Discovery of the biogenesis of membrane vesicle in E. coli upon PHB production
Figure 1A shows the synthetic pathway for production of PHB in Escherichia coli. PHB is synthesized by sequential enzyme reactions of 3HB-CoA-precusor supplying steps catalyzed by β-ketothiolase (PhaARe) and NADPH-dependent acetoacetyl-CoA reductase (PhaBRe), and polymerizing step catalyzed by PHB synthase (PhaCRe). The finding of MV biogenesis in our microbial system has been motivated by the reproducible appearance of a lot of bubbles during the aerobic cultivation of the PHB-producing recombinant strain of E. coli. Namely, when the recombinant strain carrying the PHB synthetic pathway, BW/PHB (+) (BW25113 harboring pGEM-phaCReAB)6 was cultivated, we observed a lot of foam in the Sakaguchi flask (Fig. 1B). As shown in Table 1, the cellular content of PHB was 60 ± 4 wt% in the LB medium containing 2% glucose. On the other hand, we confirmed that this phenomenon was not observed for BW/PHB (−) harboring the inactivated-PHB synthase gene [phaCRe(C319A)]6 and the parent strain E. coli BW25113 (BW) with no plasmid (Fig. 1B). Therefore, the phenomenon was specific for PHB-producing strain. This prompted us to further investigate the foam by several analyses. As a result, it was suggested that the foam contains a lipid at least that was changed to be dark, when the sample was exposed to a fixation-liquid (osmium tetroxide) for transmission electron microscopic observation (unpublished data). Then, we carried out the microscopic observation of the culture samples containing BW/PHB (+) and BW at 48 h culture. As the result, several particles (approximately 100 nm scale) were budded on the surface of single cell of BW/PHB (+) using scanning electron microscopy (SEM) (Fig. 1C). Based on these morphological observations, we postulated that such a kind of budded particles would be a membrane vesicle (MV) referring the previous papers7.
The primary findings of MV biogenesis in E. coli producing PHB. (A) Schematic illustration of a metabolic pathway for production of PHB. Acetyl-CoA (Ac-CoA) supplied from glucose via glycolysis is converted into acetoacetyl-CoA (AcAc-CoA) through function of heterologously expressed PhaARe. AcAc-CoA is converted into 3-hydroxybutyrate-CoA (3HB-CoA) through function of PhaBRe. 3HB-CoA is utilized as a monomer for the polymerization by PhaCRe. When the inactive mutant PhaC(C319A) is replaced to PhaCRe, the production of PHB is not observed. (B) The foam observed during cultivation. (C) SEM images of the cell surface of three recombinant constructs: BW/PHB (+), BW/PHB (−) and BW. Black arrowheads denote MV-like particles budded from the cell surface. Scale bars represent 1 μm.
In the next, we attempted to isolate and purify the MV-like particles by using iodixanol density-gradient ultracentrifugation according to the following scheme8. After density-gradient ultracentrifugation, the particles-contained fraction was analyzed based on the staining by using a fluorescent FM1-43 FX (data not shown). Figure 2A shows the comparison in the quantity of particles-contained band in the centrifuge tubes (BW/PHB (+), BW/PHB (−) and BW). The extraction of MVs from the sample of BW/PHB (+) was also monitored based on fluorescent intensity of FM1-43 FX (Fig. 2B). Transmission electron microscopic (TEM) analysis also revealed the existence of MVs in the collected fraction (Fig. 3A). The TEM image shows the presence of a mixture of single-layered and multilayered MVs. Next, the diameter of MVs was measured by Nanosight tracking analysis, and the size distribution was plotted (Fig. 3B). The average particle size of MVs from the BW/PHB (+) cells was 93.2 ± 3.1 nm, consistent with that observed in the parent strain of E. coli BW25113, as seen in the previous study9,10. In addition, predicted outer membrane protein bands approximately at 37 kDa (OmpF/C, and OmpA) which provide an index of MV generation9, were detected by SDS-PAGE analysis (Fig. 3C). These results suggested that the BW/PHB (+) cells released MVs into the culture medium. Finally, we have concluded the biogenesis of MV.
Comparison in the amount of the MV-fractions isolated from culture supernatant of BW/PHB (+), BW/PHB (−) and BW. (A) MV purified by density gradient-ultracentrifugation. The black arrow indicates MV fraction for BW/PHB (+). (B) Comparison of MV production. The data are shown as the mean ± standard deviation from three independent experiments.
Microscopic and biochemical characterization of the MVs. (A) TEM images of MV fractions of BW/PHB (+). Scale bar represents 100 nm. (B) The particle size distribution was analyzed by NanoSight tracking analysis. (C) Protein profiles of MV preparations from BW/PHB (+), BW/PHB (−) and BW. Purified MV samples were analyzed by SDS-PAGE using 12.0% acrylamide gel and stained with CBB. Estimated proteins of the main bands are shown on the right. Lane M represents the marker.
The PHB intracellular accumulation-triggered MV biogenesis, PIA-MVP, is a complete new from both research fields, PHB and MV. So far, no one has recognized this unexpected PIA-MVP during cultivation of the recombinant E. coli producing PHB that so many researchers usually conduct. From the side of a long research history on MV biogenesis, PIA-MVP is a different principle and approach. In order to address the molecular mechanism of PIA-MVP, we have focused on spaciotemporal relationship of both supramolecules through morphological change of the cells as follows.
Time courses of cell growth, intercellular accumulation of PHB and MV release
The PIA-MVP has been demonstrated in the recombinant PHB-producing E. coli by several biochemical analyses, as described above. Then, to better understand the relationship of both supramolecules, we conducted the time courses for production of PHB and MV as well as cell growth, using the BW/PHB (+) during the cultivation times (up to 72 h). As shown in Fig. 4, the PHB production was associated with cell growth. The released MVs were quantified with a fluorescent FM1-43 FX according to a previously described method with minor modifications11. It seemed that MVs began to be released at a little bit of delay (24–48 h) after the PHB-accumulation was reached the saturation level. This suggests a tight relationship between MVs secretion and PHB-accumulation based on the fact that the release of MV was initiated in response to the PHB accumulation. This should be an important finding in considering the PIA-MVP established in this study.
Time course profiles of MV production, PHB production and cell growth by BW/PHB (+). BW/PHB (+) was cultivated in LB medium containing 2.0% glucose at 30 °C for 72 h.
Relationship between PHB accumulation level and MV production level
To further investigate the relationship between both supramolecules, we next quantitatively analyzed the intracellularly accumulated content of PHB and the amount of released MVs. First, we attempted to control the PHB accumulation level by varying the glucose concentration as a carbon source. Figure 5A shows a good glucose concentration-dependent production of PHB. It means that the PHB production level can be fine-tuned by controlling the glucose concentration. Also, the amount of the released MVs was in good proportion to the PHB production level as shown in Fig. 5B. Surprisingly, we obtained a linear relationship between PHB production level and MV production level with an extremely high correlation coefficient of R2 = 0.99625, as shown in Fig. 5C. Accordingly, it should be noted that the MV secretion can be fine-tuned by regulating the glucose concentration. This glucose-controllable approach would be very effective for investigating the mechanism PIA-MVP and wide-range of applications, as mentioned below.
Relationship between MV production and intracellular PHB contents. (A) PHB production and (B) MV production of BW/PHB (+) under five different glucose concentrations (0–2.0%) for 48 h cultivation. (C) The relationship between the PHB production level and MV production level. The MV production clearly increased with increasing intracellular PHB production. The data are shown as the mean ± standard deviation from three independent experiments.
Morphological change of the cells associated with PHB production
In the next, we investigated the cell itself probably affected by the enhancements in PHB production and MV secretion tightly associated with the concentration of added glucose. As shown in Fig. 6A, the cell volume was increased in a proportional manner while increasing the glucose concentrations ranging from 0 to 2%. The enlargement of single cells can be probably accounted for by the internal pressure caused by the intracellular accumulation of PHB. Such a glucose concentration dependency was not observed for PHB non-producing strain at all (Supplementary Fig. S1). Therefore, it can be concluded that glucose-dependent morphological change was specifically occurred in the BW/PHB (+).
Morphological change of the BW/PHB (+) cells by varying glucose concentration. Cells were cultivated under five different glucose concentrations (0% to 2.0%) at 30 °C for 48 h cultivation. (A) Microscopic images of the BW/PHB(+) cells. Scale bars, 5 μm. (B) Flow cytometry to determine change in cell size. The intensity of forward scatter signals and back scatter signals are related to cell size and granularity, respectively.
In addition, the flow-cytometry allowed us to analyze a great number of cells independently. The distribution of cell size can be statistically evaluated by two parameters, signal intensities of forward scatter and back scatter. Forward scatter and back scatter correspond to the cell size and granularity, respectively. The pattern showing upper-right shift means the increase in the cell size. As shown in Fig. 6B, both forward scatter and back scatter signals were increased with increasing the glucose concentration, indicated that the relative cell size was increased depending on the glucose concentration, consistent with as mentioned above. The dose-dependent enlargement of the cells reminds us to reconfirm the frequency of blebbed forms of MV appeared on the cell surface. Therefore, these three physiological parameters were finely governed by glucose concentration. Notably, the glucose concentration-dependent PHB production is a key trigger for the cell enlargement and MV biogenesis. In short, it can be concluded that PIA-MVP spatiotemporally takes place via cellular morphological changes. This should be a crucial finding for establishing the molecular mechanism of PIA-MVP.
A hypothetical model for PIA-MVP
In case of Gram-negative bacteria like E. coli, MV biogenesis is promoted through several pathways such as imbalance of peptidoglycan and membrane synthesis12. Especially, an envelope stress in the membrane have been established well for MV production13,14,15. The envelope stress is caused by disorder of the linkage networking between three structured biomacromolecules, outer membrane-peptidoglycan-inner membrane. Accordingly, many spontaneous and artificial mutations closely related to hypervesiculation are noticeably distributed in the cell9,10.
On the other hand, PIA-MVP has provided us with a different principle in which a trigger for MV formation is PHB accumulation itself. Namely, there is a linear relationship between the amount of released MVs and PHB production level, associated with accompanying the morphological change of the cells. Thus, it might be considered that the change in cell volume correlates well with the internal pressure, as it were an envelope stress, induced by PHB accumulation (Fig. 7). As illustrated in Fig. 7, the secretion level of MVs can be controlled by the concentration of glucose externally added.
A hypothetical model for the Polymer Intracellular Accumulation-triggered system for MV Production (PIA-MVP). A hypothetical model for PIA-MVP is made based on the following these three steps. (Step 1) PIA-MVP would take place via the internal pressure caused by PHB accumulation. (Step 2) the internal pressure may give a physical perturbation to the linkage network formed between three structurered biomacromolecules, outer membrane-peptidoglycan-inner membrane. Consequently, a disordered state would be occurred in the periplasmic space as an envelope stress, causing MV biogenesis. (Step 3) single-layer MVs (Outer MVs, OMVs) and multilayer MVs (Outer/Inner MVs, OIMVs) frequently occur. The illustration was drawn by the drawing tool in Microsoft PowerPoint ver. 16.56 (Microsoft).
It is often argued that the formation of single-layer MV (Outer MV, OMV) and multilayer MV (Outer/Inner MV, OIMV) takes place depending on the given physiological situations in Gram-negative bacteria12. As mentioned above (Fig. 3A), in our PIA-MVP system, there are a mixture of OMVs and OIMVs in the culture supernatant. Taking the advantage of the OIMVs formation would be utilized as a cargo for encapsuling intracellularly polymerized products of interest such as protein, nucleic acids and polyesters like PHB.
Status and significance of PIA-MVP
It is of interest to consider a physiological relationship between MV formation with PHB production. The artificial E. coli system lacks the native surface of carbonosomes as it can be found in PHB-producing native bacteria represented by Ralstonia eutropha or Peudomonas putida. Hence, the hydrophobic surface of the PHB produced in E. coli is at least partially exposed to the cytoplasm where it might come in contact with the cytoplasmic membrane, in particular because PHB granules in E. coli tend to localize close to the cell poles16. In case of a larger-cell-sized Caryophanon latum, PHB granules of Nile-red stained living cells were frequently found at or close to the cytoplasmic membrane at the early stages of PHB accumulation, as revealed by TEM17. After cell lysis, it was seen that PHB granules were in some frequent associated with MVs. Throughout the cell cycle, physical contact of PHB with the cytoplasmic membrane was observed from the view of the electron-translucent structures. These natural ecosystems would provide any insights into our artificial system PIA-MVP.
In this study, we present that the MV biogenesis took place in completely different principle in terms of the coupling with intracellular accumulation of PHB in native state of the wild-type strain with maintaining growth ability (Supplementary Fig. S2) and without any mutation. This should be a characteristic feature that is distinguishable from the other reported cases7. Also, PIA-MVP is a versatile platform methodology for efficient MV production that can be combined with the conventional strategies, genetical mutations9,10 and external addition of chemicals like glycine7 properly causing MV biogenesis. It might be feasible to substitute PHB with other type of biopolymers such as lactate-based polymer18. MVs are now attractive as a potential proteoliposomal carrier of vaccines from the viewpoints of the advantages of nano-sized admixtures, their immunological properties, and their structural stability12. In near future, PIA-MVP would contribute to utilization of MVs towards the wide range of applications.
