Pseudomonas Hydrogenothermophila Research Articles

Structure of Cytochrome c552 from a Moderate Thermophilic Bacterium, Hydrogenophilus thermoluteolus: Comparative Study on the Thermostability of Cytochrome c

We have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; Pseudomonas aeruginosa (PAc(551)) growing at 37 degrees C and Hydrogenobacter thermophilus (HTc(552)) at 72 degrees C and showed that only five residues primarily differentiate their stabilities. For a more comprehensive study, we found Hydrogenophilus thermoluteolus (Pseudomonas hydrogenothermophila) growing at 52 degrees C and showed the moderate stability of the cytochrome c from this bacterium (PHc(552)). To explore the stabilization mechanisms, the crystal structure of PHc(552) was determined by X-ray analysis. The solution structure of HTc(552) elucidated previously by NMR was refined using distributed computational implementation. Furthermore, the recently reported crystal structure of HTc(552) has become available [Travaglini-Allocatelli, C. et al. (2005) J. Biol. Chem. 280, 25729-25734]. When the structures of these three cytochromes c were combined, this revealed that the five residues, corresponding to those mentioned above, determine the difference of stabilities among them as well. These facts suggested the stabilization mechanisms as follows: (1) improved van der Waals interactions by packing optimization at the N-terminal helix, (2) attractive electrostatic interactions with the heme propionate group, and (3) favorable van der Waals interaction with the heme. This comparative study, by supplementing the structural information of PHc(552) with its complementary feature, demonstrates that just a small number of amino acid residues determine the overall molecular stability by means of additivity of the effects of their substitutions. It is interesting that, in naturally occurring proteins, these adaptation strategies are accommodated by these bacteria to survive in the wide range of thermal conditions.

Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase gene cluster from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus, and phylogeny of the fructose 1,6-bisphosphate aldolase encoded by cbbA in the cluster.

Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila). cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria. cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coli without cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteolus was in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.

Hydrogenophilus thermoluteolus gen. nov., sp. nov., a thermophilic, facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium.

The taxonomic positions of 'Pseudomonas hydrogenothermophila' strain TH-1 and 'Flavobacterium autothermophilum' strain TH-4 were studied by 16S rDNA sequencing. These organisms are Gram-negative, strictly aerobic, thermophilic, facultatively chemolithoautotrophic hydrogen-oxidizing rods and have a DNA G + C content of 63-65 mol%. The major isoprenoid quinone is ubiquinone-8 and 3-hydroxy decanoic acid (3-OH C10:0) is the major 3-hydroxy cellular fatty acid. A phylogenetic analysis based on 16S rDNA sequences placed strains TH-1T and TH-4 in the beta-subclass of the Proteobacteria. The taxonomic characteristics of these organisms are different from those of previously described aerobic, facultatively chemolithoautotrophic, hydrogen-oxidizing bacteria that belong to the beta-subclass of Proteobacteria. On the basis of the information described above, a new genus and species, Hydrogenophilus thermoluteolus gen. nov., sp. nov., is described to include both strains. The type strain is strain TH-1T (= IFO 14978T).

The nirQ gene, which is required for denitrification of Pseudomonas aeruginosa, can activate the RubisCO from Pseudomonas hydrogenothermophila

Two putative ATP-binding proteins encoded in the gene cluster for the Calvin cycle of Pseudomonas hydrogenothermophila ( cbbQ) and for the denitrification of Pseudomonas aeruginosa ( nirQ) have been found to be similar. The cbbQ gene has been shown to activate the RubisCO from P. hydrogenothermophila in E. coli. The nirQ was functionally substituted for cbbQ. The nirQ gene restored the anaerobic growth and the NOR activity of the nirQOP mutant of P. aeruginosa, while the cbbQ gene did not.

Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans.

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

Genes encoding RubisCO in Pseudomonas hydrogenothermophila are followed by a novel cbbQ gene similar to nirQ of the denitrification gene cluster from Pseudomonas species

The cbbL and cbbS genes, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), were cloned and sequenced from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The cbbL gene encoded a 474-amino-acid (aa) protein (53 285 Da); cbbS encoded a 124-aa protein (14656 Da). An ORF found downstream from the cbbLS genes encoded a 267-aa protein (29 565 Da) which had no similarity to cbbX located downstream from cbbLS from Alcaligenes eutrophus and Xanthobacter flavus. This gene, called cbbQ, was highly similar to the nirQ gene of the denitrification gene cluster from P. aeruginosa and P. stutzeri.

Purification and characterization of membrane-bound hydrogenase from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1.

A membrane-bound hydrogenase was purified aerobically by one step using a hydroxyapatite column after solubilization by acetone treatment from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The enzyme consists of two polypeptides of 63 and 31 kDa, respectively. The amino-terminal amino acid sequences of both subunits were homologous to membrane-bound type [Ni-Fe] hydrogenases from other origins. The thermostability under a hydrogen gas atmosphere is highly stable at 50 degrees C, which is the optimum temperature for the cell growth.

Purification and some properties of ribulose-1,5-bisphosphate carboxylase/oxygenase from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1

Abstract Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TII-1. The enzyme was an L 8 S 8 -type hexadecamer with a molecular mass of 560 kDa. The specific activity of the purified enzyme was about 6.5 μmol/mg and stable even by heating at 60°C for 30 min. The K m values for RuBP, CO 2 , and Mg ++ were 1.03 μM, 0.086 mM and 0.92 mM, respectively.

Electron Transport System in Pseudomonas hydrogenothermophila Strain TH-1, a Thermophilic Hydrogen-oxidizing Bacterium.

The electron transport system in Pseudomonas hydrogenothermophila strain TH-1, a thermophilic, aerobic hydrogen-oxidizing bacterium, was studied using a membrane fraction, an ubiquinone-depleted membrane fraction (Q(-) MF), and an ubiqunone-reconstituted membrane fraction (Q(+) MF). From spectrophotometric and TLC analyses, cytochromes b and c, ubiquinone-8 (Q8), and cytochrome o were detected as respiratory components in the membrane fraction of P. hydrogenothermophila. Spectrophotometric analyses also showed that reduction of all the components in the membrane fraction depends on a membrane-bound hydrogenase reaction. In Q(-) MF, reduction of cytochromes b and c depending on membrane-bound hydrogenase reaction could not be observed. However, the reduction was restored in Q(+) MF. From these results, a possible electron transport system in P. hydrogenothermophila strain TH-1 is proposed.

Distribution of hydroxypolyamines, amino-propylcadaverine, and spermine in thermophilic, hydrogen-oxidizing pseudomonads.

A slightly thermophilic hydrogen bacterium, Pseudomonas hydrogenothermophila, was found to contain 2-hydroxyputrescine, 2-hydroxyspermidine, diaminopropane, putrescine, cadaverine, spermidine, aminopropylcadaverine and spermine. A mesophilic hydrogen-oxidizing pseudomonad, Pseudomonas hydrogenovora contained putrescine, 2-hydroxyputrescine, and spermidine. The polyamine distribution pattern of the latter is rather common in the beta subclass of Proteobacteria. The occurrence of 2-hydroxyspermidine, aminopropylcadaverine and spermine is unique in the thermophile.

Excretion of L-tryptophan by analogue-resistant mutants of Pseudomonas hydrogenothermophila TH-1 in autotrophic cultures.

Cells of a thermophilic hydrogen bacterium, Pseudomonas hydrogenothermophila TH-1 were treated with N-methyl-N′-nitro-N-nitrosoguanidine and resulting mutants resistant to tryptophan analogues were selected under autotrophic culture conditions (energy source, H2; carbon source, CO2). A mutant strain, 7922, which was resistant to 2000 µg/ml of 5-methyltryptophan and 200–500 µg/ml of 5-fluorotryptophan, was obtained by two step mutations. This mutant excreted 38–70 µg/ml of tryptophan into flask culture broth and a maximum of 200 µg/ml into jar fermentor broth.

Growth and Taxonomy of Thermophilic Hydrogen Bacteria

Growth characteristics of thermophilic hydrogen bacteria TH–1 and TH–4 were exarnined and taxonomic studies of these organisms were performed. Both strains assimilated various organic acids but not the tested sugars. Autotrophic growth utilizing hydrogen, oxygen and carbon dioxide was more rapid than heterotrophic growth on organic acid media. With pyruvate as the sole carbon source, TH–1 grew more rapidly under oxy-hydrogen atmosphere (H2: O2=4: 1) than in air. Neither TH-1 nor TH–4 fit the description of any previously described species; and they are thus proposed as new species, Pseudomonas hydrogenothermophila nov. sp. Goto, Kodama and Minoda and Flavobacterium autothermophilum nov. sp. Goto, Kodama and Minoda, respectively.