Molecular biology of hydrogen utilization in aerobic chemolithotrophs.

KEY WORDS: aerobic hydrogenotrophs, hydrogenase, nickel metalloproteins, hydrogenase genes, processing, regulation

CONTENTS

 
INTRODUCTION                                                   352 
PHYLOGENETIC DIVERSITY OF AEROBIC [H.sub.2]-OXIDIZING BACTERIA 352 
ECOLOGICAL AND PHYSIOLOGICAL SIGNIFICANCE OF [H.sub.2] 
OXIDATION                                                      353 
ENZYMOLOGY OF NICKEL-CONTAINING HYDROGENASES                   355 
  General Characteristics                                      355 
  Membrane-Bound Hydrogenases                                  356 
  The NAD-Reducing Multimeric Hydrogenases                     358 
ORGANIZATION OF HYDROGENASE GENES AND FUNCTION OF THE GENE 
        PRODUCTS                                               359 
  Plasmid-Linked Hydrogenase Genes                             359 
  Gene Nomenclature and Functional Units                       361 
  MBH Structural Genes                                         361 
  MBH Accessory Genes                                          366 
  SH Genes                                                     366 
BIOSYNTHESIS AND MATURATION                                    368 
  Nickel Uptake                                                368 
  Nickel Incorporation and Processing                          369 
REGULATION OF HYDROGENASE GENE EXPRESSION                      371 
RETROSPECT AND CONCLUDING REMARKS                              375 

The aerobic bacteria capable of obtaining energy from the oxidation of [H.sub.2] form a heterogenous group that includes both facultative and obligate chemolithotrophs and representatives of both gram-negative and gram-positive genera. [H.sub.2]-oxidizing aerobes inhabit such diverse biotypes as soil, oceans, and hot springs. The oxidation of [H.sub.2] in these bacteria is catalyzed by [NiFe] metalloenzymes called hydrogenases. The hydrogenases studied so far belong to two families: dimeric, membrane-bound enzymes (MBH) coupled to electron transport chains and tetrameric, cytoplasmic NAD-reducing enzymes (SH). N[i.sup.2+] is an essential component of the active site contained in the large subunit of the MBH enzymes. The genes for the MBH enzymes are located in conserved clusters of accessory genes, some of which encode maturation functions and hydrogenase-related redox proteins. Maturation of both types of hydrogenase is apparently complex, involving specific nickel incorporation and proteolytic processing steps. In Alcaligenes eutrophus and Rhodobacter capsulatus, hydrogenase expression is regulated by transcriptional activators belonging to the response-regulator family.

INTRODUCTION

More than 60 years ago Stephenson & Stickland (152) discovered that bacteria can both evolve [H.sub.2] and utilize it to reduce artificial and physiological substrates. In the 1970s considerable effort was devoted to the purification and biochemical characterization of hydrogenase enzymes. Progress was also made toward an understanding of the complex physiological roles of hydrogenases. Three major achievements mark the development of hydrogenase research in the past decade: (a) the establishment of genetic systems for many [H.sub.2]-oxidizing bacteria, which opened avenues for molecular studies; (b) the discovery that nickel is an essential constituent of many hydrogenases; and (c) the isolation of novel [H.sub.2] oxidizers among the aerobic hyperthermophiles, obligate lithoautotrophs, and archaebacterial species. A comprehensive treatment of the aerobic chemolithotrophs is beyond the scope of this review. Instead this survey focuses on Alcaligenes eutrophus, the most extensively studied representative of the true chemolithotrophs. It reviews the physiology, biochemistry, genetics, and molecular biology of [H.sub.2] oxidation in this bacterium and discusses the related [H.sub.2]-oxidizing systems of representatives of the aerobic photosynthetic and diazotrophic bacteria. The reader is referred to recent reviews (1)(2)(12)(41)(42)(45)(46)(101)(113)(130)(131)(145) (146)(162)(163) on related subjects for additional information.

PHYLOGENETIC DIVERSITY OF AEROBIC [H.sub.2]-OXIDIZING BACTERIA

The coupling of [H.sub.2] oxidation to the reduction of molecular [O.sub.2], catalyzed by the enzyme hydrogenase, is found in representations of taxonomically diverse groups of aerobic bacteria, in the so-called hydrogen (knallgas) bacteria (4) (5)(12), in [N.sub.2]-fixing bacteria (41), and in photosynthetic microorganisms (162). Most of these bacteria are mesophiles. A few species, including Bacillus schlegelii, Bacillus tusciae, Calderobacterium hydrogenophilum, Hydrogenobacter thermophilus (4), and the novel isolate Streptomyces thermoautotrophicus (59), are thermophiles. [H.sub.2] oxidizers have been isolated from marine environments. The latter organisms include a mesophilic obligately lithoautotrophic halophile, a knallgas bacterium from trophosome tissue of the hyperthermal-vent animal Riftia (4), and the hyperthermophile Aquifex pyrophilus. 16S rRNA sequence analysis showed that the latter is the deepest phylogenetic branch off of the eubacterial domain (72).

The group of aerobic [H.sub.2]-oxidizing bacteria is physiologically defined and includes species belonging to 5 gram-positive and more than 15 gram-negative genera. Most of the latter are representatives of the [alpha] and [beta] subdivisions of the proteobacteria (5). Most [H.sub.2]-oxidizing organisms have high G+C genomes (>60 mol%). The highest and lowest values are found in thermophiles (4) (59).

Phototrophic bacteria such as Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) are included in this survey because some species can utilize [H.sub.2] for chemolithoautotrophic growth in the dark at decreased [O.sub.2] tension (100). Chemolithoautotrophic growth with [H.sub.2] as the energy source has been reported for aerobic free-living [N.sub.2]-fixing bacteria, some of which were initially isolated as hydrogen bacteria (5). A few strains of Bradyrhizobium japonicum (formerly Rhizobium japonicum) oxidize [H.sub.2] during symbiotic [N.sub.2] fixation, and in the free-living state they grow as chemolithoautotrophs utilizing [H.sub.2] and C[O.sub.2] as the sole sources of energy and carbon, respectively (41). [H.sub.2]-oxidizing activity has also been found in symbiotic cells of certain strains of Rhizobium, e.g. Rhizobium leguminosarum (28)(41) and in the obligately aerobic [N.sub.2]-fixing Azotobacter vinelandii (73) and Azotobacter chroococcum (164). In contrast to the classical [H.sub.2]-oxidizing bacteria, strains of the latter two groups do not fix C[O.sub.2] autotrophically (5), and [H.sub.2] appears to be utilized as a supplementary energy source. Thus, although Rhizobium and Azotobacter spp. do not represent chemolithotrophs sensu stricto, they are included in this survey because detailed molecular analyses indicate that their [H.sub.2] metabolism is closely related to that of the chemolithoautotrophs.

ECOLOGIGAL AND PHYSIOLOGICAL SIGNIFICANCE OF [H.sub.2] OXIDATION

The geothermal habitats of the thermophilic [H.sub.2]-oxidizing bacteria provide little organic substance but are often rich in [H.sub.2] and reduced sulfur compounds. Such environments offer appropriate conditions for obligate lithoautotrophs (145). Hydrogenobacter thermophilus was the first obligately chemolithoautotrophic aerobic [H.sub.2]-oxidizing bacterium reported in the literature (81). Recently, another representative, Streptomyces thermoautotrophicus, was described (59). Thiobacillus ferrooxidans may also belong to this group, because [H.sub.2] autotrophy has been demonstrated in some strains of this acidophile (32). [H.sub.2] is not the only energy source used by obligate lithoautotrophs: H. thermophilus can oxidize thiosulfate (9), S. thermoautotrophicus uses CO (59), and T. ferrooxidans is well known for its ability to grow on sulfur compounds, ferrous iron, or sulfidic ores (82). The newly isolated Thiobacillus plumbophilus was shown to oxidize [H.sub.2] in addition to PbS and [H.sub.2]S (31).

The habitat of the majority of facultatively [H.sub.2]-oxidizing bacteria is less clearly defined. They are ubiquitously distributed in soil and water. The [H.sub.2] concentration in these biotopes is fairly low, because only traces of the anaerobically produced [H.sub.2] escape to oxyc environments (145). Of the total flux of [H.sub.2] formation, up to 5% is released as a byproduct of [N.sub.2] fixation (26) (41) and should be available to aerobic bacteria. The major sink of [H.sub.2] is the soil, where most of the [H.sub.2] is oxidized by microorganisms. In light of the high threshold concentration of [H.sub.2] (varying between 1.5 and 178 ppm) and the low affinity for the substrate (apparent [K.sub.m] 1--60 [micro]M [H.sub.2]) (25)(146), it is not clear to what extent hydrogen bacteria participate in the [H.sub.2] cycle. Nevertheless, [H.sub.2] autotrophy does provide an alternative mode of nutrition for the facultative chemolithoautotrophs (145). In Alcaligenes eutrophus, the [H.sub.2]-oxidizing enzyme system is derepressed during substrate limitation, supporting the notion that [H.sub.2] is utilized as a supplementary source of energy and as a reductant under starvation conditions (48)(50)(52). Because the natural environments of facultative lithotrophs are very limited in nutrients, these bacteria probably use organic and inorganic substrates concomitantly.

Adaptation to shortage of nutrients may explain the extremely versatile metabolism of facultative chemolithotrophs. They are able to utilize alternative inorganic compounds and a wide range of organic substances (12)(46). For instance, most of the CO-oxidizing species, with a few exceptions, are hydrogenotrophs using [H.sub.2] as reductant (112). Reduced sulfur compounds such as thiosulfate can be oxidized by some hydrogen bacteria, e.g. Paracoccus denitrificans (53).

[H.sub.2] evolution is an obligate step of the [N.sub.2]-fixation process, consuming about 27% of the electron flux. The amount of [H.sub.2] released into the atmosphere depends on the total [H.sub.2]-recycling capacity of the rhizobia (39). B. japonicum bacteroids oxidize [H.sub.2], thereby increasing the metabolic efficiency of aerobic diazotrophy (41). A similar physiological function probably accounts for the presence of [H.sub.2]-oxidizing activity in Azotobacter sp., which recycle the [H.sub.2] evolved by nitrogenase to increase the production of ATP and to protect nitrogenase against [O.sub.2] under carbon or phosphate limitation (164)(172). Furthermore, A. vinelandii can grow mixotrophically with [H.sub.2] and mannose and the energy derived from [H.sub.2] oxidation facilitates the uptake of the sugar (105).

ENZYMOLOGY OF NICKEL-CONTAINING HYDROGENASES

General Characteristics

Hydrogenases (hydrogen: acceptor oxidoreductase, EC 1.18.99.1 and 1.12.2.1) catalyze reversible redox reactions with molecular [H.sub.2] according to the following equation: [H.sub.2] [equivalence] 2[H.sup.+] + 2[e.sup.-]. Structure and catalytic properties of hydrogenases have been extensively reviewed in past years (1)(2)(41)(42)(46)(101)(130)(131)(146)(162)(165); therefore only a few general characteristics are summarized below as this review focuses on the enzymes from those aerobic lithotrophs that have been explored genetically.

With the exception of a novel hydrogenase found in methanogenic archaea (175), all hydrogenases characterized so far are iron-sulfur proteins that fall into two groups. One type contains only iron, and the second type contains nickel in addition to iron. Iron-only hydrogenases seem to be limited to a few anaerobic organisms (1)(2).

Bartha & Ordal (8) were the first to report nickel-dependent chemolithoautotrophic growth of A.eutrophus. Later studies showed that this nickel requirement was related to formation of active hydrogenase (48) and that nickel was a constitutent of both hydrogenases present in this organism (54)(55). In 1980, Lancaster (94) observed novel electron paramagnetic resonance (EPR) signals in the membrane fraction of methanogens and tentatively assigned them to Ni(III) species. Graf & Thauer (63) demonstrated the presence of one Ni atom per molecule hydrogenase from Methanobacterium thermoautotrophicum. Since then, an increasing number of Ni-containing hydrogenases have been characterized from various sources including sulfatereducing bacteria, enterics, phototrophic bacteria, methanogens, and hyperthermophilic archaebacterial species (1)(2)(42)(130)(131)(165). So far, all of the hydrogenases from aerobic [H.sub.2]-oxidizing bacteria belong to the family of [NiFe] enzymes (23)(41)(46)(101)(146)(150). Yamazaki (171) reported the presence of selenium (in the form of selenocysteine) in addition to iron, nickel, and acid-labile sulfide in Methanococcus vannielii. Representatives of this subgroup of [NiFe] hydrogenases have since been found in a few methanogens and sulfate-reducing bacteria (38)(42).

The content of nonheme iron in the nickel-containing hydrogenases is reportedly in the range of 4--40 g atom/mol (130). A minimum of one [Fe-S] cluster per nickel center has been extrapolated from current data. The significance of [3Fe-4S] found in various [NiFe] hydrogenases remains unknown. The Ni center is supposed to be the catalytic site of [H.sub.2] activation (14) and has been shown to be located in the large subunit of the enzyme (70) (71).

The structure of the Ni center is unknown. EPR and X-ray absorption spectroscopy (EXAFS) studies suggest that Ni is coordinated to a varying number (one to six) of sulfur atoms (2)(130). In the presence of [O.sub.2], most of [NiFe] hydrogenases are purified in the inactive, "unready" state that correlates to a…