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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PAO1</td>
			<td></td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>LESB58</td>
			<td></td>
			<td>6</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&#181;m filter to sterilize and store it in -20&#176;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&#181;m filter to sterilize and store it in -20&#176;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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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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

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Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

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.

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...