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

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&#160;phage production from a PAO1 LES&#934;2 lysogen culture grown under non-inducing conditions was used to determine the impact of spontaneous LES&#934;2 induction. The phage density was at its lowest point with a mean of ~2.61 x 106 plaque-forming units (PFU)&#183;mL&#8722;1 2 h after subculture in fresh medium during the early exponential phase of growth, suggesting that lysogeny was the dominant state. The LES&#934;2 titer rapidly increased to a mean of ~2.4 x 108 PFU&#183;mL&#8722;1 within 4 h and reached the highest density after 6 h (mean of ~5.83 x 109 PFU&#183;mL&#8722;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, LES&#934;2 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 &#8805; 9 passed quality control and were converted to cDNA.
The annotated LES&#934;2 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 &#60; 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 &#60; 0.01) and from ~ 6.05 x 102 to ~5.68 x 105 copies (Wilcoxon signed-rank test: p &#60; 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 &#60; 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 &#8220;noise&#8221; 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="Figure 1" class="xfigimg" src="/files/ftp_upload/64945/64945fig01.jpg" /> 
Figure 1: The protocol for creating the rifampicin-resistant indicator host (Created with BioRender.com). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64945/64945fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64945/64945fig02.jpg" /> 
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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64945/64945fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64945/64945fig03.jpg" /> 
Figure 3: The experimental design for sampling induced and un-induced cultures for RNA isolation.&#160;(Created with BioRender.com)&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64945/64945fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64945/64945fig04.jpg" /> 
Figure 4: Temporal enumeration of spontaneous induction. Temporal enumeration of spontaneous LES prophage production using the PFU from the PAO1 &#934;2 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&#183;mL&#8722;1 in LB; the dark blue points indicate the PFU&#183;mL&#8722;1 in LB. The spontaneous release of the &#966;2 infective phage by the lysogens is at the lowest measurable level at 2 h post-inoculation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64945/64945fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64945/64945fig05.jpg" /> 
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&#8211;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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64945/Table 1.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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64945/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics at JoVE.com

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics

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, ...

understanding-impact-temperate-bacteriophages-on-their-lysogens

Universidad de Cartagena UDEC

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JoVE Journal

Biology

1.7K Views.  University of Liverpool. 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.

Video: Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics

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

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

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

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.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Immunology and Infection

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

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.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

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

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

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

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant ...

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

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

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

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 &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Exploring Gut Phage-Bacteria Dynamics in Murine Models Using T4 Phage

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

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Author Spotlight: Investigating Bacteriophage-Induced Immune Responses in Gnotobiotic Mice

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.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

Using Bacterial LPS to Eliminate Bacteriophages from Salmonella Cultures

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Author Spotlight: Efficiently Eliminating Bacteriophages from Infected Salmonella Cultures Using Lipopolysaccharides

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and ...

Detection of Bacteriophages in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Alex Wassimi
Viruses are a unique group of biological entities that infect both eukaryotic and prokaryotic organisms. They are obligate parasites that have no metabolic capacity, and in order to replicate, rely on host metabolism to produce viral parts that self-assemble inside host cells.
Viruses are ultramicroscopic&#8212;too small to be viewed with the light microscope, visible only with the greater resolution of the electron microscope. A viral particle consists of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat, known as a capsid, composed of protein subunits or capsomers. In some more complex viruses, the capsid is surrounded by an additional lipid envelope, and some have spike-like surface appendages or tails.
Viruses that infect the intestinal tract of humans and animals are known as enteric viruses. They are excreted in feces and can be isolated from domestic wastewater. Viruses which infect bacteria are known as bacteriophages, and those which infect coliform bacteria are called coliphages (Figure 1). The phages of coliform bacteria are found anywhere coliform bacteria are found.
<img alt="Figure 1" src="/files/ftp_upload/10190/10190fig1.jpg" /> 
Figure 1. Coliphage T2.

Plaque Assay: Phage Enumeration in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Alex Wassimi
Viruses are a unique group of ...

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Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
Transduction is a form of genetic exchange between bacteria that utilizes bacteriophages, or phages, a class of virus that infects exclusively prokaryotic organisms. This form of DNA transfer, from one bacterium to another by way of a phage, was discovered in 1951 by Norton Zinder and Joshua Ledererg, who termed the process &#34;transduction&#34; (1). Bacteriophages were first discovered in 1915 by British bacteriologist Frederick Twort, then independently discovered again in 1917 by French-Canadian microbiologist Felix d&#39;Herelle (2). Since then, the structure and function of these phages have been widely characterized (3), dividing these phages into two classes. The first of these classes are the lytic phages which upon infection multiply within the host bacterium, disrupting the bacterial metabolism, lysing the cell, and releasing progeny phage (4). As a result of this anti-bacterial activity and the increasing prevalence of antibiotic-resistant bacteria, these lytic phages have recently proved useful as a substitute treatment for antibiotics. The second of these classes are the lysogenic phages which can either multiply within the host via the lytic cycle or enter a quiescent state in which their genome is integrated into that of the host (Figure 1), a process known as lysogeny, with the ability for phage production to be induced in multiple later generations (4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10517/10517fig1.jpg" /> 
Figure 1: Infection of host cell by bacteriophage. Adsorption by the phage to the bacterial cell wall via interactions between the tail fibers and receptor (purple). Once on the cell surface, the phage is irreversibly attached to the bacterial cell using the base plate (black) which is moved to the cell wall by the contractile sheath (yellow). Phage genome (red) then enters the cell and integrates into the host cell genome.
While bacterial transduction is a naturally occurring process, using modern technology it has been manipulated for the transfer of genes into bacteria in the laboratory setting. By inserting genes of interest into the genome of a lysogenic phage, such as phage, one is able to transfer these genes into the genomes of bacteria and consequently express them within these cells. While other methods of gene transfer, such as transformation, use a plasmid for gene transfer and expression, the insertion of the phage genome into that of the recipient bacterium not only has the potential to confer new traits to this bacterium, but also allows for naturally occurring mutations and other factors of the cellular environment to alter the function of the transferred gene.
Compared to other methods of horizontal gene transfer, such as conjugation, transduction is fairly flexible in the criteria required for donor and recipient cells. Any genetic element that can fit inside the genome of the phage being used can be transferred from any strain of donor bacteria to any strain of recipient bacteria as long as both are permissive to the phage, requiring the expression of necessary phage receptors on the cell surfaces. Once this gene is moved out of the donor genome and packaged into the phage, it can be transferred to the recipient. Following transduction, it is necessary to select for recipient bacteria that contain the gene of interest have to be selected for. This could be done by use of a genetic marker, such as a FLAG-tag or polyhistidine-tag, to mark the gene of interest, or the intrinsic function of the gene, in the case of genes that encode for antibiotic resistance. Additionally, PCR could be used to further confirm successful transduction. By using primers for a region within the gene of interest and comparing signal to a positive control, bacteria that has the gene of interest, and a negative control, bacteria that underwent the same steps as the transduction reaction with no phage. While bacterial transduction is a useful tool in molecular biology, it has and continues to play an important role in the evolution of bacteria, particularly with regard to the recent rise of antibiotic resistance.
In this experiment, bacterial transduction was used to transfer the gene encoding for resistance to the antibiotic ampicillin from the W3110 strain of E.coli to the J53 strain via the P1 bacteriophage (5). This experiment consisted of two main steps. First, the preparation of P1 phage containing the ampicillin resistance gene from the donor strain. Second, the transfer of this gene to the recipient strain by transduction with the P1 phage (Figure 1). Once carried out, the successful transfer of the ampicillin resistance gene could be determined by qPCR (Figure 2). If transduction was successful, the J53 strain of E. coli would be resistant to ampicillin, and the gene conferring this resistance detectable by qPCR. If unsuccessful, there would be no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10517/10517fig2.jpg" /> 
Figure 2: The confirmation of successful transduction by qPCR. By comparing the Cq values detected for the gene of interest from the transduction reaction and the negative control reaction, and normalizing these values against a housekeeping gene, one was able to confirm that bacterial transduction was successful.

Phage Transduction: Transferring Ampicillin Resistance in E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...

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Microbiology

Lytic Cycle of Bacteriophages

Bacteriophages, also known as phages, are specialized viruses that infect bacteria. A key characteristic of phages is their distinctive &ldquo;head-tail&rdquo; morphology. A phage begins the infection process (i.e., lytic cycle) by attaching to the outside of a bacterial cell. Attachment is accomplished via proteins in the phage tail that bind to specific receptor proteins on the outer surface of the bacterium. The tail injects the phage&rsquo;s DNA genome into the bacterial cytoplasm. In the lytic replication cycle, the phage uses the bacterium&rsquo;s cellular machinery to make proteins that are critical for the phage&rsquo;s replication and dispersal. Some of these proteins cause the host cell to take in water and burst, or lyse, after phage replication is complete, releasing hundreds of phages that can infect new bacterial cells.

<h4>Phage Therapy</h4>

Since the early 20th century, researchers have recognized the potential value of lytic bacteriophages in combating bacterial infections in crops, humans, and agricultural animals. Because each type of phage can infect and lyse only specific types of bacteria, phages represent a highly specific form of anti-bacterial treatment. This quality stands in contrast to the familiar antibiotic drugs that we often take for bacterial infections, which are typically broad-spectrum treatments that kill both pathogenic and beneficial bacteria. The widespread use of broad-spectrum antibiotics has caused the evolution of bacterial resistance to whole classes of these drugs, rendering once treatable infections potentially deadly. As more pathogenic bacteria evolve resistance to antibiotics, narrow-spectrum phage therapy may become a useful alternative. Because phages are highly specific in the bacteria that they infect, the evolution of resistance to phages would also be limited to the particular strain of bacteria.

However, several obstacles must be overcome for phage therapy to become a viable alternative to antibiotics. For instance, the high specificity of phages is also a drawback, because different phages would be needed for each species of the bacterial pathogen or even strain of bacteria within a pathogenic species. It would, therefore, be difficult to produce phages for many different bacterial infections at a large scale. Furthermore, because of phage specificity, it would be necessary to either know the particular bacterial strain that is causing an infection or use a cocktail of multiple different phages in the treatment and hope that one of them matches the pathogenic bacteria. Despite these drawbacks, phage therapy remains an active area of research.

Bacteriophages, also known as phages, are specialized viruses that infect bacteria. A key characteristic of phages is their distinctive ...

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Viruses

KEY TERMS AND CONCEPTS

Lysogenic Cycle of Bacteriophages

In contrast to the lytic cycle, phages infecting bacteria via the lysogenic cycle do not immediately kill their host cell. Instead, they combine their genome with the host genome, allowing the bacteria to replicate the phage DNA along with the bacterial genome. The incorporated copy of the phage genome is called the prophage. Some prophages can re-activate and enter the lytic cycle. This often occurs in response to a perturbation, such as DNA damage, but can also transpire in the absence of external cues.

<h4>Lysogenic Conversion</h4>

In some cases, the genes encoded by prophages can alter the phenotype of the infected bacterium, a process known as lysogenic conversion. Some phages encode proteins or toxins called virulence factors that can facilitate bacterial infections. Through lysogenic conversion, normally non-pathogenic bacteria can become highly virulent via infection by a phage carrying virulence factors.

For example, such phages are largely responsible for the pathogenicity of the bacterial species that cause botulism (Clostridium botulinum), diphtheria (Corynebacterium diphtheriae), and cholera (Vibrio cholerae). Without lysogenic conversion, these bacteria do not usually cause disease.

A particularly well-studied example of lysogenic conversion is that of the Escherichia coli strain O157:H7. Several massive food recalls have stemmed from contamination by E. coli O157:H7. This strain of E. coli has been infected by a phage that encodes Shiga-like toxin (Stx), which can cause intestinal bleeding and kidney failure. In the lysogenic cycle, Stx is not produced, and the bacteria do not cause disease. The phage must re-enter the lytic cycle for Stx to be produced. Unfortunately, certain antibiotics can trigger the induction of the prophage and consequent Stx production, making treatment of these infections difficult. Current research is investigating novel therapies that prevent initiation of the prophage.

Prophage and Lysogenic Cycle of Bacteriophages

In contrast to the lytic cycle, phages infecting bacteria via the lysogenic cycle do not immediately kill their host cell. Instead, they combine their ...

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics

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Chapters in this video

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

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

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

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

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

Bacteriophage Removal from Infected Salmonella Cultures

Detection of Bacteriophages in Environmental Samples

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Lytic Cycle of Bacteriophages

Lysogenic Cycle of Bacteriophages