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).     

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.     

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.     

Bocavirus Infection Induces Mitochondrion-Mediated Apoptosis and Cell Cycle Arrest at G2/M Phase

Bocavirus is a newly classified genus of the family Parvovirinae. Infection with Bocavirus minute virus of canines (MVC) produces a strong cytopathic effect in permissive Walter Reed/3873D (WRD) canine cells. We have systematically characterized the MVC infection-produced cytopathic effect in WRD cells, namely, the cell death and cell cycle arrest, and carefully examined how MVC infection induces the cytopathic effect. We found that MVC infection induces an apoptotic cell death characterized by Bax translocalization to the mitochondrial outer membrane, disruption of the mitochondrial outer membrane potential, and caspase activation. Moreover, we observed that the activation of caspases occurred only when the MVC genome was replicating, suggesting that replication of the MVC genome induces apoptosis. MVC infection also induced a gradual cell cycle arrest from the S phase in early infection to the G₂/M phase at a later stage, which was confirmed by the upregulation of cyclin B1 and phosphorylation of cdc2. Cell cycle arrest at the G₂/M phase was reproduced by transfection of a nonreplicative NS1 knockout mutant of the MVC infectious clone, as well as by inoculation of UV-irradiated MVC. In contrast with other parvoviruses, only expression of the MVC proteins by transfection did not induce apoptosis or cell cycle arrest. Taken together, our results demonstrate that MVC infection induces a mitochondrion-mediated apoptosis that is dependent on the replication of the viral genome, and the MVC genome per se is able to arrest the cell cycle at the G₂/M phase. Our results may shed light on the molecular pathogenesis of Bocavirus infection in general.The Bocavirus genus is newly classified within the subfamily Parvovirinae of the family Parvoviridae (21). The currently known members of the Bocavirus genus include bovine parvovirus type 1 (BPV1) (17), minute virus of canines (MVC) (57), and the recently identified human bocaviruses (HBoV, HBoV2, and HBoV3) (4, 7, 36).MVC was first recovered from canine fecal samples in 1970 (10). The virus causes respiratory disease with breathing difficulty (14, 32, 49) and enteritis with severe diarrhea (11, 39), which often occurs with coinfection with other viruses (39), spontaneous abortion of fetuses, and death of newborn puppies (14, 29). Pathological lesions in fetuses in experimental infections were found in the lymphoid tissue of the lung and small intestine (14). MVC was isolated and grown in the Walter Reed/3873D (WRD) canine cell line (10), which is derived from a subdermoid cyst of an irradiated male dog (10). The full-length 5.4-kb genome of MVC was recently mapped with palindromic termini (60). Under the control of a single P6 promoter, through the mechanism of alternative splicing and alternative polyadenylation, MVC expresses two nonstructural proteins (NS1 and NP1) and two capsid proteins (VP1 and VP2). Like the NS1 proteins of other parvoviruses, the NS1 of MVC is indispensable for genome replication. The NP1 protein, which is unique to the Bocavirus genus, appears to be critical for optimal viral replication, as the NP1 knockout mutant of MVC suffers from severe impairment of replication (60). A severe cytopathic effect during MVC infection of WRD cells has been documented (10, 60).The HBoV genome has been frequently detected worldwide in respiratory specimens from children under 2 years old with acute respiratory illnesses (2, 34, 55). HBoV is associated with acute expiratory wheezing and pneumonia (3, 34, 55) and is commonly detected in association with other respiratory viruses (34, 55). Further studies are necessary, however, to identify potential associations of HBoV infection with clinical symptoms or disease of acute gastroenteritis (7, 36). The full-length sequence of infectious MVC DNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ214110","term_id":"219665308","term_text":"FJ214110"}}FJ214110) that we have reported shows 52.6% identity to HBoV, while the NS1, NP1, and VP1 proteins are 38.5%, 39.9%, and 43.7% identical to those of HBoV, respectively (60).The cytopathic effect induced during parvovirus infection has been widely documented, e.g., in infections with minute virus of mice (MVM) (13), human parvovirus B19 (B19V) (58), parvovirus H-1 (25, 52), and BPV1 (1). In Bocavirus, cell death during BPV1 infection of embryonic bovine tracheal cells has been shown to be achieved through necrosis, independent of apoptosis (1). B19V-induced cell death of primary erythroid progenitor cells has been shown to be mainly mediated by an apoptotic pathway (58) in which the nonstructural protein 11kDa plays a key role (16). In contrast, the MVM-induced cytopathic effect has been revealed to be mediated by NS1 interference with intracellular casein kinase II (CKII) signaling (22, 44, 45), a nonapoptotic cell death. Oncolytic parvovirus H-1 infections can induce either apoptosis or nonapoptotic cell death, depending on the cell type (25, 40). Therefore, the mechanisms underlying parvovirus infection-induced cell death vary, although NS1 has been widely shown to be involved in both apoptotic and nonapoptotic cell death. The nature of the cytopathic effect during Bocavirus MVC infection has not been studied.Parvovirus replication requires infected cells at the S phase. Infection with parvovirus has been revealed to accompany a cell cycle perturbation that mostly leads to an arrest in the S/G₂ phase or the G₂/M phase during infection (30, 33, 42, 47, 65). MVM NS1 expression induces an accumulation of sensitive cells in the S/G₂ phase (6, 46, 47). Whether MVC infection-induced cell death is accompanied by an alternation of cell cycle progression and whether the viral nonstructural protein is involved in these processes have not been addressed.In this study, we found, in contrast with other members of the family Parvoviridae, expression of both the nonstructural and structural proteins of MVC by transfection did not induce cell death or cell cycle arrest. However, the cytopathic effect induced during MVC infection is a replication-coupled, mitochondrion-mediated and caspase-dependent apoptosis, accompanied with a gradual cell cycle arrest from the S phase to the G₂/M phase, which is facilitated by the MVC genome.     

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.     

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.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated,Low-Dose Vaginal Challenge

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV_mac239 or SIV_mac251_UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV_mac251_UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) is an urgent public health priority, but remains a formidable scientific challenge. Passive transfer experiments in macaques demonstrate neutralizing antibodies can prevent infection by laboratory-engineered simian-human immunodeficiency virus (SHIV) strains (6, 33, 34, 53, 59). However, no current vaccine approach is capable of eliciting antibodies that neutralize primary isolates with neutralization-resistant envelope glycoproteins. Virus-specific T-cell responses can be elicited by prime-boost strategies utilizing recombinant DNA and/or viral vectors (3, 10, 11, 16, 36, 73, 77, 78), which confer containment of viral loads following challenge with SHIV_89.6P (3, 13, 66, 68). Unfortunately, similar vaccine regimens are much less effective against SIV_mac239 and SIV_mac251 (12, 16, 31, 36, 73), which bear closer resemblance to most transmitted HIV-1 isolates in their inability to utilize CXCR4 as a coreceptor (18, 23, 24, 88) and inherent high degree of resistance to neutralization by antibodies or soluble CD4 (43, 55, 56). Live, attenuated SIV can provide apparent sterile protection against challenge with SIV_mac239 and SIV_mac251 or at least contain viral replication below the limit of detection (20, 22, 80). Due to the potential of the attenuated viruses themselves to cause disease in neonatal rhesus macaques (5, 7, 81) and to revert to a pathogenic phenotype through the accumulation of mutations over prolonged periods of replication in adult animals (2, 35, 76), attenuated HIV-1 is not under consideration for use in humans.As an experimental vaccine approach designed to retain many of the features of live, attenuated SIV, without the risk of reversion to a pathogenic phenotype, we and others devised genetic approaches for producing strains of SIV that are limited to a single cycle of infection (27, 28, 30, 38, 39, 45). In a previous study, immunization of rhesus macaques with single-cycle SIV (scSIV) trans-complemented with vesicular stomatitis virus (VSV) G elicited potent virus-specific T-cell responses (39), which were comparable in magnitude to T-cell responses elicited by optimized prime-boost regimens based on recombinant DNA and viral vectors (3, 16, 36, 68, 73, 78). Antibodies were elicited that neutralized lab-adapted SIV_mac251_LA (39). However, despite the presentation of the native, trimeric SIV envelope glycoprotein (Env) on the surface of infected cells and virions, none of the scSIV-immunized macaques developed antibody responses that neutralized SIV_mac239 (39). Therefore, we have now introduced Env modifications into scSIV that facilitate the development of neutralizing antibodies.Most primate lentiviral envelope glycoproteins are inherently resistant to neutralizing antibodies due to structural and thermodynamic properties that have evolved to enable persistent replication in the face of vigorous antibody responses (17, 46, 47, 64, 71, 75, 79, 83, 85). Among these, extensive N-linked glycosylation renders much of the Env surface inaccessible to antibodies (17, 48, 60, 63, 75). Removal of N-linked glycans from gp120 or gp41 by mutagenesis facilitates the induction of antibodies to epitopes that are occluded by these carbohydrates in the wild-type virus (64, 85). Consequently, antibodies from animals infected with glycan-deficient strains neutralize these strains better than antibodies from animals infected with the fully glycosylated SIV_mac239 parental strain (64, 85). Most importantly with regard to immunogen design, animals infected with the glycan-deficient strains developed higher neutralizing antibody titers against wild-type SIV_mac239 (64, 85). Additionally, the removal of a single N-linked glycan in gp120 enhanced the induction of neutralizing antibodies against SHIV_89.6P and SHIV_SF162 in a prime-boost strategy by 20-fold (50). These observations suggest that potential neutralization determinants accessible in the wild-type Env are poorly immunogenic unless specific N-linked glycans in gp120 and gp41 are eliminated by mutagenesis.The variable loop regions 1 and 2 (V1V2) of HIV-1 and SIV gp120 may also interfere with the development of neutralizing antibodies. Deletion of V1V2 from HIV-1 gp120 permitted neutralizing monoclonal antibodies to CD4-inducible epitopes to bind to gp120 in the absence of CD4, suggesting that V1V2 occludes potential neutralization determinants prior to the engagement of CD4 (82). A deletion in V2 of HIV-1 Env-exposed epitopes was conserved between clades (69), improved the ability of a secreted Env trimer to elicit neutralizing antibodies (9), and was present in a vaccine that conferred complete protection against SHIV_SF162P4 (8). A deletion of 100 amino acids in V1V2 of SIV_mac239 rendered the virus sensitive to monoclonal antibodies with various specificities (41). Furthermore, three of five macaques experimentally infected with SIV_mac239 with V1V2 deleted resisted superinfection with wild-type SIV_mac239 (51). Thus, occlusion of potential neutralization determinants by the V1V2 loop structure may contribute to the poor immunogenicity of the wild-type envelope glycoprotein.Here we tested the hypothesis that antibody responses to scSIV could be improved by immunizing macaques with strains of scSIV engineered to eliminate structural features that interfere with the development of neutralizing antibodies. Antibodies to Env-modified strains were selectively enhanced, but these did not neutralize the wild-type SIV strains. We then tested the hypothesis that immunization might prevent infection in a repeated, low-dose vaginal challenge model of heterosexual HIV-1 transmission. Indeed, while all six naïve control animals became infected, two of eight immunized animals remained uninfected after 20 weeks of repeated vaginal challenge. Relative to the naïve control group, reductions in peak and set point viral loads were statistically significant in the immunized animals that became infected.     

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.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

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.