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.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

The Monothiol Single-Domain Glutaredoxin Is Conserved in the Highly Reduced Mitochondria of Giardia intestinalis

The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.Giardia intestinalis is a parasitic protist that was considered amitochondrial until recently (7). Although typical ATP-producing mitochondria are not present, related organelles, named mitosomes, were discovered in this organism (29). Mitosomes and mitochondria have a number of similarities; most notably, both are surrounded by a double membrane (29), they have a common mode of protein import and maturation (6, 24), and both harbor key components of the FeS cluster assembly machinery (29). These similarities indicate that mitosomes are highly reduced forms of mitochondria (7, 30), even though alternative evolutionary scenarios are still being discussed (17).FeS clusters are cofactors of a number of FeS proteins involved mainly in electron transport, energetic metabolism, synthetic pathways, and biological sensing (12). Most importantly, the FeS protein Rli1 is indispensable for rRNA processing and ribosome biogenesis (15, 31). Consequently, the biogenesis of FeS clusters is an essential process for all cells from bacteria to human cells (15). In most nonplant eukaryotes, the crucial part of this biosynthetic pathway occurs in the mitochondrion or mitochondrion-related organelles (14, 24, 28). Studies of Saccharomyces cerevisiae mitochondria showed that the FeS cluster assembly is centered on IscU, a metallochaperone that serves as a scaffold for a new FeS cluster. The cysteine desulfurase IscS (in S. cerevisiae named Nfs1) forms a heterodimer with Isd11. This heterodimer catalyzes the mobilization of sulfur for the FeS cluster. The delivery of iron is most likely regulated by frataxin (1). Reducing equivalents, which are required during FeS cluster biogenesis, are provided by a short electron transport chain, including the mitochondrial [2Fe2S] ferredoxin and ferredoxin:NADH reductase. Finally, a transient FeS cluster is transferred from IscU (Isu1/2) to apoproteins by the action of the Hsp70 (Ssq1) and Hsp40 (Jac1) chaperones and the proteins IscA (Isa1/2) and Iba57 (15). This last step also requires a monothiol class glutaredoxin (Grx5) with a characteristic CGFS active site motif (20). This class of glutaredoxins catalyzes the reduction of disulfide bonds in proteins converting glutathione (GSH) to GSH disulfide. It has been recently demonstrated that dimeric monothiol glutaredoxins can coordinate a [2Fe2S] cluster via the cysteine residue of the active site of each monomer and the cysteines of two GSH molecules (20). Although the exact role of Grx5 remains to be elucidated, it was hypothesized that, in the final step of iron sulfur cluster biogenesis, the FeS cluster that is transiently formed on an IscU scaffold protein is transferred to a Grx5 dimer in a GSH-dependent manner and is subsequently passed on to the target apoproteins (20).The ability of Giardia mitosomes to assemble FeS clusters on apoferredoxin has been demonstrated (29), indicating that all necessary components of the FeS cluster assembly machinery are present in these organelles. However, only three components known from S. cerevisiae mitochondria have been shown to be localized to mitosomes so far (IscS, IscU, and [2Fe2S] ferredoxin), while some others, including Isd11, frataxin, and GSH, were reported to be absent in Giardia (4, 5, 21). In this study we identified monothiol glutaredoxin in a mitosome-enriched fraction of G. intestinalis (Gigrx) using a proteomic approach. The mitosomal localization of Gigrx was confirmed by expression of the tagged protein in Giardia cells. The protein contains an unusually long N-terminal extension, which is involved in targeting the protein to the organelle, while the C-terminal part contains conserved residues required for the coordination of an FeS cluster. Phylogenetic analysis showed the close relationship between Gigrx and its mitochondrial homologues, which is consistent with the proposed mitochondrial origin of the iron-sulfur cluster assembly machinery in G. intestinalis.     

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).     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

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

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

Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most potent toxins known in nature and are characterized as category A select agents since they are considered potential bioterrorism threats (3). BoNTs can be distinguished immunologically into seven serotypes by using homologous antitoxins, designated A to G. BoNT/A is of particular interest, since it is frequently implicated in cases of botulism and is a significant threat in bioterrorism (1, 10).BoNT is a 150-kDa protein composed of a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bond and noncovalent molecular interactions (24). The heavy chain (H) has two functional domains, a transmembrane domain and a receptor binding domain. The light chain (L) is a zinc-dependent protease which specifically cleaves one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors, resulting in the blockage of evoked acetylcholine release at the skeletal neuromuscular junction (8).Previous studies have found that the bont genes of all strains of C. botulinum and neurotoxigenic strains of Clostridium butyricum and Clostridium baratii have a set of genes located upstream of the bont and ntnh genes that are organized as gene clusters (5, 7, 23). The two known primary types of clusters are (i) a hemagglutinin (ha) cluster and (ii) an orfX cluster with open reading frames (ORFs) of unknown functions. The ha cluster consists of genes encoding HA17, HA33, HA70, BotR, and NTNH. The orfX cluster consists of genes encoding ORFX3, ORFX2, ORFX1, P47, P21, and NTNH. Previous studies indicate that BoNT/A subtypes possess either a ha cluster or an orfX cluster associated with their expressed bont gene, depending on the subtype and strain (5, 11, 13-15, 33).It has been shown that the BoNT complex can form stable toxin complexes (TCs) of various sizes, including LL-TC (∼900 kDa), L-TC (∼500 kDa), and M-TC (∼300 kDa) composed of various combinations of HA proteins, NTNH, and BoNT (19, 21, 23, 29, 31, 34). M-TC contains BoNT and NTNH but has no HA proteins, whereas LL-TC and L-TC contain different ratios of the BoNT, NTNH, and HA proteins (21, 22, 29, 34). The biological and structural roles of the complex proteins are not completely characterized, although it has been proposed that they serve the role of protecting BoNT from harsh conditions, including pH, salt, temperature, and digestive enzymes, and that they assist BoNT translocation across the intestinal epithelial layer (2, 6, 17). A recent report indicated that the nontoxic proteins serve as adjuvants and contribute to the immunogenicity of BoNT/A (25).The production of botulinum TCs is known to vary with different serotypes and strains, medium composition, and culture conditions (21, 24, 31). The LL-TC has only been observed in proteolytic strains (group I). Serotype A to D strains produce M-TC and L-TC in their culture medium, while serotype E and F strains produce only M-TC (17, 18).In 1986, a Japanese group isolated four HA-negative C. botulinum strains from infant botulism cases that produced only M-TC (300 kDa). They assigned the strains to subtype A2 (14, 30). In 2004, our laboratory confirmed on a genomic level that the BoNT/A2 subtype contained the orfX cluster instead of the ha cluster (12). Since then, more arrangements and combinations of neurotoxin gene clusters were characterized along with more BoNT subtypes (13, 20, 33). However, the function of the orfX genes and the role of the presumptive protein products and their role in the TCs are still unknown, including whether ORFX proteins can form a TC with the expressed toxin analogous to the ha cluster proteins.In this study, the BoNT/A2 TC was purified from a native culture to determine if the orfX cluster proteins remain associated with BoNT/A2. To better understand the role of the orfX cluster genes, the orfX cluster proteins of C. botulinum A2 strains (ORFX1, ORFX3, P47, and the middle part of NTNH) was expressed using either an Escherichia coli or a C. botulinum expression system in this study. Antibodies against individual expressed orfX cluster proteins were then raised by immunizing a rabbit and mice. These antibodies were then used as probes to investigate the expression pattern of the orfX cluster genes in the native A2 culture. ORFX2, which could not be expressed, was detected by N-terminal protein sequencing.     

Polymorphic Mutation Frequencies of Clinical and Environmental Stenotrophomonas maltophilia Populations

Mutation frequencies were studied in 174 Stenotrophomonas maltophilia isolates from clinical and nonclinical environments by detecting spontaneous rifampin-resistant mutants in otherwise-susceptible populations. The distribution of mutation frequencies followed a pattern similar to that found for other bacterial species, with a modal value of 1 × 10⁻⁸. Nevertheless, the proportion of isolates showing mutation frequencies below the modal value (hypomutators) was significantly higher for S. maltophilia than those so far reported in other organisms. Low mutation frequencies were particularly frequent among environmental S. maltophilia strains (58.3%), whereas strong mutators were found only among isolates with a clinical origin. These results indicate that clinical environments might select bacterial populations with high mutation frequencies, likely by second-order selection processes. In several of the strong-mutator isolates, functional-complementation assays with a wild-type allele of the mutS gene demonstrated that the mutator phenotype was due to the impairment of MutS activity. In silico analysis of the amino acid changes present in the MutS proteins of these hypermutator strains in comparison with the normomutator isolates suggests that the cause of the defect in MutS might be a H683P amino acid change.Stenotrophomonas maltophilia is a Gram-negative, nonfermenting environmental bacterial species often isolated from the rhizosphere and from water sources (11, 12, 63). Some S. maltophilia strains have been used for bioremediation (13, 24, 73) or bioaugmentation (37). However, besides its environmental origin and potential relevance for biotechnological purposes, S. maltophilia is also a relevant human opportunistic pathogen (44) associated with a broad spectrum of clinical syndromes, such as bacteremia (79, 81), endocarditis (18), infection in cancer patients (1), and respiratory tract infections, including those suffered by cystic fibrosis (CF) patients (72, 77). One of the most problematic characteristics of S. maltophilia is its intrinsic high resistance to several antibiotics (4). This intrinsic antibiotic resistance is at least partly due to the presence in the genome of S. maltophilia (17) of genes encoding antibiotic-inactivating enzymes (6, 9, 30, 39, 42, 58) and multidrug resistance (MDR) efflux pumps (2, 3, 43, 78). More recently, a chromosomally encoded Qnr protein that contributes to the intrinsic resistance to quinolones of S. maltophilia has been described (67, 68).A clear difference between infective (clinical) and environmental (nonclinical) S. maltophilia strains has not been reported (12, 63). However, although the available data fit the concept that opportunistic pathogens have not specifically evolved to infect humans (48), this does not mean that they do not evolve during the infective process. For most acute infections, we can presume that the time of in-host evolution is probably too short to detect relevant adaptive changes. Nevertheless, the situation might be different in chronic infections, such as those involving the bronchial compartment in CF patients. In this case, the same bacterial clone can be maintained and grow inside the host for years (62). This produces strong diversification over time and in different compartments of the lung (25, 71, 80), a process in which the acquisition of a mutator phenotype is important (52). Thus, isolates derived from an initial clone but presenting different morphotypes (47), different phenotypes of susceptibility to antibiotics (26) or in the expression of virulence determinants (14, 15, 36), or with different mutation frequencies (49, 60) are recovered from each individual patient suffering chronic infections. More recently, intraclonal diversification has also been described for Pseudomonas aeruginosa causing acute infections in intubated patients (38). Taken together, this indicates that bacteria can evolve during infection.For different bacterial species, strains isolated from CF patients with chronic lung infections show high mutation frequencies (hypermutable strains) (19, 60, 61, 66), whereas hypermutators have rarely been found in isolates from acute infections (33). An explanation for this difference could be that hypermutable strains tend to be selected for in the highly compartmentalized environment of the infected lung by intensive antibiotic therapy, as well as by the stressful conditions of the habitat. This is a second-order selection process (75, 76), in which mutations are selected because they confer an advantage in clinical environments in such a way that mutator strains are selected because they can produce more mutants (both advantageous and deleterious) for selection. In cases of chronic infections that are treated, strong and maintained selective local processes might occur, either by antibiotic treatment or by the actions of the anti-infective systems of the host. Natural out-of-host open environments obviously might have local stresses. However, the intensity of selection is expected to be lower in these habitats, and a constant replacement of potentially lost organisms by migration of neighbor populations probably mitigates the local selection of mutators and favors the enrichment of bacteria presenting low mutation frequencies. In the case of chronic infections, the replacement of mutators by neighbor normomutators is unlikely, because those infections are produced by a single clone that remains for several years in the host (62). Furthermore, although the infection process presents strong evolutionary bottlenecks for bacterial populations, the human host also provides a constant temperature, reliable nutrient supplies, and a habitat largely free from predators and competitors. Thus, while hypermutation might increase the capability of bacteria to adapt to some specific challenges in the clinical environment, the cost of hypermutation in terms of deleterious mutations might also be diminished, and these effects might be mutually reinforcing.The hypothesis explored in this paper is that S. maltophilia is adapted to deal with out-of-host fluctuating environmental variations but that once the organism enters a patient as an opportunistic pathogen, its adaptive needs significantly increase due to the actions of stressful local environmental conditions, such as the immune response and, when present, antibiotics. This enhanced stress under infective conditions might result in the selection of variants with increased mutation frequencies in a second-order selection process (75, 76). To test this hypothesis, the mutation frequencies of S. maltophilia clinical isolates (obtained from CF and non-CF patients) and from the environment (nonclinical origin) were compared. Most works that have been published on the different mutation frequencies in bacterial populations have focused on the detection of strains showing a high mutation frequency (mutators). In our work, we describe for the first time the presence of mutators in clinical isolates of S. maltophilia and demonstrate that hypermutation in several of those isolates is due to defects in MutS.Nevertheless, our main goal has been the analysis of the global distribution of mutation frequencies in an ample number of samples from clinical and nonclinical environments. Our results indicate not only that mutators are more frequent in clinical S. maltophilia isolates, but also that the overall distribution of mutation frequencies is different in S. maltophilia populations with environmental or clinical origins, with a tendency toward mutation frequencies lower than the modal mutation value (hypomutators) in the environmental isolates.     

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.Bacterial electron transfer to sparingly soluble electron acceptors is a critical component of a wide variety of environmental and energy-generating processes, including biogeochemical cycling of metals, degradation of natural and contaminant organic matter, weathering of clays and minerals, biomineralization of Fe-bearing minerals, reductive precipitation of toxic metals and radionuclides, and electricity generation in microbial fuel cells (17, 33, 34). Anaerobic and facultatively anaerobic bacteria capable of respiring sparingly soluble (<10⁻²⁵ M at pH 7) Fe(III) oxides are ubiquitous in nature and may be found in marine, freshwater, and terrestrial environments, including metal- and radionuclide-contaminated subsurface aquifers (25, 34). Fe(III)-respiring prokaryotes are also deeply rooted and scattered throughout the domains Bacteria and Archaea (possibly indicating an ancient metabolic process) and include hyperthermophiles, psychrophiles, acidophiles, and extreme barophiles (34). Despite their potential environmental, energy-generating, and evolutionary significance, the molecular details of microbial Fe(III) respiration remain unclear.Fe(III)-respiring, neutrophilic bacteria are presented with a unique physiological challenge: they are required to respire anaerobically on electron acceptors found largely as sparingly soluble Fe(III) oxides presumably unable to contact periplasm- or inner membrane (IM)-localized electron transport systems. To overcome this problem, Fe(III)-respiring bacteria are postulated to employ novel respiratory strategies not found in other bacteria (e.g., aerobes, denitrifiers, sulfate-reducing bacteria, and methanogens) that respire soluble electron acceptors (17, 38). The novel respiratory strategies include (i) a direct-contact pathway in which terminal Fe(III) reductases are secreted to the cell outer membrane (OM), where they contact and deliver electrons directly to external Fe(III) oxides (18, 23, 40, 42, 48, 57, 64, 67), (ii) a two-step electron shuttling pathway in which bacterially reduced endogenous or exogenous electron shuttles deliver electrons to external Fe(III) oxides in a second (abiotic) electron transfer reaction (11, 26, 39, 45), and (iii) a two-step Fe(III) chelation (solubilization) pathway in which Fe(III) oxides are first nonreductively dissolved by endogenously synthesized organic ligands prior to reduction of the resulting soluble organic Fe(III) [Fe(III) bound to an organic molecule] complexes (36, 59).Candidate organic ligands for production of soluble organic Fe(III) during anaerobic Fe(III) oxide respiration include siderophores, the Fe(III)-chelating compounds synthesized and secreted by a wide variety of bacteria and fungi for solubilization and subsequent assimilation of otherwise inaccessible Fe(III) substrates (12, 44, 49, 63). Hydroxamate-type siderophores are produced via N⁶ hydroxylation and N⁶ acylation of l-ornithine and, in some cases, cyclization to macrocyclic ring structures (13). The macrocyclic siderophores bisucaberin and putrebactin, for example, are two structural analogs of the cyclic bis(hydroxamate) siderophore alcaligin, synthesized by Aliivibrio salmonicida and Shewanella putrefaciens strain 200, respectively (27, 32, 65). After transport across the cell envelope via a TonB-dependent pathway, Fe(III) is subsequently released from the Fe(III)-siderophore complex by ligand exchange reactions promoted by siderophore ligand hydrolysis and/or protonation or by Fe(III)-siderophore reduction and release of Fe(II) to acceptor ligands (9, 66).The main objectives of the present study were to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are synthesized by Fe(III)-siderophore biosynthesis systems and (ii) if Fe(III)-siderophore reductases are required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. The experimental strategy for this study included (i) identification of genes encoding the siderophore biosynthesis and Fe(III)-siderophore reductase systems in the S. oneidensis genome, (ii) generation of in-frame deletions in the corresponding siderophore biosynthesis and Fe(III)-siderophore reductase genes, (iii) tests of the resulting siderophore biosynthesis mutants for production of siderophores and soluble organic Fe(III) during aerobic and anaerobic Fe(III) oxide respiration, and (iv) tests of the resulting Fe(III)-siderophore reductase mutants for respiration of soluble organic Fe(III) as an anaerobic electron acceptor.     

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.     

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3, 22, 23, 38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7, 16, 17, 27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gram-negative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (γ-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-positive bacteria rely on different thiol redox buffers based on cysteine, the novel 398-Da bacillithiol (BSH), or coenzyme A (CoA) (15, 52). To maintain the reduced state of the cytoplasm, most bacteria use enzymatic systems for disulfide bond reduction such as the thioredoxin (Trx) system, which is highly conserved in gram-negative bacteria (3, 10). The Trx system consists of thioredoxin (TrxA) and the NADPH-dependent thioredoxin reductase (TrxB).Any imbalance in the cellular redox state caused by ROS elicits expression of a repertoire of different proteins, commonly under the control of a redox-sensing regulator: for example, OxyR in Escherichia coli and PerR, OhrR, SarZ, and Spx in B. subtilis and S. aureus, respectively (11, 12, 41, 55, 58, 64-66). The subsequently induced proteins detoxify ROS and restore and protect the normal physiological redox state in the cell.Besides ROS and reactive nitrogen species, so-called “reactive electrophilic species” (RES) affect the thiol redox balance. RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2, 43, 45, 46, 53, 66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine. Exposure of cells to diamide induces the oxidative as well as the electrophile stress response in B. subtilis (43, 45, 53). The toxicity of diamide is based on disulfide bond formation (40), which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay (32, 64). It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins.However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8, 13, 33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35, 47) and signal transduction (39, 42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).Cysteine, BSH, and CoA are also dominant LMW thiols in S. aureus (52). In this study, we have investigated in more detail the extents of S thiolations and inter- and intramolecular disulfide bond formation of B. subtilis and S. aureus in response to disulfide stress. The results showed that exposure to diamide leads to S thiolations in S. aureus. Using a nonreducing/reducing sodium dodecyl sulfate (SDS) diagonal electrophoresis approach, proteins with intermolecular disulfide bonds could be distinguished from proteins with intramolecular disulfide bonds (57). The results support that the majority of reversible thiol oxidations are based on S thiolations rather than disulfide bonds between proteins. Depletion of the free cysteine pool in B. subtilis after exposure to diamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshF_Lm) was expressed in B. subtilis, enabling GSH biosynthesis (29). Although GSH production does not enhance the resistance to oxidative stress in B. subtilis, it participates in the formation of S thiolations.