Once a stationary population of Era depleted cells was obtained, we wanted to evaluate the effect of depletion on ribosome maturation. Towards this, we first performed ribosome profiling to study the ribosome population in Era depleted cells. The WtLon cells displayed a normal ribosome distribution with significant amounts of 70S particles and minor amounts of 30S and 50S particles (Fig.4.5A). To our surprise, like WtLon, EraLon cells also displayed a significant fraction of 70S particles and minor quantities of 30S and 50S population (Fig.
4.5B). These observations were in stark contrast with the observed growth defect. In order to understand these contrasting observations, we tested the translation capacities of the Era depleted cells by studying the protein production kinetics of β-galactosidase (bgal) (Dalbow and Young, 1975; Schleif et al., 1973). Towards this, WtLon or EraLon cells were transformed with a vector carrying an IPTG inducible copy of bgal encoding gene. WtLon cells displayed significant protein build up within minutes of protein induction (Fig.4. 5C). Surprisingly, EraLon failed to show any build-up of bgal activity (Fig. 4.5C), indicating lack of protein production in these cells. These observations indicate that Era depletion somehow compromises the translation capacity of cells. These observations also explain the arrest of cell growth even with a significant 70S population.
Figure 4.5: Era depletion compromises cellular translation capacity
(A) Ribosome profiles for WtLon cells. Peaks observed for the respective particles have been indicated.
(B) Ribosome profiles for EraLon cells. Peaks observed for the respective particles have been indicated.
(C) Translation kinetics for the production of bgal in WtLon or EraLon cells. The activity measured in Miller units (Y axis) is traced over time (X-axis).
Discussion
In this part of the thesis, we presented a methodology to conditionally deplete essential proteins that can enable functional characterization in a true physiological setting. Utilizing components of the M. florum ribosome rescue system (Gur and Sauer, 2008b; Janssen and Hayes, 2012), we offer a proof-of-concept by depleting the essential RAF Era in a tunable manner. The strategy relies on the use of the ssrA tag which, when recognized by the Mf-Lon protease triggers degradation of the tagged protein. Conventional methods used to deplete essential genes involve depletion by modifying the regulatory regions of the genes of interest (Kaufmann and Knop, 2011). These engineered regulatory regions provide altered expression levels or the possibility of conditional expression using appropriate inducers or triggers (Cruz-Bustos et al., 2017; Minden et al., 2000; Petrillo et al., 2014; Seeliger et al.,
2012). In addition, emerging methods to block gene expression in bacteria employ Cas9 mediated transcription repression (Peters et al., 2016). The Cas9-targeting RNA complex binds to the promoter regions of the target gene occluding the entry of RNA polymerase thus stalling transcription. The aforementioned methods although established, produce artefacts like off-target hits or creates a polarity effect, affecting the expression of downstream genes.
Additionally, the methods require extensive optimization for designing target binding RNA.
Overcoming these difficulties, the methodology devised here, mediates targeted protein exhaustion after its translation, leaving the other genetic circuits and the mRNA intact, avoiding leaky expression or any polarity effects. The relatively simple method required the creation of a simple genetic circuit that is turned ON by adding an inducer to trigger the production of Lon protease. Based on previous reports, the system can achieve nearly complete degradation of a protein within 2.5-4 hrs (Cameron and Collins, 2014) of Lon protease induction. However, given the culture conditions, the authors did not find viable cell counts after depletion of essential genes. A major advancement made in our study lies in achieving stable populations of cells lacking the essential proteins. Our protein based depletion strategy circumvents undesirable side effects of Era depletion previously achieved by deleting the rnc locus (Britton et al., 1997; Britton et al., 2002) and produces a stable cell line for focused study. Secondly, Lon mediated depletion also avoids the previously reported pleiotropic effects rising due to a collateral depletion of RNase III, an enzyme essential for processing of rRNA, tRNA and some mRNAs in E. coli (Nikolaev et al., 1974; Régnier and Grunberg-Manago, 1989, 1990).
Although currently it is difficult to comment on the exact stage of cellular arrest achieved here because of the unresolved nature of cell cycle regulation in bacteria (Zheng et al., 2016). The microscopic examination hints towards a mixed population which consists of severely elongated as well as cells in two cell stage (Fig. 4.2) indicating disruption of the cell division mechanisms. Further, the phenotypic characterization (Fig. 4.2) and translation kinetics (Fig. 4.5) of Era deficient cells reinforced its role in translation regulation, ribosome biogenesis as well as cell cycle progression, which are in line with the previous reports(Britton et al., 1997; Britton et al., 2002; Lerner and Inouye, 1991). However, the translation arrest observed despite a significant 70S population is indeed intriguing and calls for structural characterisation of the 70S particles accumulating upon Era depletion. It is possible that these
particles contain structural defects and thus are unable to engage in protein synthesis.
Alternatively, it is also plausible that, due to the extensive regulatory mechanisms that govern translation under biotic or abiotic stress, a pool of 70S particles are sequestered from protein synthesis. Such ribosomes would also be recalcitrant to ribosome degradation pathways and thus can be employed for restoring protein synthesis when stress is relieved. A tantalizing fate of such ribosomes could be to drive protein synthesis during the rise of suppressor mutants that were ultimately observed in Era depleted cells.
Here, our initial efforts have utilized an arabinose operated genetic circuit but greater control of lon expression can be achieved using other inducers like rhamnose, which confer greater linearity between inducer concentration and expressed protein (Giacalone et al., 2006;
Kelly et al., 2018). In conclusion, this chapter marks the completion of the pipeline for RAF discovery and characterization. The methodology developed in this chapter complements the BioID and BiFC pipeline by creating conditional null mutants of essential RAFs.
Summary
A thorough characterization of RAFs involves studying ribosome assembly intermediates that accumulate in cells devoid of the respective RAFs. However, this analysis is not feasible for RAFs that are essential for survival of an organism. Hence, in this chapter we have attempted to address this problem by developing a methodology to specifically degrade essential RAFs using a Lon protease. We demonstrate regulatable degradation of Era to achieve a stable population of Era deficient cells. These cells displayed a severe slowdown in growth and protein synthesis further highlighting its role in cell cycle control and regulation of protein synthesis. This Lon mediated depletion of essential RAFs can complement the BioID and BiFC mediated characterization of RAFs in bacteria.
Chapter V
Characterization of ribosome quality control mechanisms in bacteria
The work embodied in this chapter is under preparation for publication.