3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase,Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO₂ fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.In the thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula, CO₂ fixation proceeds via a 3-hydroxypropionate/4-hydroxybutyrate cycle (8, 23, 24, 28) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales and in mesophilic Crenarchaea (Cenarchaeum sp. and Nitrosopumilus sp.) of marine group I. The cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (11, 16, 17, 19, 20, 32, 33). It involves the carboxylation of acetyl-coenzyme A (CoA) to malonyl-CoA by the biotin-dependent acetyl-CoA carboxylase. Malonyl-CoA is reduced via malonate semialdehyde to 3-hydroxypropionate (1), which is further reductively converted to propionyl-CoA (3). Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by a propionyl-CoA carboxylase that is similar or identical to acetyl-CoA carboxylase. In fact, only one copy of the genes for the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, suggesting that this is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (24). (S)-Methylmalonyl-CoA is epimerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other members of the Sulfolobales. Enzymes are the following: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionyl-CoA synthetase (3-hydroxypropionate-CoA ligase, AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinate semialdehyde reductase (NADPH); 12, 4-hydroxybutyryl-CoA synthetase (4-hydroxybutyrate-CoA ligase, AMP-forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The two steps of interest are highlighted.In Chloroflexus succinyl-CoA is converted to (S)-malyl-CoA, which is cleaved by (S)-malyl-CoA lyase to acetyl-CoA (thus regenerating the CO₂ acceptor molecule) and glyoxylate (16). Glyoxylate is assimilated into cell material by a yet not completely resolved pathway (37). In Metallosphaera succinyl-CoA is converted via 4-hydroxybutyrate to two molecules of acetyl-CoA (8), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA and two bicarbonates into succinyl-CoA, and the second part converts succinyl-CoA to two acetyl-CoA molecules.The reductive conversion of 3-hydroxypropionate to propionyl-CoA requires three enzymatic steps: activation of 3-hydroxypropionate to its CoA ester, dehydration of 3-hydroxypropionyl-CoA to acryloyl-CoA, and reduction of acryloyl-CoA to propionyl-CoA. In C. aurantiacus these three steps are catalyzed by a single large trifunctional enzyme, propionyl-CoA synthase (2). This 200-kDa fusion protein consists of a CoA ligase, a dehydratase, and a reductase domain. Attempts to isolate a similar enzyme from M. sedula failed. Rather, a 3-hydroxypropionyl-CoA synthetase was found (3), suggesting that the other two reactions may also be catalyzed by individual enzymes.Here, we purified the missing enzymes 3-hydroxypropionyl-CoA dehydratase and acryloyl-CoA reductase from M. sedula, identified the coding genes in the genome of M. sedula and other members of the Sulfolobales, produced recombinant enzymes as proof of function, and studied the enzymes in some detail. A comparison with the respective domains of propionyl-CoA synthase from C. aurantiacus indicates that the conversion of 3-hydroxypropionate to propionyl-CoA via the 3-hydroxypropionate route has evolved independently in these two phyla.     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

Pseudomonas putida harbors two ferredoxin-NADP⁺ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k_cat/K_m value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.Commonly, Fprs are ubiquitous, monomeric, reversible flavin enzymes. Fprs evidence a profound preference for NADP(H) over NAD(H) (3). They harbor a prosthetic flavin cofactor (FAD) and catalyze the reversible electron exchange between NADPH and either ferredoxin (Fd) or flavodoxin (Fld) (4, 5). In oxygenic photosynthesis, the Fd is reduced by the photosystem and subsequently passes electrons on to NADP⁺ via the Fpr. This reaction provides the cellular NADPH pool required for CO₂ assimilation and other biosynthetic processes (4, 5). In heterotrophic organisms such as bacteria, reduced ferredoxin, owing to the reverse enzymatic activity of the Fpr, can donate an electron to several Fd-dependent enzymes, such as nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase, allowing ferredoxin to function in a variety of systems, including oxidative stress (1, 4, 5).Iron is the fourth most abundant element in the natural environment and exists primarily as an oxidized form, Fe(III), which has very low solubility under neutral pH conditions (9, 34) and thus presents problems in terms of bioavailability. However, ferrous iron, of Fe(II), is soluble and available at neutral pH in bacterial cytosol (34). Most bacteria secrete siderophores, which are natural chelators of ferric iron. After they bind to ferric iron, that complex enters the bacteria and releases ferric iron into the cytosol in ferric or ferrous form (9). In the bacterial cytosol, ferric iron must be reduced to ferrous form, and thus ferric reductase is essential to bacterial iron utilization.Commonly, prokaryotic ferric reductases are divided into two groups—namely, the bacterial and archaeal types (34). The typical bacterial type ferric reductase is Escherichia coli Fre, which also functions as a flavin reductase. In other words, the ferric reductase can reduce free flavin as flavin reductase, rather than having the flavin cofactor as a prosthetic group in E. coli (38). The archaeal ferric reductase harbors a flavin cofactor in the enzyme and thus does not require a flavin carrier for ferric reduction (26, 34). E. coli Fre includes a Rosmann folding structure at the NAD(P) binding region, whereas the archaeal ferric reductase (FeR) of Archaeoglobus fulgidus does not evidence that folding structure (6, 34). Many bacterial ferric reductases utilize free flavins, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and riboflavin, as electron carrier and, NADH (NAD) or NADP as electron donors to ferric reductase (14, 34). However, reduced ferric iron by reduced free flavin gives rise to the Fenton reaction, which generates the hydroxyl radical within the cell (20, 38). The Fenton reaction is known to generate hydroxyl radicals from ferrous iron and hydrogen peroxide (20). The hydroxyl radical is the most reactive radical and can damage DNA, proteins, and membrane lipids (16, 20, 34, 38). Therefore, the fine-tuning of ferric reduction regulation is required for the survival of bacterial cells.Many Pseudomonas strains, including Pseudomonas putida, a gram-negative soil model bacteria, and Pseudomonas aeruginosa, a human pathogen bacteria, do not harbor annotated ferric reductase within their genome sequences. Commonly, the pathogens compete with the host for available iron, whichis crucial for their survival within the host. Thus, studies of P. aeruginosa regarding iron utilization, siderophores, and ferric reduction are considered to be essential for a better understanding of human infections (9, 19). Studying the physiology and ecology of P. putida also provides us with a new framework for elucidating the basis of the metabolic versatility and environmental stress response of soil microorganisms. Thus, the study of ferric reductase in strains of Pseudomonas at the molecular level is certainly required. From the structural perspective, ferric reductases are generally considered to be contained within the structurally diverse ferredoxin-NADP⁺ reductase (Fprs; EC 1.18.1.2) superfamily, which is frequently involved in the transfer of electrons between Fd/Fld and NADP(H) (2, 15, 34). Thus, we tested the role of the Fpr as a ferric reductase using free flavin (FMN or FAD), NADH, or NADPH as electron donors, and ferric-citrate or ferric-EDTA as terminal electron acceptors (37). We determined that FprA could efficiently utilize NADPH in ferric reduction. Rather, FprB could use NADH as an electron donor and may perform a crucial role as a NADH-dependent ferric reductase under iron stress conditions.     

Staphylococcus aureus NrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of deoxyribonucleotides during the cell cycle (29). Three main classes of RNRs are known. Class I RNRs are oxygen-dependent enzymes, class II RNRs are oxygen-independent enzymes, and class III RNRs are oxygen-sensitive enzymes. Class I RNRs are divided into two subclasses, subclasses Ia and Ib.Staphylococcus aureus is a Gram-positive facultative aerobe and a major human pathogen (24). S. aureus contains class Ib and class III RNRs, which are essential for aerobic and anaerobic growth, respectively (26). The class Ib NrdEF RNR is encoded by the nrdE and nrdF genes: NrdE contains the substrate binding and allosteric binding sites, and NrdF contains the catalytic site for ribonucleotide reduction. The S. aureus nrdEF genes form an operon containing a third gene, nrdI, the product of which, NrdI, is a flavodoxin (5, 33). Many other bacteria such as Escherichia coli (16), Lactobacillus lactis (17), and Mycobacterium and Corynebacterium spp. possess class Ib RNR operons that contain a fourth gene, nrdH (30, 44, 50), whose product, NrdH, is a thiol-disulfide redoxin (16, 17, 40, 43, 49). More-complex situations are found for some bacteria, where the class Ib RNR operon may be duplicated and one or more of the nrdI and nrdH genes may be missing or located in another part of the chromosome (29).NrdH proteins are glutaredoxin-like protein disulfide oxidoreductases that alter the redox state of target proteins via the reversible oxidation of their active-site dithiol proteins. NrdH proteins function with high specificity as electron donors for class I RNRs (9, 16-18). They are widespread in bacteria, particularly in those bacteria that lack glutathione (GSH), where they function as a hydrogen donor for the class Ib RNR (12, 16, 17). In E. coli, which possesses class Ia and class Ib RNRs, NrdH functions in vivo as the primary electron donor for the class Ib RNR, whereas thioredoxin or glutaredoxin is used by the class Ia NrdAB RNR (12, 17). NrdH redoxins contain a conserved CXXC motif, have a low redox potential, and can reduce insulin disulfides. NrdH proteins possess an amino acid sequence similar to that of glutaredoxins but behave functionally more like thioredoxins. NrdH proteins are reduced by thioredoxin reductase but not by GSH and lack those residues in glutaredoxin that bind GSH and the GSH binding cleft (39, 40). The structures of the E. coli and Corynebacterium ammoniagenes NrdH redoxins reveal the presence of a wide hydrophobic pocket at the surface, like that in thioredoxin, that is presumed to be involved in binding to thioredoxin reductase (39, 40). NrdI proteins are flavodoxin proteins that function as electron donors for class Ib RNRs and are involved in the maintenance of the NrdF diferric tyrosyl radical (5, 33). In Streptococcus pyogenes, NrdI is essential for the activity of the NrdHEF system in a heterologous E. coli in vivo complementation assay (33). Class Ib RNRs are proposed to depend on two specific electron donors, NrdH, which provides reducing power to the NrdE subunit, and NrdI, which supplies electrons to the NrdF subunit (33).In this report we identify an open reading frame (ORF) in S. aureus encoding an NrdH-like protein with partial sequence relatedness to the E. coli, Salmonella enterica serovar Typhimurium, L. lactis, and C. ammoniagenes NrdH proteins. In contrast to these bacteria, the S. aureus nrdH gene does not form part of the class Ib RNR operon. The S. aureus NrdH protein differs in its structure from the canonical NrdH in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that in vitro, S. aureus NrdH reduces protein disulfides and is an electron donor for the homologous class Ib NrdEF ribonucleotide reductase.     

Characterization of the PduS Cobalamin Reductase of Salmonella enterica and Its Role in the Pdu Microcompartment

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B₁₂ (adenosylcobalamin, AdoCbl)-dependent fashion. Salmonella obtains AdoCbl by assimilation of complex precursors, such as vitamin B₁₂ and hydroxocobalamin. Assimilation of these compounds requires reduction of their central cobalt atom from Co³⁺ to Co²⁺ to Co⁺, followed by adenosylation to AdoCbl. In this work, the His₆-tagged PduS cobalamin reductase from S. enterica was produced at high levels in Escherichia coli, purified, and characterized. The anaerobically purified enzyme reduced cob(III)alamin to cob(II)alamin at a rate of 42.3 ± 3.2 μmol min⁻¹ mg⁻¹, and it reduced cob(II)alamin to cob(I)alamin at a rate of 54.5 ± 4.2 nmol min⁻¹ mg⁻¹ protein. The apparent K_m values of PduS-His₆ were 10.1 ± 0.7 μM for NADH and 67.5 ± 8.2 μM for hydroxocobalamin in cob(III)alamin reduction. The apparent K_m values for cob(II)alamin reduction were 27.5 ± 2.4 μM with NADH as the substrate and 72.4 ± 9.5 μM with cob(II)alamin as the substrate. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) indicated that each monomer of PduS contained one molecule of noncovalently bound flavin mononucleotide (FMN). Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD, supporting a role in cobalamin reduction in vivo. Further studies demonstrated that the PduS protein is a component of the Pdu microcompartments (MCPs) used for 1,2-PD degradation and that it interacts with the PduO adenosyltransferase, which catalyzes the terminal step of AdoCbl synthesis. These studies further characterize PduS, an unusual MCP-associated cobalamin reductase, and, in conjunction with prior results, indicate that the Pdu MCP encapsulates a complete cobalamin assimilation system.Coenzyme B₁₂ (adenosylcobalamin, AdoCbl) is an indispensable cofactor for a variety of enzymes that are widely distributed among microbes and higher animals (2, 55). Organisms obtain AdoCbl by de novo synthesis or by assimilation of complex precursors, such as vitamin B₁₂ (cyanocobalamin, CN-Cbl) and hydroxocobalamin (OH-Cbl), which can be enzymatically converted to AdoCbl. De novo synthesis occurs only in prokaryotes, but the assimilation of complex precursors is more widespread, taking place in many microbes and in higher animals (56). A model for the assimilation of CN-Cbl and OH-Cbl to AdoCbl, based on work done in a number of laboratories, is shown in Fig. ​Fig.1.1. CN-Cbl is first reductively decyanated to cob(II)alamin (22, 30, 68). Next, cob(II)alamin is reduced to cob(I)alamin, and ATP:cob(I)alamin adenosyltransferase (ATR) transfers a 5′ deoxyadenosyl group from ATP to cob(I)alamin to form AdoCbl (10, 11, 28, 29, 35, 63, 64, 72). Studies indicate that prior to reduction cob(II)alamin binds the ATR and undergoes a transition to the 4-coordinate base-off conformer (41, 48, 59, 61, 62). Transition to the 4-coordinate state raises the midpoint potential of the cob(II)alamin/cob(I)alamin couple by about 250 mV, facilitating reduction (60). OH-Cbl assimilation occurs by a similar pathway except that the first step is reduction of OH-Cbl to cob(II)alamin by cobalamin reductase or by the reducing environment of the cell (19, 69).Open in a separate windowFIG. 1.Cobalamin assimilation and recycling pathway. Many organisms are able to take up CN-Cbl and OH-Cbl and convert them to the active coenzyme form, AdoCbl. This process involves reduction of the central cobalt atom of the corrin ring followed by addition of a 5′ deoxyadenosyl (Ado) group via a carbon-cobalt bond. The Ado group is unstable in vivo, and AdoCbl breaks down to form OH-Cbl. Consequently, cobalamin recycling is required for AdoCbl-dependent processes, and recycling uses the same pathway that functions in the assimilation of cobalamin from the environment. PPPi, triphosphate.The pathway used for the assimilation of OH-Cbl and CN-Cbl is also used for intracellular cobalamin recycling. During catalysis the adenosyl group of AdoCbl is periodically lost due to by-reactions and is usually replaced by a hydroxyl group, resulting in the formation of an inactive OH-Cbl enzyme complex (66). Cobalamin recycling begins with a reactivase that converts the inactive OH-Cbl-enzyme complex to OH-Cbl and apoenzyme (43, 44). Next, the process described in Fig. ​Fig.11 converts OH-Cbl to AdoCbl, which spontaneously associates with apoenzyme to form active holoenzyme (43, 44, 66). In the organisms that have been studied, cobalamin recycling is essential, and genetic defects in this process block AdoCbl-dependent metabolism (3, 16, 29).Salmonella enterica degrades 1,2-propanediol (1,2-PD) via an AdoCbl-dependent pathway (27). 1,2-PD is a major product of the anaerobic degradation of common plant sugars rhamnose and fucose and is thought to be an important carbon and energy source in natural environments (38, 46). Twenty-four genes for 1,2-PD utilization (pdu) are found in a contiguous cluster (pocR, pduF, and pduABB′CDEGHJKLMNOPQSTUVWX) (7, 27). This locus encodes enzymes for the degradation of 1,2-PD and cobalamin recycling, as well as proteins for the formation of a bacterial microcompartment (MCP) (7). Bacterial MCPs are simple proteinaceous organelles used by diverse bacteria to optimize metabolic pathways that have toxic or volatile intermediates (6, 13, 14, 71). They are polyhedral in shape, 100 to 150 nm in cross section (about the size of a large virus), and consist of a protein shell that encapsulates sequentially acting metabolic enzymes. Sequence analyses indicate that MCPs are produced by 20 to 25% of all bacteria and function in seven or more different metabolic processes (14). The function of the Pdu MCP is to confine the propionaldehyde formed in the first step of 1,2-PD degradation in order to mitigate its toxicity and prevent DNA damage (7, 23, 24, 51). Prior studies indicate that 1,2-PD traverses the protein shell and enters the lumen of the Pdu MCP, where it is converted to propionaldehyde and then to propionyl-coenzyme A (CoA) by AdoCbl-dependent diol dehydratase (DDH; PduCDE) and propionaldehyde dehydrogenase (PduP) (8, 33). Propionyl-CoA then exits the MCP into the cytoplasm, where it is converted to 1-propanol or propionate or enters central metabolism via the methylcitrate pathway (25, 47). The shell of the Pdu MCP is thought to limit the diffusion of propionaldehyde in order to protect cytoplasmic components from toxicity. The Pdu MCP was purified, and 14 major polypeptide components were identified (PduABB′CDEGHJKOPTU), all of which are encoded by the pdu locus (23). PduABB′JKTU are confirmed or putative shell proteins (23, 24, 51). PduCDE and PduP catalyze the first 2 steps of 1,2-PD degradation as described above (7, 8, 23, 33). The PduO and PduGH enzymes are used for cobalamin recycling. PduO is an adenosyltransferase (29), and PduGH is a homolog of the Klebsiella DDH reactivase, which mediates the removal of OH-Cbl from an inactive OH-Cbl-DDH complex (43, 44). However, a reductase which is also required for cobalamin recycling was not previously identified as a component of the Pdu MCP (23). This raises the question of how cobalamin is recycled for the AdoCbl-dependent DDH that resides within the Pdu MCP.Prior studies indicated that the PduS enzyme (which is encoded by the pdu locus) is a cobalamin reductase (52). Very recently PduS was purified from S. enterica and shown to be a flavoprotein that can mediate the reduction of 4-coordinate cob(II)alamin bound to ATR but was not further characterized (40). In this study, PduS from S. enterica is purified and more extensively characterized, including identification of its cofactor requirements and kinetic properties. In addition, we show that PduS is a component of the Pdu MCP. This finding in conjunction with prior work indicates that, in addition to 1,2-PD degradative enzymes, the Pdu MCP encapsulates a complete cobalamin recycling system.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T₇₃ was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.Monoterpenes are a class of isoprenoids of increasing industrial and clinical interest usually produced by plants. They are used as aromatic additives in the food and cosmetics industries and are also important components in wine aroma. Moreover, certain monoterpenes display antimicrobial, antiparasitic, and antiviral properties as well as a plethora of promising health benefits (for recent reviews, see references 2, 7, 15, 28, and 30 and references cited therein). To date, many studies have focused on plant metabolic engineering of monoterpene production (for selected reviews, see references 1, 14, 19, 29, and 35 and references cited therein), and few studies have been carried out on microorganisms (9, 21, 22, 34, 38). Efficient microbial production of these metabolites could provide an alternative to the current methods of chemical synthesis or extraction from natural sources. In this regard, a considerable number of studies have shown the utility of Saccharomyces cerevisiae as a valuable platform for sesquiterpene, diterpene, triterpene, and carotene production (references 5, 10, 23, 26, 30, 31, 32, and 33 and references cited therein). However, all the efforts dedicated to the improvement of isoprenoid yields in S. cerevisiae have been performed using conventional laboratory strains, and there are no studies concerning natural or industrially relevant isolates.In recent years, many genes that encode plant monoterpene synthases (MTS), a family of enzymes which specifically catalyze the conversion of the ubiquitous C₁₀ intermediate of isoprenoid biosynthesis geranyl pyrophosphate (GPP) to monoterpenes, have been characterized. Such is the case with the LIS gene (codes for S-linalool synthase) of Clarkia breweri, the first MTS-encoding gene to be isolated (13). In contrast to plants, S. cerevisiae cannot produce monoterpenes efficiently, mainly due to the lack of specific pathways involving MTS. However, GPP is formed as a transitory intermediate in the two-step synthesis of farnesyl pyrophosphate (FPP), catalyzed by FPP synthase (FPPS) (Fig. ​(Fig.1),1), and some natural S. cerevisiae strains have been shown to possess the ability to produce small amounts of monoterpenes (8). Whether this occurs through unspecific dephosphorylation of a more available endogenous pool of GPP and subsequent bioconversions is not known. In addition, it has recently been established that S. cerevisiae has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions (22, 34).Open in a separate windowFIG. 1.Simplified isoprenoid pathway in S. cerevisiae, including the branch point to linalool. Dotted arrows indicate that more than one reaction is required to convert the substrate to the product indicated. Dashed arrows indicate the engineered steps. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase.Here we present the process for selecting and optimizing yeast strains for foreign monoterpene production. We have chosen the C. breweri LIS gene as a prototype because, when heterologously expressed in S. cerevisiae, it specifically results in the production of linalool (3,7-dimethyl-1,6-octadien-3-ol; a floral scent and bioactive acyclic monoterpene identified in numerous fruits and flowers) and no other by-products (22). Two S. cerevisiae strains of different origins have been selected and their endogenous mevalonate (MVA) pathways engineered to enhance the production of linalool. These strategies might be employed to produce any other recombinant monoterpene in S. cerevisiae by expressing the appropriate monoterpene synthase.     

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).     

Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase,Regulates the Dimorphic Switch in Schizosaccharomyces pombe

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.Eucaryotic cells are able to define and maintain a particular cellular organization and thus cellular morphology by executing programs modulated by internal and external signals. For example, signals generated within a cell are required for the selection of the growth zone after cytokinesis in the fission yeast Schizosaccharomyces pombe or the emergence of the bud in Saccharomyces cerevisiae (37, 44, 81). Cellular morphogenesis is also subject to regulation by a wide variety of external signals, such as growth factors, temperature, hormones, nutrient limitation, and cell-cell or cell-substrate contact (13, 34, 66, 75, 81). Both types of signals will lead to the selection of growth zones accompanied by the reorganization of the cytoskeleton.The ability to alter the growth form in response to environmental conditions is an important virulence-associated trait of pathogenic fungi which helps the pathogen to spread in and survive the host''s defense system (7, 32). Alteration of the growth form in response to extrinsic signals is not limited to pathogenic fungi but is also found in the model yeasts S. cerevisiae and S. pombe, in which it appears to represent a foraging response (1, 24).The regulation of polarized growth and the definition of growth zones have been studied extensively with the fission yeast S. pombe. In this cylindrically shaped organism, cell wall biosynthesis is restricted to one or both cell ends in a cell cycle-regulated manner and to the septum during cytokinesis (38). This mode of growth requires the actin cytoskeleton to direct growth and the microtubule cytoskeleton to define the growth sites (60). In interphase cells, microtubules are organized in antiparallel bundles that are aligned along the long axis of the cell and grow from their plus ends toward the cell tips. Upon contact with the cell end, microtubule growth will first pause and then undergo a catastrophic event and microtubule shrinkage (21). This dynamic behavior of the microtubule plus end is regulated by a disparate, conserved, microtubule plus end group of proteins, called the +TIPs. The +TIP complex containing the EB1 family member Mal3 is required for the delivery of the Tea1-Tea4 complex to the cell tip (6, 11, 27, 45, 77). The latter complex docks at the cell end and recruits proteins required for actin nucleation (46, 76). Thus, the intricate cross talk between the actin and the microtubule cytoskeleton at specific intracellular locations is necessary for cell cycle-dependent polarized growth of the fission yeast cell.The intense analysis of polarized growth control in single-celled S. pombe makes this yeast an attractive organism for the identification of key regulatory components of the dimorphic switch. S. pombe multicellular invasive growth has been observed for specific strains under specific conditions, such as nitrogen and ammonium limitation and the presence of excess iron (1, 19, 50, 61).Here, we have identified an evolutionarily conserved key regulator of the S. pombe dimorphic switch, the Asp1 protein. Asp1 belongs to the highly conserved family of Vip1 1/3 inositol polyphosphate kinases, which is one of two families that can generate inositol pyrophosphates (PP) (17, 23, 42, 54). The inositol polyphosphate kinase IP6K family, of which the S. cerevisiae Kcs1 protein is a member, is the “classical” family that can phosphorylate inositol hexakisphosphate (IP₆) (70, 71). These enzymes generate a specific PP-IP₅ (IP₇), which has the pyrophosphate at position 5 of the inositol ring (20, 54). The Vip1 family kinase activity was unmasked in an S. cerevisiae strain with KCS1 and DDP1 deleted (54, 83). The latter gene encodes a nudix hydrolase (14, 68). The mammalian and S. cerevisiae Vip1 proteins phosphorylate the 1/3 position of the inositol ring, generating 1/3 diphosphoinositol pentakisphosphate (42). Both enzyme families collaborate to generate IP₈ (17, 23, 42, 54, 57).Two modes of action have been described for the high-energy moiety containing inositol pyrophosphates. First, these molecules can phosphorylate proteins by a nonenzymatic transfer of a phosphate group to specific prephosphorylated serine residues (2, 8, 69). Second, inositol pyrophosphates can regulate protein function by reversible binding to the S. cerevisiae Pho80-Pho85-Pho81 complex (39, 40). This cyclin-cyclin-dependent kinase complex is inactivated by inositol pyrophosphates generated by Vip1 when cells are starved of inorganic phosphate (39, 41, 42).Regulation of phosphate metabolism in S. cerevisiae is one of the few roles specifically attributed to a Vip1 kinase. Further information about the cellular function of this family came from the identification of the S. pombe Vip1 family member Asp1 as a regulator of the actin nucleator Arp2/3 complex (22). The 106-kDa Asp1 cytoplasmic protein, which probably exists as a dimer in vivo, acts as a multicopy suppressor of arp3-c1 mutants (22). Loss of Asp1 results in abnormal cell morphology, defects in polarized growth, and aberrant cortical actin cytoskeleton organization (22).The Vip1 family proteins have a dual domain structure which consists of an N-terminal “rimK”/ATP-grasp superfamily domain found in certain inositol signaling kinases and a C-terminal part with homology to histidine acid phosphatases present in phytase enzymes (28, 53, 54). The N-terminal domain is required and sufficient for Vip1 family kinase activity, and an Asp1 variant with a mutation in a catalytic residue of the kinase domain is unable to suppress mutants of the Arp2/3 complex (17, 23, 54). To date, no function has been described for the C-terminal phosphatase domain, and this domain appears to be catalytically inactive (17, 23, 54).Here we describe a new and conserved role for Vip1 kinases in regulating the dimorphic switch in yeasts. Asp1 kinase activity is essential for cell-cell and cell-substrate adhesion and the ability of S. pombe cells to grow invasively. Interestingly, Asp1 kinase activity is counteracted by the putative phosphatase domain of this protein, a finding that allows us to describe for the first time a function for the C-terminal part of Vip1 proteins.     

Identification of the Polyhydroxyalkanoate (PHA)-Specific Acetoacetyl Coenzyme A Reductase among Multiple FabG Paralogs in Haloarcula hispanica and Reconstruction of the PHA Biosynthetic Pathway in Haloferax volcanii

Genome-wide analysis has revealed abundant FabG (β-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the ΔfabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteria have been characterized in detail (10, 15, 20, 25, 26, 37), the genes involved in PHA biosynthesis in haloarchaea were not recognized until recently, when the PHA synthase genes were identified and characterized for Haloarcula marismortui, Haloarcula hispanica, and Haloferax mediterranei (6, 19). These archaeal PHA synthases are all composed of two subunits, PhaE and PhaC. They are homologous to the class III PHA synthases from bacteria but have a longer C-terminal extension in the PhaC subunit. Nevertheless, the pathway of supplying the PHA precursors has not yet been clarified for any haloarchaeal strain.Both H. mediterranei and H. hispanica are able to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources, despite the content of the (R)-3-hydroxyvalerate (3-HV) monomer of PHBV in H. mediterranei (10 to 13 mol%) (4, 19) being much higher than that in H. hispanica (∼3 mol%) (19). Conversely, the bacteria Ralstonia eutropha and Synechocystis sp. strain PCC6803, which possess class I and III PHA synthases, respectively, accumulate just poly(3-hydroxybutyrate) (PHB) when the 3-HV-related carbon sources (i.e., propionate and valerate) are not supplied (30). In these two bacteria, the biosynthesis of the (R)-3-hydroxybutyrate coenzyme A [(R)-3-HB-CoA] precursor is conducted by two steps. First, two acetyl-CoA molecules are condensed into one acetoacetyl-CoA molecule by the enzyme β-ketothiolase (PhaA). The acetoacetyl-CoA is then reduced to (R)-3-HB-CoA by a PHA-specific acetoacetyl-CoA reductase (PhaB). The resulting (R)-3-HB-CoA is subsequently incorporated into PHB, catalyzed by PHA synthases (26, 36).Both PhaB and FabG belong to the short-chain dehydrogenase/reductase (SDR) superfamily, whose members are homologous in sequence and have several conserved motifs (27, 29). Interestingly, although FabGs naturally reduce 3-ketoacyl-ACP to form (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis, a few FabGs also recognize 3-ketoacyl-CoA and hence function in PHA biosynthesis. For example, the FabG proteins of Escherichia coli and Pseudomonas aeruginosa have been demonstrated to supply precursors for PHA biosynthesis in recombinant E. coli cells (21, 22, 32, 35). In addition, several FabG paralogs may have evolved a distinct function, to be responsible only for PHA accumulation. This situation was observed in Synechocystis sp. strain PCC6803, where the originally annotated FabG (12) was renamed PhaB after an understanding of its function in PHA biosynthesis (36).Genome-wide analysis of H. marismortui ATCC 43049 (1) revealed eight FabG paralogs in this haloarchaeon. Similarly, multiple fabG paralog genes (fabG1 to fabG6) were also observed in the newly sequenced genome of H. hispanica (our unpublished data). In this study, we demonstrate that fabG1, but not the other five fabG paralogs, encodes the PHA-specific acetoacetyl-CoA reductase in H. hispanica. It is responsible for providing (R)-3-HB-CoA for PHA biosynthesis in Haloarcula species, and interestingly, this enzyme also functions well in Haloferax volcanii, endowing this PHA-defective strain with the ability to accumulate PHA when cotransformed with PHA synthase genes.     

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase,an Enzyme Involved in the Metabolism of Daidzein,from Equol-Producing Lactococcus Strain 20-92

Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.Isoflavones are flavonoids present in various plants and are known to be abundant in soybeans and legumes. These compounds have been called phytoestrogens because their chemical structure is similar to that of the female sex hormone, estrogen. Isoflavones have an ability to bind to estrogen receptors and show protection against or improvement in several sex hormone-dependent diseases, such as breast cancer, prostate cancer, menopausal disorder, lower bone density, and hypertension, due to their weak agonistic or antagonistic effects (1, 19, 27).Daidzein is one of the main soy isoflavonoids produced from daidzin by the glucosidase of intestinal bacteria (17). Equol is a metabolite produced from daidzein by the enterobacterial microflora (5). Recently, equol has attracted a great deal of attention because its estrogenic activity is more potent than that of other isoflavonoids, including daidzein (27). It is well known that individual variation exists in the ability of these enteric microflora to produce equol and that less than half the human population is capable of producing equol after ingesting soy isoflavones (3). Therefore, to increase the production of equol in the enteric environment of each individual, the development of probiotics using safe bacteria which have the ability to produce equol from daidzein is ongoing.Lactococcus strain 20-92 (Lactococcus 20-92; 30a) is an equol-producing lactic acid bacterium isolated from the feces of healthy humans by Uchiyama et al. (30). This bacterium is spherical and Gram positive and is a strain of L. garvieae. The application of Lactococcus 20-92 in probiotics is advantageous because L. garvieae is not pathogenic or toxic to humans.To date, other bacterial strains that are capable of transforming daidzein to dihydrodaidzein or equol have been isolated (9, 21, 22, 23, 29, 32, 36, 37). Daidzein is thought to be metabolized by human intestinal bacteria to equol or to O-desmethylangolensin via dihydrodaidzein and tetrahydrodaidzein (14, 15, 22, 32); however, neither the enzymes involved in the metabolism of daidzein to equol nor even the metabolic pathway has been clarified fully for equol-producing bacteria.In this study, we purified an enzyme from Lactococcus 20-92 that assisted in the conversion of daidzein to dihydrodaidzein. Furthermore, we cloned the L-DZNR gene and expressed the active recombinant enzyme in E. coli.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Flavodiiron Protein from Trichomonas vaginalis Hydrogenosomes: the Terminal Oxygen Reductase

Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen.Flavodiiron proteins (FDPs) constitute a recently established superfamily of soluble enzymes, thus far exclusively found in anaerobic and facultative aerobic organisms (2, 19, 54). Originally, the function ascribed to these proteins was the reduction of molecular oxygen to water as reported for Desulfovibrio gigas rubredoxin:oxygen oxidoreductase, the first thoroughly characterized protein of this type. This protein was found to utilize electrons derived from glycolysis for safe, four-electron reduction of dioxygen, thus protecting the anaerobic bacterium from the deleterious effects of oxidative stress (19). Later, some of these proteins were also shown to be involved in the reduction of nitric oxide in addition to their oxygen-reducing activity, thereby probably protecting the microbial organism against NO released during the immune response of the higher eukaryote host. The ratio of FDP activity toward oxygen and NO may differ substantially in various organisms; in some cases, FDP is almost exclusively reactive with oxygen, in others it is reactive with NO (20, 21, 43).FDPs are modular proteins, with flavodoxin-like and metallo-β-lactamase-like domains as their core modules. This two-domain structure is found in the simplest and most common members of the family, named class A FDPs. These proteins are the terminal elements of a multicomponent electron transporting chain that uses the reducing power of NAD(P)H to reduce and detoxify dioxygen and/or nitric oxide (41). Proximal electron donors to most class A FDPs are soluble electron transfer proteins. In the class A FDP rubredoxin:oxygen oxidoreductase from the sulfate-reducing bacterium Desulfovibrio gigas, the electron donor is a small protein, rubredoxin, that itself is reduced by an NADH:rubredoxin oxidoreductase (9, 10, 22). Besides rubredoxin, roles for other iron-sulfur flavoproteins in electron transport to FDPs have been suggested in several Archaea (41); coenzyme F₄₂₀H₂ is the electron donor for the FDP in the methanogenic archaeon Methanothermobacter marburgensis (44). The members of other FDP classes have additional domains fused to the C terminus that participate in electron transfer from the ultimate donor molecule [NAD(P)H] to the terminal electron acceptor (41).While originally believed to be restricted solely to prokaryotes, recent progress in genome sequencing projects have revealed homologous protein sequences in the genomes of several “amitochondriate” anaerobic protists, mostly with parasitic lifestyles, such as Trichomonas, Giardia, Entamoeba, Spironucleus, and a free-living Mastigamoeba (1, 2, 33, 42). Giardia intestinalis is the only eukaryotic organism to have had data on its FDP published recently. In line with what is known for the prokaryotic homologues, the giardial protein was shown to possess high oxygen (but not NO)-reducing activity and was therefore proposed to participate in protection against oxidative stress (13).Trichomonas vaginalis is an anaerobic (or microaerophilic) protozoan parasite causing human trichomoniasis, the most common nonviral sexually transmitted infection (38), for which oxygen concentrations higher than those encountered in situ in the vagina (i.e., concentrations above ∼60 μM) are toxic (17). The glucose metabolism of T. vaginalis is compartmentalized; while the reactions of classical glycolysis producing lactate, as well as the branch resulting in the formation of glycerol (8, 48) occur in the cytosol, a substantial portion of glycolytic carbon is diverted into the hydrogenosome, a mitochondrion-related organelle where the reactions of extended glycolysis produce additional ATP by oxidative decarboxylation of pyruvate (47, 48). Typical in the trichomonad hydrogenosome is the presence of the iron-sulfur (FeS) cluster-containing enzymes pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, and the electron carrier ferredoxin, which are involved in the generation of molecular hydrogen using electrons released from pyruvate (36). PFOR and hydrogenase are highly oxygen-sensitive enzymes (29, 32), and it is likely that the sensitivity of trichomonads to oxygen could at least in part be due to the inactivation of these key hydrogenosomal proteins.T. vaginalis must cope with low oxygen concentrations in its natural environment and, accordingly, possesses defense mechanisms to combat oxidative damage caused by oxygen itself or by reactive oxygen species that arise either enzymatically or when the reduced prosthetic groups of enzymes such as flavins and FeS clusters come into contact with oxygen. Most eukaryotes utilize glutathione as a key redox buffer and antioxidant, but trichomonads lack this and similar thiols (17). Cysteine has been suggested as a major reducing buffer and antioxidant (17), and it is believed that the organism relies upon cytosolic NADH oxidase (reducing oxygen to water) and NADPH oxidase (reducing oxygen to hydrogen peroxide) to prevent the permeation of oxygen into the hydrogenosomes (31). Proteins of the peroxiredoxin cascade (11) are also important for cytosolic peroxide detoxification. The identified defense mechanisms of hydrogenosomes include superoxide dismutase activity (17, 30) and recently found putative peroxidases that might provide protection against peroxides (39), but the protein that was suggested long ago to be responsible for oxygen uptake and detoxification has never been identified (6).We describe here the properties of a class A FDP from T. vaginalis hydrogenosomes and suggest its role in the metabolism of oxygen and protection of the organelle.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.