Molecular characterization of the phaECHm genes, required for biosynthesis of poly(3-hydroxybutyrate) in the extremely halophilic archaeon Haloarcula marismortui

Abstract

Although many haloarchaea produce biodegradable polyhydroxyalkanoates (PHAs), the genes involved in PHA synthesis in the domain of Archaea have not yet been experimentally investigated yet. In this study, we revealed that Haloarcula marismortui was able to accumulate poly(3-hydroxybutyrate) (PHB) up to 21% of cellular dry weight when cultured in a minimal medium with excessive glucose and identified the phaE(Hm) and phaC(Hm) genes, probably encoding two subunits of a class III PHA synthase. These two genes were adjacent and directed by a single promoter located 26 bp upstream of the transcriptional start site and were constitutively expressed under both nutrient-rich and -limited conditions. Interestingly, PhaC(Hm) was revealed to be strongly bound with the PHB granules, but PhaE(Hm) seemed not to be. Introduction of either the phaE(Hm) or phaC(Hm) gene into Haloarcula hispanica, which harbors highly homologous phaEC(Hh) genes, could enhance the PHB synthesis in the recombinant strains, while coexpression of the both genes always generated the highest PHB yield. Significantly, knockout of the phaEC(Hh) genes in H. hispanica led to a complete loss of the PHA synthase activity. Complementation with phaEC(Hm) genes, but not a single one, restored the capability of PHB accumulation as well as the PHA synthase activity in this phaEC-deleted haloarchaeon. These results indicated that the phaEC genes are required for biosynthesis of PHB and might encode an active PHA synthase in the Haloarcula species.

FIG. 1.

TEM images of H. marismortui ATCC 43049 cells grown at 37°C for 72 h in different media: (A) nutrient-rich AS-168 medium; (B) minimal medium supplemented with 2% glucose and 0.5% Casamino Acids (MGC medium); and (C) minimal medium supplemented with 2% glucose (MG medium). Bar, 0.5 μm.

FIG. 2.

Profiles of PHB accumulation and cell growth of H. marismortui ATCC 43049 cultivated in MG medium in a 5-liter fermentor (NBS Bioflo 110) at 37°C for about 10 days. The PHB content and cell density, as the optical density at 600 nm (OD₆₀₀), were monitored. This figure represents a typical result of three independent experiments.

FIG. 3.

Partial alignments of the amino acid sequences of PhaC (A) and PhaE (B) subunits from H. marismortui ATCC 43049 (Hm), H. walsbyi DSM 16790 (Hw), Synechococcus sp. strain MA19 (MA19), Ectothiorhodospira shaposhnikovii (Es), Allochromatium vinosum (Av), and Thiocystis violacea (Tv). Amino acids are given in standard one-letter abbreviations, and the numbers indicate the positions of the amino acids within the respective proteins. The conserved residues are darkly shaded, and the residues identical in five of the six (for PhaC) or three of the four (for PhaE) are lightly shaded. The lipase-like box, highly conserved motif of class III synthase, and PhaE box are indicated. The conserved catalytic triad residues are shown with asterisks. GenBank accession numbers are as follows: PhaC_Hm, YP_137339; PhaC_Hw, YP_658052; PhaC_MA19, AAK38139; PhaC_Es, AAG30259; PhaC_Av, S29274; PhaC_Tv, AAC60430; PhaE_Hm, YP_137338; PhaE_Es, AAG30260; PhaE_Av, P45372; PhaE_Tv, AAC60429. The corresponding PhaE sequences were not identified in H. walsbyi DSM 16790 and Synechococcus sp. strain MA19.

FIG. 4.

Transcription analysis of phaEC_Hm genes. (A) Structure and organization of the phaEC_Hm genes in H. marismortui. The putative promoter containing a TATA box (double underlined) and a transcription factor B recognition element (single underlined) and the translational start codon of phaE_Hm (boxed) are indicated. F1, F2, and F3 represent the locations of the RT-PCR products (shown in panel C) in the phaEC_Hm genes. (B) Mapping of the transcriptional start site of phaEC_Hm by primer extension. The relevant sequence is shown on the right. The transcriptional start site is indicated with an arrow. Lane 1, H. volcanii/pWL102, negative control; lane 2, H. volcanii/pWLEC; lanes CTAG, standard sequencing reaction mixture to size the mapping signals. (C) RT-PCR products of partial phaE_Hm (F1; 314 bp), phaEC_Hm (F2; 434 bp), and phaC_Hm (F3; 341 bp) sequences. The following templates were used in the PCR: lane 1, total RNA without reverse transcriptase; lane 2, H. marismortui genomic DNA; lane 3, reverse transcripts of total RNA isolated from cells grown in AS-168 medium; lane 4, reverse transcripts of total RNA isolated from cells grown in MG medium; lane M, DNA marker.

FIG. 5.

Western blot analysis of cellular extracts and PHA granules from H. marismortui ATCC 43049 with antiserum against PhaE_Hm (A) or PhaC_Hm (B). Lane 1, crude extracts from cells grown in AS-168 medium; lane 2, crude extracts from cells grown in MG medium; lane 3, proteins from isolated PHA granules. The purified PhaE_Hm-His₆ (CK1) and PhaC_Hm-His₆ (CK2) from E. coli were used as controls. H. marismortui ATCC 43049 was cultivated at 37°C for 72 h.

FIG. 6.

GC analysis of PHB accumulation in H. hispanica PHB-1 recombinants. The 3HB peaks are the actual peaks of 3-hydroxybutyrate methylester, the methanolysis product of PHB (see Materials and Methods). (A) H. hispanica PHB-1 harboring pWLE; (B) H. hispanica PHB-1 harboring pWLfdxC; (C) H. hispanica PHB-1 harboring pWLEC; (D) PHB standard (Sigma). The peak at 4.85 min represents the methylester product of an internal standard (1 ng of benzoic acid). The H. hispanica PHB-1 recombinants were cultivated in MG medium at 37°C for 96 h.