LAS PROMESAS HECHAS A LOS PADRES

Studying spontaneous prophage induction by measuring the titer of phages in culture supernatants is a simple and effective method that has been employed for decades (Lwoff, 1953). However, the titer of induced phage is an indirect measurement of spontaneous induction, as the result relies on the secondary infection of an indicator strain by the induced phages, rather than the induction event itself. For instance, defective prophages may be inducible and capable of cell lysis, but their induction would be missed by phage-titering based methods as the resulting phage-particles are uninfectious. In order to better understand the prolific spontaneous induction phenotype of the BTP1 prophage, a fluorescence reporter system was developed to allow study of BTP1 induction at the single-cell level.

As BTP1 is a lambda-like prophage, the transcriptional architecture of prophage induction could be predicted based on the Lambda molecular switch paradigm (summarised in Figure 1.8). Upon induction, loss of repressor function putatively allows transcription to initiate from the promoters PL, upstream of the gene encoding for the n antitermination gene (STMMW_03521), and PR, upstream of the gene encoding for the cro regulatory gene (STMMW_03551). Theoretically it would be possible to use a plasmid-based system involving a fluorescent marker under the control of the PL or PR promoters in order to visualise prophage induction. However, plasmid-based systems are highly likely to affect prophage induction dynamics, as multiple copies of the PL or PR promoters would titrate the repressor protein away from the native promoter. Additionally, as a number of tightly controlled molecular events have to occur following the initiation of prophage induction by transcription at PL or PR,this event in itself is not necessarily indicative of successfully completed prophage induction. In light of these drawbacks, a plasmid-based system was not adopted to study the induction of BTP1 and instead, a chromosomal system was developed. After initial transcription from PL and PR, it is predicted based on studies in Lambda that the N protein antiterminates both the n and cro transcripts to allow transcription of the ‘early’ genes, from n to int (STMMW_03521 to STMMW_03341) and from cro to q (STMMW_03551 to STMMW_03691). Subsequently, the Q protein is thought to antiterminate a small transcript (pR’) occurring prior to the lysis genes. In the BTP1 prophage, there are 2 tRNA genes between the q gene and the lysis genes, therefore it is unclear how and where Q-mediated antitermination occurs. However, Q protein synthesis likely results in the transcription of the lysis and structural genes, known collectively as the phage ‘late’ genes (STMMW_03711 to STMMW_03901). The late

95 genes are the final genes transcribed in prophage induction, and were chosen as the best marker for prophage induction. Initially, a system was developed as shown in Figure 3.10. A gfp construct consisting of a promoter-less gfp+ gene from the pZEP08 plasmid (Hautefort et al., 2003) and the FRT-flanked kanamycin resistance gene from the pKD4 plasmid (Datsenko and Wanner, 2000) was designed (Figure 3.10B). The construct was amplified to contain 50 bp flanking ends corresponding to an intergenic position in the late genes (Chapter 2.6.4), recombined into the chromosome using the lambda-red recombineering methodology (Datsenko and Wanner, 2000), resulting in transcription of gfp+ _{with the late gene operon (Figure 3.10A). Importantly, the} kanamycin gene was orientated in the opposite direction to the gfp+ _{to prevent the}

aberrant transcription of late genes downstream of the construct in non-induced cells. Once the construct was inserted into the chromosome the kanamycin gene was removed by FLP-mediated recombination of the FRT sites. Description of the methods used to construct the reporter systems can be found in Chapter 2.6.4. Though the system functioned correctly, and fluorescent cells were visualised upon chemical induction, the fluorescence signal was transient and lost around 1 hour after chemical induction (data not shown). It is likely that the loss of fluorescence signal shortly after induction is the result of cell lysis and phage release at the end of the induction process (schematic shown in Figure 3.11). Consequently, this construct could only be used to measure the number of cells undergoing induction at any one time, rather than to measure the total number of cells in the population that have spontaneously induced.

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Figure 3.10 Illustration of initial GFP reporter system for BTP1 induction. A. Genetic context of the gfp+ in the final construction. B. Architecture of the gfp+ kanamycin resistance construct used to create the florescence reporter system on the chromosome of D23580. Methods used to develop the construct can be found in Chapter 2.6.4.

97 Figure 3.11 Schematic illustration of the expected fate of induced cells

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A second system was developed using the same construct, but instead of using an intergenic position, the entire lysis gene cluster was replaced with the gfp+ construct (Figure 3.12). As the synthesis of the lysis proteins are likely to be essential for cell lysis, loss of these genes would allow induced cells to remain fluorescent in the culture, without loss of signal (schematic shown in Figure 3.13). A time-course of chemical induction of the D23580 ∆lysis::gfp+ strain is shown in Figure 3.14. This system was used to study the natural spontaneous induction of D23580 cultures. Overnight cultures of D23580 ∆lysis::gfp+ were screened for fluorescent cells. As expected, the majority of the population were non-fluorescent, short rods, resembling healthy stationary phase Salmonella bacteria. However, it was also possible to find highly fluorescent cells in the culture (Figure 3.15). The fluorescent cells in the overnight culture of D23580 ∆lysis::gfp+ _{were observed to be highly heterologous in}

size; some resembled the non-fluorescent bacteria in size, whilst others resembled large filaments (an example is shown in Figure 3.15). As it was previously observed that BTP1 spontaneous induction occurs throughout the growth curve, this dimorphism in size may correspond to the growth-phase the cell was in when it became induced; exponentially growing cells that become induced may form large filaments, whilst cells that become induced towards stationary phase may remain as single cells.

The visualisation of fluorescent cells in the overnight culture of the D23580 ∆lysis::gfp+

shows that this approach can be used to study the cell population in which the BTP1 prophage becomes spontaneously induced, without subsequent cell lysis. In the future these constructs could be used to accurately enumerate the induced fraction of the population using flow cytometry techniques, though optimisation will be required to control for the impact of heterogeneity of fluorescent cell sizes upon cell detection and sorting. In addition, similar constructs could be made in other prophages, allowing comparison of the rate of spontaneous induction of the lysogenic population, and the factors that contribute to spontaneous prophage induction.

99 Figure 3.12 Illustration of the Δlysis::gfp+ _{construct for BTP1 induction. Genetic}

context of the gfp+ _{in the final construction. The gfp}+ _{gene replaces the entirety of the} lysis gene cluster.

WT

Δl

ysis::

g

100

Figure 3.13 Schematic illustration of the expected fate of induced cells containing the Δlysis::gfp+ _{reporter construct.}

101 Figure 3.14 Time course of D23580 BTP1 ∆lysis::gfp+ _{cell fluorescence post}

chemical induction. Samples were collected and fixed at 5 minute intervals after addition of mitomycin C (Chapter 2.3.6). Transmitted light and fluorescent light images of the same field visualised using a 40X objective are shown (Chapter 2.3.6).

102

Figure 3.15 Example of elongated fluorescent cell morphology found in overnight culture of D23580 ∆lysis::gfp+_{. Both transmitted light and fluorescent light}

images of the same field using a 100X oil immersion objective are shown. Microscopy methods can be found in Chapter 2.3.6.

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