Characterization and identification of the proteins bound to two types of polyhydroxyalkanoate granules in Pseudomonas sp. 61-3

Figures & data

Fig. 1. Organization of pha and phb loci in Pseudomonas sp. 61-3.

Notes: The genes located on pha and phb loci in Pseudomonas sp. 61-3 are involved in the biosynthesis of P(3HB-co-3HA) and P(3HB), respectively. In pha locus, the genes encoding PHA synthase 1 (PhaC1), PHA depolymerase (PhaZ), PHA synthase 2 (PhaC2), an unknown function protein (PhaD), and PHA granule-associated proteins (PhaI and PhaF) are located. In phb locus, the genes encoding a putative negative regulator protein (PhbF) related to the transcription of phbP gene, phasin (PhbP), an unknown function protein (ORF), a putative regulator protein (PhbR) related to the transcription of phbBAC, NADPH-dependent acetoacetyl coenzyme A reductase (PhbB), β-ketothiolase (PhbA), and PHB synthase (PhbC) are located.

Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid	Relevant characteristics	Source or reference
Strains
Pseudomonas sp. 61-3	Wild type	JCM 10015^30^)
Pseudomonas sp. 61-3 (phbC::tet)	Inactivation of chromosomal phbCPs by integration of Tc^r; phbCPs-negative mutant	^30^)
Pseudomonas sp. AC1-TnK	phaC1Ps-negative mutant, phaC1Ps::kan (Tn10), Km^r	This study
Pseudomonas sp. BCG-TcGm	Inactivation of chromosomal phbCPs and phaGPs by integration of Tc^r and Gm^r, respectively; phbCPs- and phaGPs-negative mutant	^33^)
E. coli DH5α	deoR endA1 gyrA96 hsdR17 (rK^− mK^+) relA1 supE thi-1 ∆(lacZYA-argFV169) ø80∆lacZ∆M15F^–λ^−	Clontech
E. coli S17-1	recA and tra genes of plasmid RP4 integrated into the chromosome; auxotrophic for proline and thiamine	^34^)
E. coli S17-1 (λpir)	π protein encoded by R6K integrated into chromosome	^35^)
plasmids
pBluescript II KS^+	Ap^r lacPOZ T7 and T3 promoter	Stratagene
pT7Blue T-vector	Ap^r, lacPOZ	Novagen
pJASc22	pJRD215 derivative; phaC1Ps	^30^)
pJKSc54-phab	pJRD215 derivative; phaPs promoter, phaC1Ps, phbRe promoter, phbARe, phbBRe	^36,37^)
pJKSc46-pha	pJRD215 derivative; phaPs promoter, phaC1Ps, phbARe, phbBRe	^36,37^)
pBSEX22	pBluescript II KS^+ derivative; phaPs promoter, phaC1Ps	^36,37^)
pBSL180	Ap^r, Km^r, R6K replicon, suicide, lacI^q, tnp (Tn10), mob^+, IS10	^35^)
pSLBE13dC1	pBSL180 derivative containing the 1.3-kb BglII-EcoRI fragment of pBSEX22	This study

Fig. 2. SDS-PAGE analysis of native PHA granules isolated from the recombinant strains of Pseudomonas sp. 61–3. Lane 1, molecular weight markers; lane 2, Pseudomonas sp. 61–3 (phbC::tet); lane 3, Pseudomonas sp. 61–3 (phbC::tet)/pJASc22; lane 4, Pseudomonas sp. 61–3 (phbC::tet)/pJKSc46-pha; lane 5, Pseudomonas sp. 61–3 (phbC::tet)/pJKSc54-phab; lane 6, Pseudomonas sp. AC1-TnK; and lane 7, Pseudomonas sp. BCG-TcGm/pJKSc54-phab.

Table 2. Relationship of the monomer composition of PHA accumulated by recombinant strains of Pseudomonas sp. 61-3 and the granule-associated proteins.

Strain	plasmid	PHA composition (mol%)		Molecular weight of granule-associated protein (kDa)
		3HB (C4)	3HA (C6–C12)
Pseudomonas sp. 61-3 (phbC::tet)	none	30	70	62	48	36		18
Pseudomonas sp. 61-3 (phbC::tet)	pJASc22	49	51	62	48	36		18
Pseudomonas sp. 61-3 (phbC::tet)	pJKSc46-pha	66	34	62	48	36		18
Pseudomonas sp. 61-3 (phbC::tet)	pJKSc54-phab	87	13	62	48	36	24	18
Pseudomonas sp. AC1-TnK	none	95	5	(69)	(48)		24
Pseudomonas sp. BCG-TcGm	pJKSc54-phab	99	1	62	(48)		24

Notes:

Cells were cultivated at 28 ºC for 48 h or 72 h (Pseudomonas sp. AC1-TnK) in MS medium containing 2% (wt./vol) glucose as a sole carbon source. Minor bands are indicated in parentheses. 3HB, 3-hydroxybutyrate; 3HA, medium-chain-length 3-hydroxyalkanoate units (C₆–C₁₂).

Fig. 3. The localization model of the proteins associated with polyester granules accumulated in (A) Pseudomonas sp. 61–3 (phbC::tet)/pJKSc46-pha, (B) Pseudomonas sp. 61–3 (phbC::tet)/pJKSc54-phab, (C) Pseudomonas sp. AC1-TnK, and (D) Pseudomonas sp. BCG-TcGm/pJKSc54-phab.

Matsusaki H, Manji S, Taguchi K, Kato M, Fukui T, Doi Y. Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3. J. Bacteriol. 1998;180:6459–6467.

 PubMed Web of Science ®Google Scholar

Matsumoto K, Matsusaki H, Taguchi S, Seki M, Doi Y. Cloning and characterization of the Pseudomonas sp. 61-3 phaG gene involved in polyhydroxyalkanoate biosynthesis. Biomacromolecules. 2001;2:142–147.

 PubMed Web of Science ®Google Scholar

Simon R, Priefer U, Pühler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. BioTechnology. 1983;1:784–791.10.1038/nbt1183-784

 Web of Science ®Google Scholar

Alexeyev MF, Shokolenko IN. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of Gram-negative bacteria. Gene. 1995;160:59–62.10.1016/0378-1119(95)00141-R

 PubMed Web of Science ®Google Scholar