Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C₄-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C₄-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter Glt_Ph from Pyrococcus horikoshii.The DctA family is one of several diverse families of secondary transporters that catalyze the uptake of C₄-dicarboxylates from the Krebs cycle in bacteria (16, 27). In Escherichia coli, DctA mediates the uptake of succinate, fumurate, and malate under aerobic conditions; genomic disruption of dctA in E. coli prevents growth with malate or fumarate as the sole carbon source, and the mutant grows poorly on succinate (5). Similarly, a dctA knockout mutant of Bacillus subtilis cannot grow with succinate or fumarate as the sole carbon source (1). DctA plays a major role in the symbiotic relationship between nitrogen-fixing rhizobia (43) and root nodule-forming plants (30, 37, 38). Transport assays with Sinorhizobium meliloti cells showed previously that in addition to succinate, malate, and fumarate, orotate is transported and that a range of other substrates such as succinamic acid and succinamide may be transported, because they inhibit the transport of orotate (42). In Corynebacterium glutamicum, malate transport by DctA is inhibited by α-ketoglutarate, oxaloacetate, and glyoxylate, indicating that these compounds may be substrates also (41).DctA transporters belong to a large family of secondary transporters (the DAACS [dicarboxylate/amino acid:cation symporter] family), which also comprises well-characterized glutamate/aspartate transporters and neutral amino acid transporters (32, 33). While DctA-type dicarboxylate transporters are found only in bacteria, glutamate/aspartate transporters of the DAACS family are found both in prokaryotes (e.g., GltT in Bacillus stearothermophilus, GltP in E. coli, and Glt_Ph in Pyrococcus horikoshii [2, 7, 34]) and in higher eukarya, where they play a pivotal role in the reuptake of the excitatory neurotransmitter glutamate from the synaptic cleft (4). Neutral amino acid (alanine, serine, and threonine) transporters are found in mammals (see, e.g., references 36 and 44) as well as bacteria (17).Secondary transporters of the DAACS family use (electro)chemical gradients of cations across the membrane to drive transport. The type of cotransported ions varies among family members: eukaryotic glutamate transporters couple the transport of glutamate to the symport of one proton and three sodium ions and the antiport of one potassium ion (24, 45). Bacterial and archaeal glutamate transporters utilize either sodium ions or protons for symport (2) and are independent of potassium ions (28, 31). The bacterial and mammalian neutral amino acid transporters are sodium ion coupled. Glutamate/aspartate transporters and bacterial serine/threonine transporters (SstTs) are electrogenic, but mammalian neutral amino acid transporters are obligate electroneutral amino acid antiporters (44).Insight into the structure-function relationships of the DAACS family members has greatly increased since crystal structures of the P. horikoshii aspartate transporter Glt_Ph have been determined (2, 29, 40). The protein consists of eight membrane-spanning helices and two reentrant regions (helical hairpins HP1 and HP2) (40). The C-terminal part of the protein (helices 7 and 8 and HP1 and HP2) is most strongly conserved with respect to other family members and binds the substrate and cotransported ions, with HP1 and HP2 functioning as lids that allow alternating access to the substrate- and ion-binding sites from either side of the membrane (3, 29). Glt_Ph forms a homotrimeric complex in which each protomer functions independently of the other subunits (11, 12, 18, 19, 23). The fold and oligomeric state are likely to be conserved throughout the family.Whereas the transport mechanisms of bacterial glutamate and neutral amino acid transporters of the DAACS family have been studied extensively in vitro, the C₄-dicarboxylate transporters of the DAACS family (DctA proteins) have been studied using whole cells only. To fully characterize these transporters, in vitro activity assays using either membrane vesicles or proteoliposomes containing purified protein are necessary. In such assays, the internal and external buffer compositions can be controlled, thus allowing manipulation of the chemical ion gradients and the electrical potential across the membrane. Here, we present the first biochemical characterization of a DctA family member in membrane vesicles. We have studied the DctA homologue from B. subtilis, which is annotated as DctP (1) but which we propose to rename DctA to reflect the homology to other DctA proteins. B. subtilis DctA (DctA_Bs) has 30 to 32% sequence identity to the aspartate transporter Glt_Ph and human excitatory amino acid transporter (EAAT) family members, over 40% sequence identity to the characterized bacterial glutamate transporters from E. coli and B. stearothermophilus, and 41 and 56% identity to DctA homologues from C. glutamicum and E. coli, respectively. We determined the substrate specificity of DctA_Bs, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns.     

Requirement of Polyphosphate by Pseudomonas fluorescens Pf0-1 for Competitive Fitness and Heat Tolerance in Laboratory Media and Sterile Soil

Knowledge of the genetic basis for bacterial survival and persistence in soil is a critical component in the development of successful biological control strategies and for understanding the ecological success of bacteria. We found a locus specifying polyphosphate kinase (ppk) and a nonpredicted antisense RNA (iiv8) in Pseudomonas fluorescens Pf0-1 to be necessary for optimal competitive fitness in LB broth culture and sterile loam soil. Pf0-1 lacking ppk and iiv8 was more than 10-fold less competitive against wild-type Pf0-1 in sterile loam soil low in inorganic phosphate. Studies indicated that ppk, and not iiv8, was required for competitive fitness. No role for iiv8 was identified. While a ppk and iiv8 mutant of Pf0-1 did not have increased sensitivity to osmotic, oxidative, and acid stress, it was more sensitive to elevated temperatures in laboratory medium and during growth in sterile soil. ppk was shown to be part of the Pho regulon in P. fluorescens, being upregulated in response to a low external P_i concentration. Of importance, overproduction of polyphosphate in the soil environment appears to be more deleterious than production of none at all. Our findings reveal a new role for polyphosphate (and the need for proper regulation of its production) in competitive fitness of P. fluorescens in laboratory and soil environments.Soils are complex environments, presenting microbial inhabitants with a range of challenges which must be met if they are to survive and persist. Nutrients, pH, water content, and temperature can all affect survival of bacteria in soil (44). An understanding of the ecological success of microbes in natural environments such as soil requires knowledge of the mechanisms underlying adaptation and persistence. This appreciation for adaptive mechanisms is also important for the successful use of microbes for environmental applications, such as biocontrol or bioremediation (29, 47). Pseudomonas species, which are frequently isolated from soil environments, have a large complement of regulatory genes which are thought to permit rapid responses to environmental changes (42). For example, the two-component pair phoB-phoR responds to change in the concentration of exogenous inorganic phosphate (P_i) (25, 46). The regulatory targets of such systems likely include genes that are critical for adaptation and thus are important for long-term success within a fluctuating environment.Many Pseudomonas species are being investigated as potential biocontrol agents because of their ability to produce compounds that are inhibitory to plant-pathogenic fungi (10). In addition, some Pseudomonas species have plant growth-promoting activity independent of their antifungal activity, making them very attractive in biocontrol (45). Despite the frequency with which Pseudomonas species are isolated from soils and the interest in developing biocontrol applications, the determinants of their environmental success are not well characterized (8).Genes that are upregulated in a particular environment are likely to be important for success in that environment (28). Indeed, a number of environmentally regulated genes, identified using in vivo expression technology (IVET), have been shown to be important for success in the environment from which they were isolated (for examples, see references 3, 8, 19, and 20). We have been investigating the genetic basis of persistence of the soil isolate Pseudomonas fluorescens Pf0-1. Using an IVET screen, we found 22 sequences upregulated during growth in sterile loam soil (40). Three of these IVET-identified genes were shown to be involved in colonization of sterile soil. Here we report the analysis of an additional IVET-identified locus, termed iiv8 (40). The sequence upregulated in soil did not match any gene predicted in the Pf0-1 genome annotation (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000094","term_id":"253992019","term_text":"CP000094"}}CP000094) and was antisense to a predicted polyphosphate (poly P) kinase gene (Pfl01_5464). In a previous study, we demonstrated that a nonpredicted IVET-identified antisense gene specified a protein of importance in colonization of sterile soil (39). Kim and Levy (16) recently showed that at a sense/antisense locus, at which the antisense gene was identified by IVET, the sense gene had a role in soil fitness.poly P is a polymer of inorganic phosphate found ubiquitously and is important for a number of processes in bacteria (reviewed in reference 36). For example, in Escherichia coli, loss of poly P production is associated with defects in stationary-phase survival and with tolerance of osmotic, oxidative, and heat stress (30). In Vibrio cholerae poly P is important for motility (33), surface attachment (27), and tolerance of low pH and oxidative and osmotic stress (12). Proposed functions of poly P include chelation of metals, phosphate storage, substitution for ATP, and maintenance of pH (reviewed in reference 18). In Pseudomonas aeruginosa PAO1, poly P plays a role in numerous traits including motility (32); biofilm formation, quorum sensing, and virulence (34); and carbenicillin tolerance and maintenance of normal cellular ultrastructure (7). In E. coli, poly P production is part of the Pho regulon (1), a suite of genes upregulated in response to low phosphate by the response regulator PhoB. When P_i is limiting, PhoB is phosphorylated by PhoR and can then activate the Pho regulon. When P_i is in excess, PhoR-mediated phosphorylation of PhoB is prevented by an interaction with the Pst (phosphate-specific transport) system. This regulatory system is widely conserved and has been demonstrated to be present in P. fluorescens Pf0-1. The Pho regulon is constitutively derepressed in pst mutants because of the inability to prevent phosphorylation of PhoB. phoB mutants cannot activate the Pho regulon, and hence, Pho regulon genes are repressed under all conditions (25).The objective of this study was to determine the importance of the overlapping gene pair ppk and iiv8 in stress tolerance and fitness. Studies of bacteria in soil sought to extend our understanding of survival and fitness traits of soil bacteria.     

Gut-Associated Denitrification and In Vivo Emission of Nitrous Oxide by the Earthworm Families Megascolecidae and Lumbricidae in New Zealand

Previous studies have documented the capacity of European earthworms belonging to the family Lumbricidae to emit the greenhouse gas nitrous oxide (N₂O), an activity attributed primarily to the activation of ingested soil denitrifiers. To extend the information base to earthworms in the Southern Hemisphere, four species of earthworms in New Zealand were examined for gut-associated denitrification. Lumbricus rubellus and Aporrectodea rosea (introduced species of Lumbricidae) emitted N₂O, whereas emission of N₂O by Octolasion cyaneum (an introduced species of Lumbricidae) and emission of N₂O by Octochaetus multiporus (a native species of Megascolecidae) were variable and negligible, respectively. Exposing earthworms to nitrite or nitrate and acetylene significantly increased the amount of N₂O emitted, implicating denitrification as the primary source of N₂O and indicating that earthworms emitted dinitrogen (N₂) in addition to N₂O. The alimentary canal displayed a high capacity to produce N₂O when it was supplemented with nitrite, and alimentary canal contents contained large amounts of carbohydrates and organic acids indicative of fermentation (e.g., succinate, acetate, and formate) that could serve as sources of reductant for denitrification. nosZ encodes a portion of the terminal oxidoreductase used in denitrification. The nosZ sequences detected in the alimentary canals of L. rubellus and O. multiporus were similar to those retrieved from soil and were distantly related to sequences of uncultured soil bacteria and genera common in soils (i.e., Bradyrhizobium, Azospirillum, Rhodopseudomonas, Rhodospirillum, Pseudomonas, Oligotropha, and Sinorhizobium). These findings (i) suggest that the capacity to emit N₂O and N₂ is a general trait of earthworms and not geographically restricted, (ii) indicate that species belonging to different earthworm families (i.e., Megascolecidae and Lumbricidae) may not have equal capacities to emit N₂O, and (iii) also corroborate previous findings that link this capacity to denitrification in the alimentary canal.Earthworms are dominant members of the soil fauna and affect the structure and fertility of soils (5, 20, 22, 23). Various species of European earthworms belonging to the family Lumbricidae (e.g., Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) emit dinitrogen (N₂) and the greenhouse gas nitrous oxide (N₂O), and their burrowing activities and feeding habits in combination with in situ conditions can influence the emission of nitrogenous gases from soils that they inhabit (1, 2, 13, 17, 25, 27, 39).The microbiology of the earthworm alimentary canal has been addressed in numerous studies (3, 4, 6, 9, 14, 16, 32). The alimentary canal of the earthworm is anoxic, in marked contrast to the aerated material that earthworms ingest (14, 39). Anoxia and other in situ conditions of the alimentary canal appear to stimulate soil microbes capable of surviving under anaerobic conditions during passage through the gut (3, 4). Soils are rich in denitrifying bacteria (37), and the capacity of European earthworms to emit nitrogenous gases has been attributed primarily to the in situ activity of ingested denitrifying bacteria that appear to be highly active under the anoxic conditions of the earthworm alimentary canal (12, 15, 17, 25, 39). However, it is not known if the capacity to emit nitrogenous gases is a general trait of earthworms independent of their taxonomic family or geographic location. The main objectives of this study were to examine the capacity of Southern Hemisphere earthworms in New Zealand to emit N₂O and to determine if this capacity was linked to denitrifying bacteria in the alimentary canal.     

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

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

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).     

Uncultivated Magnetotactic Cocci from Yuandadu Park in Beijing,China

In the present study, we investigated a group of uncultivated magnetotactic cocci, which was magnetically isolated from a freshwater pond in Beijing, China. Light and transmission electron microscopy showed that these cocci ranged from 1.5 to 2.5 μm and contained two to four chains of magnetite magnetosomes, which sometimes were partially disorganized. Overall, the size of the disorganized magnetosomes was significantly smaller than that arranged in chains. All characterized magnetosome crystals were elongated (shape factor = 0.64) and fall into the single-domain size range (30 to 115 nm). Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the enriched bacteria were a virtually homogeneous population and represented a novel lineage in the Alphaproteobacteria. The closest cultivated relative was magnetotactic coccoid strain MC-1 (88% sequence identity). First-order reversal curve diagrams revealed that these cocci had relatively strong magnetic interactions compared to the single-chain magnetotactic bacteria. Low-temperature magnetic measurements showed that the Verwey transition of them was ∼108 K, confirming magnetite magnetosomes, and the delta ratio δ_FC/δ_ZFC was >2. Based on the structure, phylogenetic position and magnetic properties, the enriched magnetotactic cocci of Alphaproteobacteria are provisionally named as “Candidatus Magnetococcus yuandaducum.”Magnetotactic bacteria (MTB) can mineralize intracellular nanosized iron oxides or sulfides called magnetosomes, which in most MTB are normally single-domain (SD) magnetite with a narrow range of grain sizes from 30 to 120 nm (3). The chain configuration of magnetosomes renders MTB able to navigate the oxic-anoxic interface in chemically stratified environments by swimming along the Earth''s magnetic field (13). Diverse MTB, including coccoid, spirillar, rod-shaped bacteria and multicellular magnetotactic prokaryotes with one, two, or more chains of magnetosomes, thrive in a broad range of aquatic environments, which sometimes even are dominant strains of the microbial biomass in sediment (10, 47). Based on their phylogeny, all currently known MTB can be divided into two taxonomic groups: Proteobacteria and Nitrospira phyla (2).When MTB die, the magnetosomes can be preserved in sediment as fossil magnetosomes (or magnetofossils) (6). Fossil magnetosomes have been found in lacustrine and deep-sea sediments (6, 35, 43, 51), which are stable carriers of natural remanence and may play substantial contributions to the bulk magnetization of sediments due to their SD sizes (6, 19, 26, 32, 33). Moreover, since most known MTB are microaerophilic or anaerobic and are concentrated in the oxic-anoxic transition zone, the presence and characteristics of MTB species in vertically stratified sediments can be used as a potential paleoenvironmental proxy (19, 44, 45). However, how to identify bacterial magnetite or greigite (Fe₃S₄) in sediments is still challenging. Recently, characterizing the magnetic properties of MTB has attracted increasing interests because magnetic techniques are fast and effective in distinguishing bacterial crystals from abiogenic magnetic minerals in sediments (11, 26, 27, 33, 38).In spite of their wide distribution and abundance in aquatic environments, most MTB are intractable, and so far only a few of them, e.g., Magnetospirillum gryphiswaldense strain MSR-1 (41), M. magnetotacticum strain AMB-1 (17), M. magnetotacticum strain MS-1 (4), and magnetotactic coccoid strain MC-1 (24), can be grown in pure culture. Until recently, most insights into the molecular characterizations and magnetic properties of MTB have been based on pure cultures, which have a single magnetosome chain per cell (10, 11, 19, 20, 25, 27, 28, 37, 38, 42, 52). However, knowledge of uncultivated MTB, especially strains with multiple chains of magnetosomes that are commonly encountered in natural environments, remains limited. In the present study, we investigated a population of uncultivated magnetotactic cocci with multiple magnetosome chains, which were abundant in the pond in Yuan Dynasty Capital City Wall Relics Park (Yuandadu Park) in Beijing, China, in order to characterize their morphological features, phylogenetic positions, and magnetic properties and finally to classify them in a provisional Candidatus taxon.     

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.     

Diversity of Formyltetrahydrofolate Synthetases in the Guts of the Wood-Feeding Cockroach Cryptocercus punctulatus and the Omnivorous Cockroach Periplaneta americana

We examined the diversity of a marker gene for homoacetogens in two cockroach gut microbial communities. Formyltetrahydrofolate synthetase (FTHFS or fhs) libraries prepared from a wood-feeding cockroach, Cryptocercus punctulatus, were dominated by sequences that affiliated with termite gut treponemes. No spirochete-like sequences were recovered from the omnivorous roach Periplaneta americana, which was dominated by Firmicutes-like sequences.The guts of wood-feeding termites and Cryptocercus punctulatus cockroaches share an unusual pattern of electron flow, as high rates of CO₂-reductive acetogenesis typically supplant methanogenesis as the terminal electron sink (2, 3). Past studies have shown that from 10 to 30% of gut acetate produced in environments of termites and wood-feeding cockroaches is microbially generated from CO₂ (3, 28), ultimately powering 18 to 26% of the host insect''s own respiratory energy metabolism (25). Nevertheless, most termites emit methane (2), and termite emissions constitute approximately 4% of the global methane budget (27). Cockroaches have been proposed to represent an additional source of note (9). Interestingly, methanogenic termites and cockroaches exhibit increased acetogenesis following addition of exogenous H₂ (3, 29). This suggests that these insects are host to a robust population of bacteria that are capable of homoacetogenesis but may be primarily using alternative electron donors (and other substrates and pathways) in vivo.Acetogenic bacteria belonging to two bacterial phyla, Firmicutes and Spirochaetes, have been isolated from the guts of termites (1, 4, 11, 12, 14). Several surveys have targeted and used the gene for formyltetrahydrofolate synthetase (FTHFS), a key gene in the Wood-Ljungdahl pathway of acetogenesis (16), as a potential marker for the pathway (15, 18). For the wood-feeding termites that have been examined, the studies have revealed an abundance of FTHFS sequences that form a coherent phylogenetic cluster, together with genes from homoacetogenic termite gut spirochetes belonging to the genus Treponema (24, 26, 30). This suggests that treponemes may be among the more abundant of the homoacetogens active in these environments.Little is known about the population structure and biology of CO₂-reducing, acetogenic bacteria in the guts of either omnivorous or wood-feeding cockroaches. The wood-feeding cockroach Cryptocercus hosts an abundance of flagellate protozoa closely related to those believed to dominate polysaccharide fermentation in the guts of termites (5, 6, 22), suggesting that at least one key environmental niche is filled by similar microbes in both termites and Cryptocercidae. Additionally, Cryptocercidae cockroaches, like termites, house diverse spirochetes and are the site of intense CO₂ reduction into acetate (3, 7). Possibly, spirochetes capable of CO₂ reduction into acetate are present in the hindguts of cockroaches. However, no evidence has yet been presented for the existence of homoacetogenic treponemes in environments other than the guts of termites, and FTHFS surveys of human (21) or horse (15) fecal matter and bovine rumen samples (20) revealed only Firmicutes-like and other FTHFS alleles that are distinct from those comprising the termite treponeme cluster.Here, by examining FTHFS gene diversity in Cryptocercus punctulatus and Periplaneta americana guts, we endeavored to learn more about the distribution and origins of homoacetogenic treponemes (and their genes) that are found in wood-feeding termites. In particular, we wished to ascertain whether FTHFS genes present in either of the two cockroaches are termite treponeme-like and, if so, whether analysis reveals any obvious signal indicating recent or ancient lateral community transfer events between insect lineages.     

Detection,Distribution, and Organohalogen Compound Discovery Implications of the Reduced Flavin Adenine Dinucleotide-Dependent Halogenase Gene in Major Filamentous Actinomycete Taxonomic Groups

Halogenases have been shown to play a significant role in biosynthesis and introducing the bioactivity of many halogenated secondary metabolites. In this study, 54 reduced flavin adenine dinucleotide (FADH₂)-dependent halogenase gene-positive strains were identified after the PCR screening of a large collection of 228 reference strains encompassing all major families and genera of filamentous actinomycetes. The wide distribution of this gene was observed to extend to some rare lineages with higher occurrences and large sequence diversity. Subsequent phylogenetic analyses revealed that strains containing highly homologous halogenases tended to produce halometabolites with similar structures, and halogenase genes are likely to propagate by horizontal gene transfer as well as vertical inheritance within actinomycetes. Higher percentages of halogenase gene-positive strains than those of halogenase gene-negative ones contained polyketide synthase genes and/or nonribosomal peptide synthetase genes or displayed antimicrobial activities in the tests applied, indicating their genetic and physiological potentials for producing secondary metabolites. The robustness of this halogenase gene screening strategy for the discovery of particular biosynthetic gene clusters in rare actinomycetes besides streptomycetes was further supported by genome-walking analysis. The described distribution and phylogenetic implications of the FADH₂-dependent halogenase gene present a guide for strain selection in the search for novel organohalogen compounds from actinomycetes.It is well known that actinomycetes, notably filamentous actinomycetes, have a remarkable capacity to produce bioactive molecules for drug development (4, 6). However, novel technologies are demanded for the discovery of new bioactive secondary metabolites from these microbes to meet the urgent medical need for drug candidates (5, 9, 31).Genome mining recently has been used to search for new drug leads (7, 20, 42, 51). Based on the hypothesis that secondary metabolites with similar structures are biosynthesized by gene clusters that harbor certain homologous genes, such homologous genes could serve as suitable markers for distinct natural-product gene clusters (26, 51). A wide range of structurally diverse bioactive compounds are synthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) systems in actinomycetes, therefore much attention has been given to revealing a previously unrecognized biosynthetic potential of actinomycetes through the genome mining of these genes (2, 3, 22). However, the broad distribution of PKS and NRPS genes and their high numbers even in a single actinomycete complicate their use (2, 3). To rationally exploit the genetic potential of actinomycetes, more and more special genes, such as tailoring enzyme genes, are being utilized for this sequence-guided genetic screening strategy (20, 38).Tailoring enzymes, which are responsible for the introduction and generation of diversity and bioactivity in several structural classes during or after NRPS, PKS, or NRPS/PKS assembly lines, usually include acyltransferases, aminotransferases, cyclases, glycosyltransferases, halogenases, ketoreductases, methyltransferases, and oxygenases (36, 45). Halogenation, an important feature for the bioactivity of a large number of distinct natural products (16, 18, 30), frequently is introduced by one type of halogenase, called reduced flavin adenine dinucleotide (FADH₂)-dependent (or flavin-dependent) halogenase (10, 12, 35). More than 4,000 halometabolites have been discovered (15), including commercially important antibiotics such as chloramphenicol, vancomycin, and teicoplanin (43).Previous investigations of FADH₂-dependent halogenase genes were focused largely on related gene clusters in the genera Amycolatopsis (33, 44, 53) and Streptomyces (8, 10, 21, 27, 32, 34, 47-49) and also on those in the genera Actinoplanes (25), Actinosynnema (50), Micromonospora (1), and Nonomuraea (39); however, none of these studies has led to the rest of the major families and genera of actinomycetes. In addition, there is evidence that FADH₂-dependent halogenase genes of streptomycetes usually exist in halometabolite biosynthetic gene clusters (20), but we lack knowledge of such genes and clusters in other actinomycetes.In the present study, we show that the distribution of the FADH₂-dependent halogenase gene in filamentous actinomycetes does indeed correlate with the potential for halometabolite production based on other genetic or physiological factors. We also showed that genome walking near the halogenase gene locus could be employed to identify closely linked gene clusters that likely encode pathways for organohalogen compound production in actinomycetes other than streptomycetes.     

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

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

Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases

The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S₅₀s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 μg/ml and 0.29 μg/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley α-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (∼0.05 μg in 50 mg of leaves) were far lower than the S₅₀ determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.Bacterial plant diseases are a source of great losses in the annual yields of most crops (5). The agrochemical methods and conventional breeding commonly used to control these bacterially induced diseases have many drawbacks. Indiscriminate use of agrochemicals has a negative impact on human, as well as animal, health and contributes to environmental pollution. Conventional plant-breeding strategies have limited scope due to the paucity of genes with these traits in the usable gene pools and their time-consuming nature. Consequently, genetic engineering and transformation technology offer better tools to test the efficacies of genes for crop improvement and to provide a better understanding of their mechanisms. One advance is the possibility of creating transgenic plants that overexpress recombinant DNA or novel genes with resistance to pathogens (36). In particular, strengthening the biological defenses of a crop by the production of antibacterial proteins with other origins (not from plants) offers a novel strategy to increase the resistance of crops to diseases (35, 39, 41). These antimicrobial peptides (AMPs) include such peptides as cecropins (2, 15, 20, 23-24, 27, 31, 42, 50), magainins (1, 9, 14, 29, 47), sarcotoxin IA (35, 40), and tachyplesin I (3). The genes encoding these small AMPs in plants have been used in practice to enhance their resistance to bacterial and fungal pathogens (8, 22, 40). The expression of AMPs in vivo (mostly cecropins and a synthetic analog of cecropin and magainin) with either specific or broad-spectrum disease resistance in tobacco (14, 24, 27), potato (17, 42), rice (46), banana (9), and hybrid poplar (32) have been reported. The transgenic plants showed considerably greater resistance to certain pathogens than the wild types (4, 13, 24, 27, 42, 46, 50). However, detailed studies of transgenic tomatoes expressing natural cecropin have not yet been reported.The tomato (Solanum lycopersicum) is one of the most commonly consumed vegetables worldwide. The annual yield of tomatoes, however, is severely affected by two common bacterial diseases, bacterial wilt and bacterial spot, which are caused by infection with the Gram-negative bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, respectively. Currently available pesticides are ineffective against R. solanacearum, and thus bacterial wilt is a serious problem.Cecropins, one of the natural lytic peptides found in the giant silk moth, Hyalophora cecropia (25), are synthesized in lipid bodies as proteins consisting of 31 to 39 amino acid residues. They adopt an α-helical structure on interaction with bacterial membranes, resulting in the formation of ion channels (12). At low concentrations (0.1 μM to 5 μM), cecropins exhibit lytic antibacterial activity against a number of Gram-negative and some Gram-positive bacteria, but not against eukaryotic cells (11, 26, 33), thus making them potentially powerful tools for engineering bacterial resistance in crops. Moreover, cecropin B (CB) shows the strongest activity against Gram-negative bacteria within the cecropin family and therefore has been considered an excellent candidate for transformation into plants to improve their resistance against bacterial diseases.The introduction of genes encoding cecropins and their analogs into tobacco has been reported to have contradictory results regarding resistance against pathogens (20). However, subsequent investigations of these tobacco plants showed that the expression of CB in the plants did not result in accumulation of detectable levels of CB, presumably due to degradation of the peptide by host peptidases (20, 34). Therefore, protection of CB from cellular degradation is considered to be vital for the exploitation of its antibacterial activity in transgenic plants. The secretory sequences of several genes are helpful, because they cooperate with the desired genes to enhance extracellular secretion (24, 40, 46). In the present study, a natural CB gene was successfully transferred into tomatoes. The transgenic plants showed significant resistance to the tomato diseases bacterial wilt and bacterial spot, as well as with a chemically synthesized CB peptide.