Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber

The first polyhydroxyalkanoic acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404. The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored the ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61,371. The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthases of A. eutrophus and Pseudomonas oleovorans. Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel. A Mr 61,000 protein was identified as the PHA synthase of R. ruber by N-terminal amino acid sequence determination.     

Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.

Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.     

Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.     

Identification of nitrogenase and carboxylase genes in the photosynthetic bacteria and cloning of a carboxylase gene from Rhodopseudomonas sphaeroides

The presumptive genes for the ribulose 1,5-bisphosphate carboxylase large subunit and for nitrogenase-specific components from Rhodopseudomonas sphaeroides and several other photosynthetic bacteria were identified and located by interspecific probing. Restriction digests of R. sphaeroides genomic DNA were hybridized under stringent conditions to cloned DNA from Rhodospirillum rubrum (plasmid pRR2119 carrying the carboxylase gene) and Klebsiella pneumoniae (pSA30 carrying the nitrogenase genes). The nitrogenase probe hybridized with different signal intensities to several distinct HindIII, BglII, EcoRI, BamHI and PvuII fragments of R. sphaeroides 2.4.1.DNA. The carboxylase probe hybridized to only single R. sphaeroides 2.4.1.DNA fragments produced with all five restriction enzymes. A 3000-bp EcoRI-BamHI R. sphaeroides 2.4.1.DNA fragment carrying the presumptive gene for the large subunit of ribulose 1,5-bisphosphate carboxylase was cloned into pBR322 and positively identified by probing with a 32P-labeled internal PstI fragment of the Rhodospirillum carboxylase gene.     

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.     

Comparison of phbC genes cloned from Ralstonia eutropha and Alcaligenes latus for utilization in metabolic engineering of polyhydroxyalkanoate biosynthesis

Two cloned phbC genes, encoding polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha and from Alcaligenes latus, were transformed into a PHB-negative mutant of R. eutropha. The expression characteristics of both genes were compared for the biosyntheses of PHB and its copolymers. Each phbC gene had different characteristics not only in the biosyntheses of PHB, poly(3-hydroxybutyrate-3-hydroxy-valerate), and poly(3-hydroxybutyrate-4-hydroxybutyrate) but also in the resulting morphology of PHB granules.     

Physiology and molecular genetics of poly(β-hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus

The Alcaligenes eutrophus genes for beta-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(beta-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.     

Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8.

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.     

浑球红细菌谷氨酸合酶基因(glt)的克隆和图谱分析

利用转座子Tn5随机插入诱变筛选得到12株浑球红细菌(Rhodobacter sphaeroides)氨同化缺陷突变株(Asm~-)。这些突变株胞内均无GOGAT活性,同时它们均无固氮酶活性(Nif~-),并且具有氮代谢多效性缺失表型(Ntr~-)。将含有Azorhizobium sesbaniae ORS571的完整glt基因的质粒pHB10转入突变株中能互补上述表型。通过筛选携带Tn5的R-prime质粒克隆了glt::Tn5片段。Southern杂交证明所克隆glt::Tn5片段与E. coli的gltBD基因有同源性。用此片段与以pLAFR3为载体所构建的R. sphaeroides 601基因文库进行菌落原位杂交筛选到了携带glt基因的cosmid pLT27。pLT27能互补所有12株R.sphaeroides氨同化缺陷突变株。酶切分析表明在该cosmid中插人的染色体DNA片段大小约为26.5kb。以pRK415为载体亚克隆了4.0kb与10.5kh的pLT27的Hindlll酶切片段,分别命名为pLTRK271与pLTRK272。pLTRK272能互补变种GT6、GT10、GT11,pLTRK…     

Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: Evidence that the gene is both plasmid and chromosomally located

Abstract The polyhydroxyalkanoic acid (PHA) synthase gene ( phaC_Ac ) of a species of Acinetobacter isolated from an activated sludge treatment plant was cloned by heterologous complementation in a poly-β-hydroxybutyrate (PHB) negative mutant of Alcaligenes eutrophus . Nucleotide sequence analysis of phaC_Ac revelaed an open reading frame of 1770 bp with potential to encode a 67.7 kDa protein. The deduced amino acid sequence displays high similarity to other PHA synthase proteins. Probing with an internal region of phaC_Ac revealed that the PHA sythase gene may be present in more than one copy and may occur at both plasmid and chromosomal locations in Acinetobacter spp. This is the first organisms for which evidence has been presented to suggest that a gene involved in PHA metabolism is plasmid-encoded. Purification of PHB granules from sucrose gradients identified proteins of 38 kDa, 41 kDa and 64 kDa which may have a role in PHB metabolism.     

A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydroxyalkanoic acids (HA _mcl ) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HA _scl ), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB^–4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HA _mcl and was expressed in GPp104 but not in PHB^–4. Correspondence to: A. Steinbüchel     

Reductive effect of H(2) uptake and poly-beta-hydroxybutyrate formation on nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides according to light intensity

Nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides under photoheterotrophic conditions is reduced directly by the hydrogenase activity catalyzing H(2) uptake and indirectly by energy-demanding metabolic processes such as poly-beta-hydroxybutyrate (PHB) formation. H(2) accumulation of R. sphaeroides was examined during cell growth under illumination of 15, 7, and 3 W/m(2). Mutations in either hupSL (H(2)-uptake hydrogenase) or phbC (PHB synthase) had no effect on nitrogenase activity. The nitrogenase activity of R. sphaeroides grown at 15 W/m(2), however, was 70% higher than that of cells grown at 3 W/m(2), while the H(2)-uptake hydrogenase activity was approximately 3-fold higher in the same comparison. Accordingly, H(2) uptake by hydrogenase, monitored by measuring the difference in H(2) accumulation between a hupSL-deletion mutant and the corresponding parental strain, appeared to reach a maximum level as illumination was increased to 15 W/m(2). On the other hand, the surplus energy due to lack of PHB formation led to a fixed increase in H(2) accumulation independent of light intensity, reflecting the fact that the cellular PHB content was not changed significantly depending on light intensity. Therefore, H(2) uptake by hydrogenase should be suppressed to achieve higher H(2) accumulation of R. sphaeroides, especially at 15 W/m(2).     

Poly(3-Hydroxybutyrate) Synthesis Genes in Azotobacter sp. Strain FA8

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.     

An insertion of Escherichia coli transposable element IS1K into the site immediately before tetracycline-resistance determinant of Bacillus subtilis chromosomal DNA fragment in cloning in E. coli

In cloning in Escherichia coli C600 of a 4.5-kbp HindIII DNA fragment with the tetracycline-resistance determinant (tetBS908) from Bacillus subtilis GSY908 chromosome using a plasmid vector, a 5.2-kbp HindIII DNA fragment was also isolated at a ratio of 2 to 89. The two independently obtained 5.2-kbp fragments were an insertion derivative of the 4.5-kbp fragment and carried E. coli transposable element ISlK, which was inserted at the same site immediately before tetBS908 in the same direction. For the ISlK insertions, the 8-bp sequence CAAATTTT was used as a target, this having no similarity to any published sequences.     

Biodeuteration of poly(beta-hydroxybutyrate).

The formation of poly(beta-hydroxybutyrate), PHB, by Rhodobacter sphaeroides and Alcaligenes eutrophus was studied using the following carbon sources and solvents: (1), acetate in H2O; (2), D3-acetate in H2O; (3), acetate in 90 to 92% D2O; and (4), D3-acetate in 90 to 92% D2O. The growth of Rb. sphaeroides cultured under condition (2) showed no apparent deuterium isotope effect, while considerably slowed growth in the presence of D2O was observed under conditions (3) and (4). In all cases, the PHB produced under deuterium enriched conditions was of high molecular weight. Interestingly, comparatively high volumetric formation of partially deuterated PHB was obtained using culture condition (4) for A. eutrophus. Fourier transform infrared spectroscopy (FT-i.r.), pyrolysis gas chromatography mass spectrometry (PGC-m.s.), and nuclear magnetic resonance (n.m.r.) were used to establish the extent and distribution of deuterium in the PHB samples produced. Partially deuterated PHB was obtained in each case, using a deuterium enriched culture. Considerable differences in the extent and distribution of deuterium were found between micro-organisms and culture conditions.