Rickettsia felis Infection in a Common Household Insect Pest,Liposcelis bostrychophila (Psocoptera: Liposcelidae)

Many species of Rickettsia are well-known mammalian pathogens transmitted by blood-feeding arthropods. However, molecular surveys are continually uncovering novel Rickettsia species, often in unexpected hosts, including many arthropods that do not feed on blood. This study reports a systematic molecular characterization of a Rickettsia infecting the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae), a common and cosmopolitan household pest. Surprisingly, the psocid Rickettsia is shown to be Rickettsia felis, a human pathogen transmitted by fleas that causes serious morbidity and occasional mortality. The plasmid from the psocid R. felis was sequenced and was found to be virtually identical to the one in R. felis from fleas. As Liposcelis insects are often intimately associated with humans and other vertebrates, it is speculated that they acquired R. felis from fleas. Whether the R. felis in psocids causes disease in vertebrates is not known and warrants further study.Many species of Rickettsia are well-known mammalian pathogens that are transmitted by blood-feeding arthropods via bites or feces and can cause mild to fatal diseases in humans (33). Some species are also considered potential bioterrorism agents (4). Most Rickettsia research has focused on pathogens that are found in two closely related species groups, the typhus and spotted fever groups, such as Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia typhi, the causal agents of epidemic typhus, Rocky Mountain spotted fever, and murine typhus, respectively (3, 4, 33). However, recent surveys suggest that Rickettsia bacteria are much more widespread than previously suspected and that they are being detected in novel hosts, the vast majority of which are arthropods, including many that do not feed on blood (29, 45).The number of new rickettsial species that cause diseases in humans is rapidly increasing (33). One such species that has been generating much interest in recent years is Rickettsia felis, the causative agent of a murine typhus-like disease (1, 2, 13, 16, 17, 28, 44). The disease is often unrecognized, and even though it is considered clinically mild, it can cause severe illness and death in older patients and in cases of delayed diagnosis (2). R. felis was identified only in 1990 (1) and has since been found worldwide in fleas, where it is maintained transovarially and can reach high infection rates (e.g., 86% to 94% in cat fleas) (2, 3, 44), as well as in ticks and mites (34). While experimental infections have confirmed that R. felis is transmitted to vertebrate hosts via blood feeding and that R. felis occurs in an infectious extracellular state (39), it is not known whether transmission can also occur through contamination of broken skin by infected vector feces, as in R. typhi (3, 34).A number of features distinguish R. felis from species in both the typhus and spotted fever groups. Lately, it has been proposed that R. felis be in its own group, allied with Rickettsia akari and Rickettsia australis, the causal agents of rickettsial pox and Queensland tick typhus, respectively, and a number of recently discovered strains infecting insects that do not feed on blood (16, 17, 29, 45). Moreover, R. felis was the first Rickettsia species shown to have a plasmid (28). While plasmids now appear to be quite widespread in the genus, the R. felis plasmid stands out with respect to its relatively large size and distinctive gene content (5, 6, 9, 14, 17).This study reports that a common and cosmopolitan insect, the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae) harbors R. felis. Liposcelids are the closest free-living relatives of parasitic lice (19) and are well-known for their close proximity to humans, particularly as pests in houses and grain storage facilities (8, 41). Through 16S rRNA gene sequencing, L. bostrychophila was recently shown to harbor a strain of Rickettsia (29, 30, 42). A systematic molecular characterization of this Rickettsia was conducted, demonstrating that it is authentic R. felis. Furthermore, the psocid symbiont plasmid was sequenced and was shown to be virtually identical to the plasmid from R. felis that infects cat fleas.     

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, “Candidatus Rickettsia amblyommii,” R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two “Ca. Rickettsia amblyommii” isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of “Ca. Rickettsia amblyommii,” R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.The alphaproteobacteria of the genus Rickettsia (Rickettsiales; Rickettsiaceae) have undergone the reductive genome evolution typical of obligate intracellular bacteria, resulting in A/T-rich genomes (1.1 × 10⁶ to 1.5 × 10⁶ bp) with a high content of pseudogenes undergoing elimination (3, 10, 20, 26). Initial sequencing of rickettsial genomes focused on the important arthropod-borne pathogens Rickettsia prowazekii, Rickettsia conorii, and Rickettsia typhi and appeared to confirm the prevailing belief that plasmids were absent and transposons were rare among Rickettsia spp. (2, 28, 39, 44). As mobile genetic elements in bacteria, plasmids and transposons drive horizontal gene transfer (HGT) and the acquisition of virulence determinants and environmental adaptive traits (30, 43, 60, 70). Subsequent sequencing of the Rickettsia felis genome revealed the surprising presence of abundant transposase paralogs and the 63-kbp pRF plasmid, with 68 open reading frames (ORFs) encoding predicted proteins, as well as a 39-kbp deletion form, pRFδ (45). Although pRF was suggested to be conjugative, it was initially thought to be unique among the rickettsiae, a reasonable inference given that plasmids are uncommon among the reduced genomes of obligate intracellular bacteria and were previously unknown in the Rickettsiales (3, 4, 13). However, a phylogenetic analysis implied an origin for pRF in ancestral rickettsiae and the possible existence of other rickettsial plasmids (28), which was soon confirmed by the cloning of the 23.5-kbp pRM plasmid from Rickettsia monacensis (6). Some of the 23 ORFs on pRM had close pRF homologs, and both plasmids carried transposon genes and the molecular footprints of transposition events associated with HGT from other bacterial taxa.The discoveries of pRF and pRM made obsolete the long-held dogma that plasmids were not present in members of the genus Rickettsia and implied a source of unexpected genetic diversity in the reduced rickettsial genomes, particularly if potentially conjugative plasmids carrying transposon genes proved to be common among members of the genus. That hypothesis gained credence when pulsed-field gel electrophoresis (PFGE) and Southern blot surveys (7) using plasmid gene-specific probes demonstrated plasmids in Rickettsia helvetica, “Candidatus Rickettsia hoogstraalii” (38), and Rickettsia massiliae and possible multiple plasmids in “Candidatus Rickettsia amblyommii” (71) isolates. The same study demonstrated the loss of a plasmid in the nonpathogenic species Rickettsia peacockii during long-term serial passage in cultured cells and the absence of a plasmid in Rickettsia montanensis M5/6, an isolate with a long laboratory passage history. Genome sequencing of R. massiliae and Rickettsia africae revealed the 15.3-kbp pRMA and 12.4-kbp pRAF sequences, with 12 and 11 ORFs, respectively, that were more similar to those of pRF than to those of pRM (11, 24).The absence of plasmids in R. montanensis and important Rickettsia pathogens maintained as laboratory isolates has left unresolved the question of the true extent of plasmid distribution among Rickettsia spp. Until recently, the genus was thought to consist of closely related species, known chiefly as typhus and spotted fever pathogens transmitted by lice, fleas, mites, and ticks (31). It is now apparent that many, and possibly most, Rickettsia spp. inhabit a diverse range of arthropods that do not feed on blood, as well as leeches, helminths, crustaceans, and protozoans, suggesting an ancient and complex evolutionary history (54). A multigene phylogenetic analysis of the Rickettsiales resulted in a “molecular clock” which indicated that the order arose from a presumably free-living ancestor and then adapted to intracellular growth during the appearance of metazoan phyla in the Cambrian explosion (76). A transition to a primary association with arthropods followed during the Ordovician and Silurian periods. The genus Rickettsia arose approximately 150 million years ago and evolved into several clades, including the early-diverging hydra and torix lineages associated with leeches and protozoans. A rapid radiation occurred about 50 million years ago in the arthropod-associated lineages (76).Whole-genome sequencing has led to a revision of phylogenetic relationships among Rickettsia spp. associated with blood-feeding arthropods (10, 26, 28). A newly defined ancestral group (AG) contains the earliest-diverging species, Rickettsia bellii and Rickettsia canadensis, while R. prowazekii and R. typhi, transmitted by lice and fleas, respectively, constitute the typhus group (TG). A proposed transitional group (TRG), consisting of the mite-borne Rickettsia akari, the flea-borne R. felis, and the tick-borne Rickettsia australis, bridges the genotypic and phenotypic differences between the TG and the much larger spotted fever group (SFG), consisting of tick-borne rickettsiae (28). However, some presumptive SFG rickettsiae remain poorly characterized and are of uncertain phylogenetic status, while the accumulation of genomic data from rickettsiae found in a diverse range of invertebrate hosts may have profound impacts on the currently understood phylogeny of rickettsiae associated with blood-feeding arthropods. For example, it appears that the above AG and TRG species have many close relatives in insects (76). Despite the recent phylogenomic advances, the genetic and host-adaptive mechanisms underlying the evolution of arthropod-transmitted pathogens of vertebrates from ancestral Rickettsia spp., including any possible role of plasmids, remain poorly understood.In this report, we have taken advantage of recent isolations of rickettsiae from North and South America to conclusively demonstrate that low-copy-number plasmids are indeed common in low-passage isolates of AG, TRG, and SFG rickettsiae. The only exceptions were multiple isolates of R. parkeri, obtained from ticks and human eschar biopsy specimens and newly recognized as a mildly pathogenic SFG rickettsia (49, 50, 52, 79), and the previously characterized species R. montanensis (7). We confirmed that some Rickettsia isolates harbor more than one plasmid by cloning and sequencing multiple plasmids from “Ca. Rickettsia amblyommii” isolates AaR/SC and Ac/Pa, and we obtained PCR- and gel-based evidence that supported genome sequence evidence for the existence of multiple plasmids in REIS, the rickettsial endosymbiont of Ixodes scapularis. Phylogenetic analysis provided strong evidence for multiple plasmid incompatibility groups and possible multiple origins of plasmid-carried parA genes in the genus Rickettsia. Other than genes encoding plasmid replication initiation and partitioning proteins, the newly sequenced “Ca. Rickettsia amblyommii” plasmids resembled the previously sequenced rickettsial plasmids in sharing limited similarities in coding capacity (6, 7, 22). However, we have previously drawn attention to the presence of hsp genes, encoding α-crystalline small heat shock proteins, as a conserved feature of most rickettsial plasmids that may play a role in host adaptation (7). Phylogenetic analysis indicated that the hsp2 genes were plasmid specific, while the hsp1 genes found on four rickettsial plasmids may have been acquired by a chromosome-to-plasmid transfer event in a TRG-like species.     

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

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

The Rhodococcus opacus PD630 Heparin-Binding Hemagglutinin Homolog TadA Mediates Lipid Body Formation

Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.While the majority of eubacteria (24, 33), and indeed many archaea (22, 33), store carbon as polyhydroxyalkanoate (PHA), a small subset of organisms, primarily actinomycetes, are capable of storing carbon in the form of triacylglycerol (TAG). TAG biosynthesis and storage has been observed in members of the genera Mycobacterium, Rhodococcus, Streptomyces, Nocardia, and others (4, 6, 11, 12, 19, 20, 36). Of these organisms, TAG biosynthesis and storage has been most extensively studied for the Gram-positive, non-spore-forming actinomycete Rhodococcus opacus, strain PD630 (1-6, 11, 12, 19, 20, 25, 36, 38-41).Several studies have demonstrated that R. opacus PD630 is capable of accumulating up to 76% of its cell dry weight (CDW) as TAG (summarized in reference 3). As is the case for PHA biosynthesis, TAG accumulation occurs during nitrogen starvation when carbon is in excess (1-3, 27, 41). Paralleling PHA biosynthesis further, TAG is stored in R. opacus PD630 in distinct inclusion bodies, termed lipid bodies (2, 3, 25, 38, 40). While several studies have sought to identify the underlying molecular and biochemical mechanisms behind TAG biosynthesis and storage in the form of lipid bodies, very little is known concerning these processes.We sought to identify genes and their products that are essential for lipid metabolism in R. opacus PD630. Utilizing a forward genetic approach, we identified a conserved hypothetical gene, termed herein tadA (triacylglycerol accumulation deficient), which is predicted to encode a protein with sequence similarity to the heparin-binding hemagglutinin (HbhA) family of proteins from the genus Mycobacterium. The tadA::Tn5 mutant accumulates 30 to 40% less TAG than the parental strain. We demonstrate that this deficiency is most likely the result of altered lipid body formation and morphology. Through biochemical studies, we further demonstrate that the predicted heparin-binding activity of this protein is essential for its activity both in vivo and in vitro. To our knowledge, this is the first protein shown to regulate lipid body assembly and maturation in prokaryotes.     

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.     

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

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

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

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

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