Genetics of Paracoccus denitrificans

Steinrücke, Peter; Ludwig, Bernd

doi:10.1016/0378-1097(93)90505-v

Cited by 45 publications

(37 citation statements)

References 240 publications

(275 reference statements)

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“…1, the nucleotide sequence of a 2,712-bp region of the 3.6-kb insert is shown. The GϩC content of the determined sequence was 66.5 mol%, which was close to that (66 to 68 mol%) reported for P. denitrificans DNA (29). From computer analysis for protein-coding regions, one open reading frame (ORF) was found.…”

Section: Resultssupporting

confidence: 80%

“…In nucleotide sequence analysis of the upstream region of the phaC Pd gene, the E. coli 70 -dependent promoter-like sequences (7) could not be found. Instead, sequences resembling promoter regions of several genes from P. denitrificans (29) were found; the most plausible promoter regions for the ORF were 5Ј-TCGGGC-3Ј (nucleotides 554 to 559) for the Ϫ35 region and 5Ј-GCAGGC-3Ј (nucleotides 577 to 582) for the Ϫ10 region, respectively. These regions were separated by 17 nucleotides.…”

Section: Resultsmentioning

confidence: 99%

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Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans

et al. 1996

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A 3.6-kb EcoRI-SalI fragment of Paracoccus denitrificans DNA hybridized with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthase gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis of this region showed the presence of a 1,872-bp open reading frame (ORF), which corresponded to a polypeptide with a molecular weight of 69,537. Upstream of the ORF, a promoter-like sequence was found. Escherichia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of ␤-ketothiolase and acetoacetylcoenzyme A reductase genes of A. eutrophus. The ORF was designated phaC Pd . A plasmid vector carrying the phaC Pd -lacZ fusion gene downstream of the promoter-like sequence expressed ␤-galactosidase activity in P. denitrificans. When a multicopy and broad-host-range vector carrying the ORF along with the promoter-like sequence was introduced into P. denitrificans, the PHA content in the cells increased by twofold compared with cells carrying only a vector sequence.Poly(3-hydroxyalkanoates) (PHAs) synthesized by bacteria have received great attention as sources for biodegradable plastic materials (4). Poly(3-hydroxybutyrate) [P(3HB)] is the most investigated PHA (4,8,16). Interest in a copolyester composed of 3HB and 3-hydroxyvalerate (3HV) [P(3HB-co-3HV)] has been growing recently because its physical properties are suitable for developing biodegradable plastics with wide utility.Methylotrophic bacteria able to grow on methanol as a sole carbon and energy source are attractive for producing useful metabolites because methanol is an inexpensive feed stock. Some researchers have used methylotrophic bacteria for producing P(3HB) on the basis of biological engineering (30, 31). Recently, we first found that a facultatively methylotrophic bacterium, Paracoccus denitrificans, was able to synthesize the P(3HB-co-3HV) copolyester extremely rich in its 3HV unit (more than 98 mol%) in the presence of both methanol and n-amyl alcohol (34, 35).Genes responsible for PHA synthesis have been isolated and characterized from several bacteria (8,9,12,13,15,16,19,23,24,26,28,36). In Alcaligenes eutrophus, three enzymes, ␤-ketothiolase, acetoacetyl-coenzyme A (CoA) reductase, and PHA synthase, function to synthesize P(3HB) (15, 18). They are encoded by phaA, phaB, and phaC, respectively. Although these genes are closely located on a chromosomal DNA in A. eutrophus, their organization seems to be different in microorganisms. Recently, Valentin et al. isolated and characterized the PHA synthase gene of a methylotrophic bacterium, Methylobacterium extorquens (36), which was the first instance in which this was done for methylotrophic bacteria. However, very little is known regarding PHA synthesis at the molecular level. In this study, we report the characterization of the PHA synthase gene of P. denitrificans and the increase in synthesis of PHA by its recombinant strain. MATERIALS AND METHODSBacterial strains, plasmids,...

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans

et al. 1996

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“…However, the idea that one copy might be located extrachromosomally had to be rejected, because the strain used in our studies does not bear plasmids (data not shown). This is in contrast to the situation in P. denitrificans, where a second gene copy of the cytochrome c oxidase subunit I was reported to be located on a megaplasmid (57,58). Since the regulatory regions are identical and the transcription pattern is unambiguous (Fig.…”

Section: Discussionmentioning

confidence: 72%

The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization

et al. 1997

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A terminal quinol oxidase has been isolated from the plasma membrane of the crenarchaeon Acidianus ambivalens (DSM 3772) (formerly Desulfurolobus ambivalens), cloned, and sequenced. The detergent-solubilized complex oxidizes caldariella quinol at high rates and is completely inhibited by cyanide and by quinolone analogs, potent inhibitors of quinol oxidases. It is composed of at least five different subunits of 64.9, 38, 20.4, 18.8, and 7.2 kDa; their genes are located in two different operons. doxB, the gene for subunit I, is located together with doxC and two additional small open reading frames (doxE and doxF) in an operon with a complex transcription pattern. Two other genes of the oxidase complex (doxD and doxA) are located in a different operon and are cotranscribed into a common 1.2-kb mRNA. Both operons exist in duplicate on the genome of A. ambivalens. Only subunit I exhibits clear homology to other members of the superfamily of respiratory heme-copper oxidases; however, it reveals 14 transmembrane helices. In contrast, the composition of the accessory proteins is highly unusual; none is homologous to any known accessory protein of cytochrome oxidases, nor do homologs exist in the databases. DoxA is classified as a subunit II equivalent only by analogy of molecular size and hydrophobicity pattern to corresponding polypeptides of other oxidases. Multiple alignments and phylogenetic analysis of the heme-bearing subunit I (DoxB) locate this oxidase at the bottom of the phylogenetic tree, in the branch of heme-copper oxidases recently suggested to be incapable of superstoichiometric proton pumping. This finding is corroborated by lack of the essential amino acid residues delineating the putative H ؉ -pumping channel. It is therefore concluded that A. ambivalens copes with its strongly acidic environment simply by an extreme turnover of its terminal oxidase, generating a proton gradient only by chemical charge separation.

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“…Analysis of this sequence revealed three open reading frames (ORFs) designated ORFA, ccdA, and ORFB. These ORFs revealed coding characteristics according to codon preference analysis (50). ORFA was separated from ccdA by 48 nucleotides.…”

Section: Resultsmentioning

confidence: 99%

Identification of ccdA in Paracoccus pantotrophus GB17: Disruption of ccdA Causes Complete Deficiency in c -Type Cytochromes

Bardischewsky

Friedrich

2001

J Bacteriol

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A transposon Tn5-mob insertional mutant of Paracoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently. ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn5-mob was inserted into ccdA. The deduced ORFA product showed no similarity to any protein in databases. However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of P. pantotrophus is 42% identical to a protein predicted by ORF2, and its location within the sox gene cluster coding for lithotrophic sulfur oxidation suggested a different function.The neutrophilic facultatively lithoautotrophic gram-negative bacterium Paracoccus pantotrophus grows aerobically with a large variety of carbon sources and with molecular hydrogen or thiosulfate as energy source, and nitrate serves as electron acceptor under anaerobic conditions (41). P. pantotrophus (formerly Paracoccus denitrificans and Thiosphaera pantotropha [31,38,41]) has a branched electron transfer network. The electron flux is regulated by the reduction of the Q pool (33). While the transfer of electrons by the terminal cytochrome oxidases aa 3 and cbb 3 results in a translocation of six protons (6H ϩ /2e Ϫ ) (36, 54), the reduction of a terminal electron acceptor by the quinol oxidase o results in a translocation of only four protons (4H ϩ /2e Ϫ ) (39). To identify the components involved in energy transformation by oxidation of reduced inorganic sulfur compounds (Sox) of P. pantotrophus GB17, genetic studies were initiated by the isolation of mutants. Transpositional mutagenesis with Tn5-

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Genetics of Paracoccus denitrificans

Cited by 45 publications

References 240 publications

Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans

Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans

The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization

Identification of ccdA in Paracoccus pantotrophus GB17: Disruption of ccdA Causes Complete Deficiency in c -Type Cytochromes

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