Prophages are bacteria phages, that have integrated into bacterial genomes. We are using a transcriptomic approach to comprehensively explore the impact of prophages on the biology of their bacterial hosts. The methods described here address the key challenge of spontaneous induction of the prophage lytic cycle.
Careful optimization of culture conditions enable clean profiling of gene expression during the prophage state. Prophages are found in most bacterial pathogens, but the significance is not usually understood. These tools can illuminate the role of prophages as regulators of multiple bacterial functions.
There are nearly limitless prophage host relationships that remain unstudied. This represents a huge untapped resource of potential therapeutic targets that these protocols can help to predict. The principle challenge is finding the right culture condition to balance lysogen growth and spontaneous prophage induction.
The well-known secondary challenge, lyson, maintaining the RNA integrity for downstream studies. To begin set up fresh lysogen and indicator host cultures by inoculating the overnight cultures in 100 milliliters of LB at a ratio of one to 100. Incubate at 37 degrees Celsius with shaking at 180 RPM.
To monitor lysogen growth, collect a one milliliter sample every hour from the point of inoculation for eight hours. Serially, dilute the culture by adding 100 microliters into 900 microliters of the LV medium. Vortex at the maximum speed, and continue the dilution series from 10 to the minus one to 10 to the minus nine.
Spot 10 microliters of the required dilution onto an LB auger plate. Allow to dry and incubate at 30 degrees Celsius for 18 to 24 hours. After incubation, count the number of colonies and calculate the number of viable bacterial cells using the given formula.
Next, to enumerate the infective phage particles in the temporal sample, add 100 microliters of log phase rifampicin resistant indicator host cells to the molten top auger, along with rifampicin. Spot 10 microliters of serially diluted lysogen culture onto the inoculated top auger layer. Allowed to dry, and incubate at 37 degrees Celsius for 18 to 24 hours.
After incubation, count the number of plaques and calculate the infective phage particles using the given formula. Label the first flask as un-induced and the others as induced, along with the time points when each sample should be harvested. Then subculture the overnight grown lysogenic cultures in 80 milliliters of LB at a ratio of one to 100 in eight 250 milliliter flasks.
Incubate the flasks at 37 degrees Celsius with shaking at 180 RPM. After 90 minutes, when the optical density at 600 nanometers is between 0.1 and 0.2, add four microliters of 1%glacial acetic acid to the un-induced flask. Add the 80 milliliter culture from the un-induced flask to 720 milliliters of LB and immediately add 160 milliliters of stop solution before placing the flask on ice for 30 minutes to stabilize the RNA transcripts.
Next, add norfloxacin at a final concentration of one microgram per milliliter to the induced labeled flask containing 80 milliliters of culture. Mix well and incubate at 37 degrees Celsius with shaking at 180 RPM for one hour. Allow the cells to recover by adding 80 milliliters of induced culture to 720 milliliters of LB.Harvest the bacterial cells from each flask every 10 minutes from zero to one hour by adding a stop solution.
Centrifuge at 10, 000 G for 15 minutes at four degrees Celsius and discard the supernatant. Before gently re-suspending the pellet in the residual liquid using the adjustable automatic pipette. Transfer the suspension to a 1.5 milliliter micro centrifuge tube and centrifuge at 13, 000 G for one minute at four degrees Celsius.
Discard the residual supernatant and seal the microfuge tube, before flash freezing the pellets into liquid nitrogen. Add one milliliter of Trizol to each frozen pellet, and homogenize the suspension by pipetting. After identifying a set of target genes that can act as markers for each stage of replication of the phage of interest, amplify each of the target genes from the genomic DNA using relevant primers.
Perform PCR using the amplification conditions described in the text manuscript. After PCR, using a PCR purification kit purify each amplicon and clone them in a TA cloning vector as per the manufacturer's instructions. Verify the sequence of each cloned product by Sanger sequencing.
After calculating the copy number of four individual plasmids, prepare a standard template for each marker gene by serially diluting the plasmid DNA in nuclease free water. Add one microliter of CDNA and respective plasmid standards for each target in a 96 well plate. And perform quantitative PCR according to the manufacturer's instructions.
After PCR, use the Excel program to plot the log DNA copy number versus the cycle threshold. Perform a linear regression calculation to display the coefficient of determination and a linear equation. Next, estimate the copy number for each target using the linear equation derived from the linear regression.
Then calculate the efficacy of the PCR amplification using the parameters from the linear regression of the standard curve and the given equation. After validating primers in terms of their percent efficiency, calculate the absolute copy number of the DNA. Temporal enumeration of spontaneous less prophage production suggested the lowest PFU numbers per cell at two hours and the highest PFU numbers per cell at six hours.
The lysogen was then most stable at two hours. So this condition was used for QRTPCR profiling. A marked increase in the expression of the cro gene, an early marker of lytic replication from 2.31 times 10 to the ninth copies in un-induced cultures to 3.02 times 10 to the 11th copies 30 minutes post induction was observed.
Similarly, P and O proteins, the mid-stage marker of lytic replication also showed significant upregulation from 1.74 times 10 to the eighth to 1.25 times 10 to the 10th copies, and from 6.05 times 10 to the second to 5.68 times 10 to the fifth copies respectively. The late marker of the lytic replication cycle displayed a considerable increase in expression in un-induced cultures to 30 minutes post induction. Among the tested internal controls, RPOD had the most stable expression compared to 16 SRNA or ProC genes.
Compared to the markers for lytic replication, the expression of the C-one gene was relatively stable. Still, the copy number of the C-one gene was reassuringly high in the un-induced cultures, compared to the markers for lytic replication. No good data or interpretations can come from a badly prepared RNA library.
Controlling spontaneous induction and RNA integrity are the most important things. Temporal dynamics of any gene can be studied using expression profiling. Tailoring the right experimental conditions and time points is important for correctly mapping biological responses to any stimulus.
This technique has identified previously unknown interactions between two distinct prophage host partnerships. Since most bacteria house unstudied prophages, this approach could help to discover many novel therapeutic targets or applications.
