Thermococcus kodakarensis Genetics: TK1827-Encoded β-Glycosidase,New Positive-Selection Protocol,and Targeted and Repetitive Deletion Technology

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second β-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ∼7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of β-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.Members of the Thermococcales, hyperthermophilic Euryarchaea, grow readily under laboratory conditions and are the focus of many basic and applied research projects (2, 8). Their investigation has, however, been limited by the lack of genetics, and it was a seminal advance therefore when Thermococcus kodakarensis (formerly Thermococcus kodakaraensis [1]) was shown to be naturally competent and to recombine added DNA into its genome (21, 23). T. kodakarensis is attractive as an experimental system as a fermentative heterotroph that grows rapidly on a variety of different substrates (1), optimally at 85°C, including substrates from which it generates substantial levels of hydrogen (11). The T. kodakarensis genome sequence and genome microarray assays are established (7, 12, 18) and, since the discovery of transformation (21), inactivation and manipulation of chromosomal genes has revealed novel biochemical pathways, facilitated in vivo evaluations of the archaeal gene expression machinery, and simplified enzyme purifications (3, 5, 6, 9, 10, 12, 15-20, 22-24, 28). For complementation assays and to facilitate heterologous gene expression in T. kodakarensis, shuttle plasmids have been constructed that replicate and confer selectable phenotypes in both T. kodakarensis and Escherichia coli (19), and TK1761 expression has been established as a reporter system that can be used to identify and quantify regulatory elements in T. kodakarensis (18, 20).During the development of the TK1761 reporter system, a T. kodakarensis strain, designated TS416, was constructed with a nonsense mutation in TK1761 (18). This mutation had no discernible effects on growth, confirming that TK1761 was not an essential gene, but lysates of T. kodakarensis TS416 retained a low level β-glycosidase activity. T. kodakarensis apparently, therefore, had a second β-glycosidase, but since this activity was very low and remained constant with changes in TK1761 expression, its presence did not compromise the use of TK1761 expression as a reporter system in the laboratory media used. The existence of a second β-glycosidase did, nevertheless, raise a potential concern for TK1761 assays in cells grown under different conditions in which expression of the gene encoding this second β-glycosidase might not be constant. To address this, we purified and characterized the second β-glycosidase and, to eliminate the concern for the reporter assay, we constructed a T. kodakarensis strain with both TK1761 and the gene (TK1827) that encodes the second β-glycosidase deleted.To construct the double-deletion strain, we developed a new selection/counterselection protocol and have extended this into a procedure that can be used to delete any nonessential gene from the T. kodakarensis genome. A two-gene cassette has been constructed that can be integrated into the T. kodakarensis chromosome at any desired locus by homologous recombination of flanking genes. Expression of the cassette provides a positive selection for transformants and confers sensitivity to 6-methyl purine (6MP^s). Mutants then isolated that are spontaneously resistant to 6-methyl purine (6MP^r) have both the cassette and the adjacent target gene(s) precisely deleted.Sato et al. (21, 23) established the T. kodakarensis transformation protocol by selecting transformants of tryptophan (trpE) and uracil (pyrF) auxotrophs that grew on minimal medium without tryptophan or uracil. Overexpression of the hydroxy-methylglutaryl-coenzyme A reductase encoded by PF1848, cloned from Pyrococcus furiosus, was later found to confer to resistance to simvastatin (15) and mevinolin (19), allowing the selection of T. kodakarensis transformants on rich media that contain either antibiotic. Mutants spontaneously resistant to these antibiotics do, however, occur at experimentally significant frequencies, and these are very expensive reagents for routine use and prohibitively expensive for incorporation into large preparative cultures. With this in mind, to develop an alternative selection that might be used in nutrient-rich media, we used the 6MP cassette system to delete TK0149. As predicted (6), the T. kodakarensis ΔTK0149 strain generated was an agmatine auxotroph that only grows in nutrient-rich media when agmatine is added. When transformed with DNA expressing TK0149, transformants of this strain can be selected directly on standard nutrient-rich media, and complementation of the ΔTK0149 mutation can be used to maintain the presence of an expression plasmid in T. kodakarensis cells grown in large-volume, rich-medium cultures for enzyme purification.     

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.     

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).     

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.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

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.     

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.     

In Vitro Analysis of Virus Particle Subpopulations in Candidate Live-Attenuated Influenza Vaccines Distinguishes Effective from Ineffective Vaccines

Two effective (vac⁺) and two ineffective (vac⁻) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac⁺ variants were distinguished from the vac⁻ variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac⁺) vaccines.Live-attenuated vaccines are considered more effective than their inactive or single-component counterparts because they activate both the innate and adaptive immune systems and elicit responses to a broader range of antigens for longer periods of time (2, 10, 25, 28). Influenza virus variants with alterations in the reading frame of the nonstructural NS1 protein gene (delNS1), which express truncated NS1 proteins, characteristically induce enhanced yields of type I interferon (IFN) relative to the yields of their isogenic parental virus encoding full-length NS1 proteins (11, 13, 21, 33, 39). Many of these delNS1 variants have proved to be effective as live-attenuated influenza vaccines (LAIVs), providing protection against challenge virus in a broad range of species (33, 46), including chickens (39, 44). The IFN-inducing capacity of the virus is considered an important element in the effectiveness of LAIVs (33). In that context, influenza viruses are intrinsically sensitive to the antiviral action of IFN (31, 32, 36), although they may display a nongenetic-based transient resistance (36). In addition, IFN sensitizes cells to the initiation of apoptosis by viruses (42) and by double-stranded RNA (40), which may be spontaneously released in the course of influenza virus replication (14). Furthermore, IFN functions as an adjuvant to boost the adaptive immune response in mammals (3, 4, 11, 26, 41, 43, 46) and in chickens when administered perorally in the drinking water of influenza virus-infected birds (19). This raises the question: does the enhanced induction of IFN by delNS1 variants suffice to render an infectious influenza virus preparation sufficiently attenuated to function as an effective live vaccine? To address that question, we turned to a recent report that described the selection of several variants of influenza virus with a common backbone of A/TK/OR/71-SEPRL (Southeast Poultry Research Laboratory) that contained NS1 protein genes which were unusual in the length and nature of the amino acid residues at the C termini of the truncated NS1 proteins that they expressed because of the natural introduction of a frameshift and stop codon by the deletion in the NS1 protein gene (44). delNS1 variants were isolated from serial low-inoculum passages of TK/OR/71-delNS1[1-124] (H7N3) in eggs (44). Four of these genetically stable plaque-purified variants, each encoding a truncated NS1 protein of a particular length, were tested as a candidate LAIV in 2-week-old chickens. Two of the delNS1 variants were effective as live vaccines (double deletions [D-del] pc3 and pc4) (phenotypically vac⁺), and two were not (D-del pc1 and pc2) (phenotypically vac⁻) (44), despite only subtle differences in their encoded delNS1 proteins. Why were they phenotypically different?The present study addresses this question by analyzing and comparing the different virus particles that constitute the subpopulations of these two effective (vac⁺) and two ineffective (vac⁻) live vaccine candidates. These analyses are based on recent reports in which noninfectious but biologically active particles (niBAPs) in subpopulations of influenza virus particles were defined and quantified (20, 21, 29). The study described in this report reveals several quantitative and qualitative differences between the particle subpopulations of the four candidate LAIVs, including the different types of IFN-induction dose-response curves, the quantum (maximum) yields (QY) of IFN induced, the efficacy of the interferon-inducing particles (IFPs), the replication efficiency of the virus, and the size of the plaques that they produced. Evidence is presented that the in vitro analysis of virus particle subpopulations may be useful to distinguish vac⁺ from vac⁻ LAIV candidates and provide a basis for identifying and enhancing the performance of particles with desirable phenotypes.     

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.     

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.