PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum
© Yoshida et al.; licensee BioMed Central Ltd. 2013
Received: 1 September 2013
Accepted: 9 December 2013
Published: 11 December 2013
Bradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-β-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding acetoacetylCoA reductase, five phbC paralogs encoding PHB synthase, two phaZ paralogs encoding PHB depolymerase, at least four phaP phasin paralogs for stabilization of PHB granules, and one phaR encoding a putative transcriptional repressor to control phaP expression.
Quantitative reverse-transcriptase PCR analyses of RNA samples prepared from cells grown using three different media revealed that PHB accumulation was related neither to redundancy nor expression levels of the phbA, phbB, phbC, and phaZ paralogs for PHB-synthesis and degradation. On the other hand, at least three of the phaP paralogs, involved in the growth and stabilization of PHB granules, were induced under PHB accumulating conditions. Moreover, the most prominently induced phasin exhibited the highest affinity to PHB in vitro; it was able to displace PhaR previously bound to PHB.
These results suggest that PHB accumulation in free-living B. japonicum USDA110 may not be achieved by controlling production and degradation of PHB. In contrast, it is achieved by stabilizing granules autonomously produced in an environment of excess carbon sources together with restricted nitrogen sources.
KeywordsBradyrhizobium japonicum Phasin PHB
Poly-β-hydroxybutyrate (PHB) is a polymer used for the storage of carbon and energy in a large variety of prokaryotes. It is accumulated in the cytoplasm if a carbon source is provided in excess and if any other essential nutrient is limited . PHB belongs to the polyesters class of polymers, which is of interest as an industrial plastic because of its biodegradability and origin from renewable resources. Microbial PHB synthesis is a promising strategy for the production of bioplastics and offers a promising opportunity to transition toward a future-oriented bioeconomy .
Most species of rhizobia synthesize PHB and accumulate it in intracellular granules . In some species, PHB accumulation can exceed 50% of the cell’s dry weight [4, 5]. Various ways that rhizobia can use PHB to benefit their plant hosts have been proposed. For instance, it was proposed that PHB utilization could sustain the oxygen demand of the bacteroids during darkness; thus, contributing to the preservation of nodule activity and the continuation of nitrogen fixation at high rates . PHB may also fuel the differentiation of rhizobia into nitrogen-fixing bacteroids . In addition, rhizobia may simply degrade PHB in ways that enhance their own fitness. PHB may provide the energy and carbon required for bacterial reproduction, or for stress tolerance required within senescing nodules or after symbiotic rhizobia escape into the soil and transition to the free-living state.
Biochemically, PHB synthesis can compete with nitrogen fixation . In addition, a negative correlation was observed between the rate of nitrogen fixation and PHB accumulation [8, 9]. A mutant of Rhizobium etli, that did not accumulate PHB, was shown to significantly fix more nitrogen than the isogenic wild type [10, 11], whereas non-fixing nifH mutants of R. etli and Bradyrhizobium japonicum accumulated more PHB than their isogenic nitrogen-fixing parental strains. There is a conflict between rhizobia and legumes over the rate of PHB accumulation, due to the metabolic tradeoff between nitrogen fixation and PHB accumulation. Therefore, PHB biosynthesis and accumulation in species of rhizobia may be controlled to balance the tradeoff, but the mechanism underlying this control has not yet been fully explained.
One of the best studied microorganisms with respect to PHB biosynthesis and accumulation is the Gram-negative bacterium Ralstonia eutropha. It synthesizes PHB using the three PHB synthetic genes: phbA, which encodes 3-ketoacyl-CoA thiolase; phbB, which encodes acetoacetyl CoA reductase; and phbC, which encodes the enzyme PHB synthase. PHB degradation, however, is performed by PHB depolymerase, which is encoded by phaZ. Phasins, encoded by phaP, are a class of low-molecular-mass amphipathic proteins that form a layer at the surface of the PHB granule and stabilize it . The R. eutropha possesses at least four phaP paralogs identified so far . Expression of the major phasin, encoded by phaP1, is regulated by the transcriptional repressor PhaR [17, 18]. Under conditions less favorable for PHB biosynthesis, PhaR binds to the phaP1 promoter region to repress transcription of this gene. After the onset of PHB biosynthesis, when the nascent PHB granules gradually form, PhaR leaves the promoter and binds to the granules so that phaP1 is transcribed and translated. During the later stages of PHB accumulation, PhaR is estimated to bind no longer to the granules as it is displaced by PhaP1 phasin. The displaced PhaR returns to bind to the phaP1 promoter and represses transcription again .
Most members of the Rhizobiaceae are known to possess single copies of the PHB biosynthesis genes. For instance, strains of Sinorhizobium meliloti, the symbionts of alfalfa, regarded as one of the model organisms to study symbiotic nitrogen fixation, are characterized to have a single set of the genes for PHB metabolism, namely phbA, phbB, phbC, and phaZ[19, 20], whereas two paralogous genes, phaP1 and phaP2, encode functional phasins . On the other hand, strains of B. japonicum, the symbionts of soybean, are known to accumulate a large amount of PHB , and the B. japonicum USDA110 genome was found to contain five paralogs of phbC, as well as two paralogs of phbAB. This genetic redundancy may suggest a functional importance that has not yet been fully elucidated. In this study, we examined the expression profiles of the paralogs relevant to PHB metabolism in free-living B. japonicum cells under PHB accumulating and non-accumulating conditions. Then, we identified the phasin-encoding paralogs from the genomic information , and used a variety of tools to investigate their involvement in PHB accumulation.
Results and discussion
B. japonicumcandidate genes involved in metabolism and PHB accumulation
Expression profile of the candidate genes in free-living cells
The transcription profile of phaP and phaR involved in PHB accumulation was also examined using qRT-PCR (Figure 4B). In contrast to the PHB-metabolic genes, induction of some of the phaP encoding putative phasins correlated with PHB accumulation. Among the four phaP, phaP4 was most prominently induced under PHB-accumulating conditions in YEM medium reaching levels up to 40 times greater than that of the control, sigA, which encodes the house-keeping sigma factor. These results imply that phaP4 may play an important role in PHB accumulation. When cultured in YEM, phaP1 and phaP2 were induced to levels up to 10 times greater than the control, implying that phaP1 and phaP2 may also have roles in PHB accumulation. In PSY medium, both phaP1 and phaP4 were induced to lower levels, which may be relevant to the lower PHB accumulation seen in this medium (Figure 3B). On the other hand, expression of phaR was kept at a low level and only barely enhanced upon PHB accumulation, which is consistent with the self-regulation model proposed in R. eutropha. Transcription of phaP3 was almost constant and as low as that of phaR, and thus this paralog might be irrelevant to PHB accumulation under these conditions.
When all these results are considered, it is conceivable that PHB accumulation in B. japonicum during free-living growth may not depend on either the redundancy or expression levels of the genes for PHB synthesis and degradation. Instead, it seems probable that the major mechanism allowing B. japonicum to accumulate large amounts of PHB may be the formation of PHB granules stabilized by phasins.
The four PhaP phasins and PhaR bound to PHB with different affinities
phaP1, phaP2, phaP3, phaP4, and phaR were cloned individually into Escherichia coli and expressed as N-terminally His6-tagged fusion proteins. For unknown reason, the His6-tag fusions could not be purified by the conventional affinity chromatography. Therefore, the crude extracts of E. coli cells containing the fusions were used directly in the PHB binding experiment. Because the N-terminus of each fusion protein contained the same single His6-tag, we assumed that each His6-tag equally reacts with the anti-His6-tag antibody, presumably regardless of fusion partner, and the signal intensities on immunoblots probed for the His6-tag were used to represent the amounts of the phasin fusions contained in the extracts.
We have not experimentally assessed the actual repressor function of PhaR; these experiments will be performed and reported later. In addition, to confirm the importance of phaP4 and phaR, we attempted to construct knockout of these, as well as the other phaP. However, for unknown reasons, repeated attempts were not successful. We have considered the construction of B. japonicum mutants overexpressing these genes to see the effects not only during free-living growth but also during symbiosis with the host plant. The results of these experiments would be reported in the near future.
B. japonicum USDA110 accumulated intracellular PHB during free-living culture in the presence of excess carbon sources together with restricted nitrogen sources. Its genome contains redundant paralogs that could be involved in PHB biosynthesis and degradation, but only one or two of each paralog family was found to be expressed during free-living growth. In addition, expression of the PHB metabolic genes was not correlated with PHB accumulation. Thus, it is conceivable that PHB accumulation during free-living growth is independent of redundancy or expression levels of PHB metabolic genes. Instead, it was found that some of the four phaP encoding phasins were induced upon PHB accumulation. All the four phasins exhibited some PHB binding in vitro. PhaP4 showed the highest affinity for PHB and could be responsible for the majority of PhaP function. Furthermore, PhaP4 was able to compete for PHB binding with PhaR, which is its plausible transcriptional repressor and possesses high affinity to PHB. PhaP4 is able to expel PhaR and stabilize the PHB granule. Therefore, in free-living B. japonicum, carbon sources in excess relative to nitrogen sources enlarge the pool of substrates for PHB synthesis, such as acetyl-CoA and acetate. This could allow elevation in levels of intracellular PHB, which is recognized by PhaR repressor. This recognition triggers induction of phasins, including PhaP4 and maybe some others. Phasins then autonomously stabilize the accumulated PHB granules. This proposed mechanism resembles the mechanism proposed in R. eutropha.
Bacterial strains, plasmids, primers, and culture conditions
Bacterial strains and plasmids
Strains and plasmids
Relevant genotypes and derivation
Source and reference
supE44, DlacU169, hsdR17, recA1, endA1, gyrA96, thi-1, relA1
F - ompT hsdSb (rb - mb - ) gal dcm (DE3)
Protein expression vector, kanamycin resistant
pET28b carrying phaP1
pET28b carrying phaP2
pET28b carrying phaP3
pET28b carrying phaR
Protein expression vector, ampicillin resistant
pColdII carrying phaP4
Quantification of PHB
USDA101 cells in the cultures were harvested by centrifugation, washed once in 50 mM Tris–HCl (pH 8.0) containing 1 M NaCl, and then suspended in 10 mM Tris–HCl (pH 8.0) containing 5 mM 2-mercaptoethanol, 5 mM ethylenediaminetetraacetic acid, 10% (w/v) glycerol, and 0.02 mM phenylmethylsulfonyl fluoride. The cells were subsequently disrupted by sonication in an ice bath. An aliquot (0.1 mL) of the solution was mixed with 1.2 mL of 5% (w/v) sodium hypochlorite, and incubated at 37°C for 1 h. After centrifugation, the pellet was successively washed with 1 mL aliquots of water, acetone, and 99.5% ethanol. PHB contained in the dried pellet was extracted three times with 0.1 mL of chloroform at 50°C, and the chloroform extracts were combined in a tube (0.3 mL in total). After evaporating the chloroform, the remaining substances were dissolved in 1 mL of concentrated H2SO4, and the absorbance of the solution was measured at 235 nm. Various amounts of commercially available PHB solid powder (Sigma Aldrich, St. Louis, MO) were treated using the procedure described above to produce a standard curve, which was then used to quantify PHB according to the absorbance.
Plasmid construction and production of the recombinant proteins
Sequence (designed restriction sites are underlined)
Total RNA was extracted from USDA110 cells using TRIZOL RNA isolation reagents (Life Technologies, Carlsbad, CA), treated with DNase I (Roche diagnostics, Basel, Switzerland), and then purified using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). The qRT-PCR was performed as follows. An aliquot (1 μg) of the total RNA sample was reverse transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). The resulting cDNA (75 ng) was mixed with THUNDERBIRD SYBR qPCR Mix (Toyobo) and specific primer pairs (RTgene name-F/R listed in Table 2, e.g., RTphbA1-F and RTphbA1-R), and then analyzed using a Thermal Cycler Dice Real Time System MRQ (Takara Bio, Otsu, Japan) according to the suppliers’ procedures.
PHB binding assay and immunoblot analysis
The crude extract containing the recombinant protein was adequately diluted and mixed with a suspension containing various amounts of crystalline PHB solid powder (Sigma Aldrich) in 10 mM Tris–HCl (pH 7.5). The mixture was incubated on ice for 90 min, and then centrifuged to pellet the PHB/protein complexes. After washing with 10 mM Tris–HCl (pH 7.5), the pellet was suspended in loading buffer and applied to an 18% SDS-PAGE gel. Proteins separated by electrophoresis were transferred onto a polyvinylidene fluoride membrane that was subsequently probed with an Anti-His6 (2) antibody (Roche) and goat anti-mouse IgM-HRP (Santa Cruz Biotech, Santa Cruz, CA), and visualized with ImmunoStar LD (Wako Pure Chemical Industries, Osaka, Japan).
Microsoft Excel Spreadsheet has been used for data processing. When needed, data were subjected to one-way analysis of variance followed by the Tukey–Kramer multiple comparison test.
Peptone salts yeast
Quantitative reverse-transcriptase PCR
SDS-polyacrylamide gel electrophoresis.
The authors are grateful to Hisayuki Mitsui, Tohoku University, for providing B. japonicum USDA110, and to Akihiro Motokubota for his technical assistance. This work was financially supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan: in part by Special Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas; by the Advanced Low-Carbon Technology Research and Development Program; and by Grants-in-Aid from the NC-CARP project. The authors would like to thank Enago (http://www.enago.jp) for the English language review.
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