Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis

In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs.Hydrogenases are the key enzymes involved in the metabolism of H₂, catalyzing the following chemical reaction: 2H⁺ + 2e⁻ ↔ H₂. Hydrogenases can be classified into [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenases, based on their distinctive functional core containing the catalytic metal center (11, 17).The genomic analysis of Thermococcus onnurineus NA1, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area, revealed the presence of several distinct gene clusters encoding seven [NiFe]-hydrogenases and one homolog similar to Mbx (membrane-bound oxidoreductase) from Pyrococcus furiosus (1, 6, 8, 12). According to the classification system of hydrogenases by Vignais et al. (17), three hydrogenases (one F₄₂₀-reducing and two NADP-reducing hydrogenases) belong to group 3 [NiFe]-hydrogenases, and four hydrogenases belong to group 4 [NiFe]-hydrogenases. The group 4 hydrogenases are widely distributed among bacteria and archaea (17), with Hyc and Hyf (hydrogenase 3 and 4, respectively) from Escherichia coli (19), Coo (CO-induced hydrogenase) from Rhodospirillum rubrum (4), Ech (energy-converting hydrogenase) from Methanosarcina barkeri (7), and Mbh (membrane-bound hydrogenase) from P. furiosus (6, 10, 12) being relatively well-characterized hydrogenases in this group. One of the four group 4 hydrogenases from T. onnurineus NA1 was found to be similar in sequence to that of P. furiosus Mbh (10).     

A Second Soluble Hox-Type NiFe Enzyme Completes the Hydrogenase Set in Thiocapsa roseopersicina BBS

Three functional NiFe hydrogenases were previously characterized in Thiocapsa roseopersicina BBS: two of them are attached to the periplasmic membrane (HynSL and HupSL), and one is localized in the cytoplasm (HoxEFUYH). The ongoing genome sequencing project revealed the presence of genes coding for another soluble Hox-type hydrogenase enzyme (hox2FUYH). Hox2 is a heterotetrameric enzyme; no indication for an additional subunit was found. Detailed comparative in vivo and in vitro activity and expression analyses of HoxEFUYH (Hox1) and the newly discovered Hox2 enzyme were performed. Functional differences between the two soluble NiFe hydrogenases were disclosed. Hox1 seems to be connected to both sulfur metabolism and dark/photofermentative processes. The bidirectional Hox2 hydrogenase was shown to be metabolically active under specific conditions: it can evolve hydrogen in the presence of glucose at low sodium thiosulfate concentration. However, under nitrogen-fixing conditions, it can oxidize H₂ but less than the other hydrogenases in the cell.Hydrogenases are metalloenzymes involved in microbial hydrogen metabolism. A great variety of them have been identified and studied in various microorganisms and grouped on the basis of their metal content as NiFe, FeFe, and iron-sulfur cluster free hydrogenases (10, 42, 43). The basic protein structure of NiFe hydrogenases is heterodimeric, while FeFe hydrogenases are mostly composed of a single amino acid chain with multiple iron-sulfur clusters (28, 43, 44). Well-defined maturation proteins assist for the assembly and activation of hydrogenase enzymes; NiFe hydrogenases require a more complex accessory machinery than FeFe enzymes (2, 3, 24).Thiocapsa roseopersicina BBS is a photosynthetic purple sulfur bacterium belonging to the Chromatiaceae family (4). It prefers to utilize reduced sulfur compounds for anaerobic photochemolithoautotrophic growth, but simple organic substrates such as glucose or acetate can be also used as extra carbon, energy, and electron sources. It can be cultivated under aerobic (nonphotosynthetic) conditions in the presence of organic compounds. In the absence of other nitrogen sources, it is able to fix molecular nitrogen; this process is accompanied by H₂ production. T. roseopersicina was earlier shown to possess at least three NiFe hydrogenases varying in their in vivo functions, localizations, and compositions. Hyn and Hup hydrogenases are attached to the membrane facing the periplasmic side (6, 18, 30). Hyn is a bidirectional enzyme with extraordinary stability (17). Recent study has demonstrated that the HynSL subunits are physiologically connected to cellular redox processes via the Isp1 and Isp2 proteins, which play an essential role in electron transfer (27). The second membrane-associated enzyme, Hup, is involved in H₂ oxidation and shows homology to uptake hydrogenases, which recycle H₂ produced by the nitrogenase enzyme complex or present in the environment. Next to the hydrogenase small and large subunits (HupSL), a b-type cytochrome, HupC, was demonstrated to be part of the in vivo active enzyme as a transmitter of electrons to the quinone pool (27). In several bacteria, e.g., Rhodobacter capsulatus (7) and Ralstonia eutropha (15, 20), the expression of the hydrogenase(s) was shown to be regulated by the hydrogen level in the environment. The genes encoding the hydrogen-sensing system also exist in T. roseopersicina (hupUV, hupT, and hupR), but the hupTUV genes proved to be silent in the wild-type strain—only hupR is expressed—which is why expression of hupSL genes is constitutive (16).A Hox-type soluble hydrogenase was also identified in T. roseopersicina (31); it is a representative of the bidirectional heteromultimeric cytoplasmic NiFe hydrogenases (37, 39). Enzymes belonging to this group are basically composed of two moieties: hydrogenase (HoxYH) and diaphorase (HoxFU) heterodimers. Additional subunits were identified in few cases. In R. eutropha H16, two HoxI proteins completing the Hox complex were suggested to provide a binding domain for NADPH (5). HoxE has been identified as the fifth subunit of heteropentameric NAD⁺-reducing Hox hydrogenases in several cyanobacteria, Allochromatium vinosum and T. roseopersicina (21, 31, 37). In-frame deletion of the hoxE gene ceased both the H₂-producing and -oxidizing activities of Hox in vivo, but these were not affected in vitro. Consequently, an electron transfer role of the HoxE subunit was suggested (31, 32).The possibility of the presence of further hydrogenases in T. roseopersicina was noted few years ago (31). In the hynSL hupSL hoxH triple-mutant strain ({"type":"entrez-nucleotide","attrs":{"text":"GB112131","term_id":"336599610","term_text":"GB112131"}}GB112131), a small in vivo and in vitro hydrogenase activity could be measured under photomixotrophic growth conditions (both CO₂ and organic compounds are used for growth) at the late growth phase. This residual activity could not be detected in the hypF mutant strain (M539). Since HypF protein has an essential role in the maturation process of all NiFe hydrogenases (9), these results suggested the presence of a previously unknown hydrogenase. Here we describe the identification and characterization of the second Hox-type hydrogenase, emphasizing the functional similarities and differences between the two soluble enzymes of this bacterium. In order to distinguish between the two Hox-type enzymes unequivocally, the HoxEFUYH complex will be renamed Hox1 and the newly described Hox2FUYH enzyme is called Hox2.     

Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O₂-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.Hydrogen is a promising energy carrier for the future (10). Photosynthetic microbes such as cyanobacteria have attracted considerable attention, because they can split water photolytically to produce H₂. However, one major drawback of the processes is that their H₂-evolving hydrogenases are extremely sensitive to O₂, which is an inherent by-product of oxygenic photosynthesis. Thus, transfer of O₂-tolerant [NiFe] hydrogenases into cyanobacteria might be one approach to overcome this O₂ sensitivity issue. A small number of O₂-tolerant hydrogenases has been identified (9, 21, 47). However, they tend to favor H₂ uptake over evolution. Searching for novel O₂-tolerant [NiFe] hydrogenases from environmental microbes therefore becomes an important part of the effort to construct such biophotolytic systems.The oceans harbor an abundance of microorganisms with H₂ production capability. Traditionally, new hydrogenases have been screened only from culturable organisms. However, since only a few microbes can be cultured (14), many of them have not been identified, and their functions remain unknown. Metagenomics is a rapidly growing field, which allows us to obtain information about uncultured microbes and to understand the true diversity of microbes in their natural environments. Metagenomics analysis provides a completely new approach for identifying novel [NiFe] hydrogenases from the oceans in a culture-independent manner. The Global Ocean Sampling (GOS) expedition has produced the largest metagenomic data set to date, providing a rich catalog of proteins and protein families, including those enzymes involved in hydrogen metabolism (45, 52, 56-58). Putative novel [NiFe] hydrogenase enzymes that were identified from marine microbial metagenomic data in these expeditions can be examined to find potentially important new hydrogenases. Because source organisms for metagenomic sequences are not typically known, these hydrogenases have to be heterologously expressed in culturable foreign hosts for protein and functional analyses.Unlike most proteins, hydrogenases have a complex architecture and must be assembled and matured through a multiple-step process (7, 11). Hydrogenases are divided into three distinct groups based on their metal contents (54): Fe-S cluster-free hydrogenases (22, 23, 48), [FeFe] hydrogenases (1, 12, 25), and [NiFe] hydrogenases (2, 3, 55). [NiFe] hydrogenases are heterodimers composed of a large subunit and a small subunit, and their NiFe catalytic centers are located in the large subunits (2, 15, 19, 40). A whole set of accessory proteins are required to properly assemble the catalytic centers (7). The accessory protein HypE first interacts with HypF to form a HypF-HypE complex, and the carbamyl group linked to HypF is then dehydrated by HypE in the presence of ATP to release the CN group that is transferred to iron through a HypC-HypD-HypE complex (6). The origin of the CO ligand that is also bound to the iron is not clear, and possibly it comes from formate, formyl-tetrahydrofolate, or acetate. The liganded Fe atom is inserted into the immature large subunit, in which HypC proteins function as chaperones to facilitate the metal insertion (5, 34, 36). Ni is delivered to the catalytic center by the zinc-metalloenzyme HypA that interacts with HypB, a nickel-binding and GTP-hydrolyzing protein. The final step in the maturation process is endoproteolytic cleavage. Once the nickel is transferred to the active site, the endopeptidase, such as HyaD or HynD, cleaves the C-terminal end of the large subunit (33, 43), which triggers a conformational change of the protein so that the Ni-Fe catalytic center can be internalized.Heterologous expression of functional [NiFe] hydrogenases has been demonstrated in several studies (4, 18, 31, 39, 44, 50), suggesting that it could be a feasible approach to express novel hydrogenases from the environment for functional analysis. In this study, we sought to prove the concept that metagenomically derived environmental DNA can give rise to a functional [NiFe] hydrogenase through expression in a foreign host and that novel [NiFe] hydrogenases from environmental microbes can be studied in a culture-independent manner. We cloned environmental DNA that harbors the genes of a putative novel hydrogenase that shows strong homology to a known O₂-tolerant hydrogenase, HynSL, from the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (21, 28, 41, 59). We heterologously expressed the two structural genes (hyaA and hyaB) and two accessory genes (hupH and hyaD) of this novel environmental hydrogenase in T. roseopersicina, a foreign host that may already have the necessary machinery required to process the environmental hydrogenase since it carries the homologous hydrogenase HynSL. We analyzed the new hydrogenase protein and its functions. In addition, we compared the maturation mechanisms between the two homolog hydrogenases by performing complementation experiments.     

Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.Molecular hydrogen (H₂) is important in intermediary ecosystem metabolism (i.e., processes that link input to output) in wetlands (7, 11, 12, 33) and other anoxic habitats like sewage sludges (34) and the intestinal tracts of animals (9, 37). H₂-producing fermenters have been postulated to form trophic links to H₂-consuming methanogens, acetogens (i.e., organisms capable of using the acetyl-coenzyme A [CoA] pathway for acetate synthesis) (7), Fe(III) reducers (17), and sulfate reducers in a well-studied moderately acidic fen in Germany (11, 12, 16, 18, 22, 33). 16S rRNA gene analysis revealed the presence of Clostridium spp. and Syntrophobacter spp., which represent possible primary and secondary fermenters, as well as H₂ producers in this fen (11, 18, 33). However, H₂-producing bacteria are polyphyletic (30, 31, 29). Thus, a structural marker gene is required to target this functional group by molecular methods. [Fe-Fe]-hydrogenases catalyze H₂ production in fermenters (19, 25, 29, 30, 31), and genes encoding [Fe-Fe]-hydrogenases represent such a marker gene. The objectives of this study were to (i) develop primers specific for highly diverse [Fe-Fe]-hydrogenase genes, (ii) analyze [Fe-Fe]-hydrogenase genes in pure cultures of fermenters, acetogens, and a sulfate reducer, (iii) assess [Fe-Fe]-hydrogenase gene diversity in H₂-producing fen soil enrichments, and (iv) evaluate the limitations of the amplified [Fe-Fe]-hydrogenase fragment as a phylogenetic marker.     

Function of Ech Hydrogenase in Ferredoxin-Dependent,Membrane-Bound Electron Transport in Methanosarcina mazei

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei Δech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C₁ compounds.The aceticlastic pathway of methanogenesis creates approximately 70% (10) of the biologically produced methane and is of great ecological importance, as methane is a potent greenhouse gas. Organisms using this pathway to convert acetate to methane belong exclusively to the genera Methanosarcina and Methanosaeta. The two carbon atoms of acetate have different fates in the pathway. The methyl moiety is converted to methane, whereas the carbonyl moiety is further oxidized to CO₂ and the electrons derived from this oxidation step are used to reduce ferredoxin (Fd) (6). During methanogenesis from methylated C₁ compounds (methanol and methylamines), one-quarter of the methyl groups are oxidized to obtain electrons for the reduction of heterodisulfide (27). A key enzyme in the oxidative part of methylotrophic methanogenesis is the formylmethanofuran dehydrogenase, which oxidizes the intermediate formylmethanofuran to CO₂ (7). The electrons are transferred to Fd. It has been suggested that reduced ferredoxin (Fd_red) donates electrons to the respiratory chain with the heterodisulfide (coenzyme M [CoM]-S-S-CoB) as the terminal electron acceptor and that the reaction is catalyzed by the Fd_red:CoM-S-S-CoB oxidoreductase system (7, 24). The direct membrane-bound electron acceptor for Fd_red is still a matter of debate; for the Ech hydrogenase, a reduced ferredoxin-accepting, H₂-evolving activity has been observed for Methanosarcina barkeri (20), which implies that the H₂:CoM-S-S-CoB oxidoreductase system is involved in electron transport (13). Direct electron flow from the Ech hydrogenase to the heterodisulfide reductase has not been shown to date (20, 21). In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought to be involved in the aceticlastic pathway of methanogenesis as an acceptor for Fd_red (8, 10, 17). The Methanosarcina mazei genome, however, contains genes coding for the Ech hydrogenase, but this species lacks the Rnf complex (5).To investigate whether the Ech hydrogenase is the only means by which M. mazei channels electrons from Fd_red into the respiratory chain, a mutant lacking the Ech hydrogenase (M. mazei Δech mutant) was constructed. Electron transport experiments using Fd_red as the electron donor and CoM-S-S-CoB as the electron acceptor were conducted with wild-type and mutant membranes to gain deeper insight into the actual membrane-bound protein complexes that accept electrons from Fd_red. Furthermore, an in-depth characterization of the growth and trimethylamine (TMA) consumption of the Δech mutant was performed, which provided insight into the in vivo role of Ech hydrogenase.     

A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ_H joints and compare them with those present in later life. The embryo DJ_H joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ^−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.The adaptive immune system is characterized by the great diversity of its antigen receptors, which result from the activities of enzymatic complexes that cut and paste the genomic DNA of antigen receptor loci. The nonhomologous end-joining (NHEJ) machinery is then recruited to repair the double-strand DNA breaks (DSBs) inflicted by the products of the recombination-activating genes (RAGs) (45, 65). Within B cells, each immunoglobulin (Ig) receptor represents a singular shuffling of two heavy (H) and two light (L) chains, which are derived from the recombination of V, D, and J gene segments of the IgH locus and of V and J for IgL (71). Besides these combinatorial possibilities, most Ig variability derives from extensive processing of the coding ends, including exonucleolytic trimming of DNA ends, together with the addition of palindromic (P) nucleotides templated by the adjacent germ line sequence and of nontemplated (N) nucleotides secondary to the activity of the terminal deoxynucleotidyl transferase (TdT), a lymphoid-specific member of family X of DNA polymerases (reviewed in reference 56). During B-lineage differentiation, IgH rearrangements occur before those of the IgL locus, and D-to-J_H rearrangements precede V-to-DJ_H rearrangements (62). DJ_H joints are formed in any of the three open reading frames (ORFs). ORF1 is predominantly used in mature Igs, ORF2 is transcribed as a Dμ protein that provides negative signals to the B-cell precursors, and ORF3 frequently leads to stop codons (32, 33, 37). Germ line V, D, and J gene segments display short stretches of mutually homologous nucleotides (SSH), which are frequently used in gene rearrangements during perinatal periods, when N additions are absent (27, 32, 55, 57). The actual Ig V-region repertoires represent both the results of the NHEJ process associated with genomic VDJ recombination and those of antigen-independent and -dependent selection events. Although the core NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNA ligase IV) are by themselves able to join RAG-induced, incompatible DNA ends, family X DNA polymerases can be recruited to fill gaps created by imprecise coding ends with 3′ overhangs (DNA polymerase μ [Polμ] and Polλ) and/or to promote diversity through the addition of N nucleotides (TdT) (34, 56).The lymphoid differentiation pathways and clonotypic repertoires are developmentally regulated and differ between the embryo-fetal and adult periods (2, 44, 68). The perinatal B cells result from a wave of B lymphopoiesis occurring during the last third of mouse gestation (13, 14, 21, 70). Perinatal V_H gene usage differs from that predominating in the adult (1, 69), and the former VDJ joints rarely display N additions, leading to V-region repertoires enriched in multi- and self-reactive specificities (36, 40). The program of B-cell differentiation starts at embryonic days 10 to 11 (E10 to E11) in embryo hematopoietic sites, after the emergence of multipotent progenitors (at E8.5 to E9.5) (18, 19, 23, 31, 51, 73). DJ_H rearrangements were detected in these early embryos, whereas full VDJ_H sequences were not observed before E14 (14, 18, 51, 66), when VJκ rearrangements were also found (63). The earliest mouse DJ_H/VDJ_H Ig sequences analyzed to date corresponded to late fetuses (E16) (14, 53). We reasoned that the true baseline of the Ig rearrangement process occurs in midgestation embryos, when the first DJ_Hs are not yet transcribed and, consequently, not subjected to selection and are conditioned only for the evolutionarily established and developmentally regulated usage of distinct NHEJ machineries.We report here the sequence profiles of the earliest embryo E10 to E12 DJ_H joints. Unexpected frequencies of embryonic DJ_H joints bearing N nucleotides, in the absence of detectable TdT expression, were found. Moreover, the embryo DJ_H joints lacking N nucleotides (N⁻) used fewer SSH to recombine than newborn DJ_Hs, and these SSH were widely dispersed along the embryo D sequences, in contrast to the most joint-proximal ones, which predominated in newborn DJ_Hs. Considering that Polμ is the closest relative of TdT (42% amino acid identity) (22), which is able to introduce N nucleotides in vitro (4, 22, 34, 39, 49) and to join DNA ends with minimal or even null complementarity (17, 58), and that it is expressed in early-embryo organs, we decided to investigate its putative contribution to the first embryo DJ_H joints. The DJ_H joints obtained from Polμ^−/− embryos (48) showed a significant reduction of N nucleotides compared to wild-type (WT) embryos. Moreover, highly preserved DJ_H joints (with <3 deleted nucleotides) were selectively depleted in the Polμ^−/− mouse embryos, while the remaining DJ_Hs preferentially relied upon longer stretches of homology for end ligation. These findings support the idea that Polμ is active during early-embryo DJ_H rearrangements and that both its template-dependent and -independent ambivalent functions may be used to fill in small nucleotide gaps generated after asymmetric hairpin nicking and also to extend coding ends via a limited TdT-like activity.     

Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase

The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H₂ sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O₂-tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH_PsaE) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H₂ production capacity of the purified MBH_PsaE fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH_PsaE with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH_PsaE-PS I complex formation and its light-driven H₂ production capacity by means of spectroelectrochemistry.Molecular hydrogen (H₂) is often discussed as an alternative source of energy (13, 22, 26, 41). It is a highly energetic, renewable, and zero-carbon dioxide emission fuel; however, it is produced mainly from fossil resources. One intriguing possibility for sustainable H₂ production is the development of cellular systems in which the light-driven oxygenic photosynthesis is efficiently coupled to hydrogen production by hydrogenase (1, 21, 36).During the process of oxygenic photosynthesis, photosystem II (PS II), a thylakoid membrane (TM)-embedded multiprotein complex, utilizes solar energy to oxidize water into dioxygen (O₂), protons, and electrons. The electrons released by PS II are further conducted through an electron transport chain consisting of plastoquinones, the cytochrome b₆f complex, and either plastocyanin or cytochrome c₆ to the chlorophyll (Chl) dimer P₇₀₀ in photosystem I (PS I) (20, 48). During light-induced charge separation in PS I, P₇₀₀ is oxidized, leading to the reduction of the adjacent cofactor A₀ (Chl a). From there, the electrons are transmitted to the phylloquinone A₁ and subsequently to the Fe₄S₄ clusters F_X, F_A, and F_B (9) that are located at the acceptor site of PS I. The acceptor site is composed of the PsaC subunit, which harbors the iron-sulfur clusters F_A and F_B, and the two additional cofactor-free extrinsic subunits PsaD and PsaE. In the final step, the electrons are transferred from F_B to the ferredoxin (PetF), which has a midpoint potential of −412 mV (see Fig. ​Fig.1B)1B) (8, 9).Open in a separate windowFIG. 1.Models of the hydrogenase and photosystem I complexes used in this study. (A) Membrane-bound hydrogenase (MBH_wt) of Ralstonia eutropha H16. (B) Wild-type photosystem I (PS I) from Synechocystis sp. PCC 6803. (C) MBH_stop protein lacking the C-terminal anchor domain of HoxK. (D) MBH_PsaE and PS I_ΔPsaE.Hydrogenases of the NiFe and FeFe types catalyze the reversible cleavage of H₂ into protons and electrons (18, 63). For most hydrogenases, this reaction is highly sensitive to O₂ and leads to the reversible or even irreversible inactivation of the enzyme (49, 66, 67). A prominent exception is the oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16, which catalyzes H₂ conversion in the presence of O₂ (42, 65). The MBH consists of large subunit HoxG (67 kDa), harboring the NiFe active site, and small subunit HoxK (35 kDa), bearing three FeS clusters (Fig. ​(Fig.1)1) (32). Both cofactor-containing subunits are completely assembled within the cytoplasm and become subsequently translocated through the cytoplasmic membrane by the twin-arginine translocation (Tat) system. This transport is guided by a specific Tat signal peptide that is located at the N terminus of small subunit HoxK (53). The MBH is then connected to the membrane via the hydrophobic C-terminal “anchor” domain of HoxK, which provides the electronic connection to the diheme cytochrome b, HoxZ (5, 57). All structural, accessory, and regulatory genes for the synthesis of active MBH are arranged in a large, megaplasmid-borne operon (7, 11, 14, 29, 33, 38, 58).The concept of light-driven hydrogen production has been investigated in numerous studies (for reviews, see references 3, 21, and 23), including one involving direct electron transfer from PS I to the free form of hydrogenase in vitro (45). In a preliminary attempt, the MBH from R. eutropha was recently directly fused to PsaE (creating MBH_PsaE) (28). The fusion protein was partially purified and subjected to in vitro reconstitution with PS I lacking PsaE (PS I_ΔPsaE) (54) for light-driven hydrogen production. This concept was based on the previous observation that PS I lacking the peripheral subunit PsaE is fully reconstituted in vitro simply by the addition of independently purified PsaE protein (12).In the present communication, we describe a novel purification procedure for R. eutropha MBH_PsaE that yields homogeneous, functionally active MBH_PsaE. Additionally, a new method for efficient and fast purification of Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) His-tagged PS I was established. Finally, the pure proteins MBH_PsaE and PS I_ΔPsaE were successfully subjected to in vitro reconstitution.     

Role of the Hya Hydrogenase in Recycling of Anaerobically Produced H2 in Salmonella enterica Serovar Typhimurium

Double and triple uptake-type hydrogenase mutants were used to determine which hydrogenase recycles fermentatively produced hydrogen. The Δhyb Δhya and Δhyd Δhya double mutants evolved H₂ at rates similar to that of the triple mutant strain, so Hya alone oxidizes the bulk of H₂ produced during fermentation. When only Hya was present, no hydrogen production was observed in nutrient-limited medium. H₂ uptake assays showed that Hya can oxidize both exogenously added H₂ and formate hydrogen lyase-evolved H₂ anaerobically. Even after anaerobic growth, all three uptake-type hydrogenases could function in the presence of oxygen, including using O₂ as a terminal acceptor.Due to the anticipated scarcity of fossil fuels, there has been a surge of interest in H₂ production for alternative energy means. Numerous studies have attempted to engineer H₂-producing organisms, such as photosynthetic bacteria, cyanobacteria, and Escherichia coli, to produce maximal amounts of H₂ while minimizing the H₂-oxidizing capability of the organism (4, 5, 9, 10, 19). Hydrogenase expression and activity are controlled by multiple regulatory pathways and respond to fluctuations in pH, oxygen levels, and availability of metabolites and metal cofactors (17). In addition, the presence of hydrogen uptake hydrogenases decreases the net H₂ yield even under conditions that favor H₂ production. It is therefore important to understand the interactions between H₂-oxidizing enzymes (i.e., respiratory hydrogenases) and H₂-producing enzymes.Gene sequence analysis has revealed that many enteric bacteria contain the genes necessary for hydrogen production and oxidation. The E. coli hydrogenases have been studied extensively, while Salmonella enterica serovar Typhimurium hydrogenases have been studied to a lesser extent. Both E. coli and Salmonella serovar Typhimurium contain the hydrogen-oxidizing hydrogenases Hya and Hyb. Salmonella serovar Typhimurium also contains Hyd, which is another hydrogen-oxidizing hydrogenase (2, 13, 15). Hyc and Hyf are hydrogen-evolving hydrogenases that are present in both E. coli and Salmonella serovar Typhimurium, although it is unknown whether Hyf is functional (1).The Salmonella serovar Typhimurium hydrogenases are important for cellular metabolism. Hyc produces H₂ in order to remove excess reductant generated during mixed-acid fermentation. Hyc and formate dehydrogenase constitute the formate hydrogen lyase (FHL) complex (16), which oxidizes formate to produce CO₂ and H₂ (12). The hyb genes in E. coli and Salmonella serovar Typhimurium are expressed at high levels under anaerobic respiration conditions, and Hyb probably contributes to energy conservation (11, 15, 20). Hyb oxidizes H₂ and generates electrons, which are passed through the electron transport chain to terminal acceptors such as fumarate. The protons generated contribute to the proton-motive force. The role of Hya is not as well characterized. Hya may be used to recycle Hyc-produced H₂, since the hya operon is expressed at high levels during fermentative growth, or it may play a role in acid stress resistance (6, 14, 20, 21). The hyb genes are expressed at high levels under aerobic conditions in Salmonella serovar Typhimurium, and Hyb may couple H₂ oxidation to O₂ reduction (20).Redwood et al. recently examined the roles of uptake-type hydrogenases on net hydrogen production in E. coli (10). Cells were pregrown aerobically or anaerobically with formate and then allowed to ferment in anaerobic bottles. H₂ gas was collected, and other fermentation products were measured. They found that H₂ production increased by 37% in an hya hyb double mutant (compared to that in the wild type) that was grown overnight aerobically with formate. This increase in production was associated with the loss of hyb and not hya. Therefore, in E. coli, Hyb may be responsible for recycling fermentatively produced H₂.In this study, we measured the effect of uptake-type hydrogenase mutations on H₂ production in Salmonella serovar Typhimurium. We found that the majority of H₂-recycling activity in fermenting cells was dependent on the presence of hya, and having only Hya was sufficient to prevent any detectable H₂ evolution. These results demonstrate yet another difference between H₂ metabolism in E. coli and H₂ metabolism in Salmonella serovar Typhimurium.     

In Vitro Kinetics of Prebiotic Inulin-Type Fructan Fermentation by Butyrate-Producing Colon Bacteria: Implementation of Online Gas Chromatography for Quantitative Analysis of Carbon Dioxide and Hydrogen Gas Production

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of five butyrate-producing clostridial cluster XIVa colon bacteria grown on acetate plus fructose, oligofructose, inulin, or lactate were performed. A gas chromatography method was set up to assess H₂ and CO₂ production online and to ensure complete coverage of all metabolites produced. Method accuracy was confirmed through the calculation of electron and carbon recoveries. Fermentations with Anaerostipes caccae DSM 14662^T, Roseburia faecis DSM 16840^T, Roseburia hominis DSM 16839^T, and Roseburia intestinalis DSM 14610^T revealed similar patterns of metabolite production with butyrate, CO₂, and H₂ as the main metabolites. R. faecis DSM 16840^T and R. intestinalis DSM 14610^T were able to degrade oligofructose, displaying a nonpreferential breakdown mechanism. Lactate consumption was only observed with A. caccae DSM 14662^T. Roseburia inulinivorans DSM 16841^T was the only strain included in the present study that was able to grow on fructose, oligofructose, and inulin. The metabolites produced were lactate, butyrate, and CO₂, without H₂ production, indicating an energy metabolism distinct from that of other Roseburia species. Oligofructose degradation was nonpreferential. In a coculture of R. inulinivorans DSM 16841^T with the highly competitive strain Bifidobacterium longum subsp. longum LMG 11047 on inulin, hardly any production of butyrate and CO₂ was detected, indicating a lack of competitiveness of the butyrate producer. Complete recovery of metabolites during fermentations of clostridial cluster XIVa butyrate-producing colon bacteria allowed stoichiometric balancing of the metabolic pathway for butyrate production, including H₂ formation.The implementation of 16S rRNA gene-based analytical techniques in the ongoing exploration of the microbial diversity of the human colon ecosystem has both broadened and sharpened the prevailing image of its population (17, 24, 32). While a rather conservative perception of the composition of the colon microbiota has dominated gut research for several decades (36), recent studies have revealed the importance of previously largely neglected bacterial groups and have reduced historically numerically overestimated subpopulations to their actual (marginal) size (8, 22, 52). The human colon has been shown to be a remarkably selective environment, which is reflected by a rather shallow microbial diversity (32). Species belonging to the bacterial divisions Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria make up more than 98% of the bacterial population of the human colon (2, 17, 24). However, this superficial uniformity only covers an overwhelming diversity at the lower taxonomic levels; the human colon has been estimated to harbor between 500 and 1,000 species, representing over 7,000 strains, with up to 80% of them considered uncultivable using presently available methodologies (14, 28, 53).Assessing identity and abundance of the major microbial groups composing the colon microbiota is a first and indispensable step toward a better understanding of the ecosystem of the large intestine (48). However, defining a complex ecosystem such as the human colon requires more than the construction of a catalog of its members (32). A major challenge of gastrointestinal microbiology lies in linking phylogenetic subgroups with particular ecological habitats and niches (7, 8, 23). The latter requires further development of highly discriminating 16S rRNA gene-targeted probes to monitor spatial bacterial distribution, combined with renewed efforts toward species isolation through the application of innovative cultivation methods and media, and extensive metabolic characterization of representative strains (19, 35, 48).Recently, a global ecological approach, combining efforts in probe development (1, 27), species isolation (3), and metabolic characterization (4, 11, 15, 20), has led to the identification of a functional group of microorganisms, composed of species belonging to the clostridial clusters IV and XIVa, that are responsible for colon butyrate production. As butyrate is regarded as a key metabolite for the maintenance of colon health, this functional subunit of the colon microbiota could have a major influence on human well-being and might be considered as a target for prebiotic dietary interventions (25, 35, 45). Some recently described lactate- and/or acetate-converting colon butyrate producers have been reported to be able to degrade prebiotic inulin-type fructans, although the kinetics of their respective breakdown mechanisms have hardly been investigated (10, 20). The enhancement of colon butyrate production observed after consumption of oligofructose or inulin (6, 31, 40)—the so-called butyrogenic effect—as well as the limited stimulatory effect of these prebiotics on the clostridial cluster IV and XIVa colon populations (16, 30) have been attributed to cross-feeding with bifidobacteria, which are still considered the primary fructan degraders (5, 38). Anaerostipes caccae as well as Roseburia spp. have been shown to be able to (co)metabolize end products of bifidobacterial fructan fermentation (lactate and/or acetate) or to grow on short oligosaccharides and monosaccharides released by Bifidobacterium spp. during fructan degradation (4, 20).Recently, many clostridial cluster IV and XIVa butyrate producers characterized in detail have been shown to produce gases, mainly CO₂ and H₂ (12, 15, 20, 46). Consequently, they might be responsible for an enhancement of gas production as a result of fructan fermentation, through either cross-feeding or direct degradation of inulin-type fructans (15, 16). Indeed, inulin-type fructan consumption has been reported to cause some gastrointestinal discomfort related to gas production—essentially, flatulence and bloating (43)—while bifidobacteria, the main beneficiaries of dietary fructan intake, do not produce gases (19, 49). Although CO₂ and H₂ production by colon butyrate producers could have implications for human intestinal well-being, (in vitro) production has not been satisfactorily monitored up to now, probably due to limited availability of a performant apparatus for (online) gas analysis (15, 20). Moreover, the currently proposed pathway for colon butyrate production does not provide a conclusive quantitative link between bacterial (co)substrate metabolism and H₂ formation (11).This study investigated the kinetics of inulin-type fructan degradation by representatives of the genera Anaerostipes and Roseburia. A method based on online gas chromatography (GC) was developed to assess gas production qualitatively and quantitatively in a continuously sparged fermentation vessel for complete coverage of metabolite production. The competitiveness of inulin-degrading butyrate producers was investigated through coculture fermentations with Bifidobacterium longum subsp. longum LMG 11047, a strain representing a highly competitive cluster of bifidobacteria that share both high fructose consumption and oligofructose degradation rates and are able to perform partial breakdown of inulin (18, 20). A stoichiometrically balanced pathway for butyrate production, including H₂ production, is proposed.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

Anaerobic Biodegradation of n-Hexadecane by a Nitrate-Reducing Consortium

Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H₃₄-hexadecane, fully deuterated hexadecane (d₃₄-hexadecane), or H₃₄-hexadecane and NaH¹³CO₃. Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H₂₉]pentadecanoic acid, [H₂₅]tridecanoic acid, [1-¹³C]pentadecanoic acid, [3-¹³C]heptadecanoic acid, [3-¹³C]10-methylheptadecanoic acid, and d₂₇-pentadecanoic, d₂₅-, and d₂₄-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-¹⁴C]hexadecane was demonstrated based on the recovery of ¹⁴CO₂ in active cultures.Linear alkanes account for a large component of crude and refined petroleum products and, therefore, are of environmental significance with respect to their fate and transport (38). The aerobic activation of alkanes is well documented and involves monooxygenase and dioxygenase enzymes in which not only is oxygen required as an electron acceptor but it also serves as a reactant in hydroxylation (2, 16, 17, 32, 34). Alkanes are also degraded under anoxic conditions via novel degradation strategies (34). To date, there are two known pathways of anaerobic n-alkane degradation: (i) alkane addition to fumarate, commonly referred to as fumarate addition, and (ii) a putative pathway, proposed by So et al. (25), involving carboxylation of the alkane. Fumarate addition proceeds via terminal or subterminal addition (C-2 position) of the alkane to the double bond of fumarate, resulting in the formation of an alkylsuccinate. The alkylsuccinate is further degraded via carbon skeleton rearrangement and β-oxidation (4, 6, 8, 12, 13, 21, 37). Alkane addition to fumarate has been documented for a denitrifying isolate (21, 37), sulfate-reducing consortia (4, 8, 12, 13), and five sulfate-reducing isolates (4, 6-8, 12). In addition to being demonstrated in these studies, fumarate addition in a sulfate-reducing enrichment growing on the alicyclic alkane 2-ethylcyclopentane has also been demonstrated (23). In contrast to fumarate addition, which has been shown for both sulfate-reducers and denitrifiers, the putative carboxylation of n-alkanes has been proposed only for the sulfate-reducing isolate strain Hxd3 (25) and for a sulfate-reducing consortium (4). Experiments using NaH¹³CO₃ demonstrated that bicarbonate serves as the source of inorganic carbon for the putative carboxylation reaction (25). Subterminal carboxylation of the alkane at the C-3 position is followed by elimination of the two terminal carbons, to yield a fatty acid that is one carbon shorter than the parent alkane (4, 25). The fatty acids are subject to β-oxidation, chain elongation, and/or C-10 methylation (25).In this study, we characterized an alkane-degrading, nitrate-reducing consortium and surveyed the metabolites of the consortium incubated with either unlabeled or labeled hexadecane in order to elucidate the pathway of n-alkane degradation. We present evidence of a pathway analogous to the proposed carboxylation pathway under nitrate-reducing conditions.     

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.The genera Thermoanaerobacter and Thermoanaerobacterium contain bacteria which are thermophilic, obligate anaerobes that specialize in polysaccharide and carbohydrate fermentation, producing primarily l-lactic acid, acetic acid, ethanol, CO₂, and H₂ (24, 27, 49). Taxonomically, they are distinguished from other anaerobic thermophilic clostridia by the ability to reduce thiosulfate to hydrogen sulfide or elemental sulfur (21). The majority of characterized Thermoanaerobacter and Thermoanaerobacterium strains have been isolated from hot springs and other thermal environments (20-22, 38, 47); however, they have also been isolated from canned foods (4, 10), soil (48), paper mills and breweries (41, 43), and deep subsurface environments (5, 13, 35), suggesting a somewhat ubiquitous environmental presence.Representatives of the Thermoanaerobacter and Thermoanaerobacterium genera have been considered for biotechnological applications, such as conversion of lignocellulosic biomass to ethanol (8, 27) or other fuels and chemicals (3, 24). However, the branched fermentation pathways of these organisms generally require modification for industrial application. Several studies have investigated manipulating bioprocess and growth conditions to alter end product ratios and yields, but this has not resulted in reliable conditions to maximize the yield of a single end product (18, 25). Genetic engineering is likely necessary for commercial application of Thermanaerobacter or Thermoanaerobacterium species (26, 27, 44). As genetic systems for these bacteria have emerged (28, 45), increased product yields have been demonstrated by gene knockout of l-lactate dehydrogenase (9, 14), phosphotransacetylase and acetate kinase (40), and hydrogenase (39). Despite this recent progress, genetic transformation is still considered the greatest barrier for engineering these organisms (44).In contrast, some of the bacteria most amenable to genetic manipulation are those exhibiting natural competence; for example, work with the naturally competent Streptococcus pneumoniae first established DNA as the molecule containing inheritable information (42). Naturally competent organisms are found in many bacterial phyla, although the overall number of bacteria known to be naturally competent is relatively small (16).The molecular mechanisms of natural competence are often divided into two stages: early-stage genes that encode regulatory and signal cascades to control competence induction, and late-stage genes that encode the machinery of DNA uptake and integration (16). The Gram-positive late-stage consensus mechanism for DNA uptake and assimilation, elucidated primarily through work with Bacillus subtilis, occurs through several molecular machinery steps. First, DNA is believed to interact with a type IV pilus (T4P) or pseudopilus that brings it into close proximity of the cell membrane. The precise mechanism of this phenomenon is unclear; although components of the T4P in both Gram-positive and Gram-negative bacteria have been shown to bind DNA (7, 19), in specific studies, a full pilus structure has been either not observed or shown not to be essential during natural competence (6, 36). Two proteins, ComEA and ComEC, are then involved in creation and transport of single-stranded DNA across the membrane, where it is subsequently bound by CinA-localized RecA and either integrated into the genome or replicated at an independent origin, as for plasmid DNA (6).Here, we report that several Thermoanaerobacter and Thermoanaerobacterium strains are naturally competent, characterize growth conditions conducive to natural competence, and identify genes in Thermoanaerobacterium saccharolyticum JW/SL-YS485 required for competence exhibition.