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.     

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).     

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).     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Allele-Specific H3K79 Di- versus Trimethylation Distinguishes Opposite Parental Alleles at Imprinted Regions

Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the “histone code” at imprinted genes.Imprinted genes are defined by the characteristic monoallelic silencing of either the paternally or maternally inherited allele. Most imprinted genes exist in imprinted gene clusters (10), and these clusters are usually associated with one or more differentially methylated regions (DMRs) (27, 65). DNA methylation at DMRs is essential for the allele-specific expression of most imprinted genes (31). Maternal or paternal allele-specific DNA methylation of a subset of DMRs (germ line DMRs) is gamete specific (27, 39). These maternal or paternal methylation differences are established during oogenesis or spermatogenesis, respectively, by the de novo DNA methyltransferases Dnmt3a and Dnmt3b together with Dnmt3L (5, 26, 48). The gamete-specific methylation differences set the stage for the parental allele-specific action of germ line DMRs, some of which have been shown to control the monoallelic expression of the associated genes in the respective domains (11, 34, 36, 53, 66, 71-73, 77). These DMRs are called imprinting control regions (ICRs).Two recurring themes have been reported for ICR action. ICRs can function as DNA methylation-regulated promoters of a noncoding RNA or as methylation-regulated insulators. Recent evidence suggests that both of these mechanisms involve chromatin organization by either the noncoding RNA (45, 50) or the CTCF insulator protein (17, 32) along the respective imprinted domains. The CTCF insulator binds in the unmethylated maternal allele of the H19/Igf2 ICR and blocks the access of the Igf2 promoters to the shared downstream enhancers. CTCF cannot bind in the methylated paternal ICR allele; hence, here the Igf2 promoters have access to the enhancers (4, 18, 24, 25, 62). When CTCF binding is abolished in the ICR of the maternal allele, Igf2 expression becomes biallelic, and H19 expression is missing from both alleles (17, 52, 58, 63). Importantly, CTCF is the single major organizer of the allele-specific chromatin along the H19/Igf2 imprinted domain (17). Significantly, CTCF recruits, at a distance, Polycomb-mediated H3K27me3 repressive marks at the Igf2 promoter and at the Igf2 DMRs (17, 32).A role for chromatin composition is suggested in the parental allele-specific expression of imprinted genes. Repressive histone tail covalent modifications, such as H3K9me2 H3K9me3, H4K20me3, H3K27me3, and the symmetrically methylated H4R3me2 marks, are generally associated with the methylated DMR alleles, while activating histone tail covalent modifications, such as acetylated histone tails and also H3K4me2 and H3K4me3, are characteristic of the unmethylated alleles (7-9, 12-15, 17, 21, 33, 35, 43, 44, 51, 55, 56, 67, 69, 74, 75). Importantly, the maintenance of imprinted gene expression depends on the allele-specific chromatin differences. ICR-dependent H3K9me2 and H3K27me3 enrichment in the paternal allele (67) is required for paternal repression of a set of imprinted genes along the Kcnq1 imprinted domain in the placenta (30). Imprinted Cdkn1c and Cd81 expression depends on H3K27 methyltransferase Ezh2 activity in the extraembryonic ectoderm (64). Similarly, H3K9 methyltransferase Ehmt2 is required for parental allele-specific expression of a number of imprinted genes, including Osbpl5, Cd81, Ascl2, Tfpi2, and Slc22a3 in the placenta (44, 45, 70).There is increasing evidence that covalent modifications, not only in the histone tails but also in the histone globular domains, carry essential information for development and gene regulation. The H3K79 methyltransferase gene is essential for development in Drosophila (60) and in mice (22). H3K79 methylation is required for telomeric heterochromatin silencing in Drosophila (60), Saccharomyces cerevisiae (47, 68), and mice (22). The H4K91 residue regulates nucleosome assembly (76). Whereas mutations at single acetylation sites in the histone tails have only minor consequences, mutation of the H4K91 site in the histone H4 globular domain causes severe defects in silent chromatin formation and DNA repair in yeast (37, 42, 76).Contrary to the abundant information that exists regarding the allele-specific chromatin composition at DMRs of imprinted genes, no information is available about the parental allele-specific marking in the histone globular domains at the DMRs. We hypothesized that chromatin marks in the globular domains of histones also distinguish the parental alleles of germ line DMRs. In order to demonstrate this, we measured the allele-specific enrichment of H3K79me1, H3K79me2, H3K79me3, and H4K91ac at 11 mouse DMRs using quantitative multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays. In general, H3K79me3 was associated with the methylated allele at most DMRs, whereas the unmethylated allele showed enrichment for H3K79me1, H3K79me2, and H4K91ac. These results are consistent with the possibility that allele-specific differences in the globular domains of histones contribute to the “histone code” at DMRs.     

XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.DNA double-strand breaks (DSBs) are potentially dangerous lesions, since their misrepair may cause chromosomal translocations, gene amplifications, loss of heterozygosity (LOH), and other types of genomic instability characteristic of human cancers (7, 9, 21, 40, 76, 79). DSBs are repaired predominantly by nonhomologous end joining or homologous recombination (HR), two evolutionarily conserved DSB repair mechanisms (8, 12, 16, 33, 48, 60, 71). DSBs generated during the S or G₂ phase of the cell cycle may be repaired preferentially by HR, using the intact sister chromatid as a template for repair (12, 26, 29, 32, 71). Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DSBs, which has led to the proposal that SCR protects against genomic instability, cancer, and aging. Indeed, a number of human cancer predisposition genes are implicated in SCR control (10, 24, 45, 57, 75).HR entails an initial processing of the DSB to generate a free 3′ single-stranded DNA (ssDNA) overhang (25, 48, 56). This is coupled to the loading of Rad51, the eukaryotic homolog of Escherichia coli RecA, which polymerizes to form an ssDNA-Rad51 “presynaptic” nucleoprotein filament. Formation of the presynaptic filament is tightly regulated and requires the concerted action of a large number of gene products (55, 66, 68). Rad51-coated ssDNA engages in a homology search by invading homologous duplex DNA. If sufficient homology exists between the invading and invaded strands, a triple-stranded synapse (D-loop) forms, and the 3′ end of the invading (nascent) strand is extended, using the donor as a template for gene conversion. This recombination intermediate is thought to be channeled into one of the following two major subpathways: classical gap repair or synthesis-dependent strand annealing (SDSA) (48). Gap repair entails the formation of a double Holliday junction, which may resolve into either crossover or noncrossover products. Although this is a major pathway in meiotic recombination, crossing-over is highly suppressed in somatic eukaryotic cells (26, 44, 48). Indeed, the donor DNA molecule is seldom rearranged during somatic HR, suggesting that SDSA is the major pathway for the repair of somatic DSBs (26, 44, 49, 69). SDSA terminates when the nascent strand is displaced from the D-loop and pairs with the second end of the DSB to form a noncrossover product. The mechanisms underlying displacement of the nascent strand are not well understood. However, failure to displace the nascent strand might be expected to result in the production of longer gene conversion tracts during HR (36, 44, 48, 63).Gene conversion triggered in response to a Saccharomyces cerevisiae or mammalian chromosomal DSB generally results in the copying of a short (50- to 300-bp) stretch of information from the donor (short-tract gene conversion [STGC]) (14, 47, 48, 67, 69). A minority of gene conversions in mammalian cells entail more-extensive copying, generating gene conversion tracts that are up to several kilobases in length (long-tract gene conversion [LTGC]) (26, 44, 51, 54, 64). In yeast, very long gene conversions can result from break-induced replication (BIR), a highly processive form of gene conversion in which a bona fide replication fork is thought to be established at the recombination synapse (11, 36, 37, 39, 61, 63). In contrast, SDSA does not require lagging-strand polymerases and appears to be much less processive than a conventional replication fork (37, 42, 78). BIR in yeast has been proposed to play a role in LOH in aging yeast, telomere maintenance, and palindromic gene amplification (5, 41, 52). It is unclear to what extent a BIR-like mechanism operates in mammalian cells, although BIR has been invoked to explain telomere elongation in tumors lacking telomerase (13). It is currently unknown whether LTGC and STGC in somatic mammalian cells are products of mechanistically distinct pathways or whether they represent alternative outcomes of a common SDSA pathway.Vertebrate cells contain five Rad51 paralogs—polypeptides with limited sequence homology to Rad51—Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3 (74). The Rad51 paralogs form the following two major complexes: Rad51B/Rad51C/Rad51D/XRCC2 (BCDX2) and Rad51C/XRCC3 (CX3) (38, 73). Genetic deletion of any one of the rad51 paralogs in the mouse germ line produces early embryonic lethality, and mouse or chicken cells lacking any of the rad51 paralogs reveal hypersensitivity to DNA-damaging agents, reduced frequencies of HR and of sister chromatid exchanges, increased chromatid-type errors, and defective sister chromatid cohesion (18, 72, 73, 82). Collectively, these data implicate the Rad51 paralogs in SCR regulation. The purified Rad51B/Rad51C complex has been shown to assist Rad51-mediated strand exchange (62). XRCC3 null or Rad51C null hamster cells reveal a bias toward production of longer gene conversion tracts, suggesting a role for the CX3 complex in late stages of SDSA (6, 44). Rad51C copurifies with branch migration and Holliday junction resolution activities in mammalian cell extracts (35), and XRCC3, but not XRCC2, facilitates telomere shortening by reciprocal crossing-over in telomeric T loops (77). These data, taken together with the meiotic defects observed in Rad51C hypomorphic mice, suggest a specialized role for CX3, but not for BCDX2, in resolving Holliday junction structures (31, 58).To further address the roles of Rad51 paralogs in late stages of recombination, we have studied the balance between long-tract (>1-kb) and short-tract (<1-kb) SCR in XRCC2 mutant hamster cells. We found that DSB-induced gene conversion in both XRCC2 and XRCC3 mutant cells is biased in favor of LTGC. These defects were suppressed by expression of wild-type (wt) XRCC2 or XRCC3, respectively, although the dependence upon ATP binding and hydrolysis differed between the two Rad51 paralogs. These results indicate that Rad51 paralogs play a more general role in determining the balance between STGC and LTGC than was previously appreciated and suggest roles for both the BCDX2 and CX3 complexes in influencing the termination of gene conversion in mammals.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

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.     

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.     

Roles of the Glyoxylate and Methylcitrate Cycles in Sexual Development and Virulence in the Cereal Pathogen Gibberella zeae

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (ΔGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the ΔGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.During the infection process, pathogenic fungi usually encounter nutrient deprivation in the host before gaining access to sufficient nutrients for successful colonization of the living tissue. To cope with a nutrient-limited environment, fungal pathogens seem to rely mostly on fatty acid metabolism for both energy supply and biosynthesis of essential molecules (29). The ability of fungi to use fatty acids as a carbon source for growth is based on the glyoxylate cycle. Fungal pathogens have been proposed to employ the glyoxylate bypass for the use of acetyl coenzyme A (CoA) units produced by the β-oxidation of even-chain-length fatty acids, probably available from host cell membranes or the lipid reservoir inside the fungal spore (7, 12, 20, 27, 28, 41, 44, 46). Recent studies suggest that the glyoxylate pathway plays an important role in fungal virulence toward both plant and animal hosts (12, 20, 27, 44, 46). The key enzymes of the glyoxylate pathway, such as isocitrate lyase (ICL), which catalyzes the cleavage of isocitrate to glyoxylate and succinate, and malate synthase, which mediates the condensation of acetyl-CoA and glyoxylate into malate, are strongly induced within the host (16, 27, 41, 44). Moreover, disruption of genes encoding either of these enzymes causes severely reduced virulence of fungal phytopathogens, including Leptosphaeria maculans (20), Magnaporthe grisea (46), Stagonospora nodorum (44), and Colletotrichum lagenarium (2), and the animal pathogen Candida albicans (27). In contrast, these glyoxylate cycle enzymes have been known to be dispensable in invasive aspergillosis caused by Aspergillus fumigatus (38, 43).During fatty acid and amino acid catabolism by fungi, propionyl-CoA can be generated along with acetyl-CoA, particularly from the breakdown of odd-chain-length fatty acids or of the amino acids valine, isoleucine, and methionine (14). Therefore, fungal pathogens may need to use or remove propionyl-CoA during the infection process because it is toxic to fungi. In fungi, propionyl-CoA is metabolized via the methylcitrate cycle, in which propionyl-CoA is oxidized to pyruvate in four enzymatic steps (4, 5, 6, 19, 30, 31, 40, 49, 50). Recently, the importance of the methylcitrate cycle in fungal virulence was demonstrated in A. fumigatus: a mutant defective in methylcitrate synthase, the first enzyme of this cycle, displayed attenuated virulence in mice and insects (19, 31). However, the role of methylisocitrate lyase (MCL), which catalyzes the last reaction in the methylcitrate cycle (i.e., the cleavage of methylisocitrate into pyruvate and succinate) in fungal virulence, has not been determined, although deletion of the MCL gene inhibits hyphal growth and conidiation in Aspergillus nidulans (4). The protein sequences of several fungal MCLs show high similarity to fungal ICLs of the glyoxylate cycle (4, 30). In the pathogenic bacterium Mycobacterium tuberculosis, the methylcitrate cycle, only when working together with the glyoxylate cycle, is involved in virulence as well as fatty acid metabolism and intracellular growth (34, 35).Here, we focused on the roles of these two cycles during disease development caused by the devastating cereal pathogen Gibberella zeae (anamorph: Fusarium graminearum). G. zeae is a ubiquitously distributed ascomycete fungus that causes major disease in cereal crops such as corn, wheat, barley, and rice (33). Severe epidemics of these diseases result in serious economic consequences due to yield losses and contamination by fungal mycotoxins (32, 33). Wind-disseminated sexual spores (ascospores), which are produced in perithecia formed on plant debris, can infect plant spikes during anthesis (13, 39, 45). Detailed studies of the G. zeae infection process on wheat and barley heads have shown that fungal hyphae on the inner surfaces of the spike penetrate epicarp cells through pits or pores and grow into the caryopses through the pericarp (21). Thus, the glyoxylate cycle, either alone or in conjunction with the methylcitrate cycle, is likely employed by G. zeae during the infection process, as in other fungus-plant interactions (20, 46). G. zeae genome searches have identified orthologs of fungal ICL and MCL genes, designated GzICL1 and GzMCL1, respectively. Here, we performed functional analyses of these genes to provide new insight into their importance in lipid metabolism during the G. zeae infection process in host plants.     

Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.