Kazuhiro Nagahama

Construction and analysis of a recombinant cyanobacterium expressing a chromosomally inserted gene for an ethylene-forming enzyme at the psbAI locus

The coding sequence of a gene for a Pseudomonas syringae ethylene-forming enzyme was inserted at the psbAI locus in a cyanobacterium, Synechococcus elongatus PCC 7942 via rps12-mediated gene replacement. The recombinant strain photoautotrophically produced ethylene at 451 nl ml(-1) h(-1) OD730(-1), but showed a depressed specific growth rate as well as a yellow-green phenotype indicating a severe metabolic stress. The rate of ethylene production in the recombinant culture decreased as a result of competition with faster growing ethylene-non-forming mutants that carried short nucleotide insertions within the coding sequence of the gene for the ethylene-forming enzyme.

Two reactions are simultaneously catalyzed by a single enzyme: The arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae

A single enzyme isolated from Pseudomonas syringae pv. phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1. In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide. In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L-delta 1-pyrroline-5-carboxylate. We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2

The gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1. The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109. Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol. wt. 39,444). In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low. However, functionally significant regions are conserved.

Classification of ethylene-producing bacteria in terms of biosynthetic pathways to ethylene

Abstract Two hundred and twenty-nine ethylene-producing strains of bacteria were identified among 757 bacterial strains which included 13 strains of chemolithotrophs. The ethylene-producing bacetria were classified into three groups, namely, l -methionine-dependent, 2-ketoglutarate-dependent and meat extract-dependent, with reference to their respective biosynthetic pathways to ethylene. Two hundred and twenty-five l -methionine-dependent strains were obtained, while the only 2-ketoglutarate-dependent strain was Pseudomonas syringae pv. phaseolicola PK2. Three strains of chemolithotrophs had ethylene-forming capacity, and Thiobacillus novellus IFO 12443 had a novel ethylene-forming system which was dependent upon the addition of meat extract into the culture medium. The ethylene-forming systems of two of the strains of Thiobacillus ferrooxidans have not yet been characterized. Several strains of non-ethylene-producing bacteria failed to produce ethylene, even when l -methionine was added to the culture medium. We examined the causes of their lack of ethylene-producing ability and judged that these strains are either NADH:Fe(III)EDTA oxidoreductase-less or methionine-uptake activity-less.

Ethylene production by strains of the plant-pathogenic bacterium Pseudomonas syringae depends upon the presence of indigenous plasmids carrying homologous genes for the ethylene-forming enzyme.

The molecular characteristics of the ethylene-forming enzymes of strains of Pseudomonas syringae were tested. The ethylene-producing activities of the nine strains as measured in vivo and in vitro were similar, except for that of P. syringae pv. mori M5. A polyclonal antibody and a DNA probe for the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 were prepared to investigate homologies among the proteins and genes for the ethylene-forming enzymes. With the exception of P. syringae pv. mori M5, eight strains tested expressed the same antigen as the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 and were homologous to DNA sequences on indigenous plasmids. Molecular masses of antigenic proteins from all ethylene-producing strains were 40 kDa. The N-terminal amino acid sequence of the purified ethylene-forming enzyme from P. syringae pv. glycinea KN130 was identical to that of the enzyme from P. syringae pv. phaseolicola PK2. These results show that the ethylene-forming enzymes encoded by the indigenous plasmid(s) in the pathogenic bacteria examined were similar.

L-Arginine is essential for the formation in vitro of ethylene by an extract of Pseudomonas syringae

Summary: A system was developed for the formation of ethylene in vitro by an extract of Pseudomonas syringae pv. phaseolicola PK2. The ethylene-forming activity of a cell-free extract of this bacterium measured in a system reported previously was almost completely lost when the cell-free extract was dialysed against potassium phosphate buffer for 24 h at 4 °C. When the fraction of cell-free extract with a molecular mass < 10 kDa (SupI) was added back to the enzyme fraction after gel filtration of the cell-free extract, the enzymic activity increased to about four times that of the gel-filtered crude enzyme. The action of SupI could be reproduced by the addition of l-arginine. The complete system for the formation of ethylene under aerobic conditions in vitro required 0·25 mm-2-oxoglutarate, 0·2 mm-FeSO4, 2 mm-DTT, 10 mm-l-histidine and 0·2 mm-l-arginine. The cofactor specificity was examined by replacing l-arginine or l-histidine with various analogues, but none of them were effective. The components of this system, with the exception of l-histidine, were similar to those of a system derived from the ethylene-producing, plant pathogenic fungus Penicillium digitatum which also produced ethylene in vitro in a reaction dependent on 2-oxoglutarate. The intermediates in the ethylene-forming reaction are postulated and the roles of l-arginine and l-histidine in the formation of ethylene by Ps. syringae are discussed.

Heterologous expression of the gene for the ethylene-forming enzyme fromPseudomonas syringae in the cyanobacteriumSynechococcus

SummaryBioconversion of atmospheric carbon dioxide to ethylene was studied in a recombinant cyanobacterium. The gene for the ethylene-forming enzyme ofPseudomonas syringae pv.phaseolicola PK2 was cloned and expressed in the cyanobacteriumSynechococcus PCC7942 R2-SPc by use of a shuttle vector pUC303. The ethylene-forming activityin vivo ofSynechococcus PCC7942 R2-SPc that carried the gene for the ethylene-forming enzyme ofP. syringae pv.phaseolicola PK2 was one-fifth of that ofE. coli JM109 that harbored the same plasmid. The enzyme accounted for 0.021% by weight of the total soluble protein inSynechococcus PCC7942 R2-SPc.

Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission.

In a dimorphic yeast, Yarrowia lipolytica, replicative plasmids can be established only in the coexistence of the replication origin (ORI) and centromere (CEN) from its own chromosomal DNA. Although six CEN sequences so far isolated from this yeast exhibit no similarity with conventional CEN DNA elements from other budding yeasts, they are confined within short regions (approximately 0.2 kb) and contain various conserved sequence blocks. We surveyed here a CEN1-1 sequence on an ORI-containing plasmid by deletion and site-directed mutagenesis, and found a partial palindrome, CCTAATTTGG designated DS9, to be an essential element for high-efficiency transformation. In particular, point mutations that alter symmetry and/or length of the palindrome abrogated the activity of CEN1-1. Gel mobility shift assay of CEN1-1 DNA fragments incubated with Y. lipolytica nuclear proteins revealed four bands corresponding to protein-DNA complexes, whereas the mutations within DS9 that disabled transformation also abolished the formation of part of these complexes, depending on particular mutations. These results demonstrate that the palindrome is a binding site for specific protein(s) necessary for plasmid transmission in Y. lipolytica.

Establishment of a Pure Culture of the Hitherto Uncultured Unicellular Cyanobacterium Aphanothece sacrum, and Phylogenetic Position of the Organism

ABSTRACT Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells ∼6 μm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.

Site-Directed Mutagenesis of Histidine Residues in the Ethylene-Forming Enzyme from Pseudomonas syringae

Abstract The roles of histidine residues in the catalysis of the transformation of 2-oxoglutarate into ethylene via the ethylene-forming enzyme (EFE) from Pseudomonas syringae were studied using site-directed mutagenesis with substitution of glutamine for ten individual histidine residues. The mutant enzymes, which were expressed in Escherichia coli , were purified to homogeneity, and assayed in vitro for K m , k cat and thermostability. The relative k cat of two mutated EFEs, H305Q and H335Q, were 40% and 60%, respectively. However, a mutation at either His-189 or His-233 caused a total loss of activity, implying that these residues play important roles in the binding of iron. The k cat values for other mutant enzymes were 11-to 55-fold less than that for the wild-type enzyme. For six partially inactive mutated EFEs (but not for H305Q or H335Q), the first order rate constants for heat inactivation at 30°C were 11-to 24-fold higher than for the wild-type enzyme. It is noteworthy that the value of the first order rate constant for heat inactivation of H268Q was identical to that of H335Q. The substitution of H268 resulted in a drastic decrease of the k cat value (relative k cat was 1.8%). This suggests that the substitution at His-268 may cause the disruption of the active site of the EFE. Heat inactivation studies with the puridied mutant enzymes revealed that some mutant enzymes, such as H168Q and H116Q, were more thermolabile than the wild-type enzyme.