Fluorescence bimolecular complementation enables facile detection of ribosome assembly defects in Escherichia coli

ABSTRACT

Assembly factors promote the otherwise non-spontaneous maturation of ribosome under physiological conditions inside the cell. Systematic identification and characterization of candidate assembly factors are fraught with bottlenecks due to lack of facile assay system to capture assembly defects. Here, we show that bimolecular fluorescence complementation (BiFC) allows detection of assembly defects that are induced by the loss of assembly factors. The fusion of N and C-terminal fragments of Venus fluorescent protein to the ribosomal proteins uS13 and uL5, respectively, in Escherichia coli facilitated the incorporation of the tagged uS13 and uL5 onto the respective ribosomal subunits. When the ribosomal subunits associated to form the 70S particle, the complementary fragments of Venus were brought into proximity and rendered the Venus fluorescent. Assembly defects that inhibit the subunits association were provoked by either the loss of the known assembly factors such as RsgA and SrmB or the presence of small molecule inhibitors of ribosome maturation such as Lamotrigine and several ribosome-targeting antibiotics and these showed abrogation of the fluorescence complementation. This suggests that BiFC can be employed as a surrogate measure to detect ribosome assembly defects proficiently by circumventing the otherwise cumbersome procedures. BiFC thus offers a facile platform not only for systematic screening to validate potential assembly factors but also to discover novel small molecule inhibitors of ribosome assembly toward mapping the complex assembly landscape of ribosome.

Introduction

In the last decade, crystal structures of ribosome rationalized decades of arduous research to understand the mechanism of protein synthesis.^1-3 However, given the pivotal role played by ribosome, much remains to be understood how this massive macromolecular machine is assembled from its constituent RNA and protein complements with such exquisite precision.^4,5 While the ribosome can be reconstituted in vitro without the involvement of accessory factors, it requires non-physiological conditions such as high salt concentration, elevated temperature and long duration of incubation.^6,7 Given that Escherichia coli divide in about 20 minutes implies that in order to stimulate the assembly process under physiological conditions inside the cell, non-ribosomal factors referred as assembly factors play a prominent role. These assembly factors are shown to transiently associate with the premature subunits and facilitate and organize the seemingly interrelated events ranging from RNA processing and folding to coordinated binding of ribosomal proteins in order to efficiently drive the maturation. Given the requirement for complex roles, it is not surprising to note that diverse classes of proteins ranging from GTPases, Helicases, Chaperones, Ribonucleases, modification enzymes viz., Methyltransferases and Pseudouridinylases and other RNA binding proteins participate in ribosome maturation.^4,8-10 Their vigilant monitoring of the ribosome maturation preclude the entry of premature subunits into the translation and secures the fidelity of the translational machinery.¹¹ It is therefore, the loss of assembly factors ensue premature subunits that are inept to associate to form functional ribosomes competent for protein synthesis and this state is captured as altered ribosome profile in the density gradient fractionation.

Despite the test tube reconstitution of ribosomes several decades ago in E. coli,^6,7 we are still far from achieving a comprehensive mechanistic understanding of the involvement and roles of assembly factors and the events leading to the maturation of ribosomes even in simple bacterial system.⁴ While there are about 200 assembly factors¹² that appear to be involved in the eukaryotes, less than 50 such factors were reported in bacteria.⁴ Since many assembly factors exhibit pleiotropic phenotype in bacteria,¹³ the functional characterization becomes challenging. Therefore, it appears that hitherto the identification of assembly factors is majorly fortuitous and sporadic. This has a potential setback of overlooking a bonafide candidate assembly factor. Therefore, in order to map the complex assembly landscape of bacterial ribosomes, systematic identification of assembly factors is needed and these efforts are fraught with lack of facile tools to detect assembly defects. Cold sensitive phenotype coupled with an altered ribosome profile in density gradient fractionation has been considered as a symptom to identify the involvement of candidate assembly factors in ribosome maturation.¹⁴ The profile of ribosomal subunits is obtained by employing laborious and time consuming sucrose density gradient ultracentrifugation and subsequent fractionation of ribosomal subunits.¹⁵ Therefore, this approach though robust and widely employed doesn't lend itself for scalability to identify potential assembly factors. Here, we have developed and deployed bimolecular fluorescence complementation (BiFC) as a facile tool with medium throughput to capture assembly defect in E. coli. The potential utility of this tool is validated by capturing the assembly defects induced by the loss of known assembly factors, viz., RsgA and SrmB in E. coli.

Results

Design of a BiFC system to monitor ribosome assembly in E. coli

The involvement of an assembly factor in ribosome assembly is typically probed whether its deficiency ensues an altered ribosome profile in sedimentation analysis along with cold sensitive growth phenotype.¹⁴ An alternative facile assay system that offers scope in scalability for systematic screening of candidate assembly factors is desirable to probe the comprehensive roles of assembly factors during the maturation. In this direction, we exploited the split-Venus fluorescence complementation^16,17 to design the assay system. Here, the fluorescent Venus protein is split into two complementary parts – the N (amino acids 1-175) and C-terminal (amino acids 155-238) fragments. When these fragments are brought together by intermolecular interactions, they associate to form the full complement that gives rise to fluorescence.

In order to adopt this concept for our assay system, from each subunit of the ribosome, one ribosomal protein (r-protein) was chosen based on its location at the interface of the 30S and 50S subunits as well as its accessibility to tagging with Venus (Fig. 1A). uS13 and uL5 conformed to such requirements and these r-proteins were fused to N (V_N) and C (V_C) terminal fragments of the Venus, respectively. We reasoned that when the r-proteins assemble onto the ribosome, the two complementary Venus fragments might come into close spatial proximity such that the fluorescence is restored. These Venus tagged r-proteins were integrated into two different loci in E. coli genome by a one-step process for cloning and integrating genes into bacterial attB sites referred as clonetegration¹⁸ and were placed under an arabinose inducible promoter for tunable as well as tight expression (vide. Fig. 1B and methods). E. coli K-12 MG1655 was used as a parent strain for all genome manipulation experiments and we refer to this strain as wild type (WT) for all comparative analyses with respect to growth phenotype and BiFC. We refer the modified strain as SLV_NC when both the Venus complements are tagged to uS13 and uL5, respectively and SV_N or LV_C when one of the complements is tagged to either uS13 or uL5 (vide. Fig. 2A for the nomenclature).

Figure 1.

BiFC scheme to monitor ribosome assembly in E. coli (A) 30S and 50S subunits are shown as ribbons in green and cyan, respectively. uS13 and uL5 are depicted on the ribosome as red and yellow ribbons, respectively. The Split-Venus fragments V_N and V_C are shown as half complementary stars in blue and magenta. uS13 from 30S and uL5 from 50S subunits (the proposed new nomenclature for r-proteins⁴⁰ is followed) were chosen for their location at the interface of the subunits and accessibility of their N and C termini for tagging with Venus fragments. This figure was rendered using UCSF Chimera.⁴¹ (B) Split-Venus fused uS13 and uL5 were cloned and integrated into the P21 and 186 phage integrations sites, respectively, in E. coli. This places the expression of these constructs under arabinose inducible promoter (pBAD). The gene regulatory regions of the integrated cassettes are indicated.

Figure 2.

Ribosomes isolated from BiFC competent strain are fluorescent. (A) Nomenclature for various strains created using different combinations of split-Venus, uS13 and uL5 to establish the corresponding BiFC system is shown. (B) A growth comparison between the WT and SLV_NC strains is shown. No difference in the growth was observed for WT and SLV_NC at 37°C. (C) Immunoblotting to confirm the expression and integration of Venus tagged uS13 and uL5 onto ribosomes is shown. These were probed with anti-GFP antibody. The upper and lower bound range of the protein marker with respect to Venus tagged uS13 (31 kDa) and uL5 (34 kDa) is shown. Lanes corresponding to ribosomes from WT and SLV_NC are indicated. The ratio of fraction of Venus tagged ribosomal proteins uS13 and uL5 (denoted as T) to Venus untagged uS13 and uL5 (denoted as U) is indicated at the bottom of each lane. (D) Fluorescence emissions from cell lysates of SV_N, LV_C, V_NC, SV_NC and LV_NC strains relative to lysate from SLV_NC strain were measured (vide. text). Cell lysates from V_NC, SV_NC and LV_NC showed comparable or slightly higher fluorescence with respect to SLV_NC lysate. (E) Fluorescence complementation is shown for crude ribosomes purified from WT, SLV_NC and V_NC strains. Lysate from V_NC and SLV_NC showed comparable fluorescence (inset). However, crude ribosomes purified from SLV_NC alone were intrinsically fluorescent. (F) Ribosome profiles for WT and SLV_NC strains are shown. The peaks pertaining to 30S, 50S and 70S are indicated. The BiFC measured from the corresponding fractions is shown for SLV_NC. The BiFC profile is in agreement with the sedimentation profile of the ribosomes. (G) In order to dissociate the subunits, crude ribosomes were treated with EDTA, RNase A and buffer containing 1mM Mg²⁺. The fluorescence intensity normalized with respect to untreated ribosomes from SLV_NC is shown. Ribosomes from SLV_NC tend to lose the ability to fluoresce upon destabilization or separation of subunits. Since ribosomes from LV_NC are not intrinsically fluorescent, they showed inert response against the treatments.

BiFC signals the maturation of ribosomes

Having uS13 and uL5 tagged with Venus, we intended to assess whether the presence of tag would alter the growth of SLV_NC with respect to WT. We observed no apparent cell growth defect for SLV_NC under the given experimental conditions suggesting that the tag doesn't impair the growth (Fig. 2B). Following this, SLV_NC cells were screened for the expression of Venus tagged uS13 and uL5 by RT-PCR and mass spectrometry (Fig. S2 & S3). After confirming the expression by RT-PCR, we also tested for the integration of the Venus tagged uS13 and uL5 onto ribosomes from SLV_NC cells by immunoblotting (Fig. 2C). It turns out that about 25% of the ribosomes harbor Venus tagged uS13 and uL5. Upon confirming the expression and integration, we proceeded to test whether the fluorescence complementation would occur in strains other than SLV_NC. We quantified the relative fluorescence (rf) that is the ratio of fluorescence from cell lysate of Venus tagged variant to fluorescence from cell lysate of SLV_NC (Fig. 2D). It turned out that though lysates from SV_N and LV_C emitted low background compared to SLV_NC(rf < 1), other variants such as V_NC, SV_NC and LV_NC were fluorescing as good as SLV_NC(rf ≥ 1). This suggests that even in the absence of uS13 or uL5, the association between V_N and V_C fragments occurs in location other than the ribosome leading to fluorescence complementation. This in turn leads to high background signal if one has to assess the complementation directly from the cell lysate.

Since we were interested in monitoring the association of the Venus tagged ribosomal subunits alone, we reasoned that the background noise could be mitigated if the free Venus tagged uS13 and uL5 were segregated from the ribosome-bound counterparts. It is worth mentioning that in order to achieve this, it was just sufficient to purify crude ribosomes by clarifying the cell lysate on an isocratic sucrose cushion. Subsequent steps of density gradient centrifugation and fractionation of ribosomal subunits, as performed for ribosomal profile generation, were obviated (vide. Methods). After purifying the crude ribosomes from SLV_NC, we tested for the presence of fluorescence signal. Purified crude ribosomes from WT and V_NC were analyzed along with SLV_NC for the fluorescence signal (Fig. 2E). WT lacks the Venus tag, whereas no complementation is expected to take place on the ribosome for V_NC. Therefore, BiFC is not expected from these variants. Indeed, though fluorescence from SLV_NC lysates was comparable to that measured from V_NC lysates (vide. Fig. 2E inset), in the modified approach, no significant fluorescence was observed for crude ribosomes purified from WT and V_NC (Fig. 2E). However, crude ribosomes isolated from SLV_NC alone exhibited significant fluorescent signal (Fig. 2E). This suggests that the background noise can be largely mitigated for BiFC analysis even using crude ribosomes that can be rapidly purified from cell lysates.

Having reduced the background noise, we set out to ask whether the presence of tagged uS13 and uL5 alleles would impair the assembly. Therefore, we performed sedimentation analysis for the ribosomes prepared from the cell lysates of SLV_NC and WT under conditions that favor association of ribosomal subunits (10 mM Mg²⁺). For WT, we observed majorly 70S and polysomes with minor amounts of 30S and 50S indicating that the association between 30S and 50S is robust. The ribosome profile for SLV_NC was similar to that of WT suggesting that the assembly is not impaired by the presence of Venus tag on uS13 and uL5 (Fig. 2F). Further, the BiFC measured from the fractionated crude ribosomes correlated well with the sedimentation profile displaying significantly high fluorescence signal for fractions corresponding to 70S and polysomes than those related to 30S and 50S. This suggests that BiFC indeed captures the association of the subunits (Fig. 2F). It is worthy to note that when the sedimentation analysis of the ribosomes was performed by loading the cell lysates directly onto the sucrose gradient, noticeable fluorescence corresponding to 30S and 50S fractions was observed (data not shown). This might have stemmed from the co-sedimentation of trace amounts of free Venus-tagged uS13 and uL5 along with the 30S and 50S subunits. In line with our earlier observation, however, when we separately purify crude ribosomes and then subjected them to sedimentation analysis, this background noise is largely alleviated (Fig. 2F). This sedimentation analysis further illuminates that free premature subunits do not contribute to the fluorescence of the purified crude ribosomes. This suggests that BiFC from crude ribosomes can be considered as a surrogate measure for subunit association (vide. SLV_NC in Fig. 2E and Fig. 2G). In addition, we also created null mutant of rpsM that codes for the non-essential S13¹⁹ against the SLV_NC background (SLV_NC ΔrpsM). In this modified strain, the Venus tagged uS13 allele remains the only source for uS13 and that allowed us to assess whether the Venus tag on uS13 would impair the assembly. Based on the correspondence between the ribosome profile and the BiFC signal, we confirmed that the Venus tag doesn't alter the assembly (Fig. S4). Similar such efforts to create null mutant of rplE that codes for the essential L5 ^20,21 didn't fructify despite several meticulous attempts. Therefore, it wasn't possible to assess the effect of tag against the SLV_NC ΔrplE background. However, based on SLV_NC and SLV_NC ΔrpsM, we believe that it is unlikely that the tag on uL5 will impact the assembly significantly.

In order to further reinforce that the BiFC signal from the purified crude ribosomes is due to the association of 30S and 50S, we destabilized the subunit association by the addition of EDTA and RNase A. This led to the reduction in the fluorescence intensity significantly (Fig. 2G). EDTA chelates the Mg²⁺ ions essential for subunit association and RNase A compromises the integrity of rRNA – both leading to the dissociation of 30S and 50S to varying extent – that was proficiently captured by the fluorescence complementation (Fig. 2G). Similar test was performed under low Mg²⁺ concentration that favors the dissociation of subunits. Here, the crude ribosomes were first purified and dialyzed against buffer containing 1 mM Mg²⁺. This showed reduction in the BiFC signal (Fig. 2G). This suggests that indeed BiFC is highly sensitive and correlates well with the dissociation of the subunits.

BiFC detects maturation defects that are prompted by the loss of assembly factors

Encouraged by the ability of the BiFC system to monitor the state of assembly, we proceeded to assess this in case of assembly defects due to the loss of known assembly factors. RsgA is a circularly permuted GTPase that is known to be involved in the assembly of small subunit (30S) in E. coli. The loss of RsgA produces slow growth and severe assembly defect that is characterized by the accumulation of premature 30S and concomitant reduction in the 70S particles.^22-25 Similarly, the loss of SrmB – a DEAD box helicase – is shown to produce a 50S assembly defect in E. coli.^26-28 We chose these factors as they represent different class of proteins – a GTPase and another a helicase – that are reasonably well characterized and shown to participate in the maturation of different subunits (30S or 50S). We generated null mutants of rsgA and srmB against the genetic background of SLV_NC. The fluorescence complementation from these mutants was assessed relative to that of SLV_NC. This showed that compared to crude ribosomes purified from SLV_NC, those from SLV_NC ΔrsgA and SLV_NC ΔsrmB strains showed drastic reduction in fluorescence complementation (less than 50% that of SLV_NC) that was more pronounced at 20°C (Fig. 3A). In line with this, SLV_NC ΔrsgA strain showed slow growth phenotype at 37°C that was further exacerbated at 20°C reinforcing the essential requirement of RsgA especially at low temperature (Fig. 3B). In order to further investigate the nature of these defects, sedimentation analysis was also performed for ribosomes from SLV_NC ΔrsgA and SLV_NC ΔsrmB strains (Fig. 3C and 3D). The ribosome profile generated from SLV_NC ΔrsgA recapitulated the earlier observation, which showed the accumulation of 30S at the expense of 70S²² (Fig. 3D). In tune with this, the BiFC profile that was generated from the corresponding fractions from the sedimentation analysis, mirrored this altered ribosome profile with drastically reduced fluorescence signal for 70S relative to SLV_NC highlighting the assembly defect (Fig. 3D). In contrast to SLV_NC ΔrsgA, despite the reduction in BiFC, the SLV_NC ΔsrmB exhibited minor growth defect both at 37°C and 20°C, which is consistent with the previous report ²⁹ (Fig. 3B). The ribosome profile generated for this strain grown at 37°C showed noticeable amounts of 40S whose appearance represents the hallmark of ΔsrmB indicating the 50S assembly defect ^26,29 (Fig. 3C). We measured BiFC for the fractionated ribosomes from this strain that showed drastically reduced fluorescence consistent with the altered ribosome profile (Fig. 3C). Strikingly, the fractions corresponding to 70S from SLV_NC ΔsrmB displayed significantly reduced fluorescence than those from SLV_NC ΔrsgA suggesting that the cause for the defect in subunit association differs between the 2 (vide. Discussion).

Figure 3.

BiFC detects assembly defects that are induced by the loss of assembly factors. (A) Relative fluorescence complementation is shown for purified ribosomes from SLV_NC ΔsrmB and SLV_NC ΔrsgA strains that were grown at 20°C and 37°C. Drastic differences in the fluorescence complementation profiles were observed at 20°C for SLV_NC ΔsrmB and SLV_NC ΔrsgA strains. (B) Growth profiles of SLV_NC ΔsrmB and SLV_NC ΔrsgA strains are shown at permissible (37°C) and non-permissible temperatures (20°C). Both SLV_NC ΔsrmB and SLV_NC ΔrsgA strains exhibited growth defect that was more prominent at 20°C. (C) and (D) Ribosome profiles for SLV_NC ΔsrmB and SLV_NC ΔrsgA cells are shown. The peaks corresponding to 30S, 40S, 50S and 70S are indicated. BiFC measured for the corresponding fractions from sedimentation analysis is also presented. (E) Null mutants complemented with respective assembly factors by exogenous expression depicted significant restoration of BiFC for the purified ribosomes at 20°C. BiFC for gene complementation is shown relative to that of SLV_NC carrying the respective plasmids and the error bars depict the standard deviation. The empty vector is depicted as p8R and the vector harboring the respective assembly factor is indicated. (F) The stress on growth due to deletion of assembly factors at 20°C was relieved by expression of the assembly factors from an inducible vector. The empty vector is depicted as p8R and the vector harbouring the respective assembly factor is indicated. The complementation experiment was conducted against wild type and deletion background of the respective assembly factor in SLV_NC.

To further reaffirm the notion that the reduced fluorescence complementation in SLV_NC ΔrsgA and SLV_NC ΔsrmB strains was due to the assembly defect that was induced by the loss of RsgA and SrmB, respectively, we exogenously complemented their expression at 20°C. The fluorescence complementation from these ribosomes was assessed relative to that of SLV_NC. This showed that the restoration of fluorescence complementation corresponds to 30% for SrmB and 60% for RsgA (Fig. 3E). In tune with this, the slow growth defect was relieved by exogenous expression of rsgA and srmB (Fig. 3F). Intriguingly, we observed in the gene complementation that though the restoration of growth defect is comparable to the wild type, the restoration of BiFC, especially for SLV_NC ΔsrmB, is not appreciable (Fig. 3E and 3F). This may be attributed to the fact that the exogenous dosage in the gene complementation experiments lacks the regulatory mechanism the corresponding chromosomal counterpart is subjected to under varying environmental cues. This is likely to impact the temporal expression of srmB in SLV_NC ΔsrmB compared to the wild type. Secondly, the over-expression of rsgA and srmB from multi-copy plasmid as against the single copy in native chromosomal locus might have some undesirable effects on the assembly that could lead to low fluorescence. It is possible that these factors could exert negligible effect on RsgA compared to SrmB giving rise to better restoration of BiFC for RsgA. Given this, it is indeed encouraging to note that BiFC can highlight such subtle differences in the roles of the assembly factors.

BiFC highlights assembly defects that are provoked by chemical perturbation too

Apart from the loss of assembly factors, a few small molecule inhibitors are shown to induce assembly defects. Therefore, we intended to assess the feasibility of deploying BiFC system for screening small molecule inhibitors of ribosome maturation. Toward this, we utilized Lamotrigine, an anticonvulsant drug that is recently shown to reversibly induce assembly defects especially at low temperatures in bacteria.¹⁴ Crude ribosomes purified from Lamotrigine treated SLV_NC exhibited low fluorescence complementation with respect to that of untreated SLV_NC at 37°C (Fig. 4A). However, those grown at 20°C showed drastic reduction in BiFC suggesting that the Lamotrigine induced assembly defect is indeed proficiently captured by BiFC (Fig. 4A). In line with the maturation defect at 20°C, Lamotrigine treated cells exhibited slow growth phenotype at 20°C than at 37°C (Fig. 4B). These observations are in consonance with earlier report.¹⁴ Encouraged by this, we proceeded to probe whether BiFC would detect antibiotics induced assembly defects too. Antibiotics viz., Chloramphenicol, Kanamycin, Neomycin, Erythromycin and Tetracycline target the protein synthesising machinery and render it inactive. This leads to the cascading effect of diminishing the production of r-proteins too whose dosage insufficiency becomes rate limiting thus provoking the ribosome assembly defects indirectly.^30,31 On the contrary, Ampicillin, which is an inhibitor of transpeptidases prevents cell wall synthesis and therefore anticipated to not induce any ribosome assembly defects. We assessed BiFC from crude ribosomes purified from antibiotics treated SLV_NC relative to that of untreated SLV_NC. Indeed, the ribosomal antibiotics drastically reduced BiFC at 20°C than at 37°C; however, Ampicillin remained neutral to BiFC both at 20°C and 37°C (Fig. 4C and 4D). This underscores that BiFC is specific for monitoring the assembly defect induced by ribosomal antibiotics. This further suggests that though BiFC highlights the deficiency in the ribosome maturation, it doesn't deconvolute whether the defect is due to error in the assembly process or indirectly caused by error in protein synthesis. This is a conceptual limitation wherein the role of BiFC is restricted to just as a “read-out” of ribosome assembly defect – like the density gradient sedimentation analysis – and it lacks the scope to uncover the underlying causal relationships. Resolving this necessitates further analyses as shown in earlier studies.¹⁴ Overall, the applicability of BiFC seems to be scalable for medium throughput platforms for screening chemical inhibitors that target the ribosome assembly either directly or indirectly and has the potential to uncover new classes of assembly inhibitors and antibiotics.

Figure 4.

BiFC displays assembly defects that are provoked by chemical inhibitors and antibiotics. (A) Fluorescence complementation profile of SLV_NC cells treated with Lamotrigine at 37°C and 20°C is presented. Major deviation in the levels of fluorescence complementation was observed at 20°C than at 37°C. BiFC for ribosomes from DMSO treated cells is shown as mock experiment. BiFC is shown relative to that of untreated SLV_NC. (B) The effect of Lamotrigine on growth of SLV_NC was also monitored at 37°C and 20°C. In line with the BiFC profile, the growth defect was more pronounced at 20°C. (C) and (D) The effect of Chloramphenicol, Kanamycin, Neomycin, Erythromycin, Tetracycline and Ampicillin on the fluorescence complementation profile at sub lethal concentrations is shown at 37°C and 20°C. BiFC is shown relative to that of untreated SLV_NC. BiFC highlights that the defects are more pronounced at 20°C for ribosome targeting antibiotics.

Discussion

Exploiting the modular nature of fluorophore maturation in Venus, we have presented an ingenious approach to track the maturation of ribosomes in E. coli. Our earlier attempts to detect BiFC directly from cell lysates didn't fructify due to the presence of background noise. This arises owing to the intrinsic limitation of segregating the free Venus tagged uS13 and uL5 from the ribosome bound counterparts. Despite this, in our modified approach, in order to detect BiFC it is sufficient to purify the crude ribosomes alone that can be accomplished rather quickly. We have further demonstrated that BiFC emanating from crude ribosomes has negligible contributions from the premature subunits (Fig. 2F, 3C and 3D). This suggests that subsequent long stages of fractionation of the ribosomal subunits under the density gradient centrifugation to generate the profile for assessing the maturation can be circumvented. This in turn minimizes the considerable time and effort to detect the assembly defect. At this point, though the requirement to purify the crude ribosomes limits the scalability to be medium throughput, we anticipate that further investigation to alleviate this background can potentially scale-up the application.

Though Venus is a stable fluorescent protein with high quantum yield, it has been reported that the presence of high concentration of chloride and nitrate ions at mildly acidic (pH 6) condition reduce the fluorescence drastically.³² Further, it was observed for YFP that the maturation of complements for BiFC is impacted around pH 7.2.³³ In our study, we observed that pH 7.4 and 60 mM NH₄Cl seem to be suitable for the fluorophore maturation and therefore we suggest that the pH range of 7.4-8.0 may be optimal for detection of ribosome assembly defect using BiFC.

Our choice of tagging uS13 and uL5 was based on their location at the interface of the ribosomal subunits. In our diverse validation experiments such as the loss of assembly factors RsgA and SrmB as well as the presence of chemical inhibitors such as Lamotrigine and antibiotics – that target the ribosome at different sites – induce assembly defects that are proficiently captured by BiFC. Nevertheless, it raises a remote possibility wherein the defects induced by hitherto unidentified assembly factors that have no effect on subunit association, may go unnoticed. However, it appears that the quality control mechanism ensures that only the mature subunits are allowed to interact and enter the translational cycle while the premature subunits are rapidly cleared from the cell.^11,34,35 In such a scenario, only the mature 30S and 50S subunits can be stable long enough to associate and turn on BiFC. Further, BiFC is highly sensitive to the spatial proximity of the chromophore complements and therefore any mild assembly defect that impacts the orientation of uS13 and uL5, despite their presence on the ribosome, can render the BiFC inactive. For example, in the case of SLV_NC ΔsrmB, we noticed that though the population of 70S is not altered as deduced from the ribosome profile, the BiFC corresponding to these fractions is significantly low (Fig. 3C). We speculate that such possibility could arise if the positioning of uS13 and uL5 is distorted in those 70S particles owing to some minor assembly defect of the subunits that seems to be tolerated with no major perturbation to growth rate. Therefore, it is highly likely that only the perfect positioning of uS13 and uL5 on the mature subunits are competent to facilitate the spatial proximity of the split-Venus to turn on BiFC with high efficiency. On the other hand, the reduction in BiFC for 70S in SLV_NC ΔrsgA is concomitant with the reduction seen in ribosomal profile (Fig. 3D). This suggests that the 70S population accrued in SLV_NC ΔrsgA harbours ribosomal subunits wherein the spatial proximity between uS13 and uL5 remains unperturbed. Since RsgA and SrmB participate in the maturation of different subunits (i.e., 30S and 50S, respectively), their mode of action is expected to differ as well. In such scenarios, it is highly encouraging to note that BiFC could capture those subtle defects that may go unnoticed in sedimentation analysis. This further suggests that in conjunction with the conventional sucrose gradient sedimentation analysis, BiFC could bring forth new insights on the roles of the assembly factors. Finally, the choice of uS13 and uL5 is not sacrosanct; it is possible that in lieu of uS13 and uL5, another r-protein pair that is located at a different region can be chosen based on their proximity on the ribosome and assayed for their ability to activate BiFC.

Earlier attempts to capture assembly defects in E. coli based on fluorescence relied on dual color fluorescence. This necessitated that the ratio of the intensity of both colors is characterized which is further subjected to variability under different experimental conditions.^31,36 In our design, this is circumvented by adopting BiFC as a surrogate measure to monitor the state of the ribosome assembly. Since the fluorophore maturation in Venus – owing to the requirement of spatial proximity of the complementary parts – takes place on the fully assembled ribosomes, BiFC analysis based on purified crude ribosomes alleviates false positives to a large extent without compromising the sensitivity. This work shows that in most instances, the BiFC “OFF” state signals the abrogation of assembly while the BiFC “ON” state indicates the proficient assembly. This simplicity enables one to assess the status of the ribosome maturation with ease and precision based on the binary state of BiFC – “ON” or “OFF.” Thus, this facile assay system can be readily adopted for systematic screening to probe the involvement of candidate assembly factors and small molecule effectors of ribosome maturation both in a quantitative and qualitative framework.

Materials and methods

Construction of bacterial strains and plasmids

The E. coli K-12 MG1655 (WT) was used as a parent strain for all genome manipulation experiments (vide. Supplementary Table S1). Genes encoding uS13 and uL5 were amplified from WT cells; araC-ara regulatory regions were amplified from vector 8R; V_N(amino acids 1-176) and V_C(amino acids 155-238) coding regions were amplified from vector pET21b-RL027A with primers containing 25-30 bases overlap between the respective constructs. Overlap extension PCR was performed to create different combinations of V_N-uS13 and V_C-uL5 fusion cassettes. V_N-uS13 fusion cassette was cloned into pOSIP-CT using Kpn1/Xho1 sites (New England Biolabs) and V_C-uL5 fusion cassette was cloned into pOSIP-KO using Kpn1/Pst1 sites (New England Biolabs). The cassettes were integrated into the genome of WT strain by a one-step process for cloning and integrating genes into bacterial attB sites referred as “clonetegration” as described elsewhere.¹⁸ After selection against the respective antibiotics, the cells were PCR screened for the integration of the given construct using gene specific primers (vide. Fig. S1).

SLV_NC strain was used as a parent strain to create null mutants of srmB, rsgA and rpsM using lambda red recombination method.³⁷ A Tetracycline cassette amplified from E. coli TKC cells with 50 bases flanks for respective genes was used to create the null mutants of SLV_NC ΔsrmB: tetA and SLV_NC ΔrsgA: tetA (vide. Fig. S5). Similarly, in order to create SLV_NC ΔrpsM: cat, the Chloramphenicol cassette was amplified from DH10B ΔrpsM pCDSSara-S13 strain (vide. Supplementary Table S1). Genes encoding SrmB and RsgA were amplified from WT cells and cloned into vector 8R using ligation independent cloning to create vectors psrmB and prsgA. Plasmid psrmB was used to transform SLV_NC and SLV_NC ΔsrmB strains and plasmid prsgA-8R was used to transform SLV_NC and SLV_NC ΔrsgA strains by chemical transformation method. All constructs were verified by sequencing.

Culture growth conditions and growth curve measurement

SLV_NC strain or variants were maintained and propagated in LB medium, supplemented with respective antibiotics: Chloramphenicol (15 µg/ml), Kanamycin (15 µg/ml), Tetracycline (10 µg/ml) and Ampicillin (100 µg/ml). All growth related studies were done in experimental replicates. Overnight grown cultures were diluted 100 times with fresh LB medium with or without respective antibiotics followed by vigorous shaking at permissible (37°C) or non-permissible (20°C) temperatures. Aliquots of 200 µL each were drawn at regular intervals and OD₆₀₀ measurements were taken in Tecan M200 micro plate reader (Tecan instruments, Mannedorf, Switzerland) in 96 well transparent flat bottom plates.

RT-PCR for confirming gene expression

Strain SLV_NC was grown in presence of 2 mM arabinose till 0D₆₀₀ reaches 0.8 and mixed with equal volume of RNAlater (Sigma-Aldrich) for 5 minutes followed by centrifugation to recover the cells. RNA from recovered cells was isolated by successive extractions using acid phenol (pH 4.5)-chloroform followed by ethanol precipitation. The extracted RNA was treated with RNase free DNase for 2 hours at 37°C followed by ethanol precipitation. Subsequently, it was resupended in RNase free water. Reverse transcription (RT) reaction was performed using SuperScript® III Reverse Transcriptase (Invitrogen) according to the manufacturer's specifications. PCR was performed using the RT reaction product as template with forward and reverse primers specific to the corresponding Venus fused uS13 or uL5.

Antibiotic and Lamotrigine treatment

Overnight grown SLV_NC strain was diluted into fresh LB medium supplemented with 2 mM arabinose and was allowed to grow at 37°C and 20°C till OD₆₀₀ reached 0.2. Subsequently, sub lethal concentrations of respective antibiotics were added and the cells were grown at 37°C or 20°C till OD₆₀₀ reached 0.6-0.8. Chloramphenicol (7µg/ml), Erythromycin (100µg/ml) and Tetracycline (7µg/ml) were obtained from Sigma-Aldrich, St. Louis, USA and Ampicillin (15µg/ml), Kanamycin (7µg/ml) and Neomycin (7µg/ml) were obtained from Himedia Laboratories, Mumbai, India. After the growth, cells were harvested for fluorescence complementation studies. In order to administer Lamotrigine (Sigma-Aldrich) treatment, cells were grown in fresh LB medium supplemented with 2 mM arabinose and 10 µM Lamotrigine (dissolved in DMSO) at respective temperatures till OD₆₀₀ reached 0.6-0.8.

SDS-PAGE for purified ribosomes

The concentrations of the ribosomes purified from SLV_NC, V_NC and WT were normalized based on absorbance at 280 nm. The proteins were separated on a 10% polyacrylamide gel by SDS-PAGE in Bio-Rad Protean II xi Electrophoresis apparatus followed by staining with Coomassie brilliant blue (vide. Fig. S3). The identity of the ribosomal proteins was analyzed using mass spectrometry.

Immunoblotting

Equal amounts of purified ribosomes from WT, SLV_NC or SLV_NC ΔrpsM cells were fractionated by SDS-PAGE and transferred to a Polyvinylidene difluoride (PVDF) membrane using a transfer apparatus according to the manufacturer's protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 130 mM, NaCl, 0.1% Tween 20) for 60 min at 4°C, the membrane was washed thrice (10 minutes each) with TBST and incubated with antibodies against GFP (Rabbit Anti-GFP antibody, Biobharti Lifesciences, Kolkata) according to the manufacturers recommendations. Membranes were washed 3 times for 10 min and incubated with a 1:10,000 dilution of horseradish peroxidase conjugated Goat anti-rabbit antibody (Merck life sciences) for 1 hour at room temperature. The membrane was then washed thrice with excess of TBST for 10 minutes each and developed with the Clarity™ Western ECL Blotting Substrate (Bio-Rad) according to the manufacturer's recommendations.

Densitometry to calculate the ratio of Venus tagged ribosomal proteins to that of the untagged ribosomal proteins was done using ImageJ software.³⁸ Calculation of pixel intensities for tagged ribosomal proteins in WT and SLV_NC was done using immunoblots while the intensities for untagged ribosomal proteins were estimated using Coomassie brilliant blue (CBB) stained gel. These were compared against standard plot generated for immunoblots and CBB stained gels to estimate the total amount of Venus tagged or untagged ribosomal proteins. The fraction of the tagged and untagged ribosomal proteins was calculated based on the total protein content loaded in each lane.

Isolation of crude ribosomes

Overnight grown cultures were diluted 100 times with fresh LB medium with or without the addition of the respective antibiotic in presence of 2 mM arabinose. WT, SLV_NC or variant strains were grown at 37°C for permissible and 20°C for non-permissible temperatures with vigorous shaking. Lamotrigine treatment was administered by growing cells at 37°C or 20°C with or without 10 µM Lamotrigine or only DMSO. Cells were grown till OD₆₀₀ reached 0.8 and incubated on ice for 15 minutes to allow formation of “run-off” ribosomes. The following procedure was adopted to ensure preparation of compact and highly active tight-coupled ribosomes.¹⁵ The cells were harvested by centrifugation, washed and resuspended in 1 ml chilled buffer A (20 mM Tris–HCl (pH 7.4), 100 mM NH₄Cl, 10.5 mM Mg(CH₃COO)₂, 0.5 mM EDTA, and 6 mM 2-mercaptoethanol). Lysis was initiated by incubating cells with 0.2 mg/ml Lysozyme, 40 U RNase free DNase, 1 mM phenylmethanesulfonyl fluoride and RNaseOUT (Invitrogen) for 1 hour on ice followed by 5-8 cycles of freeze thaw lysis. The lysate was cleared by centrifugation at 30,000g for 30 minutes. The cleared lysate was loaded onto equal volume of buffer B containing 20 mM Tris–HCl (pH 7.4), 1 M NH₄Cl, 1.1 M sucrose, 10.5 mM Mg(CH₃COO)₂, 0.5 mM EDTA and 6 mM 2-mercaptoethanol and centrifuged at 220,000g for 80 min using TLN-100 or Type 70.1 Ti rotors on the Optima TLX or Optima LX-100 XP ultracentrifuge (Beckman coulter). The precipitated ribosomes were washed twice with buffer A and resuspended in 200 µL pellet resuspension buffer C (20 mM Tris–HCl, (pH 7.4), 60 mM NH₄Cl, 10.5 mM Mg (CH₃COO)₂, 0.5 mM EDTA, and 6 mM 2-mercaptoethanol) and used immediately for further analysis.

Ribosome profile analysis

Ribosome profile analysis was done as described previously³⁹ with minor modifications. Briefly, overnight grown cultures were diluted 100 times with fresh LB medium with or without the addition of the respective antibiotic in presence of 2 mM arabinose. Cells were grown till OD₆₀₀ reached 0.6-0.8. Following this, Chloramphenicol was added to a final concentration of 100 µg/ml and cells were allowed to grow for 15 minutes. Subsequently, the cells were chilled rapidly and harvested by centrifugation. Harvested cells were used for isolation of crude ribosomes as described above. Total ribosomes concentration was quantified by measuring the absorbance at 254 nm (A₂₅₄). To analyze the polysome profiles, crude ribosomes from different strains having A₂₅₄ = 100 were loaded onto a 10-50 % (w/v) linear sucrose gradient in buffer E (20 mM Tris–HCl, (pH 7.4), 100 mM NH₄Cl, 10.5 mM Mg (CH₃COO)₂, 0.5mM EDTA and 6 mM 2-mercaptoethanol) followed by centrifugation at 210,000 g for 2 hours in TLS-55 rotor on Optima TLX ultracentrifuge. Gradients were analyzed by manually separating fractions of 100 µL each followed by measurement of absorbance at 254 nm. The peaks corresponding to the respective subunits were ascertained by isolating RNAs from the fractions and analyzing them on a denaturing agarose gel. The separated fractions were also analyzed for fluorescence complementation wherever required.

Analysis of subunit dissociation by BiFC

Crude ribosomes were incubated with 50 mM EDTA or 10 mg/ml RNase A (Sigma-Aldrich) on ice for 2 hours. After the completion of the treatment, fluorescence complementation was analyzed from RNase A or EDTA treated and untreated ribosomes. In order to decouple the ribosomal subunits gently by employing low concentration of Mg²⁺, purified crude ribosomes were resuspended or diluted in buffer D (20 mM Tris–HCl, (pH 7.4), 60 mM NH₄Cl, 1 mM Mg (CH₃COO)₂, and 6 mM 2-mercaptoethanol) along with RNaseOUT (Invitrogen) and dialyzed for 4 hours and were later analyzed for fluorescence complementation.

Measurement of fluorescence intensities

Quantification of purified ribosomes was carried out by measuring absorbance at 260 nm as a direct indicator of ribosome concentration using GeneQuant 1300 spectrophotometer (GE Healthcare). In order to assess fluorescence complementation without a concentration artifact or any inner filter effect, the ribosome concentration was normalized to 55 nM for all fluorescence measurements. All fluorescence spectrums were generated by exciting at 514 nm and scanning for emission from 524-700 nm, with averaging over 3 scans after baseline correction in FluoroMax-4 spectrofluorometer (Horiba Scientific, Edison, NJ). The slit width used for excitation and emission was 2 nm and 5 nm, respectively. The measured fluorescence intensity was further corrected for background fluorescence that arises due to other cellular components. All fluorescence experiments were performed with atleast 3 independent trials and the plots are shown from one such representative experiment. Unless specified otherwise, the relative fluorescence indicates that the fluorescence intensity of the subject is normalized with respect to that of SLV_NC. Based on this, the relative fluorescence for SLV_NC is considered as 1.

In order to identify the optimal concentration of inducer for high sensitivity fluorescence measurement, we have recorded the intensity of emission maxima for crude ribosomes isolated from SLV_NC strain that was grown in the presence of 0.2, 2 and 4 mM of arabinose (vide. Fig. S6). It was found that arabinose concentration at 2 mM evoked optimal fluorescence response and therefore all the BiFC experiments were carried out at 2 mM arabinose concentration.

Disclosure of potential confllict of interest

No potential conflict of interest were disclosed.

Acknowledgment

Vector pET21b-RL027A was a kind gift from Sheref Mansy (Addgene plasmid # 42133), pBAD Strep TEV LIC cloning vector (8R) was a kind gift from Scott Gradia (Addgene plasmid # 37506), pOSIP- KO and pOSIP-CT (Addgene plasmid # 45985, 45981) were a kind gift from Drew Endy and Keith Shearwin. E. coli TKC cells were a kind gift from Donald L Court. DH10B ΔrpsM pCDSSara-S13 strain was a kind gift from Peter G. Schultz. We acknowledge the Mass Spectrometry facility at C-CAMP, Bangalore for their services. We acknowledge the ultracentrifugation facility at Guwahati Biotech Park. We thank Nikhilesh Dey and Sumit Kinger for the technical support and all members of MAB lab for their critical comments and suggestions.

Funding

This work was supported by grants from Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India [BT/341/NE/TBP/2012, BT/PR5511/MED/29/631/2012 and BT/406/NE/U-EXCEL/2013].