BIOL2142

Factors affecting cell growth

Solutes, pH, temperature, O2, pressure, and radiation

How do microbes respond to hypertonic solution?

Water will flow out of the cell, causing the cell membrane to shrink and detach from the cell wall - can cause metabolic inactivity. Bacteria respond by producing solutes compatible with metabolism and growth, i.e. KCl and organic solutes (choline & proline), increasing internal solute concentration and osmotic concentration.

How do microbes respond to hypotonic solution?

Water will flow into the cell causing the cell to swell (protected from osmotic lysis by cell wall usually). Bacteria have mechanosensitive channels in their plasma membrane that open when the membrane stretches, allowing solutes to leave.

What is the normal osmotic concentration like in a microbe?

The solute concentration in the cell is slightly higher than the environment so the plasma membrane is tightly pressed against the cell wall.

What are halophiles?

Microorganisms that grow in or can tolerate saline conditions. Grow optimally in the presence of NaCl or other salts at a concentration of >0.2M

What are extreme halophiles?

Adapted to hypertonic saline conditions, and therefore require a salt concentration of 2M-6M and have a high concentration of potassium in the cytoplasm. Their cells walls, proteins and plasma membrane require high salt to maintain stability and activity

What is water activity?

The amount of water available to organisms, equivalent to the ratio of a solution's vapor pressure : vapour pressure of pure water. Water activity is reduced by interaction with solute molecules because the water is bound to solutes.

What is pH and how does it affect organisms?

pH = -log(H+). Different bacteria have different optimum, but they all maintain a neutral intracellular pH.

What are acidophiles? How do they maintain cytoplasmic neutrality?

Have a growth optimum between pH 0 - 5.5. They transport potassium into the cell and therefore decrease the movement of hydrogen ions into the cell. They also have hydrogen transporters.

What are neutrophiles? How do they maintain cytoplasmic neutrality?

Have a growth optimum between 5.5 and 7. They exchange potassium ions for protons using an antiport transport system. If pH

What are alkilophiles? How do they maintain cytoplasmic neutralilty?

Have a growth optimum between pH 8.5 - 11.5. They exchange internal sodium ions for external H+ ions.

What is problematic about temperature changes?

Microorganisms cannot regulate their internal temperature, and many enzymes have particular temperature optimums. High temperatures denature enzymes and disrupt cell membranes.

What are cardinal temperatures?

The minimum, optimum, and maximum temperatures at which an organism grows.

Psychrophiles

0-20 degrees C

Psychotrophs

Organisms that grow between 0-35 degrees celsius and are responsible for most food spoilage while in the refrigerator

Mesophiles

20 degrees - 45 degrees celcius.

Thermophiles

55-85 C

Hyperthermophiles

85 degrees celcius to 113 degrees celcius

How have psychrophiles and psychotrophs adapted to low temperatures?

Have highly unsaturated fats in their membrane to maintain membrane fluidity, and have solutes in their cytoplasm to lower their freezing point. Also have anti-freeze proteins.

What are adaptations of proteins, membranes, and DNA in thermophiles to high temperatures?

They have protein structures stabilised by: high numbers of hydrogen bonds, high numbers of proline AAs to decrease flexibility, and more chaperons to prevent denaturation of proteins. DNA's stabilised by histone-like proteins. Membrane's stabilised by: highly saturated, highly branched and high molecular weight lipids. Also have ether linkages which are more stable (not ester).

Aerobes

Grow in the presence of atmospheric oxygen (20%)

Obligate aerobes

require oxygen

Obligate anaerobe

usually killed by O2

Microaerophiles

requires 2-10% of O2.

facultative anaerobes

don't require O2 but grow better in its presence

aerotolerant anaerobes

grow with or without O2

How do you determine a microbes O2 profile?

Inoculate bacteria in solid agar and observe the localisation of growth. i.e. obligate aerobes will be concentrated at the top, facultative anaerobes will be found everywhere but more at the top...

What causes a sensitivity to oxygen?

Oxygen's easily reduced to toxic products, such as a superoxide radical (O2-), hydrogen peroxide (H2O2), and hydroxide radicals (OH-). Aerobes produce protective enzymes against these such as catalase (H2O2 --> H2O + O2), peroxidase (H2O2 --> H20 + NAD+) and superoxide dismutase (O2- --> H2O2+O2)

Why can't strict anaerobes tolerate oxygen?

They lack/have low quantities of enzymes removing ROS, such as superoxide dismutase and catalase.

How are strict anaerobes grown?

They're grown without O2. Can be done using: special anaerobic media with reducing agents to reduce O2 (thioglycollate and cysteine); a chamber which pumps out O2 and flushes with CO2 and N, Gaspak jars which generate CO2 and H2 to react with O2, and candle jars.

What pressure do most microbes live at?

Microbes on land and on the surface of water live at 1 atm. Some bacteria and archea live in deep sea with high hydrostatic pressure.

What are some types of ionising radiation?

X-rays and gamma rays. Short wavelength and high energy

How does ioinsing radiation affect organisms?

Disrupts the chemical structure of molecules and can cause mutations and death. Can be repaired by DNA repair mechanisms if only a small does.

What is special about Deinoccoccyus radiodurans?

Extremely resistant to ionising radiation and DNA damage because it expresses highly efficient DNA repair systems.

How do cells communicate with each other in microbial populations?

They use molecular signals in a density dependent manner called Quorum sensing. Allows bacteria to detect and respond to cell population density by gene regulation.

Explain acylhomoserine lactone in the context of quorum sensing.

Acylhomoserine lactone (AHL) is an autoinducer molecule produced by many Gram-negative organisms. It can diffuse across the p.m. in either direction. When the density is low, AHL is produced and diffuses out of the cell. When a certain density's reached, the concentration of AHL outside the cell is optimum, the gradient is reversed, and AHL diffuses into the cell. It binds to an activator protein and induces gene expression. e.g. at a certain density streptococcus become competent and can take up DNA

Give examples of quorum sensing systems

Vibrio fischeri at a certain density results in bio luminescence in squid. Pathogenicity and increased virulence factors. Biofilm formation. DNA uptake (become competent at certain density). Rhizobia and legumes - plant produces flavanoids which is taken up by rhizobia, rhizobia produces nod factor which affects root hair and causes the rhizobia to be taken up.

What are the 3 forms of DNA transfer?

Transformation - uptake of free DNA. Transduction - DNA transfer via bacteriophage. Conjugation - transfer of DNA via direct cell-to-cell contact.

Who first coined the term transformation and what was the experiment?

Griffith. Used S. pneumoniae. When the capsulated smooth strain was injected into mice, mice died. When the rough, non-capsulated strain was injected into mice, mice survived. When the heat-killed S strain was injected, mice survived. When the mixed heat-killed S strain and live R strain was injected, the mice died, and live S and R strain was found in the dead mice - therefore the nonvirulent R strain had been changed into virulent S strain (capsule = virulence).

Who showed DNA is the transforming principle and how?

Avery, MacLeod and McCarty. They plated: R cells, R cells + S strain DNA exract, R cells + S strain DNA exract + DNAase/RNAase/protease. No colonies were formed with just R cells, no colonies were forming with R cells + S DNA extract + DNAase. Transformed colonies were seen in the R cells + S strain DNA extract, R cells + S DNA + RNAase, and R cells + S DNA + protease. Therefore, transformation is only inhibited when there is no DNA present, and therefore it is the transforming principle.

What is bacterial transformation?

The uptake of free DNA by a competent cell followed by incorporation of the DNA into the recipient's genome.

What is natural transformation?

When the bacteria is naturally competent, i.e. it produces competence factors that allow it to take up DNA naturally, i.e. S. pneumoniae (at a certain density)

What is artificial transformation? How can this be achieved?

When the species is not normally competent, transformation can be done in the lab by making the cells competent. Can be made competent by: Calcium chloride treatment to make the cells more permeable to DNA; electroporation where high voltage pulses are delivered which forms pores in the cell. Electroporation is more efficient, producing more transformants.

How does transformation occur in S. pneumoniae?

Double stranded DNA binds to the cell surface receptor. An extracellular endonuclease can cut the DNA into smaller fragments. Then it cleaves one strand of the DNA which can be transported into the cell. The other strand degrades. When the DNA is inside the cell, it aligns with the homologus sequence on the bacterial chromosome, and is incorporated via homologus recombination.

How does natural transformation occur in Gram +ve bacteria?

Have a ComGC pilus which captures DNA. The pilus retracts, pulling the DNA to the ComEA, a DNA binding protein. ComEA binds the DNA, and endonuclease A degrades one strand of the DNA, whilst the other strand moves through the ComEC channel, driven by an ATP-dependent translocase called ComFA.

How does natural transformation occur in Gram -ive bacteria?

Have a PilE pilus extending from the outer membrane which captures DNA and retracts, pulling DNA through PilQ in the outer membrane. ComE, a DNA binding protein, then binds the DNA. An endonuclease breaks down 1 strands, allowing the other to pass through ComA into the cytosol (translocase here is unknown).

What is bacterial transduction?

The transfer of bacterial genes by virsuses/bacteriophages.

What is the lytic cycle?

The phages that undergo the lytic cycle are virulent phages, because the kill the host cell by replicating immediately upon entry and lysing the cell. The phage binds to the cell and injects its DNA. The phage DNA then directs synthesis of new phages, hydrolysing the host DNA and producing phage DNA and proteins. The replicated phage DNA is packaged into phage heads. The cell's lysed, releasing the phages.

What is the lysogenic cycle? What is a lysogen? What is a prophage?

Temperate phages do not immediatley kill the host, but rather they establish a relationship with the host called lysogeny (the bacteria are then called lysogens). Lysogeny is established when phage genomes get inserted into the bacterial chromosome - the genome is called a prophage.

Describe the lysogenic cycle.

Phage binds to the cell and inserts DNA. The phage DNA integrates into the host genome, forming a latent prophage within the lysogen. The prophage DNA's copied when the cell divides. Exposure to stress such as UV light triggers excison from the host chromosome. The phage then enters the lytic cycle.

What is generalised transduction?

Where any part of the bacterial genome can be transferred. It occurs during the lytic cycle of a virulent phage. Following insertion of the phage DNA and hydrolysation of the host DNA, phages assemble and pack DNA into phage heads. Sometimes during this viral assembly, fragments of the host DNA are mistakenly packaged (i.e. fragments of similar size to the phage DNA). This phage is called a generalised transducing particle, which can inject another bacterial cell and recombine with the recipient chromosome.

What is specialised transduction?

Carried out by temperate phages that have established lysogeny, such that only a specific part of the bacterial genome's transferred. When the phage DNA is excised, it can pick up some of the bacterial genes that were near the phage attachement site, therefore leaving some of the phage genes behind such that it cannot function reproduce properly. The incorrectly excised DNA is packages into a phage head and is injected into another bacterial cell, where it can be integrated as a prophage (contains virus and donor DNA) or can be crossed over so only donor DNA is incorporated.

What is an example of specialised transduction?

Carried out by the E. coli lamda phage. The phage integrates into the bacterial chromosome at specific attachment sites (att sites), forming a prophage. The att site for lamda is between the gal and bio genes so when lambda excises incorrectly to generate a specialized transducing particle, these bacterial genes are most often present. The product of cell lysis (lysate) resulting from the induction of a population of lysogenized E. coli cells contains normal phage and a few defective transducing particles. These particles are called lambda dgal if they carry the galactose utilization genes or lambda dbio if they carry the bio from the other side of the att site.

What is high frequency transduction lysate?

If there is a normal phage λ genome in the same cell, the defective phage λ carrying the a gal/bio gene can integrate. There are two bacterial phage hybrid att sites where the defective λdgal can insert phage that acts as helper phage because it helps the integration and reproduction of the defective phage. The transductants are unstable because the prophage can be excised by certain stress like UV radiation. The UV radiation results in new phages if a normal phage λ has also lysogenized this cell. Hence, induction of this double lysogen produces high frequency transduction (HFT) lysates containing about 50% λdgal and 50% phage λ. In constrast to LFT lysate, it is shown as HFT lysate because the number of HFT particles is very effective in transduction.

What is low frequency transduction lysate?

When prophage is excised out from E. coli chromosome, it sometimes takes out with it gal or bio genes.This occurs due to improper excision of integrated prophage DNA as it occurs in the formation of F factor. Thus after cell lysis resulting from induction of lysogenized E. coli, some normal phages and a few defective transducing particles are released.These particles are called λdgal (λ defective gal) because they contain galactose utilizing gene. Since the number of defective particles in cell lysate (product) is low, it is called the low frequency transduction (LFT) lysates.

What is transfection? What is transformation in eukaryotes?

When DNA/plasmid is put into eukaryotic cells. Transformation is when the cells are converted into cancer cells.

What are plasmids?

Small, autonomously replicating DNA molecules that can exist independently or integrate reversibly into the host chromosome to form episomes. Can carry a variety of genes.

What is the structure of a plasmid? Why do plasmids run differently on gel?

Small, double-stranded, usually circular DNA molecules. Can be supercoiled, open-circular duplex (1 nick on 1 strand) or linear duplex (a nick at the same place. That's why different bands are formed on agarose gel (supercoiled runs fastest).

How do plasmids replicate?

Independently of the chromosome. Begins at origin of replication and is bidirectional. Enzymes required come from host.

What is curing of plasmids?

Removal of a plasmid. Either spontaneously (i.e. if there's no selection for it) or by chemical/physical agents.

What is the F plasmid?

Conjugative plasmids/F (Fertility) Plasmid/Factor - can transfer copies of themselves to other bacteria during conjugation. One third of the genes are involved in DNA transfer (e.g. tra genes for pilus), and have insertion sequences which are also present on the bacterial chromosome and therefore can integrate via homologus recombination.

What is bacterial conjugation?

The transfer of genes between bacteria which requires direct cell to cell contact mediated by the F pilus and involves unidirectional transfer of DNA from donor to recipient. The recipient can't have the F factor.

What was the study that first showed conjugation?

Lederberg and Tatum. Took 2 auxotrophic strains (Bio-, Phe-, Cys-, Thr+, Leu+, Thi+ vs Bio+, Phe+, Cys+, Thr-, Leu-, Thi-), mixed, and plated on minimal media. Formed prototrophic colonies, i.e. synthesised all the needed. Control plates did not have any growth. Therefore, DNA transfer occured?

What study showed physical contact was necessary for bacterial conjugation?

U-tube experiment. U-tube with glass filter with pores large enough for media to move but not bacteria. Had 2 auxotrophic strains either side of the glass, i.e. Met-, Thre+ vs Met+, Thre-. Then plated each side - no colonies were formed.

What is F+ x F- mating?

Overall the F- recipient becomes F+. No chromosomal DNA is transferred. F+ sex pilus makes contact with F- recipient cell. Sex pilus contracts and pulls cells together. The type 4 secretion system is constructed and joins the cells. A relaxosome makes a cut at the origin of transfer and begins to seperate the DNA strands. The intact strand's replicated by the rolling-circle mechanism. Accessory proteins of the relaxosome is released, the DNA/relaxase complex is recognised by the coupling factor and is transferred to the secretion system which pumps DNA/relaxase to the recipient cell. As the DNA enters the recipient, it's replicated by the rolling-circle mechanism to become double stranded.

What is the rolling-circle mehcanism?

At the nick, the 3' OH end is extended, displacing the 5' tail.

What are resistance factors? Give an example.

R factors/plasmids. Have genes for resistance to antibiotics. Some are conjugative. Usually do not integrate into chromosome. e.g. R100 - contains the tra operon needed for transferal of DNA.

What are mechanisms of antibiotic resistance?

Altering the target site (e.g. erythromycin). Modifying the antibiotic so it is no longer active (e.g. chloramphenicol, penicillin/ampicillin). Preventing the antibiotic from entering the cell (e.g. tetracycline). Specifying an enzyme which provides a substitute for a host-specified enzyme which is the target of the antibiotic (e.g. sulpha drugs).

What are sulpha drugs and how do they work?

e.g. sulfanilamide. They are analogues of p-aminobenzoic acid which is part of folic acid. They get incorporated instead, therefore stopping folic acid synthesis. Doesn't affect humans because we don't synthesise our own folic acid.

What are col plasmids?

Encode colicin - a type of bacteriocin, which are proteins that destroy other bacteria, usually closely related species. Some are conjugative and some carry resistance genes.

What are virulence plasmids?

Carry virulence genes. e.g., genes that confer resistance to host defense mechanisms, or genes that encode toxins.

What are metabolic plasmids?

Carry genes for metabolic processes. e.g. genes encoding degradative enzymes for pesticides, or genes for nitrogen fixation

What is Hfr conjugation?

HFR stands for high frequency of recombination. The donor Hfr cell has F factor integrated in its genome. Donor genes are transferred to the recipient cell, and usually a complete copy of the F factor is not transferred.

Describe Hfr x F- conjugation.

The sex pilus of Hfr donor makes contact with the F- cell, and contracted to pull the cells together. Type 4 secretion system connects the cells. The DNA's nicked at the oriT (origin of replication) in the integrated F factor, and a new strand's synthesised via the rolling-circle mechanism. The DNA is then pumped into the recipient via the type 4 secretion pathway. If the cells are conjugated for a short amount of time and are agitated and seperated earlier, the entire chromosome will not be transferred and therefore an entire F factor is not transferred. Therefore, the recipient remains F-. If they stay conjugated for 100 minutes, the whole chromosome can be replicated.

What affects which gene's transferred first in Hfr x F-?

The oritentation of the integrated F factor affects whether the genes are transferred clockwise or anticlockwise.

What is F' conjugation?

Results when the F factor is incorrectly excised from the host chromosome, picking up some chromosomal genes and leaving behind some F factor genes.

Describe F' x F- conjugation.

Occurs in the same way as described. The recipient becomes F', and the whole F' plasmid is transferred.

What is a mutation

A stable, heritable change in nucleotide sequence which may or may not have an effect on the phenotype of an organism.

What is the method used to detect auxotrophic mutants?

The replica plating technique. Treat cells with a mutagen and inoculate plate with complete growth media. WT and mutant colonies form on the 'master plate'. A replica block with a velvet surface is then used to stamp the bacteria and stamp onto a replica plates - 1 with complete medium and 1 lacking a growth factor. Can compare what grows on which.

What is the carcinogenicity test/Ames test? What theory is this based on?

e.g. Salmonella histidine auxotrophs. Plate the culture on a minimal media with a small amount of histidine and minimal media with a test mutagen and a small amount of histidine. Then compare the amount of colonies formed. If the revertant rate in the presence of the suspected carcinogen is greater than in the absence, then the agent is a mutagen that may be a carcinogen. This test is based on the fact that carcinogens are usually also mutagens, so mutagenicity screens for carcinogenic potential. For this to work, the original strain should have a point mutation.

How to increase the power of the Ames test?

Use strains lacking DNA repair enzymes so the mutations caused by the mutagens cannot be fixed. Or use liver enzyme preparations to convert chemicals into their active mutagenic forms

What is genomics?

Study of molecular organization of genomes, their information content, and gene products they encode

What types of DNA sequencing are there

Sanger and next-generation. Sanger is more time-consuming and expensive.

What is bioinformatics?

Analysis of genome data using computers. It uses annotation to determine location of genes on newly sequenced genome and also provides data on protein structure and function.

What is involved in genome annotation?

Locating genes in the genome map, identifying open reading frames. Any protein with >100 codons w/o a stop codon is considered a potential gene. For a protein-coding gene, the gene should have a start codon (methionine) and a ribosome binding site.

What is BLAST software?

BLAST (basic local alignment search tool) software. Base by base comparison of 2+ gene sequences, used to assign tentative function of gene or protein structure by comparing to homologues.

What is functional genomics?

Determination of how genome works

How can you identify genes of unknown function?

Has been done with Saccharomyces cerevisiae - construction of mutant strains, each with a deletion in a specific ORF of unknown function and determine the mutantʼs phenotype used to assign tentative function for gene. This approach is also used for bacteria.

What is transcriptome analysis?

Can determine which genes are expressed at a specific time or have changed expression in response to environmental changes by directly sequencing total cellular mRNA (RNA-seq). Cellular mRNA is converted to cDNA with reverse transcriptase, adapter sequences are added to the ends of the cDNA fragments, and each fragment is sequenced. Sequence data can be used to: identify sequences by alignment with known (reference) genome sequences, or to convert sequences to amino acid sequences and compare to databases of known protein sequences.

What is proteomics?

The study of the proteome - the entire collection of proteins that an organism produces. Provides information about genome function not available from mRNA studies.

What is functional proteomics?

Information that determines what is actually happening in cells (i.e. function, interaction and regulation)

What is 2D electrophoresis?

Has 2 dimensions. The first seperates proteins based on their isoelectric point - the pH where the total charge of the protein is 0, and therefore stops moving at this point. The second dimesion seperates the proteins by molecular weight via electrophoresis (SDS-PAGE).

What is tandem mass spectrometry?

An unknown spot from 2-D gel is cut and cleaved. The fragments are analyzed by mass spectrometer, the mass of fragments is plotted, and the protein tentatively identified from probable amino acid composition.

What is metagenomics? What are the advantages?

AKA environmental genomics. The study of microbial genomes bases on DNA isolate from the environment. It's a cultivation-independent technique, which is good because most bacteria's not culturable. It allows information to be deduced about the diversity and metabolic potential of microbial communities. It takes a census of microbial populations and detemines the presence and level of classes of genes. It increases the encyclopedia of bacteria and archea by sequencing a large range of genomes.

Give some examples of metagenomics.

Can take bacteria from the intestinal colon to determine the bacteria present. You prepare the DNA from different sites and perform next-generation sequencing. Can be used to determine the microbiome of organisms (e.g. in the GI tract)

What are the 4 types of cloning vectors?

Plasmids, phages/viruses, cosmids, and artificial chromosomes.

What does a cloning factor need?

An origin of replication (2 in the case of a shuttle vector (i.e. one for replication in yeast, 1 for in bacteria)), a selectable marker (to discriminate those that have picked up the plasmid, e.g. antibiotic resistance genes), and a multicloning site/polylinker (cluster of restriction sites)

What is blue/white screening?

Used to discrimate recombinant bacteria. Relies on the activity of beta-galactosidase, which cleaves lactose to form glucose and galactose. A multiple-cloning site's placed in the lacZ sequence og the plasmid vector. A restriction enzyme's introduced which cuts the cloning vector and foreign DNA. If the foreign DNA is not inserted or is inserted in another location, then lacZ will be functional and produce blue colonies. White colonies will be formed if the vector and foreign DNA insert, i.e. recombinants.

What is a phage vector?

Engineered phage genomes genetically modified to include restriction sites. After insertion of the foreign DNA, the recombinant phage genome is packaged into the capsid and can be used to infect host cells.

What is a cosmid?

Do not exist in nature. These vectors have been constructed to contain features from both phages and plasmids - they have a selectable marker, multiple cloning sites from plasmids, and a cos site from lamda phage. They can be packaged into phage capsids and phage then introduces recombinant DNA into E. coli.