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.     

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.     

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).     

Malonic Semialdehyde Reductase,Succinic Semialdehyde Reductase,and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO₂ fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.The thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula uses a 3-hydroxypropionate/4-hydroxybutyrate cycle for CO₂ fixation (9, 28, 29, 35) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales (31) and in mesophilic marine group I Crenarchaea (Cenarchaeum sp., Nitrosopumilus sp.). This cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (15, 22-25, 41, 42). It involves the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA by a biotin-dependent acetyl-CoA carboxylase (12, 29). The carboxylation product is reduced to malonic semialdehyde by malonyl-CoA reductase (1). Malonic semialdehyde is further reduced to 3-hydroxypropionate, the characteristic intermediate of the pathway (9, 31, 35). 3-Hydroxypropionate is further reductively converted to propionyl-CoA (3), which is carboxylated to (S)-methylmalonyl-CoA by propionyl-CoA carboxylase. Only one copy of the genes encoding the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, indicating that this enzyme is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (12, 29). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, which is followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other autotrophic Sulfolobales. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase, identical to acetyl-CoA carboxylase; 8, (S)-methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH), identical to malonyl-CoA reductase; 11, succinic semialdehyde reductase (NADPH); 12, 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 highlighted steps are catalyzed by the enzymes studied here.Succinyl-CoA is converted via succinic semialdehyde and 4-hydroxybutyrate to two molecules of acetyl-CoA (9), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA molecule 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 molecule and two bicarbonate molecules into succinyl-CoA (Fig. ​(Fig.1,1, steps 1 to 9), and the second part converts succinyl-CoA to two acetyl-CoA molecules (Fig. ​(Fig.1,1, steps 10 to 16).The second part of the autotrophic cycle also occurs in the dicarboxylate/4-hydroxybutyrate cycle, which operates in autotrophic CO₂ fixation in Desulfurococcales and Thermoproteales (Crenarchaea) (27, 37), raising the question of whether the enzymes in these two lineages have common roots (37). The first part of the cycle also occurs in the 3-hydroxypropionate cycle for autotrophic CO₂ fixation in Chloroflexus aurantiacus and a few related green nonsulfur phototrophic bacteria (19, 22, 23, 32, 49).The two-step reduction of malonyl-CoA to 3-hydroxpropionate in Chloroflexus is catalyzed by a single bifunctional 300-kDa enzyme (30). The M. sedula malonyl-CoA reductase is completely unrelated and forms only malonic semialdehyde (1), and the enzyme catalyzing the second malonic semialdehyde reduction step that forms 3-hydroxypropionate is unknown. In the second part of the 3-hydroxypropionate/4-hydroxybutyrate cycle a similar reduction of succinyl-CoA via succinic semialdehyde to 4-hydroxybutyrate takes place. The enzymes responsible for these reactions also have not been characterized.In this work we purified the enzymes malonic semialdehyde reductase, succinyl-CoA reductase, and succinic semialdehyde reductase from M. sedula. The genes coding for these enzymes were identified in the genome, and recombinant proteins were studied in some detail. Interestingly, succinyl-CoA reductase turned out to be identical to malonyl-CoA reductase. We also show here that enzymes that are highly similar to succinyl-CoA reductase in Thermoproteus neutrophilus do not function as succinyl-CoA reductases in M. sedula.     

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.     

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.     

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.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

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.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

Role for CysJ Flavin Reductase in Molybdenum Cofactor-Dependent Resistance of Escherichia coli to 6-N-Hydroxylaminopurine

We have previously described a novel Escherichia coli detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (ycbX and yiiM) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the cysJ gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (CysJ₈I₄), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX “hybrid” protein from two Vibrio species is capable of compensating for a double cysJ ycbX defect in E. coli.Mutagenic base analogs are chemically modified nucleobases that can be incorporated in the cellular metabolism through purine or pyrimidine salvage pathways. Once converted to the deoxynucleoside triphosphate (dNTP) level, they may participate in DNA replication in an error-prone manner because of their ambivalent base-pairing capacity (11). Such synthetic base analogs are often used as a sensitive tool for studying DNA replication fidelity, DNA repair, or the metabolism of nucleic acid precursors. Mutagenic base analogs such as 8-oxoguanine or 3-methyladenine can also be formed in vivo as a consequence of normal cellular metabolism or produced by chemical and physical factors, such as alkylating agents or ionizing radiation.An important group of mutagenic and cytotoxic analogs are the N-hydroxylated nucleobases (or ribosides) such as 6-N-hydroxylaminopurine (HAP), 2-amino-HAP, or N⁴-hydroxycytidine (15). Specifically, HAP was found to be a very strong mutagen in bacteria and fungi, as well as mammalian cells (2, 20, 27). Some data have suggested that HAP may also be formed in vivo under oxidative stress (30) or as a by-product of certain purine salvage/interconversion pathways (5, 22).The genetic control of HAP-induced mutagenesis has been studied in some detail in the yeast Saccharomyces cerevisiae and in the bacterium Escherichia coli. In S. cerevisiae, resistance to HAP depends primarily on genes involved in adjusting and regulating the DNA or RNA precursor pools (HAM1 [ITP/XTPase], AAH1 [adenine aminohydrolase], and ADE genes involved in de novo AMP biosynthesis) (34).In E. coli, the major pathway that protects cells against HAP and related N-hydroxylated compounds is controlled by the moa, moe, and mog genes, which are required for biosynthesis of molybdenum cofactor (MoCo) (18, 19). MoCo is an essential cofactor for a varied group of oxidoreductases that are widely distributed from bacteria to humans. Chemically, MoCo is a pterin derivative (molybdopterin) that coordinates a molybdenum atom that serves as a catalytic redox center (for reviews, see references 23, 28, and 29). Based on catalytic details and sequence homology, molybdopterin-containing enzymes have been divided in four families: the xanthine oxidase family, the sulfite oxidase family, the dimethyl sulfoxide (DMSO) reductase family, and the aldehyde ferredoxin oxidoreductase family (14, 16). However, our previous studies on the MoCo-dependent resistance to HAP showed that none of the known or putative E. coli members of these families are responsible for the major HAP resistance mechanism (19). Instead, we discovered that HAP resistance is dependent on two newly described proteins, YcbX and YiiM, that are characterized by a so-called MOSC domain (molybdenum cofactor sulfurase C-terminal domain) (1, 17). This domain was first described as part of eukaryotic MoCo sulfurases (MOSs) (1), and it most likely represents a novel class of MoCo-binding domain, as indicated by studies on two mammalian MOSC-containing proteins (mARC1 and mARC2) discovered in mitochondria (12, 13).Our studies in E. coli showed that cell-free bacterial extracts were capable of converting HAP to adenine by an N-reductive reaction (17). Importantly, this conversion was entirely dependent on the presence of MoCo and the YcbX or YiiM proteins (17). Consequently, we suggested that this reduction of HAP to adenine forms the basis of the in vivo MoCo-dependent detoxification in E. coli (17). Interestingly, the mammalian MOSC-containing proteins mARC1 and mARC2 were shown to mediate the reduction of the N-hydroxylated prodrug benzamidoxime to its active amino form benzamidine (12, 13). Thus, the reduction of N-hydroxylated compounds may be a defining feature for the broadly distributed MOSC proteins (1).Our previous analyses also revealed that the E. coli ycbX and yiiM genes define two independent subpathways within the MoCo-dependent system (17). This is illustrated in the overall scheme shown in Fig. ​Fig.1.1. MoCo is synthesized in a series of steps from GTP by-products of the moa, moe, and mog operons. MoCo is then used as a cofactor for the YcbX and YiiM proteins, which reduce the N-hydroxylated compound to the corresponding amino form. The ycbX and yiiM pathways are genetically distinct as determined by epistasis experiments (17). They also differ by their substrate specificity patterns: YcbX protects most strongly against HAP, whereas YiiM has its largest effects toward hydroxylamine (NH₂OH) (17).Open in a separate windowFIG. 1.Genetic framework for the major molybdenum cofactor (MoCo)-dependent pathways of detoxification of N-hydroxylated base analogs in E. coli (17). moaA to mogA indicate the series of genes required for MoCo biosynthesis (19, 28), while ycbX and yiiM represent the two independent subpathways identified within the MoCo-dependent pathway (17). Specifically, ycbX and yiiM produce apoenzymes that react with MoCo to form the active YcbX and YiiM proteins. The diagram also indicates the differential specificity of the two subpathways toward the model N-hydroxylated compounds used in our studies: 6-N-hydroxylaminopurine (HAP), 2-amino-HAP (AHAP), and hydroxylamine (NH₂OH). For simplicity, the diagram does not distinguish between the MPT and MGD forms of MoCo (19). As shown elsewhere (19), YcbX and YiiM likely employ the MPT form. One additional, minor pathway for HAP detoxification dependent on biotin sulfoxide reductase (an MGD-requiring enzyme) is observable only in the double ycbX yiiM-deficient background and is likewise not shown here (see reference 17 for details).Prior to the establishment of this scheme of YcbX and YiiM as molybdoproteins, we had entertained certain alternative possibilities for the precise function of the ycbX and yiiM open reading frames (ORFs), including a possible role in MoCo sulfuration (which is a required modification of MoCo in certain molybdoenzymes, such as xanthine oxidase) (23, 29). This sulfuration model was ultimately eliminated (17), but certain experiments related to this hypothesis yielded interesting further clues regarding the detailed mechanisms of HAP resistance. These observations included an unexpected HAP-sensitive phenotype for cysJ mutants as well as a noted sensitization of wild-type strains to HAP by l-cysteine. In the present work, we describe these experiments and show the cysJ gene to be an essential component of the ycbX branch of HAP resistance. In a related mechanism, the observed sensitization of wild-type strains by l-cysteine results from the suppression, by l-cysteine, of the cys regulon. Overall, our experiments suggest that CysJ is a specific protein partner of YcbX and that CysJ mediates the N-reductive reaction through its NADPH:flavin oxidoreductase activity. This activity provides reducing equivalents to its partner YcbX, which ultimately performs the reduction of HAP to nontoxic adenine at its molybdocenter.