Characterization of the Replication,Transfer, and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12

We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (φZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of φZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear φZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear φZL12. φZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of φZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage φZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.Streptomyces species, a major source of antibiotics and pharmacologically active metabolites, are Gram-positive, mycelial bacteria with high G+C content in their DNA (15). They usually harbor conjugative circular and/or linear plasmids, propagating in autonomous and/or chromosomally integrated forms (14). Most Streptomyces circular plasmids reported are small (8 to 14 kb), including rolling-circle-replication (RCR) plasmids (pIJ101, pJV1, pSG5, pSN22, pSVH1, pSB24.2, pSY10, pSNA1, pSLG33, pEN2701, etc.) (12, 14) and chromosomally integrating/autonomous plasmids (SLP1 and pSAM2) (4, 27, 28). Some theta replication plasmids are of intermediate size (31 to 39 kb), such as SCP2, pFP1, and pFP11 (13, 40). These theta replication loci comprise a rep gene and an adjacent noncoding or iteron sequence, to which Rep protein binds specifically in vitro (10, 40). The occurrence of an ∼163-kb large plasmid, pSV1, in Streptomyces violaceoruber SANK95570 was confirmed (1, 37), but this plasmid could not be physically isolated by standard procedures for plasmid preparation (17). In contrast to more than 30 genes for conjugal transfer on the Escherichia coli F plasmid (20), Streptomyces plasmids usually need a single tra gene (encoding a DNA translocase containing a cell division FtsK/SpoIIIE domain) (15, 29). The transfer of Streptomyces circular plasmids involves binding of the nonnicked double-stranded DNA (dsDNA) by multimers of Tra proteins at a noncoding sequence and ATP hydrolysis-dependent translocation of this DNA through the hyphal tips of the Streptomyces mycelium (15, 32).Numerous Streptomyces phages have been described, including φC31 (22), SAt1 (26), TG1 (11), FP43 (24), φSPK1 (19), φSC623 (34), DAH2/DAH4/DAH5/DAH6 (6), and mu1/6 (9). They range in size from 36 kb (19) to 121 kb (6), with 50 to 71.2% GC content (9, 23, 35). Streptomyces phages often have a wide host range; for example, 16 of 27 Streptomyces strains are susceptible to infection by φSPK1 (19), and phage FP43 transduces species of Streptoverticillium, Chainia, and Sacchropolyspora (24). φC31 is the most-studied Streptomyces phage and cloning vector (8). The sequences of the φC31 head proteins (e.g., portal, capsid, and head protease) resemble those of other bacterial dsDNA phages, suggesting evolutionary relationships to other viruses (35).We report here the isolation and recombinational cloning of a 90,435-bp plasmid, pZL12, from endophytic Streptomyces sp. 9R-2 and the characterization of its replication and transfer. Surprisingly, the supernatant of 9R-2 liquid culture infected 9R-2 cured of pZL12 to form plaques. A cycle comprising autonomous plasmid pZL12 and lytic phage φZL12 is described.     

Biochemical Characterization of a Novel Indole Prenyltransferase from Streptomyces sp. SN-593

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (−)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min⁻¹ μM⁻¹) for l-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-l-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, ΔiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-l-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.Natural products have been an important resource for drug discovery and development. Actinomycetes have been a rich source of natural products, and a wide variety of these chemicals have been used as medicinal drugs (7, 40) and as bioprobes (56) for the elucidation of biological functions. Recently, the screening of bioactive compounds from microorganisms has often resulted in the identification of previously isolated compounds. The decreasing hit rate for new chemicals has reduced the advantage of natural product screening. However, genome sequencing of Streptomyces species highlighted numerous potential areas with metabolic diversity (4, 25, 42). The number of cryptic gene clusters was much larger than that of secondary metabolites identified from each strain. In addition, the cryptic gene clusters contained genes encoding plenty of unique modification enzymes that had the potential to expand the chemical diversity in drug seeds.To uncover cryptic gene clusters that might code for biosynthesis of secondary metabolites, genome sequence-guided metabolite identification has been performed in combination with heterologous expression, gene knockout, and complementation analyses and silent gene activation studies. Many microbial metabolites have been discovered through genome mining approaches (5, 8, 26, 29, 34, 41, 50). On the other hand, predictions of protein function are not always successful from BLAST searches, because the substrates or products of unknown enzyme reactions cannot be predicted correctly. Only the type of protein function can be annotated by a homology search. The major difficulty for the identification of cryptic gene clusters is a lack of chemical information. Most gene clusters remain dormant or less active if there are no specific chemicals or physiological signals. Therefore, the discovery of secondary metabolites that are normally expressed at very low levels opens up a strategy for addressing the functions of cryptic gene clusters or unique genes. We performed a metabolite profiling and genome draft sequence analysis of a reveromycin A-producing strain, Streptomyces sp. SN-593 (43). Based on systematic isolation of secondary metabolites, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. There are many isolation reports on 6-DMAI derivatives from Streptomyces sp. (39, 46, 48), fungi (17, 24, 49), and plants (2, 3). However, the gene responsible for dimethylallyl transfer to the C-6 indole ring has not been identified for all living organisms. Because the unique modification enzyme retains a high potential to expand the diversity of natural products, we started a homology search and cloning of the target gene. Here we report the heterologous expression and biochemical characterization of a novel indole prenyltransferase (IptA) catalyzing the transfer of a dimethylallyl group to the C-6 indole ring.     

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).     

Candicidin Biosynthesis Gene Cluster Is Widely Distributed among Streptomyces spp. Isolated from the Sediments and the Neuston Layer of the Trondheim Fjord,Norway

A large number of Streptomyces bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (can). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with can-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained can genes. The majority of the isolates studied contained large plasmids, and two can-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the can genes and candicidin production, while mating of the “cured” strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete can gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes.Actinomycete bacteria, especially those belonging to the family Streptomycetaceae, are well-known producers of secondary metabolites with diverse biological activities. Representatives of the genus Streptomyces produce a variety of antibiotics with antibacterial, antifungal, and antitumor activities. The majority of antibiotic-producing streptomycetes have been isolated from terrestrial environments, while antibiotic-producing streptomycetes from the marine sources remain largely unexplored. Therefore, studies of streptomycetes from the marine environment are important for unraveling their potential for antibiotic production. In addition, such studies might reveal the means by which antibiotic biosynthesis and resistance genes are spread in nature.It is widely acknowledged that plasmids play an important role in genetic exchange between bacterial species. Conjugative plasmids are quite common in Streptomyces strains (13), and a number of these mobile genetic elements have been characterized in detail. The characterized mobile genetic elements include both circular plasmids, such as pIJ101 from Streptomyces lividans (14) and SCP2 from Streptomyces coelicolor (2, 35), and linear plasmids, such as SLP2 from S. lividans (6) and SCP1 from S. coelicolor (38, 39). The presence of a linear plasmid in Streptomyces was first reported in 1979, and the plasmid was the 17-kb pSLA2 plasmid of Streptomyces rochei (11). SCP1 of S. coelicolor was discovered in the early 1970s (38, 39), but because of its large size (356 kb), isolation of this plasmid with conventional techniques was not possible and therefore it was not recognized as a linear plasmid until pulsed-field gel electrophoresis (PFGE) was invented. Later, SCP1 was shown to harbor a complete set of genes for biosynthesis of the antibiotic methylenomycin (21; K. F. Chater, C. J. Bruton, S. J. O''Rouke, and A. W. Wietzorrek, 5 July 2001, Patent Cooperation Treaty international application WO/2001/048228), while another linear plasmid, found in S. rochei, has been shown to contain genes for biosynthesis of both lankamycin and lankacidin (16, 19, 28, 36). Other examples of plasmids include pPZG103 carrying oxytetracycline biosynthesis genes acquired from the chromosome of Streptomyces rimosus (10) and pKSL from Streptomyces lasaliensis, which might be involved in the production of lasalocid and/or echinomycin (17, 20).Linear plasmids can be transferred between Streptomyces strains by means of conjugation, and SCP1 is an example of a conjugative linear plasmid as it is easily transferred from an SCP1⁺ strain to an SCP1⁻ strain (39). Interspecific transfer to S. lividans and Streptomyces parvulus has also been reported for this plasmid, and it was demonstrated that the recipient strains had acquired the ability to produce and be resistant to methylenomycin (12, 21). Transfer of intact linear plasmids containing mercury resistance genes from two Streptomyces strains isolated from the marine environment to S. lividans, conferring mercury resistance to the initially mercury-sensitive recipient, has been reported by Ravel et al. (32). It has also been shown that interspecific transfer of linear plasmids is possible in sterile amended soil microcosms, suggesting that mercury resistance might be spread by plasmid transfer in polluted environments (31).We report here isolation and screening of several thousand actinobacterial strains from the Trondheim fjord (Norway), which resulted in identification of producers of both known and potentially new polyene macrolides with antifungal activity. The ability to produce the polyene macrolide candicidin D was found to be widespread among the Trondheim fjord Streptomyces isolates. We also report that the candicidin biosynthesis genes (can) are present on a linear plasmid identified in one of these isolates, suggesting that the can genes might be spread by means of conjugation.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

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.     

Gene Cluster Involved in the Biosynthesis of Griseobactin,a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974

The main siderophores produced by streptomycetes are desferrioxamines. Here we show that Streptomyces sp. ATCC 700974 and several Streptomyces griseus strains, in addition, synthesize a hitherto unknown siderophore with a catechol-peptide structure, named griseobactin. The production is repressed by iron. We sequenced a 26-kb DNA region comprising a siderophore biosynthetic gene cluster encoding proteins similar to DhbABCEFG, which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA) and in the incorporation of DHBA into siderophores via a nonribosomal peptide synthetase. Adjacent to the biosynthesis genes are genes that encode proteins for the secretion, uptake, and degradation of siderophores. To correlate the gene cluster with griseobactin synthesis, the dhb genes in ATCC 700974 were disrupted. The resulting mutants no longer synthesized DHBA and griseobactin; production of both was restored by complementation with the dhb genes. Heterologous expression of the dhb genes or of the entire griseobactin biosynthesis gene cluster in the catechol-negative strain Streptomyces lividans TK23 resulted in the synthesis and secretion of DHBA or griseobactin, respectively, suggesting that these genes are sufficient for DHBA and griseobactin biosynthesis. Griseobactin was purified and characterized; its structure is consistent with a cyclic and, to a lesser extent, linear form of the trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexed with aluminum under iron-limiting conditions. This is the first report identifying the gene cluster for the biosynthesis of DHBA and a catechol siderophore in Streptomyces.Iron is an essential element for the growth and proliferation of nearly all microorganisms. In the presence of oxygen, the soluble ferrous iron is readily oxidized to its ferric form, which exists predominantly as a highly insoluble hydroxide complex at neutral pH. To overcome iron limitation, many bacteria synthesize and secrete low-molecular-weight, high-affinity ferric iron chelators, called siderophores (38, 53). Following the chelation of Fe³⁺ in the medium, the iron-siderophore complex is actively taken up by its cognate ABC transport system, and Fe³⁺ is subsequently released by reduction to Fe²⁺ and/or by hydrolysis of the siderophore (28, 32, 36). The three main classes of siderophores contain catecholates, hydroxamates, or (α-hydroxy-)carboxylates as iron-coordinating ligands, but mixed siderophores and siderophores containing other functional groups, such as diphenolates, imidazoles, and thiazolines, have also been found (16, 38).Siderophores containing peptide moieties are synthesized by proteins belonging to the nonribosomal peptide synthetase (NRPS) family (16, 38). These multimodular enzymes function as enzymatic assembly lines in which the order of the modules usually determines the order of the amino acids incorporated into the peptide (19, 34). Each module contains the complete information for an elongation step combining the catalytic functions for the activation of the amino acid by the adenylation (A) domain, the tethering of the corresponding adenylate to the terminal thiol of the enzyme-bound 4′-phosphopantetheinyl (4′-PP) cofactor by the peptidyl carrier protein (PCP) domain, and the formation of the peptide bond by the condensation (C) domain (26, 34, 52). At the end, the product is released by the C-terminal thioesterase (TE) domain by hydrolysis or by cyclization via intramolecular condensation. Each adenylation domain recognizes a specific amino acid, and its substrate specificity can be predicted by its sequence. An NRPS specificity-conferring code consisting of 10 nonadjacent amino acid residues in the A domain has been proposed (49). Exceptions to the “colinearity-rule” (19) have been discovered. For example, in the biosynthesis of the siderophores enterobactin and bacillibactin, all the modules in the NRPS are used iteratively, and the TE domain stitches the chains together into a cyclic product (35, 45). Enterobactin is the trilactone of 2,3-dihydroxybenzoyl-serine, and bacillibactin is the lactone of 2,3-dihydroxybenzoyl-glycyl-threonine.The typical siderophores produced by streptomycetes are desferrioxamines (24), and the genes encoding the enzymes for their biosynthesis have been identified (5). Recently, structurally different siderophores have been reported to be coproduced with desferrioxamines in some species, e.g., coelichelin in Streptomyces coelicolor (9, 30) and enterobactin in Streptomyces tendae (18). The genes encoding the proteins for the biosynthesis of enterobactin in S. tendae remain unknown.Here we describe the gene cluster for the biosynthesis of a new siderophore, named griseobactin, produced by Streptomyces sp. strain ATCC 700974 and some strains of Streptomyces griseus. By sequencing two cosmids isolated from a Streptomyces sp. strain ATCC 700974 genomic library, we assigned the encoded proteins to enzymes that convert chorismate to 2,3-dihydroxybenzoate (DHBA), and to proteins involved in nonribosomal peptide biosynthesis and in the export, uptake, and utilization of siderophores. Knockout mutagenesis and heterologous expression confirmed the requirement of this gene cluster for the biosynthesis of griseobactin. This is the first report on the identification of the genes responsible for DHBA and catechol siderophore biosynthesis in Streptomyces.     

Bat Guano Virome: Predominance of Dietary Viruses from Insects and Plants plus Novel Mammalian Viruses

Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.Bats belong to one of the most diverse, abundant, and widely distributed group of mammals. More than 1,100 bat species belong to the order of Chiroptera, representing approximately 20% of all mammalian species (54). Most bat species feed on insects and other arthropods, while others feed on fruit nectar, bird or mammal blood, and small vertebrates such as fish, frogs, mice, and birds (30). Of the 47 species of bats reported in the United States, most of them are insectivorous (http://www.batcon.org/).Bats are considered the natural reservoir of a large variety of zoonotic viruses causing serious human diseases such as lyssaviruses, henipaviruses, severe acute respiratory syndrome coronavirus, and Ebola virus (6, 38, 46, 59, 63, 65). Characteristics of bats, including their genetic diversity, broad geological distribution, gregarious habits, high population density, migratory habits, and long life span (30, 58), likely endow them with the ability to host diverse viruses, some of which are also able to infect humans and other mammals (41, 63).More than 80 virus species have been isolated or detected in bats using nucleic acid-based methods (6, 38, 59, 65). Viruses that have been recently discovered in bats include astroviruses, adeno-associated viruses (AAVs), adenoviruses, herpesviruses, and polyomavirus (8, 9, 13, 31, 32, 35, 37, 39, 40, 42, 61, 62, 68). For example, it was recently reported that a newly identified adenovirus isolated from bat guano was capable of infecting various vertebrate cell lines, including those of humans, monkeys, dogs, and pigs (35). With increasing human populations in previously wild areas, contact of bats with humans and with wild and domestic animals has increased, providing greater opportunities for cross-species transmissions of potentially pathogenic bat viruses. To better understand the range of viruses carried by bats, we undertook an initial characterization of the guano viromes of several common bat species in the United States.The development of massively parallel sequencing technology makes is possible to reveal uncultured viral assemblages within biological or environmental samples (11, 28). To date, this approach has been used to characterize viruses in equine feces (7), human blood (5), tissue (14), human feces (3, 4, 15, 45, 60, 67), and human respiratory secretions (64), which in turn has facilitated the discovery of many novel viruses (18, 20, 25, 33, 47, 50). In the present study, we analyzed the viruses present in guano from several bat species in California and Texas, using sequence-independent PCR amplification, pyrosequencing, and sequence similarity searches.