Type: Analytical Essays
Sample donated: Michael Long
Last updated: July 13, 2019
SaPIs encode open reading frames stl and str which are responsible for the organisation and regulation of SaPI transcription.
The stl gene is the master regulator for the SaPI life cycle, when stl is expressed, it blocks excision of the island from the chromosome and so the SaPI is maintained in its integrated, quiescent state. However, in the presence of an inducing helper phage, stl repression is deactivated (de repression) which occurs as a result of the infecting phage proteins (i.e. Sri protein acting as anti-repressor) binding to stl and removing it from the promoter region to allow for the transcription of the divergent str anti repressor. This in turn initiates the commencement of the SaPI ERP cycle. Stl de repression will not occur during the SOS response of the bacteria and so requires a phage encoded moonlighting protein to deactivate it. The replication module present within the SaPI genome consists of a primase (pri), replication inhibitor (rep) and an origin of replication (ori).
The pri-rep-ori module is highly conserved among SaPIs and is only expressed following relief of Stl repression. Expression of the module results in high frequency replication of SaPI DNA, producing concatamers; the building blocks required for SaPI packaging. The helper phage DNA packaging machinery is located on the small terminase (terS) gene which is present on all SaPIs and encoded by the LexA regulated operon. The phage genome is able to encode both the small TerS and large terminase (TerL) proteins which are critical components of the phage holoterminase complex. Expression of the TerS/TerL complex allows for the mobilisation of the genomic island by recognising and cutting a specific part of the DNA sequence within the SaPI, termed the packaging (pac) site.
The pac site resides in the centre of the phage and initiates genome packaging (which begins from the location of the integrated prophage). Cos site packaging requires the use of an endonuclease called HNH, required for in vitro cos site cleavage, through direct interaction with TerL, and also assists in phage head morphogenesis. Island induction can interfere with the life cycle of the phage by means of expressing phage packaging inhibition genes (ppi) phage transcription interference genes (pti) and capsid morphogenesis proteins (i.e. CmpA and CmpB).
These genes involved with the regulation of genomic replication and expression of structural components are expressed altogether on a tightly regulated operon within the SaPI genome. The SaPI genome (~15kbp) is around one third the size of the phage genome (~45kbp). The capsid morphogenesis proteins serve to remodel the phage capsid to produce smaller capsid heads, rendering the phage capsids eligible to uptake and mobilise the smaller SaPI genome only. This ability of remodelling the phage capsid particles has proven highly advantageous to bacteria which possess PICI elements, assisting in the prevention of lytic predation of the host cell by blocking phage replication and spread. High frequency mobilisation of the PICI elements under these conditions can be used a ‘Trojan Horse’ strategy to deliver novel genes to other bacteria in order to confer new fitness advantages both inter and intra genetically.A potential limitation of the PICI system is the acquisition of clustered regularly spaced short palindromic repeats (CRISPR), in addition to CRISPR’s accompanying protein; Cas, the CRISPR/Cas system has been discovered in ~45% of all bacterial genomes, including S. aureus (i.e.
pCasSA). The system can be passed onto bacteria from phage and plasmids, providing an adaptive immunity response in bacteria against invading DNA and RNA. This could potentially disrupt the signalling mechanisms between the helper phage and PICI elements. The CRISPR/Cas system locates and binds to a single guide RNA (sgRNA) and recruits the Cas protein to make a specific cut through the dsDNA. Alothough the cell can carry out DNA repair the repair often causes errors leading to disruption of the targeted gene or indeed, gene knock out.
A similar system has been described by Seed et al., which found the CRISPR/Cas system present in the Vibrio cholerae virus (ICP1) interfered with island excision from the chromosome and in addition had the ability to knock out the island by specifically targeting the PICI-like element (PLE) found there. However further work is necessary in order to gain a better understanding of the underlying threat imposed by the CRISPR/Cas system in terms of its effects on PICI induction.The development of portable microfluidic devices (i.e. Lab on a Chip) have been used as a low cost, environmentally friendly way to carry out rapid diagnosis of infections and disease, contaminated drinking water and much more.
Paper microfluidic analytical devices (µPADs) work through capillary action and can deliver rapid readouts (and high frequency measurements) to detect various pathogens and toxins in a multitude of samples, including humans and the environment). These devices can prove highly beneficial, especially in under developed countries where they can serve as disposable point of care (POC) devices to diagnose illness, thus replacing the need for transporting lab equipment whilst avoiding delays in results. Microfluidics has been used to carry out ‘on the spot’ tests including liver function tests, and can also be used to obtain immunoglobulin levels, glucose levels and fluorescent sensing of bacterial growth etc. Using low cost diagnostics such as paper microfluidics can save time, money and resources, effectively taking the ‘Lab on a Chip’ out of the lab and into the real world to provide continuous real time monitoring in a highly affordable and accessible way. This dual science approach, combining bacteriology and bioengineering can prove an excellent way to translate the use of the PICI elements in a low cost device to test for example, the efficiency of phage induction in vitro