounding medium

• Competence pheromones are short peptides secreted into the culture medium by dividing bacteria, and only when the density of bacteria is high will the pheromones reach sufficient levels to trigger competence.

Compare and contrast the mechanisms of natural competence and artificially induced competence

• Natural competence: involves the induction of a variety of genes whose products take part in DNA uptake

  • DNA is bound by cell-surface receptor

  • Then the bound DNA is cut into shorter segments by endonucleases, and one of the strands is completely degraded by an exonuclease

  • the resulting short single-stranded segments of DNA enter the cell and part of the incoming DNA may then displace the corresponding region of the host chromosome by recombination

• Artificially induced competence: Double-stranded DNA enters the cell through a cell wall that is seriously damaged

  • the majority of the cells that are made artificially competent are killed by the treatment

  • few survivors take up the DNA

  • Achieved using chemicals or electrical pulses

  • Temporarily disturb the cell wall allowing DNA to enter the cell

Describe how Vibrio cholerae kills other bacterial cells and steal their DNA

• The victim (bacterial cells) is stabbed by a structure known as a type VI secretion system (T6SS). This kills the target cell and results in the release of its DNA. The DNA is then imported into the attacking cell by a type IV pilus together with other components

Describe the two types of transduction

• Generalized transduction: where fragments of bacterial DNA are packaged at random and all genes have roughly the same chance of being transferred

  • the phage must not degrade the bacterial DNA

  • Lambda phage uses specific recognition sequences when packaging their DNA into the virus particle and so will not package random fragments of DNA

  • headful packaging: virus packaging mechanism that depends on the amount of DNA the head of the virus particle can hold

• Specialized transduction: certain regions of the bacterial DNA are carried preferentially

  • certain specific regions of the bacterial chromosome are favored, due to the integration of the bacteriophage into the host chromosome (read 4.2)

Describe the key steps in bacterial transformation for molecular biology

• DNA is extracted from one organism by the experimenter and offered to other cells in culture

• Cells able to take up DNA are said to be “competent.”

• no other biological macromolecules, such as proteins, are present to enclose or protect the DNA

Compare and contrast chemical and electroporation methods of bacterial transformation

• Chemical transformation: Uses Ca2+ ions and temperature shift to open up pores in the cell envelope. DNA can enter the cytoplasm of the bacterial cell during the heat shock step

  • Bacterial cells bathed in a solution containing CaCl2 are kept on ice

  • After adding DNA, the tube is moved to a warm water bath

  • DNA can enter the bacterial cell through the small pores that develop with the temperature change

  • Called heat shock → heat applied (usually 42°C for 30-120 seconds)

• Electroporation transformation: Inducing small pores in the cellular envelope with an electrical current

  • Plasmid and electrocompetent cells are mixed in a cuvette

  • Cuvette is subjected to an electrical shock and briefly disrupts the cell envelope to allow the plasmid into the cytoplasm

Discuss the importance of positive and negative controls in transformation experiments

• They allow researchers to confirm that observed results are truly due to the transformation process and not extraneous factors

• demonstrates what should happen under known conditions (positive control) and what should not happen if the transformation is not successful (negative control)