Hydrogen-oxidizing Bacterium Research Articles

Recent advances in the knowledge of the metabolism of bacterial polyhydroxyalkanoic acids and potential impacts on the production of biodegradable thermoplastics

AbstractBacteria are able to synthesize a wide variety of different thermoplastic polyhydroxyalkanoic acids (PHA). The physiology of PHA metabolism has been studied in most detail in the aerobic hydrogen bacterium Alcaligenes eutrophus. Strains of this bacterium are already used for the production of biodegradable polyesters on a technological scale. The current knowledge on the pathways for PHA synthesis, on the genes essential for PHA biosynthesis and on genes affecting the accumulation of PHA in this bacterium will be reviewed.

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.

Hydrogenobacter thermophilus: its unusual physiological properties and phylogenic position in the microbial world

Hydrogenobacter thermophilus (IAM12695) is an obligately autotrophic, thermophilic and aerobic hydrogen-oxidizing bacterium. Chemotaxonomic studies revealed various unusual features of this microorganism. H. thermophilus operates a reductive TCA cycle, which is the only confirmed example of the operation of a non-Calvin type carbon dioxide fixation pathway among aerobic organisms. Isolation of strains similar to Hydrogenobacter from different areas suggests a possible role of these microorganisms as primary producers of organic compounds from carbon dioxide in geothermal and/or aquatic environments.

Hydrogenobacter thermophilus: its unusual physiological properties and phylogenic position in the microbial world

Hydrogenobacter thermophilus (IAM12695) is an obligately autotrophic, thermophilic and aerobic hydrogen-oxidizing bacterium. Chemotaxonomic studies revealed various unusual features of this microorganism. H. thermophilus operates a reductive TCA cycle, which is the only confirmed example of the operation of a non-Calvin type carbon dioxide fixation pathway among aerobic organisms. Isolation of strains similar to Hydrogenobacter from different areas suggests a possible role of these microorganisms as primary producers of organic compounds from carbon dioxide in geothermal and/or aquatic environments.

Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta.

The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas phi. Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.

Polyamines of carbon monoxide-utilizing bacteria, Pseudomonas thermocarboxydovorans and Pseudomonas carboxydohydrogena

1. Summary A slightly thermophilic, CO-utilizing pseudomonad, Pseudomonas thermocarboxydovorans grown under both autotrophic and heterotrophic growth conditions was found to contain 2-hydroxyspermidine and 2-hydroxyputrescine in addition to putrescine, diaminopropane and spermidine. A mesophilic CO — utilzing hydrogen bacterium, Pseudomonas carboxydohydrogena contained putrescine and homospermidine under both autotrophic and heterotrophic growth conditions. Although these two carboxydobacteria are classified to the same genus Pseudomonas , the difference in their polyamine distribution patterns suggests that they may belong to different subclasses of Proteobacteria .

Nickel resistance of Alcaligenes denitrificans strain 4a-2 is chromosomally coded

A newly isolated aerobic hydrogen-oxidizing bacterium, Alcaligenes denitrificans strain 4a-2, differs from related autotrophic bacteria by containing only a single cytoplasmic, NAD-reducing hydrogenase, and by its high resistance to nickel ions, i.e. tolerance to 20 mM NiCl2. Strain 4a-2 harbors a single plasmid of about 250 kb. On helper-assisted mating of 4a-2 with Alcaligenes eutrophus strains H16,G29, and M85 nickelresistant transconjugants were selected; these did not contain the donor plasmid, however. All three transconjugants tolerated 3 to 10 mM NiCl2. The resistance was constitutively expressed. DNA/DNA hybridization showed homology with EcoRI-digested DNA of the wild type 4a-2 and transconjugants using a DNA probe containing nickel resistance genes of pMOL28. This indicated that the 4a-2 nickel resistance genes are located on the chromosome.

Effects of alkyltin compounds on hydrogen-oxidizing anaerobic bacteria

Methyl and butyltin compounds were inhibitory to all anaerobes examined, but there were great variations, depending on the specific alkyltin and bacterium. The methanogens were inhibited more strongly by methyl than by butyl derivatives; more than 50% inhibition occurred with 0.025–0.5 mM of the methyltins, whereas 0.16–1.8 mM butyltins were needed for the same level of inhibition; tri-butyltin was the least toxic.Methanosarcina barkeri was, in general, more resistant than theMethanococcus sp. andMethanobacterium bryantii. TheDesulfovibrio were more strongly inhibited by mono-methyltin than by di- and tri-methyl derivatives; butyltins were, in general, not so toxic. Mono-methyltin at 0.15 mM almost completely inhibited three of the sulfate reducers, butDesulfovibrio thermophilus required 0.7 mM for this level of inhibition. Tri-butyltin at 1.8 mM did not cause major inhibition, whereas mono- and di-butyltins were more inhibitory.Acetobacterium woodii was most affected by mono- and dimethyltins, and least by tri-methyltin and mono-butyltin. In contrast,Wollinella succinogenes was most affected by tri-methyltin. This study suggests several major groups of anaerobes thought to be involved in metal biocorrosion vary greatly in their response to alkyltins; most interesting is the relative insensitivity by methanogens and sulfate reducers to tri-butyltin, which is a major component in commercial antifouling paints. Our results differ considerably from those reported for aerobic microorganisms, which were found to be most affected by tributyltin.

Polypeptide synthesis on ribosomes of an extreme thermophilic hydrogen bacterium Calderobacterium hydrogenophilum

Ribosomes of the extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum interact with a broad spectrum of polyamines. In the absence of polyamines and at 70°C close to the growth optimum (75°C), high salt washed ribosomes lost their activity in the poly(UG)-directed polypeptide synthesis. At 70°C the in vitro system synthesized the polypeptide in the presence of spermidine, spermine or natural polyamines (tri-pentaamines) isolated from ribosomal extracts but not in the presence of putrescine. The activity of ribosomes was affected by a number of antibiotics interfering with functions of typical eubacterial 70S, such as tetracyclines, lincomycin, chloramphenicol and erythromycin. However, the ribosomes were relatively resistant to streptomycin and insensitive to 80S inhibitors, such as ricin and cycloheximide. The 30S and 50S subunits have structural features typical of eubacterial ribosomes.

A new isolate of Hydrogenobacter, an obligately chemolithoautotrophic, thermophilic, halophilic and aerobic hydrogen-oxidizing bacterium from seaside saline hot spring

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 μm. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl7-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C18:0, C18:1 and C20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.

Hydrogen-oxidizing bacteria for biomass production

The conversion of molecular hydrogen into microbial biomass using hydrogen-oxidizing bacteria, cultivated in the presence of mineral nutrients and H 2 + CO 2 as energy and carbon source, has been examined. The transformation of solar energy into electric power by a photovoltaic device and the production of molecular hydrogen from water by electrolysis was considered. The technical recovery of the process should attain 0.02, which gains over the photosynthetic efficiency expressed by the more valuable plants in the better environmental conditions.

DNA topoisomerase I from Alcaligenes eutrophus H16

Molecular and functional properties of DNA topoisomerase I isolated from a hydrogen-oxidizing bacterium, Alcaligenes eutrophus H16, were investigated. Under native conditions the enzyme forms a monomer with a relative molar mass of 98,500. A rod-like shape of the molecule was derived from the calculated frictional coefficient. The isoelectric point of the enzyme was determined to be in the range of 7.6-8.0. The enzyme activity is strictly Mg2+ dependent with an optimum at 3 mM Mg2+. The pH optimum ranges within 7.5-9.0. A. eutrophus DNA topoisomerase I activity is inhibited by M13 ssDNA, high ionic strength, polyamines, heparin and by a number of intercalating drugs.

Thermostability of cytochrome c-552 from the thermophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus

The denaturation of the c-type cytochrome of the thermophilic bacterium Hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra. The melting temperature (T1/2) of cytochrome c-552 in the presence of 1.5 M guanidine hydrochloride was 34 degrees C higher than that of the c-type cytochrome of Pseudomonas aeruginosa cytochrome c-551. Hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrome c-551 of the mesophilic bacterium P. aeruginosa, even though their amino acid sequences are 56% identical and they have numerous other similarities. However, notwithstanding these similarities between the sequences of the cytochromes c-552 and c-551 that were compared, it is very likely that these differences in stability could be due to some heretofore undefined differences in their spatial structures. It has been suggested that alpha-helix structure and electrostatic interaction could be the source of the stable spatial structure of cytochrome c-552.

Comparative studies of ribulose-1,5-biphosphate carboxylase/oxygenase from Alcaligenes eutrophusH16 cells, in the active and CABP-inhibited forms

RuBisCO ( d-ribulose-1,5-biphosphate carboxylase/oxygenase; EC 4.1.1.39) has been isolated from the autotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus H16. Combining photon correlation and sedimentation analysis transport parameters of the enzyme were investigated in the active, (E · CO 2.Mg 2+) as a ternary complex, and inactive state, (E · CO 2.Mg 2+ · CABP) as a quaternary complex, where RuBisCO is complexed with the transition state analogue CABP (2- C-carboxy- d-arabinitol-1,5-biphosphate). Within experimental error, no difference has been detected between the diffusion and sedimentation coefficients ( D 20, w 0 = 2.72(±0.07) × 10 −7 cm 2 s −1, s 20, w 0 = 17.8(±0.5)S) of active and CABP-complexed enzyme thus leading to the conclusion that the molecule, at least in solution, does not assume a different conformation when complexed with CABP.

Isolation of an obligately chemolithoautotrophic, halophilic and aerobic hydrogen-oxidizing bacterium from marine environment

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from seawater of the Shonan Coast, Kanagawa Pref., Japan. The isolate was a Gram-negative, comma-shaped rod cell measuring 0.2 to 0.5 by 1 to 2 μm. The cells occurred singly and were motile by a polar flagellum. The deoxyribonucleic acid base composition of the isolate was 44.1 mol% guanine plus cytosine. The optimal temperature for autotrophic growth on H2−O2−CO2 was around 37°C, and no growth was observed at 5° C or 45° C. The optimal pH for growth was around 6.5. NaCl was required for growth with an optimum of 0.5 M. Elemental sulfur, thiosulfate or tetrathionate was utilized, as well as molecular hydrogen, as the sole energy source. No heterotrophic growth was observed on organic media tested. To our knowledge, this is the first report of the isolation of a marine, aerobic hydrogen-oxidizing bacterium, and of an obligately chemolithoautotrophic, mesophilic and aerobic hydrogen-oxidizing bacterium.

Comparison of the NH2‐terminal amino acid sequences of the four non‐identical subunits of the NAD‐linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.

Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria

Polyclonal, monospecific antibodies were produced against the two subunits ( M r 62 000, and M r 31 000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodif usion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits ( M r 63 000, 56 000, 30 000 and 26 000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H 2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All These proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration of three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH 2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH 2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.

Utilization of cathodic hydrogen by hydrogen-oxidizing bacteria

Hydrogen is consumed by methanogenic, sulphate-reducing, and homoacetogenic bacteria and members of these bacterial groups are able to grow chemolithotrophically with hydrogen as sole energy source. Cathodic hydrogen consumption by sulphate-reducing bacteria has been proposed as one of the factors in the anaerobic corrosion of metals. Desulfovibrio spp. were able to utilize cathodic hydrogen from mild steel as the only source of energy for growth with sulphate or nitrate as terminal electron acceptor. Other hydrogen-oxidizing bacteria such as Methanospirillum hungatei, Acetobacterium woodii and Wolinella succinogenes were also able to utilize cathodic hydrogen from mild steel for energy generation and growth. Weight loss studies of mild steel coupons under different growth conditions of Desulfovibrio spp. indicated that hydrogen removal alone is not the cause of corrosion and the depolarization phenomenon probably plays a role only in the initiation of the anaerobic microbial corrosion process.

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.

Transmissibility of hydrogen oxidation (Hox) plasmid from Alcaligenes hydrogenophilus

The transmissibility of the Hox plasmid pHG21-a, transferred from Alcaligenes hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4 by conjugation, was examined in each recipient. Hox plasmid pHG21-a behaved as a self-transmissible plasmid in P. oxalaticus OX4 as in A. hydrogenophilus, but in P. oxalaticus OX1, it behaved as nonself-transmissible one, being mobilized by R-plasmids. It has been argued whether Hox plasmids of hydrogen-oxidizing bacteria are self-transmissible or nonself-transmissible, but our results showed that the transmissibility of Hox plasmid pHG21-a varied with the host.