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Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells - PubMed

Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells

G M York et al. J Bacteriol. 2001 Jul.

Abstract

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.

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Figures

FIG. 1
FIG. 1

(A) Accumulation of PhaCRe in R. eutropha wt, phaC deletion, and phaCRe C319A strains, as detected by anti-PhaCRe antibody. Proteins were separated by SDS–10% PAGE and were subjected to immunoblot analysis. Molecular-mass standards are indicated in kilodaltons. Cells from R. eutropha cultures were harvested after cultivation for 48 h in TSB or PHA(high). Bacterial samples correspond to cells from 10 μl of culture diluted to an OD600 of 1.0. Purified PhaCRe was included as a positive control. (B) Accumulation of PhaECCv in R. eutropha wt, phaECCv, phaCCv, and phaECCv C149A strains, as detected by anti-PhaECCv antibody. Samples were analyzed as described above. Purified PhaECCv was included as a positive control. Note that the data correspond to two blots (first blot, lanes 1 to 5; second blot, lanes 6 to 10).

FIG. 2
FIG. 2

(A) Accumulation of PhaP in R. eutropha wt, phaP deletion, phaC3::Tn5, phaC deletion, phaCRe C319A, phaECCv, phaCCv, and phaECCv C149A strains. Proteins were separated by SDS–15% PAGE and were subjected to immunoblot analysis for detection of PhaP. Molecular-mass standards are indicated in kilodaltons. Cells from R. eutropha cultures were harvested after cultivation for 48 h in TSB, PHA(med), and PHA(high). Bacterial samples correspond to cells from 10 μl of culture diluted to an OD600 of 1.0. Data correspond to three blots (first blot, lanes 1 to 3; second blot, lanes 4 to 15; third blot, lanes 16 to 26). Purified PhaP was included as a positive control on each blot. Blots were exposed to film for 5, 10, and 30 min. Data correspond to 10-min exposures for the first and second blots and a 30-min exposure for the third blot. (B) Measurements of OD600 for R. eutropha strains after cultivation for 48 h in TSB, PHA(med), and PHA(high). Data were extrapolated from 10-fold dilutions of cultures.

FIG. 3
FIG. 3

(A) Accumulation of GFP in wt, phaC3::Tn5, phaP-gfp, and phaP-gfp phaC3::Tn5 R. eutropha strains. Proteins were separated by SDS–15% PAGE and were subjected to immunoblot analysis for detection of GFP. Molecular-mass standards are indicated in kilodaltons. Cells from R. eutropha cultures were harvested after cultivation for 48 h in TSB, PHA(med), and PHA(high). Bacterial samples correspond to cells from 10 μl of culture diluted to an OD600 of 1.0. The E. coli strains DH5α/pGY1a+, which carries the phaP-gfp fusion on a plasmid, and DH5α/pSW213, which carries the corresponding vector lacking the phaP-gfp fusion, were included as positive and negative controls, respectively. (B) Measurements of OD600 for R. eutropha strains after cultivation for 48 h in TSB, PHA(med), and PHA(high). Data were extrapolated from 10-fold dilutions of cultures.

FIG. 4
FIG. 4

Comparison of levels of PhaP versus PHB for wt strain cultivated under PHB utilization conditions (PHA[no carbon]) for 72 h. Cells were cultivated in PHA(med) (A) or PHA(high) (B) (200 ml) for 72 h, washed, diluted fourfold, and were then cultivated in PHA(no carbon) for 72 h. Time zero corresponds to start of cultivation in PHA(no carbon). All data points represent average value for two cultures (error bars represent standard deviations).

FIG. 5
FIG. 5

Accumulation of PhaP in cultures of wt, phaC deletion, and phaP deletion strains, cultivated alone or cocultivated in pairs. Proteins were separated by SDS–15% PAGE and were subjected to immunoblot analysis for detection of PhaP. Molecular-mass standards are indicated in kilodaltons. Cells from R. eutropha cultures were harvested after cultivation for 48 h in PHA(med). Bacterial samples correspond to cells from 10 μl of culture diluted to an OD600 of 1.0. Purified PhaP was included as a positive control.

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References

    1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1994.
    1. Campos-García J, Caro A D, Nájera R, Miller-Maier R M, Al-Tahhan R A, Soberón-Chávez G. The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase which is specifically involved in rhamnolipid synthesis. J Bacteriol. 1998;180:4442–4451. - PMC - PubMed
    1. Chen C Y, Winans S C. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J Bacteriol. 1991;173:1139–1144. - PMC - PubMed
    1. Cormack B P, Valdivia R H, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP) Gene. 1996;173:33–38. - PubMed
    1. de Lorenzo V, Herrero M, Jakubzik U, Timmis K N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990;172:6568–6572. - PMC - PubMed

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