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

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.     

Amino acid sequence of Synechocystis 6714 ferredoxin: a unique structural feature of unicellular blue-green algal ferredoxin

The amino acid sequence of ferredoxin from Synechocystis 6714, a unicellular blue-green alga, was determined by a combination of conventional methods. The ferredoxin was composed of 96 amino acid residues and lacked methionine and tryptophan. The sequence was as follows: Ala-Ser-Tyr-Thr-Val-Lys-Leu-Ile-Thr- Pro-Asp-Gly-Glu-Asn-Ser-Ile-Glu-Cys-Ser-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala- Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys- Ala-Gly-Lys-Ile-Thr-Ala-Gly-Ser-Val-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp- Asp-Gln-Ile-Glu-Ala-Gly-Tyr-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Glu-Thr-His-Lys-Glu-Glu-Asp-Leu-Tyr. In an alignment of various ferredoxins with high homology from unicellular and filamentous blue-green algae, Synechocystis 6714 ferredoxin showed 4 gaps. Those between residues 9 and 10 and between residues 12 and 13 were unique for the ferredoxins from the unicellular algae Synechocystis 6714 and Aphanothece sacrum (ferredoxin I). Therefore, ferredoxins from unicellular algae were distinguishable from those of filamentous algae in terms of the presence of gaps. This feature appears to coincide with the phylogenetic division between the two types of blue-green algae.     

Ferredoxin from Aphanothece halophitica, a unicellular blue-green alga: close relationship to ferredoxins from filamentous blue-green algae and phylogenetic implications

The amino acid sequence of a ferredoxin from a unicellular blue-green alga, Aphanothece halophitica, was established by the conventional methods. Total number of residues was 98 lacking only tryptophan. A most probable phylogenetic tree was constructed for 19 algal ferredoxins on the basis of an amino acid difference matrix made from the sequence comparison. A. halophitica has been classified as a unicellular blue-green alga in the same genus to which Aphanothece sacrum belongs, but the tree indicates A. halophitica ferredoxin to be very close to those of the members of filamentous blue-green algae. The tree divides prokaryotic and eukaryotic algal ferredoxins into several groups, suggesting that the ferredoxin phylogenetic tree reflects the evolutionary trails of various algae, which is also reflected in the structural characteristics, particularly in the presence of gaps. Other notable features are presented in considering algal taxonomy.     

Isolation and characterization of the gene encoding the chloroplast-type ferredoxin component of carbazole 1,9a-dioxygenase from a putative Kordiimonas sp.

Carbazole (CAR)-degrading genes (carRAaCBaBb) were isolated from marine CAR-degrading isolate strain OC9 (probably Kordiimonas gwangyangensis) using shotgun cloning experiments and showed 35–65% similarity with previously reported CAR-degrading genes. In addition, a ferredoxin-like gene (carAc) was found downstream of carR, although it was not homologous with any reported ferredoxin components of the CAR 1,9a-dioxygenase (CARDO) system. The carAc-deduced amino acid sequence possessed consensus sequences for chloroplast-type iron-sulfur proteins for binding the [2Fe-2S] cluster. These car genes were arranged in the order of carAcRAaCBaBb, but carRAc and carAaCBaBb genes were the opposite orientation. Escherichia coli JM109 cells harboring pBOC91 (carAa) converted CAR to 2′-aminobiphenyl-2,3-diol at a ratio of 12%, and the transformation ratio of CAR increased from 12 to 100% when carAc was added, indicating that CarAc is the ferredoxin component of the CARDO system in strain OC9. This is the first finding of a chloroplast-type ferredoxin component in a CARDO system. Biotransformation tests with aromatic compounds revealed that the strain OC9 CarAaAc showed activity with polycyclic aromatic hydrocarbons and dioxin compounds and exhibited significant activity for fluorene, unlike previously reported CARDOs.     

The involvement of a multicopper oxidase in iron uptake by the green algae Chlamydomonas reinhardtii

In the unicellular green algae Chlamydomonas reinhardtii, high-affinity uptake of iron (Fe) requires an Fe(3+)-chelate reductase and an Fe transporter. Neither of these proteins nor their corresponding genes have been isolated. We previously identified, by analysis of differentially expressed plasma membrane proteins, an approximately 150-kD protein whose synthesis was induced under conditions of Fe-deficient growth. Based on homology of internal peptide sequences to the multicopper oxidase hephaestin, this protein was proposed to be a ferroxidase. A nucleotide sequence to the full-length cDNA clone for this ferroxidase-like protein has been obtained. Analysis of the primary amino acid sequence revealed a putative transmembrane domain near the amino terminus of the protein and signature sequences for two multicopper oxidase I motifs and one multicopper oxidase II motif. The ferroxidase-like gene was transcribed under conditions of Fe deficiency. Consistent with the role of a copper (Cu)-containing protein in Fe homeostasis, growth of cells in Cu-depleted media eliminated high-affinity Fe uptake, and Cu-deficient cells that were grown in optimal Fe showed greatly reduced Fe accumulation compared with control, Cu-sufficient cells. Reapplication of Cu resulted in the recovery of Fe transport activity. Together, these results were consistent with the participation of a ferroxidase in high-affinity Fe uptake in C. reinhardtii.     

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE1

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form‐genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form‐genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form‐genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form‐genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form‐genus Cyanothece were observed to be in a close relationship with our isolates.     

Proteomic analysis of the response of an acidophilic strain of Chlamydomonas sp. (Chlorophyta) to natural metal‐rich water

A proteomic approach including 2‐DE and MALDI‐TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas sp. isolated from an extreme acidic environment, Río Tinto (southwest Spain). We have analyzed the soluble proteome obtained from whole cells growing on metal‐rich natural acidic water from the river in comparison with the same strain growing in artificial BG‐11 media. The most drastic effect was the decrease in the abundance of the ribulose‐1,5‐biphosphate carboxylase as well as other enzymes related to photosynthesis. However, phytochrome B, phosphoribulokinase, and phosphoglycerate kinase were upregulated when cells were grown in metal‐rich acidic water. Besides, increased accumulation of two Hsps, Hsp70 and Hsp90 as well as other stress‐related enzymes were also found in the cells growing in natural acidic water. These results suggest that naturally occurring metal‐rich water induces a stress response in acidophilic Chlamydomonas forcing algal cells to reorganize their metabolic pathways as an adaptive response to these environmental conditions.     

A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9

Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9. 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the alpha subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11, 12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the alpha subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

Primary structure of the two (4 Fe-4 S) clusters ferredoxin from Desulfovibrio desulfuricans (strain Norway 4)

The primary structure of a ferredoxin isolated from D. desulfuricans Norway strain, which we called ferredoxin II (Fd II) has been elucidated. This ferredoxin is a dimer constituted of two identical subunits of molecular weight 6000. In ferredoxin II two (4 Fe-4 S) centers are present per subunit instead of one (Fe-S) center as is the case for the other ferredoxins isolated from Desulfovibrio and for Fd I from the same organism. The comparison of amino-acid sequences shows that ferredoxin II presents more homologies with clostridial type ferredoxin than with the ferredoxins from D. gigas and D. africanus.     

Properties and structure of the soluble ferredoxin from Synechococcus 6301 (Anacystis nidulans). Relationship to gene sequences.

Photoautotrophic cultures of the unicellular cyanobacterium Synechococcus 6301 (Anacystis nidulans) possessed a single [2Fe-2S] ferredoxin with a midpoint redox potential of -385 mV. Determination of the amino acid sequence of the ferredoxin showed that it consisted of 98 residues, with methionine and tryptophan both absent, and with only the four cysteine residues that are required to co-ordinate the iron-sulphur cluster. Comparisons with other ferredoxin sequences showed that most resemblance was to those from filamentous cyanobacteria, with up to 87% homology. There was less resemblance to the ferredoxins of unicellular cyanobacteria, with 25 differences when compared with that from another Synechococcus sp. However, the sequence of Synechococcus 6301 ferredoxin was identical with that derived for a gene sequence for a putative ferredoxin from the genotypically closely related Synechococcus 7942 (Anacystis nidulans R2). In contrast, the sequence showed substantial differences from that corresponding to a putative ferredoxin gene from Synechococcus 6301 reported by Cozens & Walker [(1988) Biochem. J. 252, 563-569].     

Isolation and characterization of the unicellular diazotrophic cyanobacterium Group C TW3 from the tropical western Pacific Ocean

A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5-3.0 μm in width and 4.0-6.0 μm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h(-1) when cultured at 30°C and 140 μmol m(-2) s(-1) of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light-dark cycle, was 7.31 × 10(-15) mol N cell(-1) day(-1) . Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.     

Recombination of a 3-chlorobenzoate catabolic plasmid from Alcaligenes eutrophus NH9 mediated by direct repeat elements.

Alcaligenes eutrophus NH9 was isolated from soil. This strain can utilize 3-chlorobenzoate (3-CB) as a sole source of carbon and energy. Most of the 3-CB-negative segregants had lost one of the plasmids present in the parent strain. The genes for catabolism of 3-CB were located within a 9.2-kb SacI fragment of this plasmid (pENH91). The genes were found to hybridize with genes for components of the modified ortho cleavage pathway from Pseudomonas putida. In one of the 3-CB-negative segregants, the plasmid had undergone the deletion of a segment with a size of about 12.5 kb that covered the catabolic genes. The deletion event seemed to be the result of reciprocal recombination between two highly homologous sequences with sizes of 2.5 kb that were present as a direct repeat at the two ends of the region that included the catabolic genes. Nucleotide sequence analysis of homologous fragments revealed a structure that resembled an insertion sequence and relatedness to IS21. During repeated subculturing of NH9 on liquid media with 3-CB, the culture was taken over by a derivative strain (designated NH9A) in which the degradative plasmid carried a duplicate copy of the 12.5-kb region that contained the catabolic genes. The duplication of these genes seemed again to have been mediated by recombination between the direct repeat sequences.