Name: Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics (Video) | JoVE Journal
Uploaded: 2024-01-05T13:20:00.000+00:00
Duration: 9 min 23 s
Description: Explore transcriptomic profiling of prophage-bacterial host interactions using optimized culture conditions and RT-qPCR. This method reveals how temperate bacteriophages regulate gene expression in lysogenic states.

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The creation of a selectable indicator host, previously used in plaque assays to more accurately quantify the spontaneous induction of Stx phage from E. coli MC106137,38,39, has been described here for P. aeruginosa phage LES&#934;2. This intervention has the added benefit of reducing the sample processing steps and time, thus enabling the simultaneous assessment of spontaneous induction rates in multiple culture conditions. There is a risk of generating other mutations during the creation of rifampicin-resistant variants40; however, in this work, the evolved strain was only used as an indicator host for the enumeration of plaques from cultures of interest and was not included in the transcriptomic analysis. As long as the selectable indicator strain remains equally susceptible to infection by the phage of interest, there is no concern about other acquired mutations. Nevertheless, no differences in the restriction fragment length polymorphism profiles were detected by the pulse field gel electrophoresis (PFGE) analysis of PAO1WT and PAO1RIF (data not shown).
When choosing host cells, it is rare to find an indicator strain that does not already harbor prophages. As a case in point, PAO1 harbors the filamentous prophage Pf4. The experimental controls for this study were designed to be able to directly examine the gene expression of specific phages (in this case, LES prophage 2) and the effects this phage has on bacterial gene expression. In the comparison of transcripts from PAO1 carrying the LES prophage 2 and lacking the LES prophage 2 (both lysogen and non-lysogen carry the endogenous Pf4), which serve as internal controls to exclude the impact of Pf4 on the host. Additionally, it has been demonstrated that Pf4 usually does not cause lysis in its host cell41 and is, therefore, not capable of confounding the results of these experiments.
It is well-established that careful quality control is crucial in sample preparation for producing meaningful omics data42. However, as previously described11, the careful characterization of prophage activity in the preparation of lysogen cultures for such studies is rarely performed. Here, we detail our systematic protocols for preparing&#160;a well-controlled and optimized set of cultures&#160;for transcriptomic studies to better explore the interactions between bacteria and temperate phages. The synchronicity of the population was controlled by bringing the culture through at least four doublings before treating it with the inducing antibiotic norfloxacin. By determining the MIC of norfloxacin for the strain in the study, we could ensure that the concentration of the inducing agent was just above the MIC for the &#8220;induction&#8221; treatment. The treated cells were then diluted 1:10 to lower the norfloxacin concentration below the MIC after the 1 h treatment in order to allow the cells to recover and complete the phage replication process, ending in the lysis of the cell and the release of infective phage progeny. The cells only enter the lytic replication cycle following the induction stimulus once the concentration of norfloxacin has been brought below the MIC during the recovery period. In this case, going above 1 &#181;g&#183;mL&#8722;1 norfloxacin means that the drug could not be effectively diluted below the MIC, as the MIC for norfloxacin for PAO1 is 0.19 &#181;g&#183;mL&#8722;1. The level of inducer dilution must be balanced with the need for lysogen recovery and the retention of the culture density for harvesting the RNA. The data discussed here demonstrate that it is possible to synchronize cultures to create samples in which lysogeny dominates, thus reducing the noise from spontaneous induction and enabling the detection of true lysogeny-driven changes in gene expression. Since the lysogenic state is predominant in the early-exponential phase of growth when the bacterial cell density is low, we suggest scaling up the cultures to harvest enough RNA for subsequent gene expression studies such as RNA-Seq.
The use of norfloxacin as an inducing agent to force cultures into the lytic cycle is well-reported43,44; however, this will also affect the expression of other bacterial genes in the process45,46. To mitigate this, RNA libraries from control wild-type cultures grown under the same inducing and non-inducing conditions should be included in RNA-Seq experiments. The use of internal controls and key marker genes to validate the stages of phage replication by qRT-PCR is also crucial for accurate comparisons. Quantitative RT-PCR profiling cannot be interpreted by comparing the absolute numbers of transcripts for each gene at various time points; it is the shape of the profile that matters. First, only one small region in the transcript for any gene has been sampled, so whether it is a short-lived or longer-lived element is unknown27. Certainly, RNA-Seq mapping of transcripts shows that the density of the mapping data varies significantly over the length of a gene. Secondly, it is the shape of the gene expression profile that should be interpreted for a marker gene associated with the lytic cycle or the lysogenic lifestyle or even uncoupled from the phage regulatory circuits11. Spontaneous induction is a real issue in lysogen culture and will always result in the expression of lytic cycle-associated genes. However, profiling does show that the genes associated with the lytic replication cycle are suppressed in their expression pre-induction (at least two log folds) and up-regulated post-induction.
The previously conducted transcriptomic analyses of Stx phage interactions with E. coli support a thorough understanding of the phage genes involved in maintaining lysogeny and triggering the lytic cycle11,17. Currently, the LES phages of P. aeruginosa have been annotated, but their key gene functions are less well understood. Transcriptomic studies will enable the re-annotation of the LES prophages and improve our understanding of the genes involved in the lysogeny and lytic cycle. Linking gene sequence to function represents a major challenge in the study of novel prophages, which further highlights the need for more studies to confirm the phage gene functions for the production of better annotation tools47. The wider application and adaptation of the protocols and extra quality control measures detailed in this video article could help in unveiling various prophage functions and, thus, improving annotation pipelines and transforming our understanding of phage and bacterial biology.

Recently, phage therapy for tackling antimicrobial resistance1&#160;and CRISPR-Cas-based gene editing2&#160;have generated renewed interest in bacteriophage research. Again, advancements in biotechnology have enabled the deeper investigation of the interactions between bacteria and phages3. However, the therapeutic use of phage (&#34;phage therapy&#34;) is hampered by concerns about phages acting as mobile genetic elements with the capacity to transfer virulence and resistance genes horizontally4. The expanse of &#34;dark matter&#34;5&#160;(genes with unknown functions) is both troubling and enticing. Dark matter is considered a gap&#160;in our understanding of phage biology and a largely untapped resource for molecular tools and potential novel therapeutics6. The development of high-throughput sequencing techniques, along with improved gene annotation7,8,9 and new peptide-folding algorithms10, is improving the detection, description, and functional prediction of phage genes. However, science is still far from validating most phages&#39; gene functions in culture or in the real world.
RNA sequencing (RNA-Seq) can globally map gene expression during phage infection and has significantly improved the understanding of both the phage and bacterial elements involved in lytic and lysogenic cycles11,12. During lysogenic processes, temperate phage genomes are integrated into bacterial DNA to become prophages13. Global gene expression profiling experiments can be used to identify prophage-restricted genes that are encoded on temperate phage genomes but only expressed during the lysogenic state11. Such genes do not encode phage structural proteins and are not involved in any phage infection processes. RNA-Seq can be used to identify those genes that are more likely to influence the biology of the bacterial host, either by inducing a gain of function or regulating the existing bacterial genes, thus often enabling the bacteria to adapt to changing environments. Therefore, the ability of prophages to act as microbial puppet masters, controlling a range of bacterial functions, could be studied.
There are two major barriers to the effective analysis of prophage-restricted gene expression. Firstly, the availability of susceptible hosts is a key issue. By definition, prophages are already incorporated into their specific host genome, so it is challenging to find a susceptible wild-type host to compare the global gene expression in the presence and absence of the prophage. This can be achieved either through the de novo infection of another susceptible host or the deletion of the prophage from the original wild-type isolate, without disrupting the rest of the host genome. The second barrier lies in the heterogeneous nature of lysogenic populations. Some prophages degrade through mutation or recombination to become &#34;cryptic&#34;, meaning they are fixed in a specific location of the bacterial genome. However, other prophages are &#34;active&#34; and can be induced into a replicative, lytic cycle spontaneously or after exposure to inducing factors. In many lysogenic cultures, the rate of spontaneous induction means that a proportion of the bacterial cells are always undergoing lytic phage replication14,15,16. A high level of expression of late phage genes in these populations masks the low-level gene expression associated with lysogen-restricted gene expression11,17. The proportion of lysogens undergoing spontaneous prophage induction may vary with the growth state, growth conditions, or other triggers. Therefore, to study the impacts of prophages upon the lysogen, spontaneous prophage induction events must be minimized as much as possible by optimizing the growth conditions to favor the lysogenic state.
This study reports the preparatory work done to investigate the influence of a set of cohabiting prophages from the Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa. Active prophages were induced and isolated from LES&#160;and used to infect the model P. aeruginosa host strain, PAO116,18,19. The whole genomes of the wild-type P. aeruginosa strain, PAO1, and its lysogen, PAO1&#934;2, were sequenced (at a depth of 30x coverage) to ensure the identity of the wild-type strain and to confirm that the lysogen was isogenic.&#160;The&#160;LES has been associated with increased morbidity and mortality in cystic fibrosis patients, and LES phages19&#160;have been suggested to aid adaptation&#160;to the cystic fibrosis lung environment16,19,20. Despite strong evidence that these prophages affect the biology of their host20,21, the majority of their gene functions are yet to be characterized, and the specific mechanisms of interaction are poorly understood. A transcriptomics approach can empirically uncover the prophage gene functions in a controlled host background. Since spontaneous induction can affect expression profiles, this article describes how to optimize the growth conditions to favor the lysogenic state. Such synchronization of cultures can be validated by real-time PCR to quantify the expression levels of key genetic markers that are associated with crucial stages of LES phage replication in PAO1. The same approach has been used previously to identify the prophage-restricted functions of Shiga-toxigenic phages that affect motility, acid resistance, and antimicrobial resistance in Escherichia coli11,17,21,22.

<table><tbody><tr><td>PAO1</td><td></td><td>&lt;sup&gt;6&lt;/sup&gt;</td><td></td></tr><tr><td>LESB58</td><td></td><td>&lt;sup&gt;6&lt;/sup&gt;</td><td></td></tr><tr><td>LES phages</td><td>Induced and purified from LESB58 using Norfloxacin.</td><td>This study</td><td></td></tr><tr><td>Lysogeny Broth (LB)</td><td>Merck</td><td>1.10285.500</td><td></td></tr><tr><td>LB Agar</td><td>Merck</td><td>1.10283.500</td><td></td></tr><tr><td>Agar Agar</td><td>Fisher</td><td>A/1080/53</td><td></td></tr><tr><td>Top Agar</td><td>0.4 g Agar Agar+2.5 g LB Broth in 100 mL water; autoclave and use.</td><td>-</td><td></td></tr><tr><td>Rifampicin</td><td>Sigma (Stock: 50 mg/mL in Methanol- Mix well and use 0.22&amp;micro;m filter to sterilize and store it in -20&amp;deg;C until use)</td><td>R3501</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher 1% (v/v) in water</td><td>10060000</td><td></td></tr><tr><td>Norfloxacin</td><td>Sigma (Stock: 25 mg/mL of 1% Glacial Acetic Acid-Mix well and use 0.22&amp;micro;m filter to sterilize and store it in -20&amp;deg;C until use;To avoid freeze thaw cycles, store as small aliquotes)</td><td>N9890</td><td></td></tr><tr><td>Phenol saturated with citrate buffer pH 4.3</td><td>Sigma</td><td>P-4682</td><td></td></tr><tr><td>Molecular Biology grade Ethanol</td><td>Fisher</td><td>16695992</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>12044977</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>11398187</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher</td><td>17150576</td><td></td></tr><tr><td>Nuclease-free H2O</td><td>Invitrogen</td><td>10526945</td><td></td></tr><tr><td>10X TURBO DNase</td><td>Ambion</td><td>AM1907</td><td></td></tr><tr><td>Qubit RNA HS, BR Kit</td><td>Invitrogen</td><td>Q10210</td><td></td></tr><tr><td>Agilent RNA 6000 Nano Kit</td><td>Agilent</td><td>5067-1511</td><td></td></tr><tr><td>SuperScriptIII first strand synthesis kit</td><td>Invitrogen</td><td>18080051</td><td></td></tr><tr><td>PCR Reagents</td><td>Bioline Mytaq Red 2X</td><td>BIO-25043</td><td></td></tr><tr><td>qPCR Reagents</td><td>Sensifast SYBR Hi Rox</td><td>BIO-92020</td><td></td></tr><tr><td>PCR purification kit</td><td>Isolate II PCR and Gel Kit</td><td>BIO-52060</td><td></td></tr><tr><td>TA cloning kit</td><td>TA Cloning Kit, with pCR 2.1 Vector, without competent cells</td><td>K202040</td><td></td></tr><tr><td>StepOne Real Time PCR system</td><td>Thermo Fisher Scientific</td><td>4376600</td><td></td></tr></tbody></table>

understanding the impact of temperate bacteriophages on their lysogens through transcriptomics

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics

Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, but others are active and can be triggered into a replicative form either spontaneously or by exposure to inducing factors. Prophages are commonly associated with the ability to confer toxin production or other virulence-associated traits on their host cell. More recent studies have shown they can play a much bigger role in altering the physiology of their hosts. The technique described here has enabled us to investigate how prophages affect gene expression in the opportunistic bacterium Pseudomonas aeruginosa.
In this work, the growth of the wild-type P. aeruginosa strain PAO1 was compared with that of isogenic lysogens carrying different combinations of prophages from the Liverpool Epidemic Strain (LES) LESB58. In a lysogen culture, a proportion of bacterial cells will be supporting lytic bacteriophage replication (spontaneous induction) with a high level of expression per cell of&#160;late phage genes, such as those associated with the assembly of phage particles,&#160;thus masking the low-level gene expression associated with lysogen-restricted gene&#160;expression. The impact of spontaneous induction can thus obscure prophage gene expression across a lysogen population.
Growth profiling experiments were used to identify spontaneous induction, which was minimal during the early exponential growth phase. This study reports how to prepare sample cultures during the early exponential growth phase and how to set up adequate controls despite low cell numbers. These protocols ensure the reliable and reproducible comparison of wild-type and lysogenic bacteria under various conditions, thus improving the transcriptomic profiling of prophage genomes and aiding in the identification of previously unrecognized prophage functions.

In this work, the direct temporal enumeration of phage production from a PAO1 LESΦ2 lysogen culture grown under non-inducing conditions was used to determine the impact of spontaneous LESΦ2 induction. The phage density was at its lowest point with a mean of ~2.61 x 106 plaque-forming units (PFU)·mL−1 2 h after subculture in fresh medium during the early exponential phase of growth, suggesting that lysogeny was the dominant state. The LESΦ2 titer rapidly increased to a mean of ~2.4 x 108 PFU·mL−1 within 4 h and reached the highest density after 6 h (mean of ~5.83 x 109 PFU·mL−1; Figure 4).
Minimal spontaneous induction was observed during the early log phase of lysogen growth (after 2 h). However, the measurable presence of phages in the culture medium was the result of many prior events, including the following: the packaging of nucleic acids into protein heads, the assembly of proteins into phage particles, and the expression of late phage genes, middle-stage phage genes, and early regulatory phage genes. It was important to catch the infected cells prior to the expression of the phage-associated replication events; hence, 90 min was chosen to let the culture grow prior to induction. To capture the gene expression profile of the PAO1, LESΦ2 lysogen samples from a culture were harvested pre-induction and post-induction over a 90 min period, as mentioned in step 3.4. This 90 min time point is well before high levels of spontaneous induction of the resident prophage are detected by the plaque assay from step 2.3.2. Since the bacterial cell density was low during early exponential growth, the culture volumes were scaled up to 800 mL to ensure ample material for the gene expression studies. The samples were collected from the uninduced culture and induced cultures every 10 min, and RNA was extracted to map the expression profile of the key markers for lysogeny and lytic replication during the bacterial growth. Total RNA was purified and validated for the absence of genomic DNA using qPCR assays targeting the 16S rRNA gene (step 6.1). The samples reaching an RIN ≥ 9 passed quality control and were converted to cDNA.
The annotated LESΦ2 genome was examined to identify genes that are well-known players in the lysogenic and lytic replication cycles of temperate phages. These identified genes were then used to validate the qRT-PCR for the expression profiling of the lysogen cycle-restricted and lytic cycle-associated genes from induced and un-induced cultures. We quantified the absolute DNA copy number and conducted a Wilcoxon signed-rank test using R36 to compare the expression levels in un-induced and induced cultures (Figure 5). A marked increase in the expression of the cro gene (an early marker of lytic replication) from ~2.31 x 109 copies in un-induced cultures to ~3.02 x 1011 copies 30 min post-induction (Wilcoxon signed-rank test: p &lt; 0.01) was observed. Similarly, O proteins and P proteins, which are mid-stage markers of lytic replication (and are predicted to be involved in phage genome replication), also showed significant upregulation from ~1.74 x 108 to ~1.25 x 1010 copies (Wilcoxon signed-rank test: p &lt; 0.01) and from ~ 6.05 x 102 to ~5.68 x 105 copies (Wilcoxon signed-rank test: p &lt; 0.01), respectively. Finally, the tail-associated structural genes were used as late markers of the lytic replication cycle. Again, we observed a significant increase in expression from ~2.31 x 106 copies in un-induced cultures to ~4.38 x 108 copies 30 min post-induction (Wilcoxon signed-rank test: p &lt; 0.01). Thus, the quantitative RT-PCR data confirmed that the gene expression of well-established marker genes for lytic replication followed the expected trend, with the early, mid, and late markers showing multiple-fold differential expression in the predicted order (Figure 5). Since the expression of the markers for lytic replication was upregulated 30 min post-recovery, this is considered as an appropriate representative time point for studying the transcriptomic landscape of active temperate phages and their bacterial hosts during the lytic cycle.
We observed some expression of lytic genes in un-induced conditions, confirming that some spontaneous induction always occurs, even in optimized cultures in which the lysogen numbers are represented with the highest ratio of CFU to released PFU in the early log phase. This means that there will always be some level of “noise” in the transcriptomics data, which reinforces the importance of carefully prepared controls, including induced and un-induced cultures. The appropriate choice of the internal control genes to determine the fold changes in expression relies on carefully examining the transcriptomics data to identify genes that are expressed at the same level in both the un-induced and induced samples. Our preliminary results suggest that rpoD was the most reliable control gene tested and had the most stable expression (~1.71 x 105 copies before induction and ~3.33 x 105 copies 30 min post-induction; Wilcoxon signed-rank test: p = 0.3594) compared to the 16S rRNA or proC genes (Figure 5). The variability of the expression of the internal controls led to the measurement of the absolute numbers of transcripts. Future examination of the transcriptomics data will support the choice of appropriate internal controls for further validation.
The cI gene was used in our gene profiling exercise, as it is a well-recognized marker of lysogeny. Compared to the markers for lytic replication, the expression of the cI gene was relatively stable (Figure 5), but the copy number of this gene was reassuringly high in the un-induced cultures compared to those of the markers for lytic replication. These data are in agreement with the low PFU numbers in the same samples, thus confirming that high repressor expression was associated with lower levels of phage production. The data reported here demonstrate that the expression of the cI transcript for this particular phage is not significantly downregulated post-induction, as seen in the Stx phages11,17. Repressor activity is normally controlled at both the transcriptional and post-translational levels, so the repressor gene can be transcribed, but the resultant protein is immediately subjected to autocleavage. Further experimentation is required to validate transcriptional and post-translational controls. Moreover, from our standard curve, the minimum detection limit of qPCR appears to be ~102 copies.
Together, our findings from plaque and qRT-PCR assays validate our strategy for culture and RNA sample preparation to generate a well-controlled input for RNA-Seq experiments. The un-induced cultures in the early-exponential phase exhibited low levels of spontaneous induction and lytic gene expression, suggesting the dominance of lysogeny. In contrast, the cultures isolated 30 min after induction showed significant increases in the expression of marker genes that indicate the dominance of lytic replication.
<img alt="Rifampicin concentration experiment diagram showing test tubes with increasing yellow tint over 7 days." class="xfigimg" src="/files/ftp_upload/64945/64945fig01.jpg" data-altupdatedat="1787025648000" data-alt="Figure 1"> 
Figure 1: The protocol for creating the rifampicin-resistant indicator host (Created with BioRender.com). <a href="https://www.jove.com/files/ftp_upload/64945/64945fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Serial dilution diagram for CFU and PFU enumeration; shows lysogen and indicator cell cultures." class="xfigimg" src="/files/ftp_upload/64945/64945fig02.jpg" data-altupdatedat="1787025648000" data-alt="Figure 2"> 
Figure 2: The experimental design for enumerating the PFU and CFU of a lysogen from the same sample. (Created with BioRender.com) <a href="https://www.jove.com/files/ftp_upload/64945/64945fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dual process of RNA extraction diagram for induced vs. uninduced bacterial cultures using norfloxacin." class="xfigimg" src="/files/ftp_upload/64945/64945fig03.jpg" data-altupdatedat="1787025648000" data-alt="Figure 3"> 
Figure 3: The experimental design for sampling induced and un-induced cultures for RNA isolation. (Created with BioRender.com) <a href="https://www.jove.com/files/ftp_upload/64945/64945fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial growth and phage interaction graph; Log PFU mL⁻¹ vs. Time (h); experimental results." class="xfigimg" src="/files/ftp_upload/64945/64945fig04.jpg" data-altupdatedat="1787025648000" data-alt="Figure 4"> 
Figure 4: Temporal enumeration of spontaneous induction. Temporal enumeration of spontaneous LES prophage production using the PFU from the PAO1 Φ2 lysogen with the concurrent CFU, n = 8 (two biological and four technical replicates); the error bars represent the standard deviation. The dark red points indicate the CFU·mL−1 in LB; the dark blue points indicate the PFU·mL−1 in LB. The spontaneous release of the φ2 infective phage by the lysogens is at the lowest measurable level at 2 h post-inoculation. <a href="https://www.jove.com/files/ftp_upload/64945/64945fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Box plot graphs showing DNA copy number variation over time in phage2 gene markers; experimental result." class="xfigimg" src="/files/ftp_upload/64945/64945fig05.jpg" data-altupdatedat="1787025648000" data-alt="Figure 5"> 
Figure 5: Absolute copy number of the target marker genes. The absolute copy number of phage marker genes confirm the predicted expression patterns, derived using RT-qPCR, of genes expected to play important roles in lysogeny and lytic cycles. The dots represent both three biological and three technical replicates (n = 9). (A)The red box represents the lysogeny marker, cI; (B) green represents the early lytic marker, cro; (C,D) blue represents the mid lytic markers, DNA replication genes; (E) magenta represents the late lytic marker, tail structural genes; (F–H) gray represents the host markers that were used as internal controls, and (I) white represents the DNA gyrase B, which was used as an induction control. The solid horizontal lines show the median of the distribution. <a href="https://www.jove.com/files/ftp_upload/64945/64945fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Primers designed in this study. The sequences of specific primers for the marker genes and internal controls used in this study are provided, along with their corresponding NCBI accession IDs. <a href="https://www.jove.com/files/ftp_upload/64945/Table%201.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Efficiency of the primers used in this study calculated using the qPCR standard curve. <a href="https://www.jove.com/files/ftp_upload/64945/Table%202.xlsx" target="_blank">Please click here to download this Table.</a>

This protocol enables the impact of prophages on their hosts to be revealed. Bacterial cultures are synchronized using conditions that best support the lysogenic state, limiting spontaneous induction. RT-qPCR unequivocally distinguishes prophage-restricted genes and those uncoupled from phage control from those that are expressed during the lytic replication cycle.

Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, ...

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, lies in, 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 LB 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 ager 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 agar, along with rifampicin. Spot 10 microliters of serially diluted lysogen culture onto the inoculated top agar 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 an 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 in 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 LES 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 qRT-PCR 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 16S RNA 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.

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Research

JoVE Journal

Biology

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Following Cell-fate in E. coli After Infection by Phage Lambda

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp.(2018)This video demonstrates preparation of a bacteriophage lysate containing transducing particles. A donor strain is infected, plaques form, and the soft agar overlay is processed with chloroform to yield a clarified lysate for downstream analysis.

Preparation of Bacteriophage Lysate for Generalized Transduction

Source: Krishnamurthi, R., et al. Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics. J. Vis. Exp. (2024).This video demonstrates the temporal enumeration of spontaneous phage induction in lysogenic Pseudomonas aeruginosa by quantifying viable bacterial cells (CFUs) and infective phage particles (PFUs) over time. By sampling at defined intervals during growth and analyzing both colony and plaque formation, the method reveals when spontaneous induction...

Measuring Spontaneous Phage Induction in Lysogenic Bacteria Over Time

Source: Krishnamurthi, R., et al. Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics. J. Vis. Exp. (2024)This video demonstrates the induction of lytic replication in a prophage integrated into the bacterial genome and the capture of gene expression changes through timed sampling for transcriptomic analysis.

Inducing the Prophage Lytic Replication for Transcriptomic Analysis

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using φBB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Understanding Cerebellar Pattern Formation

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Explore transcriptomic profiling of prophage-bacterial host interactions using optimized culture conditions and RT-qPCR. This method reveals how temperate bacteriophages regulate gene expression in lysogenic states.

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics

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