Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.Polyhydroxyalkanoates (PHAs) are polyoxoesters that are synthesized intracellularly in diverse bacteria under conditions of limitation for a nutrient other than carbon (5). The polymers are water insoluble and accumulate as intracellular granules (5). Phasins are low-molecular-weight proteins that are proposed to promote PHA synthesis in cells (9,10,15,18). Three different mechanisms for the function of phasins have been proposed. First, phasins may enhance PHA production by binding to granules and increasing the surface/volume ratio of the granules (18). Second, phasins may activate the rate of PHA synthesis by interacting directly with PHA synthase (18). Third, phasins may promote PHA synthesis indirectly by preventing growth defects associated with the binding of other cellular proteins to PHA granules (6, 18). Phasins have also been proposed to function as storage proteins (7), a role that could also conceivably affect PHA synthesis. Studies on the function of phasins in recombinant host strains (6, 9) and in vitro (3) have yielded anomalous results which hint that the timing and levels of expression of phasins may be crucial for their function. In order to understand the function of phasins, we have generated new tools and carried out a systematic study to examine the kinetics and amounts of the Ralstonia eutropha phasin PhaP in R. eutropha under a variety of growth conditions. R. eutropha is well suited for studies on phasins, given that the strain produces a PHA, poly-[(R)-3-hydroxybutyrate] (PHB), under many standard cultivation conditions (5, 17) and is amenable to genetic manipulation (8,12,14). The first genetic analysis of the role of PhaP in PHB synthesis in R. eutropha was conducted by Wieczorek et al. (18). They reported the isolation of five phaP::Tn5 mutants that were claimed to be defective in the production of PhaP and to produce half as much PHB as the wild-type (wt) strain (18...

Intracellular poly[D-(؊)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid.Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share ϳ30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (⌬phaZ1, ⌬phaZ2, ⌬phaZ3, ⌬phaZ1⌬phaZ2, ⌬phaZ1⌬phaZ3, ⌬phaZ2⌬phaZ3, and ⌬phaZ1⌬phaZ2⌬phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ϳ80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.Polyhydroxyalkanoates (PHAs) are polyoxoesters produced by a wide range of bacteria when they find themselves in an environment with an available carbon source but limited in additional nutrient(s) required for growth (9). The shortchain-length PHAs, where R is a methyl or ethyl, have properties of thermoplastics and are biodegradable (Fig. 1). Much effort has focused on understanding the biology of PHA homeostasis for several reasons. First, this understanding could lead to expression of the appropriate gene set in heterologous systems to make PHA production economically competitive with oil-based polymers. Second, understanding PHA homeostasis serves as a paradigm for understanding the mechanism of homopolymerization reactions in which the product undergoes a phase transition during its formation, generating insoluble inclusions (granules). The intracellular PHAs can be degraded when the bacteria require carbon but are in otherwise nutrientreplete conditions, and the monomers and energy released can be reused to allow the bacteria to grow (17). The insoluble PHA granules must, therefore, be biosynthesized in a controlled fashion to facilitate enzymatic degradation. A variety of proteins associated with PHA homeostasis have been identified and are being characterized (3,8,10,12,20,22). As part of our research to understand the mechanisms that control polymer size and reuse, we have been interested in identifying the intracellular depolymerases that degrade poly[D-(Ϫ)-3-hydroxybutyrate] (PHB) w...

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

SummaryWhen grown on medium supplemented with the succinoglycan-binding dye, Calcofluor, and visualized under UV light, colonies of Rhizobium meliloti (Sinorhizobium meliloti ) exoK mutants produce a fluorescent halo with a delayed onset relative to wild-type colonies. By conducting transposon mutagenesis of exoK mutants of R. meliloti and screening for colonies with even more severe delays in production of these fluorescent halos, we identified three genes, designated prsD, prsE, and exsH, which are required for the eventual production of fluorescent halos by exoK colonies. Nucleotide sequence indicates that the prsD and prsE genes encode homologues of ABC transporters and membrane fusion proteins of Type I secretion systems, respectively, whereas exsH encodes a homologue of endo-1,3-1,4-␤-glycanases with glycine-rich nonameric repeats typical of proteins secreted by Type I secretion systems. The exoK gene and the prsD /prsE /exsH genes were shown to be components of independent pathways for production of extracellular succinoglycan degrading activities and for production of low-molecular-weight succinoglycan by R. meliloti. Based on these results, we propose that ExsH is a succinoglycan depolymerase secreted by a Type I secretion system composed of PrsD and PrsE, and that the ExsH and ExoK glycanases contribute to production of low-molecular-weight succinoglycan.

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Introducing and expressing foreign genes in plants present many technical challenges that are not encountered with microbial systems. This review addresses the variety of issues that must be considered and the variety of options that are available, in terms of choosing transformation systems and designing recombinant transgenes to ensure appropriate expression in plant cells. Tissue specificity and proper developmental regulation, as well as proper subcellular localization of products, must be dealt with for successful metabolic engineering in plants..

The Rhizobium meliloti ExoK and ExsH glycanases have been proposed to contribute to production of low molecular weight (LMW) succinoglycan by depolymerizing high molecular weight succinoglycan chains in R. meliloti cultures. We expressed and purified ExoK and ExsH and determined that neither enzyme can extensively cleave succinoglycan prepared from R. meliloti cultures, although neutral͞ heat treatment and acid͞heat treatment convert succinoglycan to forms that can be cleaved efficiently by both enzymes. These results were somewhat surprising, given that the exoK ؉ and exsH ؉ genes play a crucial role in production of LMW succinoglycan in R. meliloti cultures. We demonstrated by Western blot analyses that R. meliloti expresses ExoK and ExsH, that both proteins can be detected extracellularly, and that ExsH secretion depends on the prsD ؉ ͞prsE ؉ genes, consistent with previous predictions based on mutant analyses. Furthermore, we determined that the depolymerization activities associated with purified ExoK and ExsH are comparable with exoK ؉ and exsH ؉ -dependent depolymerization activities expressed in R. meliloti cultures. We resolved the apparent contradiction between the results of our previous genetic analyses and depolymerization assays by determining that ExoK and ExsH can cleave high molecular weight succinoglycan that is being produced actively by R. meliloti, but not succinoglycan that has accumulated in cultures, to yield LMW succinoglycan. We propose that ExoK and ExsH dynamically regulate the molecular weight distribution of succinoglycan by cleaving nascent succinoglycan only during a limited period after its synthesis, perhaps before it undergoes a timedependent change in its conformation or aggregation state.