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Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (rdhA) genes in marine subsurface sediments. A total of 32 putative rdhA phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem.Scientific ocean drilling explorations have revealed that marine subsurface sediments harbor remarkable numbers of microbial cells that account for approximately 1/10 to 1/3 of all living biota on Earth (20, 25, 33). Thermodynamic calculations of pore-water chemistry suggest that subseafloor microbial activities are generally supported by nutrient and energy supplies from the seawater and/or underlying basaltic aquifers (6, 7). Although sulfate, nitrate, Fe(III), Mn(IV), and bicarbonate are known to be potential electron acceptors for anaerobic microbial respiration in marine subsurface sediments (5), the incidence of both the dissimilatory dehalorespiration pathway and microbial activity in halogenated organic substrates remains largely unknown.Previous molecular ecological studies using 16S rRNA gene sequences demonstrated that Chloroflexi is one of the most frequently detected phyla in subseafloor sediments of the Pacific Ocean margins (12-14). Some of the sequences within the Chloroflexi are closely related to sequences in the genus Dehalococcoides, which contains obligatory dehalorespiring bacteria that employ halogenated organic compounds as terminal electron acceptors (21, 29). The frequent detection of Dehalococcoides-related 16S rRNA genes from these environments implies the occurrence of dissimilatory dehalorespiration in marine subsurface sediments.In this study, we detected and phylogenetically analyzed the reductive dehalogenase homologous (rdhA) genes, key functional genes for dehalorespiration pathways, from frozen sediment core samples obtained by Ocean Drilling Program (ODP) Leg 201 (Peru margin and eastern equatorial Pacific) (7, 14); Integrated Ocean Drilling Program (IODP) Expedition 301 (Juan de Fuca Ridge flank) (8, 24); Chikyu Shakedown Expedition CK06-06 (Northwest Pacific off Japan) (20, 23); and IODP Expedition 315 (Nankai Trough Forearc Basin off Japan) (Table (Table1).1). DNA was extracted using an ISOIL bead-beating kit (Nippon Gene, Japan) and purified using a MagExtractor DNA fragment purification kit (Toyobo, Japan) according to the manufacturer''s instructions. To increase concentration, DNA was amplified by multiple displacement amplification using the phi29 polymerase supplied with a GenomiPhi kit (GE Healthcare, United Kingdom) (20). Putative rdhA genes were amplified by PCR using Ex Taq polymerase (TaKaRa, Japan) with degenerate primers RRF2 and B1R (17), dehaloF3, dehaloF4, dehaloF5, dehaloR2, dehaloR3, and dehaloR4 (32), and ceRD2S, ceRD2L, and RD7 (26) and the PCR conditions described in those studies. Amplicons of the approximate target size were gel purified and cloned into the pCR2.1 vector (Invitrogen, Japan). Sequence similarity was analyzed using FastGroupII web-based software (34), and sequences with a 95% identity were tentatively assigned to the same phylotype. Amino acid sequences were aligned by ClustalW (31), including known and putative reductive dehalogenase sequences in the genome of Dehalococcoides ethenogenes strain 195 (28), as well as several functionally characterized reductive dehalogenases from other species.

TABLE 1.

Sample locations and results of PCR amplification of rdhA
Sampling site (expedition name)LocationWater depth (m)Core sectionSediment depth (mbsf)rdh amplification resulta
1226 (ODP Leg 201)Eastern equatorial Pacific3,2971-33.2++
6-346.7++
1227 (ODP Leg 201)Southeast Pacific off Peru4271-10.3+
3-216.6+
5D-542.0
9-375.1+
1230 (ODP Leg 201)Southeast Pacific off Peru5,0861-10.3++
10-373.8
27-3209.3
1301 (IODP Expedition 301)Northeast Pacific Juan de Fuca Ridge flank off Oregon2,6561-22.5+
6-651.2
11-190.8
1D-2132.5
C9001 (JAMSTEC Chikyu Shakedown Expedition CK06-06)Northwest Pacific off Japan1,1801-11.0++
2-513.5++
9-478.5+
21-4191.5+
24-4216.8++
25-6228.9
38-7346.3
40-3358.6+
C0002 (IODP Expedition 315)Nankai Trough Forearc Basin off Japan1,9371-31.9+
1-64.7
2-49.2+
2-813.4
3-520.2+
4-530.0
8-366.6+
16-4155.4
Open in a separate windowa−, PCR product of expected size not amplified; +, PCR product of expected size weakly amplified; ++, PCR product of expected size amplified and confirmed by sequencing analysis.Putative rdhA genes were successfully detected by primer set RRF2-B1R in samples from the eastern equatorial Pacific (ODP site 1226, 3.2 and 46.7 m below the seafloor [mbsf]), the Peru margin (ODP site 1227, 0.3, 16.6, and 75.1 mbsf, and ODP site 1230, 0.3 mbsf), the Juan de Fuca Ridge flank (IODP site 1301, 2.5 mbsf), offshore from the Shimokita Peninsula of Japan (CK06-06 site C9001, 1.0, 13.5, 78.5, 191.5, 216.8, and 358.6 mbsf), and the Nankai Trough Forearc Basin off the Kii Peninsula of Japan (IODP site C0002, 1.9, 9.2, 20.2, and 66.6 mbsf) (Table (Table1).1). No amplification was observed in samples from several deep horizons at sites 1227, 1230, 1301, C9001, and C0002 (Table (Table1).1). A total of 92 clones of subseafloor putative rdhA genes were sequenced and classified into 32 phylotypes (Fig. (Fig.1).1). Phylogenetic analysis revealed that all of the detected putative rdhA sequences were related to those of Dehalococcoides.Open in a separate windowFIG. 1.Phylogenetic tree based on the deduced amino acid sequences of rdhA genes, including sequences from marine subsurface sediments. Putative rdhA sequences from marine subsurface sediments (rdhA clones 1 to 32) are marked in red, while those of the Dehalococcoides genome are marked in blue. Clonal frequencies and sequence accession numbers are indicated in parentheses. Bootstrap values from 50% to 84% and 85% to 100% are indicated by open and solid circles at the branches, respectively. Asterisks indicate the following functionally characterized rdhA genes: pceA and prdA, tetrachloroethene reductive dehalogenase; tceA, trichloroethene reductive dehalogenase; vcrA and bvcA, vinyl chloride reductive dehalogenase; dcaA, 1,2-dichloroethane reductive dehalogenase; cprA, chlorophenol reductive dehalogenase; and cbrA, chlorobenzene reductive dehalogenase. The tree was constructed by a neighbor-joining (NJ) method based on an alignment of almost-complete rdhA amino acid sequences with pairwise gap deletion on MEGA version 4.0 software (30). The resulting tree was displayed using Interactive Tree Of Life (19). The scale bar represents 0.1 substitutions per amino acid position.In the alignment of the subseafloor rdhA sequences, we observed two Fe-S cluster-binding motifs as a conserved structure of previously reported reductive dehalogenases (29). The sequences were amplified with primer RRF2 containing the N-terminal twin arginine translocation (Tat) signal sequence and primer B1R containing the rdhB genes encoding a putative dehalogenase membrane anchor protein (17). Thus, the dehalogenases of subseafloor bacteria have a structural framework similar to that of known dehalogenases from terrestrial Dehalococcoides species. However, BLASTP analysis showed that similarities among subseafloor rdhA sequences and previously reported dehalogenase sequences were generally low, ranging from 33.06% to 64.27%. Some sequences were affiliated, with relatively high bootstrap values, with subseafloor rdhA clusters I and II, which are clearly distinct from the rdhA sequences of Dehalococcoides and other known species (Fig. (Fig.1).1). In addition, we were unable to detect subseafloor rdhA genes using other primer sets targeting cprA- and pceA-like genes (26, 32). These results indicate that most subseafloor rdhA genes are distinct from those reported from terrestrial environments, a trend that corroborates the results of a metagenomic survey of subseafloor microbial communities at the Peruvian site (3). However, it is worth noting that the RRF2 and B1R primers used in this study are based on the rdhA sequences present in Dehalococcoides (17) and that sequence retrieval is probably biased by primer mismatch. It is thus likely that there are still unexplored functional genes related to the dehalorespiration pathways in marine subsurface sediments.An interesting finding of the functional gene survey is that the subseafloor rdhA homologues are preferentially detected in shallow sediments. At site C9001 off Japan, the sedimentation ratio is considerably higher than at other sites (54 to 95 cm per 1,000 years) (unpublished data), and rdhA genes were successfully detected in horizons as deep as 358 mbsf (Table (Table1).1). The rdhA genes were also detected in sediments from the open ocean at site 1226, which contained very low concentrations (<0.2%) of organic matter (7). This may be because halogenated compounds are derived not only from terrestrial environments but also from the seawater overlying the sediments. In addition, a diverse range of marine organisms, such as phytoplankton, mollusks, algae, polychaetes, jellyfish, and sponges, are known to produce halogenated organic compounds (11). For example, the amount of brominated organic compounds in the ocean has been estimated at 1 to 2 million tons per year (10). Since these halogenated compounds are generally recalcitrant or not metabolizable by aerobic microorganisms in the seawater column (15), they are effectively buried in marine subsurface sediments. In fact, debromination of brominated phenols in marine, estuarine, or intertidal strait sediments has been reported (4, 9, 16, 22), and a brominated phenol-dehalogenating microbial community has been observed in the marine sponge Aplysina aerophoba, which produces bromophenolic metabolites (1).We also observed reductive dehalogenation activity in subseafloor sediment slurry from site C0002 in the Nankai Trough (Fig. (Fig.2;2; also see the supplemental material). The slurry sample was prepared by mixing sediment samples from 1.9, 4.7, 9.2, 13.4, 20.2, 30.0, 66.6, and 155.4 mbsf. During the initial incubation with 2,4,6-tribromophenol (2,4,6-TBP) for 179 days, 2,4,6-TBP was completely converted to phenol. We then supplemented the same incubation slurry with 2,4,6-TBP and once again observed dehalogenation activity (Fig. (Fig.2A).2A). During the incubation, 2,4-dibromophenol and 4-bromophenol were produced as intermediates (Fig. (Fig.2C),2C), suggesting that ortho debromination occurred in preference to para debromination, as observed previously in marine sponge habitats (1). The maximum phenol production rate during the second incubation was calculated to be 0.094 μM per 1 cm3 of sediment per day (Fig. (Fig.2A2A).Open in a separate windowFIG. 2.Dehalogenation activities of subseafloor microbes. (A) Debromination of 2,4,6-TBP in a subseafloor sediment slurry from site C0002 in the Nankai Trough Forearc Basin. Arrow indicates the timing of 2,4,6-TBP supplementation. (B) Dechlorination of TCE in the same slurry sample. Sterilized control sediment slurries did not exhibit phenol and/or cis-DCE production (data not shown). (C) Potential debromination pathway of 2,4,6-TBP (solid arrows) and (D) potential dechlorination pathway of TCE (solid arrows) observed. The pathways indicated by dashed arrows were not observed in this experiment.Using the same sediment slurry sample, we also observed dehalogenation activity of trichloroethene (TCE), a substantial pollutant in the natural environment. During an incubation lasting more than 200 days, TCE was almost entirely converted to cis-dichloroethene (cis-DCE) (Fig. (Fig.2B).2B). The subsequent dechlorination step of cis-DCE, which is presumably from cis-DCE to monochloroethene, was not observed during the incubation. The rate of cis-DCE production was calculated as 0.045 μM per 1 cm3 of sediment per day.In conclusion, the observed molecular and activity data suggest that metabolically active dehalorespiring microbes are well represented in marine subsurface sediments and that these microbes may be widely distributed in Pacific Ocean margin sediments. Given the relatively high in vitro activity rates, we expect that subseafloor dehalorespiring microbes play important ecological roles in the biogeochemical cycles of chlorine, iodine, and bromine, as well as in halogenated carbon substrates. The distribution of in situ activity rates, chemical and geophysical constraints, metabolic characteristics of the individual dehalorespiring phylotypes, and genetic and enzymatic mechanisms of the microbes remain to be clarified. Nevertheless, the findings of this study provide new evidence of microbial functioning in the subseafloor ecosystem.  相似文献   

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The human immunodeficiency virus type 1 (HIV-1) variants that are transmitted to newly infected individuals are the primary targets of interventions, such as vaccines and microbicides, aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly of subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional 31 sexually transmitted Kenyan HIV-1 env genes, representing several recent infections with subtype A, as well as subtypes A/D, C, and D, were cloned, and their neutralization profiles were characterized. Most env variants were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in areas of endemicity. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.Most effective viral vaccines are thought to provide protection primarily by stimulating neutralizing antibodies (NAbs) to clear cell-free virus (25, 27). Because protection by NAbs requires recognition of common viral epitopes, the extreme genetic diversity of human immunodeficiency virus type 1 (HIV-1) presents a particular challenge to NAb-based vaccine approaches. Therefore, a critical starting point for studies of immune-mediated protection against HIV-1 is a collection of newly transmitted HIV-1 variants, particularly from areas of endemicity, such as sub-Saharan Africa, in order to determine whether vaccines are appropriately targeted to common epitopes from these relevant transmitted strains.During HIV-1 transmission, a bottleneck allows only one or a few variants to be transmitted to a newly infected individual (6, 9, 16, 29, 34, 37, 39), and the sensitivity of these early transmitted strains to antibody-mediated neutralization is therefore of particular interest. Newly transmitted HIV-1 variants have demonstrated significant heterogeneity in their neutralization phenotypes both within and between subtypes (2, 3, 6-8, 11, 13-15, 22, 30, 32, 36). Panels of sexually transmitted HIV-1 envelope variants (based on the envelope gene, env) have been characterized, including subtype B variants from North America, Trinidad, and Europe, subtype C variants from South Africa and Zambia, and subtype A variants from Kenya collected between 1994 and 1996 (2, 14, 15). Here, we characterize an additional 31 envelope variants from 14 subjects with sexually transmitted HIV-1 who were infected in Kenya, where subtypes A, C, and D circulate, between 1993 and 2005 (24, 31).The env genes were cloned from samples drawn 14 to 391 (median, 65) days postinfection from individuals enrolled in a prospective cohort of high-risk women in Mombasa, Kenya (19-21). Demographic characteristics of the subjects are summarized in Table Table1;1; the timing of first infection was determined by both HIV-1 serology and HIV RNA testing as described previously (12). All of the subjects were presumably infected by male-to-female transmission and displayed a range of plasma viral loads at the time of env gene cloning (Table (Table1).1). For most individuals, full-length env genes were cloned from uncultured peripheral blood mononuclear cell (PBMC) DNA, though for two individuals, clones were obtained from DNA following short-term coculture with donor PBMCs (Table (Table1).1). env genes were cloned by single-copy nested PCR with primers and PCR conditions as described previously (4, 17). We tested env genes for their ability to mediate infection by transfecting env plasmid DNA into 293T cells along with an env-deficient HIV-1 subtype A proviral plasmid, Q23Δenv, to make pseudoviral particles (17). More than 80 env clones were obtained from 16 subjects; less than one-half were functional on the basis of the infectivity of pseudoviral particles in a single-round infection of TZM-bl cells (AIDS Research and Reference Reagent Program, National Institutes of Health), as observed previously for env genes cloned from proviral sequences (17); a lower fraction of functional env genes have been reported from plasma (18). We focused on the proviral sequences here because they presumably best represent the sequence closest to that of the transmitted strains. The 31 functional env variants are described in Table Table11.

TABLE 1.

Demographic characteristics, diversities, gp120 variable-region lengths, numbers of PNGS, and accession numbers of cloned env variants
SubjectVirus subtypeSample date (mo/day/yr)dpiaPlasma VLbSourcecIndividual env clonePairwise difference (%)dVariable-loop length (aa)
No. of PNGS
GenBank accession no.
V1/V2V3V4V5gp120gp41gp41 ecto
QB726A04/16/967061,940ucPBMCQB726.70M.ENV.B30.16633536102244FJ866111
QB726.70M.ENV.C4633536102244FJ866112
QF495A05/16/0623217,050ucPBMCQF495.23M.ENV.A10.121073537113044FJ866113
QF495.23M.ENV.A31073537113044FJ866114
QF495.23M.ENV.B21133537113144FJ866115
QF495.23M.ENV.D11133537113144FJ866116
QG984A07/12/042130,300ucPBMCQG984.21M.ENV.A3NA693436112433FJ866117
QH209A10/13/051428,600ucPBMCQH209.14M.ENV.A2NA723529112444FJ866118
QH343A09/08/052140,750,000ucPBMCQH343.21M.ENV.A100.19773532152644FJ866119
QH343.21M.ENV.B5773532152644FJ866120
QH359A10/05/052132,120ucPBMCQH359.21M.ENV.C11.4843536102944FJ866121
QH359.21M.ENV.D1733535102644FJ866122
QH359.21M.ENV.E2723540132844FJ866123
QA790eA/D06/10/9620448,100ccPBMCQA790.204I.ENV.A40.36773533112544FJ866124
QA790.204I.ENV.C1773533112644FJ866125
QA790.204I.ENV.C8773533112444FJ866126
QA790.204I.ENV.E2773533112544FJ866127
QG393A2/D06/23/046017,360ucPBMCQG393.60M.ENV.A10.7603431102455FJ866128
QG393.60M.ENV.B7573431102455FJ866129
QG393.60M.ENV.B8573431102455FJ866130
QB099eC02/10/9539127,280ucPBMCQB099.391M.ENV.B10.43653529102544FJ866131
QB099.391M.ENV.C8653529102544FJ866132
QC406C07/08/9770692,320ucPBMCQC406.70M.ENV.F3NA643520112254FJ866133
QA013D10/11/95701,527,700ccPBMCQA013.70I.ENV.H10.16603429122544FJ866134
QA013.70I.ENV.M12603429122544FJ866135
QA465D08/19/935937,750ucPBMCQA465.59M.ENV.A10.24653530112844FJ866136
QA465.59M.ENV.D1653530112744FJ866137
QB857D10/16/9711014,640ucPBMCQB857.23I.ENV.B3NA683432112654FJ866138
QD435D04/06/9910017,470ucPBMCQD435.100M.ENV.A40.88693429122654FJ866139
QD435.100M.ENV.B5673429112454FJ866140
QD435.100M.ENV.E1693429122654FJ866141
Open in a separate windowadpi, days postinfection as defined by RNA testing (12).bVL, viral load on the sample date in which env genes were cloned.cucPBMC, uncultured PBMCs; ccPBMC, cocultured PBMCs.dAverage pairwise distance between the full-length env variants from a given subject. NA, not applicable because there was only one variant available from the subject.eenv variants from these two subjects were cloned from >6 months postinfection, as noted, and should not be considered true early env variants.The full-length, functional env genes were sequenced and aligned to generate a maximum likelihood phylogenetic tree with reference sequences from the Los Alamos National Laboratory HIV database, as described previously (26). Viral env clones from the same subject clustered together, and a wide spectrum of genetic diversity was observed overall (Fig. (Fig.1).1). Some women, such as subject QF495, were infected with a relatively homogeneous viral population, with average pairwise differences of only 0.12% between env variants (Table (Table11 and Fig. Fig.1).1). However, as observed previously in this cohort (16, 28, 29, 33-35), other individuals, such as subjects QH359 and QD435, were infected with more heterogeneous viral populations with average pairwise differences of 1.4% and 0.88% between variants, respectively (Table (Table11 and Fig. Fig.1).1). env genes from subtypes A (13 variants), C (3 variants), and D (8 variants), as well as A/D recombinants (4 variants) and A2/D recombinants (3 variants), were represented (Fig. (Fig.1).1). The viral subtypes were confirmed using the NCBI genotyping database (http://www.ncbi.nlm.nih.gov/).Open in a separate windowFIG. 1.Maximum likelihood phylogenetic tree of full-length sequences from early subtype A, C, D, and A/D recombinant env variants in Kenya. The 31 novel env clones from Kenyan early infections and reference sequences for subtypes A, B, C, D, and K from the Los Alamos HIV database (http://www.hiv.lanl.gov/content/index) are displayed. The phylogenetic tree was rooted with subtype K env sequences. Values at nodes indicate the percentage of bootstraps in which the cluster the right was found; only values of 70% or greater are shown.The deduced amino acid sequences revealed that all functional variants had an uninterrupted open reading frame in env except for variant QB099.391I.ENV.C8, which had a frameshift mutation within the cytoplasmic tail of gp41. There was significant heterogeneity in the length of the protein variable loops, particularly V1/V2, which ranged from 57 amino acids (aa) to 113 aa (Table (Table1).1). The V3, V4, and V5 loops also varied in length, though less dramatically (Table (Table1).1). Variants from the same subject were generally similar in their variable-loop lengths. Moderate variation was also observed in the number and position of potential N-linked glycosylation sites (PNGS) (Table (Table11).Previous analyses indicated that early subtype C env proteins had shorter variable loops than did early subtype B env proteins (13), suggesting that there are different env protein features between subtypes. Thus, to compare variable-loop lengths and the numbers of PNGS between subtypes using this expanded group of early env variants, we evaluated the 31 newly cloned variants plus an additional 15 subtype A variants (2), 19 subtype B variants (14), and 18 subtype C variants (15) from other early virus panels. In order to avoid bias, when more than one env variant was available from a subject, the average loop length or PNGS number for that subject''s env proteins was used. We did not observe significant differences in V1/V2 length, V5 length, or the numbers of PNGS between subtypes by the Kruskal-Wallis equality-of-populations rank test (Table (Table2)2) . However, there were significant differences between the V3 and V4 loop lengths of the subtypes after adjusting for multiple comparisons (Table (Table2).2). The differences in V3 length appeared to be a result of shorter V3 loops in subtype D env proteins than in early subtype B (P = 0.006) or C (P < 0.001) env proteins (Table (Table2).2). The differences in V4 length were caused by shorter V4 loops in subtype C env proteins in comparison to both subtype A and B env proteins (P < 0.001; Table Table22).

TABLE 2.

Summary of variable-loop lengths and the numbers of PNGS in gp120 and gp41 within early HIV-1 env variantsa
ParameterMedian value (25th percentile, 75th percentile) for subtype:
Kruskal- Wallis P valuebWilcoxon rank sum P values for individual comparisonsc
A (n = 11)B (n = 19)C (n = 20)D (n = 4)A vs. BA vs. CA vs. DB vs. CB vs. DC vs. D
Length
    V1/V270.3 (62, 76)70 (66, 70)65 (62, 76)66.5 (62, 69)0.210.7300.2820.2150.0510.1130.846
    V335 (34, 35)35 (35, 35)35 (34, 35)34 (34, 35)0.0010.2400.0160.1070.1410.006<0.001
    V432 (30, 36)33 (31, 34)26.5 (22, 29)29.5 (29, 31)0.00010.880<0.0010.148<0.0010.0230.056
    V511 (11, 11)10 (9, 11)10 (9, 11)11.5 (11, 12)0.0300.0960.0150.1840.6770.0990.021
No. of PNGS in:
    gp12024 (23, 28)25 (24, 26)24 (23, 25)26 (26, 27)0.200.6800.6920.2650.1460.1860.042
    gp414 (4, 5)5 (4, 5)5 (4, 5)4.5 (4, 5)0.200.0300.1790.4700.4100.4080.799
    gp41ecto4 (4, 4)4 (4, 4)4 (4, 5)4 (4, 4)0.0440.1070.0250.5500.0880.5070.201
Open in a separate windowaVariable-loop lengths and the numbers of PNGS in gp120 and gp41 within early HIV-1 env variants from subtypes A, B, C, and D characterized here and previously (2, 14, 15). n, number of samples.bKruskal-Wallis equality-of-populations rank test (based on multiple comparisons; P values of <0.007 were considered significant; significant values are presented in boldface).cWilcoxon rank sum test (based on multiple comparisons; P values of <0.008 were considered significant; significant values are presented in boldface).We then assessed the neutralization sensitivity of the pseudoviruses to antibodies in plasma from HIV-1-infected individuals and to HIV-1-specific MAbs by using the TZM-bl neutralization assay as described previously (2, 23, 38). Median inhibitory concentrations (IC50s) were defined as the reciprocal dilution of plasma or concentration of MAb that resulted in 50% inhibition of infection (2, 38). The Kenya pool was derived by pooling plasma collected between 1998 and 2000 from 30 HIV-1-infected individuals in Mombasa, Kenya, and the other three pools were derived by pooling plasma collected between 1993 and 1997 from 10 individuals from Nairobi, Kenya, and with an infection with a known subtype (A, C, or D) of HIV-1 as described previously (2).The env variants demonstrated a range of neutralization sensitivities to plasma samples, from neutralization resistant (defined as <50% neutralization with a 1:50 dilution of plasma) to neutralization sensitive with an IC50 of 333 (Fig. (Fig.2).2). Some clones, such as QF495.23M.ENV.A1, were relatively sensitive to all the plasma pools, with IC50s from 100 to 333, whereas other clones, such as QH343.21M.ENV.A10, were relatively resistant to these plasma pools, with IC50s from <50 to 85 (Fig. (Fig.2).2). The plasma pools did differ in their neutralization potencies. The Kenya pool, with a median IC50 of <50 across all viruses tested, was significantly less likely to neutralize these transmitted variants than were the subtype A, C, and D plasma pools, which had median IC50s of 110, 105, and 123, respectively (P values of <0.0001, 0.0001, and 0.001, respectively, by paired t test on log-transformed IC50s). The basis for these differences in neutralizing activity is not clear, although the location, timing, and level of immunodeficiency at the time of sample collection could have contributed to the differences in NAb levels between the pools.Open in a separate windowFIG. 2.Neutralization sensitivity of early subtype A, C, D, and A/D recombinant env variants to plasma samples and MAbs in relation to the sequences of the MAb binding sites. The env used to generate the pseudovirus tested is shown at the left, and the plasma pool or MAb tested is indicated at the top. The IC50s of each plasma sample or MAb against each viral pseudotype is shown, with darker shading indicating more potent neutralization, as defined at the bottom of the figure. Gray boxes indicate that <50% neutralization was observed at the highest dilution of plasma or concentration of MAb tested. Each IC50 shown is an average of the results from two independent neutralization assays, using pseudovirus generated in independent transfection experiments. The median IC50s from the 31 variants are shown at the bottom. Neutralization of the pseudovirus derived from the subtype B variant SF162 is shown as a control, and neutralizations of murine leukemia virus (MLV) and simian immunodeficiency virus clone 8 (SIV) are shown as negative controls. In the panels on the right, the sequences for the MAbs 2G12, 2F5, and 4E10 are displayed. For 2G12, the amino acid numbers for the five PNGS that are important for 2G12 binding are shown for each virus tested. A plus sign indicates that the PNGS at that site in the envelope sequence was preserved, and a minus sign indicates that the PNGS was deleted. A shift in the PNGS position is indicated by the amino acid position to which the PNGS shifted. All sequences were numbered relative to the HXB2 sequence. The two rightmost panels show data for the canonical 2F5 and 4E10 epitopes, with a period indicating that the amino acid is preserved.The env variants were significantly more susceptible to their subtype-matched plasma pool, with a higher mean IC50 for subtype-matched plasma samples than for unmatched plasma samples (138 versus 108, P = 0.0081, paired t test). However, a significant amount of cross-subtype neutralization was observed, as every env variant that was susceptible to the subtype-matched plasma pool was also susceptible to at least one of the other plasma pools (Fig. (Fig.2).2). Thus, although potency was enhanced when the plasma antibodies were produced in response to infection with the same subtype of HIV-1, there were shared neutralization determinants between subtypes, as has been observed previously (reviewed in reference 3).To identify potential correlates of neutralization sensitivity to the antibodies within these plasma pools, we included these 31 env variants and an additional 15 subtype A env variants we previously characterized from the same cohort with the same plasma pools (2). We did not observe a change in neutralization sensitivity during the evolution of the HIV-1 epidemic in Kenya, as no correlation was observed between neutralization sensitivity and the calendar date from which the env variants were isolated. In addition, no correlation was observed between the neutralization sensitivity of a variant to the plasma pools and the duration of estimated infection within that individual. Finally, there was no significant correlation between the neutralization sensitivity and variable-loop length or the number of PNGS. Thus, although changes in the variable-loop length or number of PNGS may alter the exposure of epitopes within the HIV-1 env protein, these changes do not appear to be the primary determinant of neutralization sensitivity.Despite relatively universal sensitivity to at least one of the pooled plasma samples, these transmitted Kenyan env variants were generally resistant to the MAbs 2G12 (provided by Hermann Katinger, Polymun Scientific) and b12 (provided by Dennis Burton, The Scripps Research Institute), as well as 2F5 and 4E10 (obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health) (Fig. (Fig.2),2), though these MAbs neutralized the subtype B env variant SF162, with IC50s similar to those reported previously (1). Subtype D strains were the most susceptible to MAbs, with 4/8 variants neutralized with <20 μg/ml of 2F5 and 2/8 neutralized with <20 μg/ml of the other MAbs. This could reflect the fact that subtype D variants are more closely related to subtype B strains (Fig. (Fig.1)1) (see reference 10), and these MAbs were all derived from subtype B-infected individuals.Among all 31 variants, 2F5 was the most broadly neutralizing, with 15/31 variants from 8/14 subjects neutralized with <20 μg/ml of this MAb. Some 2F5-resistant env variants, such as QH209.14M.ENV.A2 and QB857.110I.ENV.B3, had mutations in the canonical 2F5 binding epitopes, though other 2F5-resistant env variants such as QF495.23M.ENV.A3 and QA790.204I.ENV.A4 maintained the canonical 2F5 epitope. The results with the MAb 4E10 were similar; 4E10 neutralized only seven variants from 4 of the 14 subjects, and the presence of mutations in the 4E10 epitope, which were common, did not predict neutralization sensitivity (Fig. (Fig.2).2). For instance, the env variants QH343.21M.ENV.A10 and QH343.21M.ENV.B5 contained identical N671S and D674S mutations and QH343.21M.ENV.B5 was highly sensitive to 4E10, while QH343.21M.ENV.A10 was resistant (Fig. (Fig.2).2). Thus, for the 2F5 and 4E10 epitopes, the presumed epitopes appear to be shielded in a subset of these early non-subtype B env variants, as has been previously observed (Fig. (Fig.2)2) (1, 2, 5, 14).The MAb b12 neutralized only two variants from two subtype D-infected individuals, with no neutralization of the subtype A, C, and A/D recombinant pseudoviruses. Only four variants from two subjects were neutralized by 2G12 at <20 μg/ml, and these were the only variants that maintained all five of the PNGS within the 2G12 epitope (Fig. (Fig.2).2). Overall, the median IC50 of all the MAbs against these transmitted variants was >20 μg/ml. None of the variants was susceptible to all four MAbs (Fig. (Fig.2),2), unlike many of the early subtype B env variants characterized previously (14).In summary, these newly characterized HIV-1 env clones represent a range of neutralization sensitivities and can be used to supplement existing panels of transmitted variants, in particular, adding the first subtype D and A/D recombinant variants. Some differences between subtypes in env structure following transmission were noted, though these differences did not correlate with neutralization sensitivity. Although the significant levels of cross-subtype neutralization sensitivity observed with plasma samples indicate that some neutralization determinants were shared across subtypes, the epitopes for the MAbs b12, 2G12, 2F5, and 4E10 did not appear to be among the shared determinants. Thus, despite the fact that significant attention has focused on using vaccination to develop antibodies that resemble these MAbs in their specificity, such antibodies may not neutralize the transmitted strains that are causing most new infections worldwide. These data therefore stress the importance of evaluating transmitted variants in endemic areas when designing immunogens and evaluating vaccine and microbicide strategies.  相似文献   

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This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.Haloalkane dehalogenases (EC 3.8.1.5.) acting on halogenated aliphatic hydrocarbons catalyze carbon-halogen bond cleavage, leading to an alcohol, a halide ion, and a proton as the reaction products (7). Haloalkane dehalogenases originating from various bacterial strains have potential for application in bioremediation technologies (4, 6, 22), construction of biosensors (2), decontamination of warfare agents (17), and synthesis of optically pure compounds (19). Recent evolutionary study of haloalkane dehalogenase sequences revealed the existence of three subfamilies, denoted HLD-I, HLD-II, and HLD-III (3). In contrast to subfamilies HLD-I and HLD-II, the subfamily HLD-III is currently lacking experimentally characterized proteins. We have therefore focused on the isolation and study of two selected representatives of the HLD-III subfamily, DrbA and DmbC.The drbA gene was amplified by PCR using the cosmid pircos.a3g10 originating from marine bacterium Rhodopirellula baltica SH1, and the dmbC gene was amplified from DNA originating from obligatory pathogen Mycobacterium bovis 5033/66. Six-histidine tails were added to the C termini of DrbA and DmbC in a cloning step, enabling single-step purification using Ni-nitrilotriacetic acid resin. Haloalkane dehalogenase DrbA was expressed under the T7 promoter and purified, with a resulting yield of 0.1 mg of protein per gram of cell mass. Haloalkane dehalogenase DmbC was obtained by expression in Mycobacterium smegmatis, with a yield of 0.07 mg of purified protein per gram of cell mass.The correct folding and secondary structures of the newly prepared enzymes were verified by circular dichroism (CD) spectroscopy. Far-UV CD spectra were recorded for DrbA and DmbC enzymes and other, related haloalkane dehalogenases. All enzymes tested exhibited CD spectra with two negative features at 208 and 222 nm and one positive peak at 195 nm, which are characteristic of α-helical content (Fig. (Fig.1).1). This suggested that both new enzymes, DrbA and DmbC, were folded correctly. However, DmbC exhibited more intense negative maxima which differed from other haloalkane dehalogenases in the θ222208 ratio. This finding indicated a slight variation in the arrangement of secondary structure elements of the DmbC enzyme. Thermally induced denaturations of DrbA and DmbC were tested in parallel. Both enzymes showed changes in ellipticity during increasing temperature. The melting temperatures calculated from these curves were 45.8 ± 0.4°C for DmbC and 39.4 ± 0.1°C for DrbA. The thermostability results obtained for DrbA and DmbC were in good agreement with the range of melting temperatures determined for other, related haloalkane dehalogenases.Open in a separate windowFIG. 1.Far-UV CD spectra of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Protein concentration used for far-UV CD spectrum measurement was 0.2 mg/ml.The sizes of the purified proteins were estimated by electrophoresis under native conditions conducted using a 10% polyacrylamide gel (Fig. (Fig.2).2). More precise determination of the sizes of DrbA and DmbC was achieved by gel filtration chromatography performed on Sephacryl S-500 HR (GE Healthcare, Uppsala, Sweden), calibrated with blue dextran 2000, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (Fig. (Fig.3A).3A). Both DrbA and DmbC were eluted from the column in the fraction prior to blue dextran, indicating that both enzymes form oligomeric complexes of a size larger than 2,000 kDa (Fig. 3B and C). The haloalkane dehalogenases which have been biochemically characterized so far form monomers, except for DbjA isolated from Bradyrhizobium japonicum USDA110 (21), which shows monomeric, dimeric, and tetrameric forms according to the pH of the buffer (R. Chaloupkova, submitted for publication).Open in a separate windowFIG. 2.Native protein electrophoresis of DrbA and DmbC. Lane 1, carbonic anhydrase (29 kDa); lane 2, ovalbumin (43 kDa); lane 3, bovine albumin (67 kDa); lane 4, conalbumin (75 kDa); lane 5, catalase (240 kDa); lane 6, ferritin (440 kDa); lane 7, DrbA; lane 8, DmbC.Open in a separate windowFIG. 3.Gel filtration chromatogram of DrbA and DmbC. (A) The following calibration kit samples (0.5 ml of a concentration of 2 mg/ml protein loaded) were analyzed using 50 mM Tris-HCl with 150 mM NaCl, pH 7.5, as elution buffer: blue dextran (line 1, 9.6-ml fraction), thyroglobulin (line 2, 15.95-ml fraction), ferritin (line 3, 16.78-ml fraction), ovalbumin (line 4, 18.55-ml fraction), and RNase A (line 5, 20.08-ml fraction). (B and C) Haloalkane dehalogenase DrbA eluted in the 9.03-ml fraction (B), and haloalkane dehalogenase DmbC in the 9.31-ml fraction (C).The substrate specificities of DrbA and DmbC were investigated with a set of 30 selected chlorinated, brominated, and iodinated hydrocarbons. Standardized specific activities related to 1-chlorobutane (summarized in Table Table1)1) were compared with the activity profiles of other haloalkane dehalogenases (Fig. (Fig.4).4). DrbA and DmbC displayed similar activity patterns, with catalytic activities approximately two orders of magnitude lower than those of other known haloalkane dehalogenases (1, 5, 8-11, 13-16, 18, 20, 23). HLD-III subfamily enzymes showed a restricted specificity range and a preference for iodinated short-chain hydrocarbons. Both phenomena may be related to the composition of the catalytic pentad Asp-His-Asp+Asn-Trp, which is unique to the members of the HLD-III subfamily (3). The preference for substrates carrying an iodine substituent can be related to a pair of halide-binding residues and their spatial arrangement with the catalytic triad. These residues make up the catalytic pentad, playing a critical role in substrate binding, formation of the transition states, and the reaction intermediates of the dehalogenation reaction (12).Open in a separate windowFIG. 4.Substrate specificity profiles of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Activities were determined using a consistent set of 30 halogenated substrates (see Table Table1).1). Data were standardized by dividing each value by the sum of all activities determined for individual enzymes in order to mask the differences in absolute activities. Specific activities (in μmol·s−1·mg−1) with 1-chlorobutane are 0.0003 (DrbA), 0.0001 (DmbC), 0.0003 (DatA), 0.0133 (DbjA), 0.0010 (DbeA), 0.0128 (DhaA), 0.0231 (LinB), 0.0171 (DmbA), and 0.0117 (DhlA).

TABLE 1.

Specific activities of haloalkane dehalogenases DrbA and DmbC toward a set of 30 halogenated hydrocarbonsa
SubstrateDrbA
DmbC
Sp act (nmol product·s−1· mg−1 protein)Relative activity (%)Sp act (nmol product·s−1· mg−1 protein)Relative activity (%)
1-Chlorobutane0.2911000.122100
1-Chlorohexane0.129440.122100
1-Bromobutane0.081281.2211,000
1-Bromohexane0.181620.977800
1-Iodopropane0.143492.1981,800
1-Iodobutane0.5061742.5642,100
1-Iodohexane0.095330.244200
1,2-DichloroethaneNANANANA
1,3-DichloropropaneNANA0.01210
1,5-DichloropentaneNANA0.06150
1,2-Dibromoethane0.098340.855700
1,3-DibromopropaneNANA5.0074,100
1-Bromo-3-chloropropane0.00101.4651,200
1,3-Diiodopropane0.3581236.7165,500
2-Iodobutane0.0289NANA
1,2-DichloropropaneNANANANA
1,2-Dibromopropane0.148510.244200
2-Bromo-1-chloropropane0.091310.488400
1,2,3-TrichloropropaneNANANANA
Bis-(2-chloroethyl) etherNANANANA
ChlorocyclohexaneNANANANA
Bromocyclohexane0.0269NANA
(1-Bromomethyl)-cyclohexaneNANA0.08973
1-Bromo-2-chloroethane0.167570.11191
ChlorocyclopentaneNANANANA
4-Bromobutyronitrile0.200690.444364
1,2,3-TribromopropaneNANA0.222182
3-Chloro-2-methyl propeneNANANANA
2,3-Dichloropropene0.27695NANA
1,2-Dibromo-3-chloropropane0.01030.04436
Open in a separate windowaNA, no activity detected.Substrates 1-iodobutane and 1,3-diiodopropane, identified as the best substrates for haloalkane dehalogenases DrbA and DmbC, were used for measuring the dependency of enzyme activity on temperature and for determination of the pH optima. DrbA exhibited the highest activity with 1-iodobutane at 50°C, although above this temperature, the enzyme rapidly became inactivated. DmbC showed the highest activity toward 1,3-diiodopropane at 40°C, which is similar to the temperature determined with the haloalkane dehalogenases DmbA and DmbB (45°C), isolated from the same species (10). Irrespective of the reaction temperature, DrbA showed the maximum activity at pH 9.15. DrbA kept 80% of its activity throughout a relatively wide range of pH values (pH 7.00 and 9.91) compared to DmbC, which showed a sharp maximum at pH 8.30. The Michaelis-Menten kinetics of DrbA and DmbC determined by isothermal titration microcalorimetry were investigated with 1-iodobutane, which is an iodinated analogue of 1-chlorobutane routinely used for characterization of haloalkane dehalogenases. The low magnitudes of the Michaelis constants (Km = 0.063 ± 0.003 mM for DrbA and 0.018 ± 0.001 mM for DmbC) suggest a high affinity of both enzymes for 1-iodobutane. The catalytic constants determined with 1-iodobutane (kcat = 0.128 ± 0.002 s−1 for DrbA and 0.0715 ± 0.0004 s−1 for DmbC) suggest that the low specific activities observed during substrate screening are not due to poor affinity but are instead due to a low conversion rate.The biochemical characteristics of purified DrbA and DmbC suggest that these proteins represent novel enzymes differing from previously characterized haloalkane dehalogenases by (i) their unique ability to form oligomers and (ii) low levels of dehalogenating activity with typical substrates of haloalkane dehalogenases. This study further illustrates how genome sequencing projects and phylogenetic analyses contribute to the identification of novel enzymes. Characterization of DrbA and DmbC, belonging to the subfamily HLD-III, partially filled a gap in the knowledge of the haloalkane dehalogenase family and provided an additional insight into evolutionary relationships among its members.  相似文献   

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A fitness cost due to imbalanced replichores has been proposed to provoke chromosome rearrangements in Salmonella enterica serovars. To determine the impact of replichore imbalance on fitness, the relative fitness of isogenic Salmonella strains containing transposon-held duplications of various sizes and at various chromosomal locations was determined. Although duplication of certain genes influenced fitness, a replichore imbalance of up to 16° did not affect fitness.The bacterial chromosome is a dynamic molecule that can undergo various types of rearrangements, including inversions, translocations, and duplications. These rearrangements can alter gene order and change replichore length. Replichores are defined as the halves of the chromosome between the origin of replication and the terminus region in the vicinity of the dif site (4, 6, 10, 15). In most bacteria, both replichores are approximately the same length, with each comprising 180° around the circular chromosome. In this state, the replichores and DNA replication are balanced. Imbalance is introduced when one replichore becomes longer than the other. For example, if the replichores comprise 200° and 160° around the circular chromosome, the replichores are 20° imbalanced. Various studies over the years have investigated the effect of imbalanced DNA replication on fitness in Escherichia coli (8, 9, 16, 17). In a recent analysis, E. coli strains that contained asymmetrical interreplichore inversions were found to have growth defects when the imbalance was at least 50° (8). While these studies have demonstrated that imbalanced replichores can affect fitness, the approaches used to introduce the imbalance either do not occur or are extremely rare in nature.Duplications play important evolutionary roles because the increase in gene copy number can facilitate adaptation to certain growth conditions (20), as well as being a source for the evolution of new genes (3). Typically duplications occur at frequencies between 10−3 and 10−5 in unselected cultures (2) but are lost at rates of up to 1,000-fold more often if they do not provide a selective advantage (19). Duplications also result in replichore imbalance. However, the effect of duplications on fitness in relation to replichore balance has not been investigated.A major hurdle in studying the effects of duplications on fitness is that if the duplication is detrimental, haploid revertants will outgrow the parental strain containing the duplication. To circumvent this problem, we used a set of 11 isogenic Salmonella enterica strains with transposon-held duplications (5) (Fig. (Fig.11 and Table Table1).1). Culturing these strains in the presence of chloramphenicol selects for the duplication because if the duplication collapses, the transposon is lost and the cells become chloramphenicol sensitive. As the size of the duplications in these strains varies, so does the amount of introduced replichore imbalance, ranging from 5° to 23°. In addition, fitness effects due to the location of the duplication versus the size of the duplication were also discerned, as the collection includes duplications of similar sizes located in different regions of the chromosome.Open in a separate windowFIG. 1.Genetic map of S. enterica serovar Typhimurium LT2 showing genes used as endpoints in constructing transposon-held duplications of the regions between genes. Balanced replichores are indicated by the symmetry of the oriC-Ter axis. Duplications increase the length of one replichore relative to the other, imbalancing axis symmetry.

TABLE 1.

Properties of the S. enterica strains used in this study
StrainAliasGenotypeDuplication location (min)Duplication size (kbp)Replichore imbalance (°)Reference
MST1LT2Wild type1.911
MST3813SV4200Dup [trp248 MudP hisD9953]38-44332.111.55
MST3814SV3193Dup [hisH9962 MudP cysA1586]44-53399.713.75
MST3815SV4015Dup [cysA1586 MudP purG2149]53-56158.45.75
MST3816SV4193Dup [purG2149 MudP argA9001]56-64430.714.75
MST3817SV4194Dup [argA9000 MudP cysG1573]64-75484.616.35
MST3818SV1601Dup [cysG1573 MudP ilvA2642]75-85486.416.45
MST3819SV4195Dup [ilvA2648 MudP purA1881]85-95495.316.75
MST3820SV4142Dup [purA1881 MudP thr469]95-0248.78.85
MST3821SV1604Dup [thr469 MudP proA692]0-8367.112.65
MST3822SV1603Dup [proA692 MudQ purE2164]8-12230.28.15
MST3823SV1611Dup [purE2514 MudP purB1879]12-27723.223.35
MST1529TT11183srl-203::Tn10d(Cam)1
TYT4480rrfH::pCE36This work
MST5198rrfH::pCE36 srl-203::Tn10d(Cam)This work
Open in a separate windowThe relative fitness of these strains was investigated by measuring growth rates and by assaying growth in a mixed culture with an isogenic competitor strain. Single colony isolates streaked from frozen stocks were used to inoculate broth cultures. Growth was measured in E medium supplemented with 0.2% glucose (minimal medium), Luria-Bertani medium (LB), or LB supplemented with 1× E-salts and 0.2% glucose (LBEDO) (18). Competition assays were done in LB. Media were supplemented with chloramphenicol (20 mg/ml) to maintain the duplications, and the solid media used in competition experiments also contained 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; 40 mg/ml) to differentiate between the duplication and competitor strains.In minimal medium, most of the strains had a generation time of approximately 40 min, except for MST3813, MST3823, and MST3819, which grew somewhat slower (Fig. (Fig.2).2). In LB, the average generation times of all of the duplication-bearing strains were longer than that of the wild type. When grown in LBEDO, all of the strains had generation times of 11 to 15 min. Observed differences in growth rate were not due to lower viability as determined by plate counts. There was no correlation between duplication size and generation time in any of the three media.Open in a separate windowFIG. 2.Generation times of strains as a function of duplication size. Strains were grown in minimal medium (♦), LB (▪), or LBEDO (▴) aerobically at 37°C. Error bars show standard deviations.Competition indices were determined from mixed cultures with the isogenic competitor strain MST5198 and either the wild-type strain or one of the duplication-bearing strains. The competitor strain contained an integrated plasmid encoding a promoterless lacZ gene (7) driven by the rrnH promoter and Tn10d(Cam) conferring chloramphenicol resistance integrated into the srl locus (Table (Table1).1). Strains were grown to saturation overnight, and 10−4 dilutions were used to inoculate mixed cultures. Samples taken at t = 0 (input) and subsequent time points (output) were diluted and spot plated in triplicate. The competition index (C.I.) was calculated as follows: C.I. = (no. output Lac CFU/no. output Lac+ CFU)/(no. input Lac CFU/no. input Lac+ CFU).Data are shown for 6 and 12 h, but readings were taken at time points of up to 1 week. There was no correlation between the duplication size and the C.I. (Fig. (Fig.3).3). Most of the duplication-bearing strains competed as well as or slightly better than the wild type against the competitor strain. Two exceptions were MST3819 and MST3823, which both competed significantly more poorly against the competitor strain than did the wild-type strain (Student''s t test P < 0.05). While these two strains contain the largest duplications, the size of the duplication in MST3819 was close to the size of the duplications in MST3817 and MST3818. Strains MST3817 and MST3818 were either similar or slightly better competitors than the wild type, indicating that the fitness defect in MST3819 is not due to replichore imbalance.Open in a separate windowFIG. 3.Competition indices of the wild-type and duplication-bearing strains after 6 (▪) and 12 h (░⃞) of growth in mixed cultures with MST5198 (n = 6). Strains are in order of increasing duplication size, with the locations of duplications indicated at the top. Indices statistically significantly different (P < 0.05) from that of the wild-type strain (MST1) are marked (*). Error bars show standard deviations.To confirm that the duplications in MST3819 and MST3823 were responsible for the observed growth defects, isolates of each strain with collapsed duplications were obtained by growing cultures of each strain without chloramphenicol and then screening for chloramphenicol-sensitive derivatives. The loss of the duplication restored wild-type growth to both strains. The frequencies of duplication collapse in cultures of MST3818 and MST3819 were also compared. Since these two strains have duplications of similar sizes, the collapse frequencies of the duplicated regions, and concomitant loss of chloramphenicol resistance, were expected to be similar. While the reversion frequency of MST3818 cells was 3.5 × 10−3 cells/generation, MST3819 cells reverted with a frequency of 2.8 × 10−2 cells/generation, resulting in 97 to 98% of MST3819 cells being chloramphenicol sensitive and haploid after reaching stationary phase.In conclusion, the results of this study show that a replichore imbalance of up to 16° introduced by transposon-held duplications in the chromosome does not have a measurable effect on the fitness of S. enterica. The duplicated regions in these strains are larger than the islands acquired via horizontal gene transfer, which have been purported to disrupt replichore balance sufficiently to promote chromosome rearrangements. A duplication that introduced a replichore imbalance of 23° did have a growth defect, but whether the defect is from replichore imbalance or the gene content of the duplication could not be distinguished. This study suggests that the disruption of replichore balance by acquisition of horizontally transferred genes is not the cause of chromosome rearrangements in host-specific S. enterica serovars as hypothesized (12-14).  相似文献   

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A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans worldwide (18). Cattle are considered the primary reservoir for this pathogen and play a central role in transmission to humans (6). Healthy cattle transiently carry EHEC O157:H7 and shed the bacteria in their feces (5, 7). Human infections have been associated with the consumption of contaminated meat and milk, direct contact with cattle, and the consumption of vegetables, fruits, and water contaminated with cattle manure (6).Because of its high discriminatory power, pulsed-field gel electrophoresis (PFGE) has been widely employed as a molecular typing method in many epidemiological investigations to identify various outbreaks and routes of transmission of EHEC O157:H7 (1, 12, 15, 17). Simpson''s index of diversity (9) was reported to be >0.985 in previous studies (1, 15), supporting the identification of richness (the number of types among isolates) and evenness (the relative distribution of individual strains among the different types) of molecular typing using PFGE.Instability of the PFGE patterns of EHEC O157:H7 isolates has been reported. Changes in PFGE patterns were observed among strains after repeated subculturing and prolonged storage at room temperature (11). Loss of Shiga toxin genes and a large-scale inversion within the genome were identified as genetic events generating changes in PFGE patterns in vitro (10, 13). Shifts in the genotypes of EHEC O157:H7 clinical isolates from patients and cattle have been reported (3, 14). This phenomenon was also observed in EHEC O157:H7 experimental infections of cattle. Spontaneous curing of a 90-kb plasmid resulted in the loss of two restricted fragments from the PFGE profiles of EHEC O157:H7 isolates obtained from experimentally infected cattle (2). The purpose of the present study was to identify the genetic events affecting the PFGE patterns of EHEC O157:H7 after passage through the intestinal tract of cattle, especially for restriction fragments that are >90 kb long.Four 5-month-old Holstein steers were housed individually in climate-controlled biosafety level 2 containment barns in accordance with the guidelines for animal experimentation defined by the National Institute of Animal Health of Japan. The pens had individual floor drains and were cleaned twice daily with water and disinfectant. All animals were healthy and culture negative for EHEC O157:H7 strains, as determined by a previously described technique (2), prior to inoculation.EHEC O157:H7 strain Sakai-215 (12, 23), which was isolated from an outbreak in Sakai, Osaka Prefecture, in 1996 was used for inoculation. This strain harbors the genes encoding Stx1 and Stx2. A spontaneous resistant strain was selected with nalidixic acid in order to facilitate the recovery of this strain from fecal samples. All calves were inoculated using a stomach tube with an exponential-phase culture (109 CFU) of the nalidixic acid-resistant Sakai-215 strain. Fecal samples were collected from the four calves daily for 45 days. Fecal culturing was performed as described previously (2). Eight non-sorbitol-fermenting colonies were selected daily from each animal and identified as EHEC O157:H7 colonies by routine diagnostic methods (25).All animals were clinically normal throughout the experimental period. The EHEC O157:H7-inoculated calves (calves 1 to 4) were culture positive for the organism 24 h after inoculation. Intermittent fecal shedding by the calves was observed until 27, 32, 26, and 39 days postinoculation for calves 1, 2, 3, and 4, respectively (Fig. (Fig.1).1). The numbers of EHEC O157:H7 isolates recovered from calves 1, 2, 3, and 4 were 200, 224, 200, and 281, respectively.Open in a separate windowFIG. 1.Changes in PFGE profiles of EHEC O157:H7 isolates recovered from calves 1 (A), 2 (B), 3 (C), and 4 (D). The absence of a bar indicates that no EHEC O157:H7 was detected. The open horizontal bars under the vertical bars indicate that the eight isolates obtained on a day were obtained from the enrichment culture.A total of 905 recovered isolates in addition to the inoculated strain were used for PFGE analysis. Genomic DNA from each EHEC O157:H7 isolate was prepared using the method of Persing (Mayo Clinic, Rochester, MN) described by Rice et al. (20). Agarose-embedded chromosomal DNA was cleaved with XbaI by following the manufacturer''s instructions. PFGE was performed in a 0.85% megabase agarose gel, using a CHEF DR III apparatus (Bio-Rad Laboratories). The pulse time was increased from 12 to 35 s for 18 h. The PFGE profiles of all of the EHEC O157:H7 isolates recovered from the four calves were compared with that of the inoculated strain. The number of band differences was determined by enumerating the loss and addition of fragments (22).Two hundred eighty-nine isolates had PFGE profiles different from that of the inoculated strain, and 12 distinct PFGE profiles were identified for these isolates (Table (Table1).1). The fact that only one to three band differences were observed for the 12 profiles suggested that these isolates were closely related (22) and were variants of the inoculated strain. In addition, the pens had individual floor drains and were cleaned twice daily with water and disinfectant, which reduced the likelihood of introduction of novel EHEC O157:H7 strains. We designated the PFGE profiles A to L. PFGE profiles A, C, and H were obtained for all four calves and accounted for 30.4% of the 905 isolates recovered. Different PFGE profiles were obtained for all animals at least 2 days postinoculation (Fig. (Fig.1).1). All eight isolates from calf 2 collected on day 15 postinoculation and from calf 3 collected on days 22 and 23 postinoculation had PFGE profiles different from that of the original isolate (Fig. (Fig.1).1). The isolates that had the same PFGE profile as the inoculated strain were detected again later.

TABLE 1.

Temporal distribution of PFGE profiles of EHEC O157:H7 isolates recovered from experimentally infected cattle
PFGE profileNo. of isolates recovered at different times postinoculation from:
Total no. of isolates (%)
Calf 1
Calf 2
Calf 3
Calf 4
1 to 10 days11 to 20 days21 to 27 days1 to 10 days11 to 20 days21 to 30 days31 to 32 days1 to 10 days11 to 20 days21 to 36 days1 to 10 days11 to 20 days21 to 30 days31 to 39 days
Ina63562562465066046559454746616 (68.1)
A1119101372121324410191612181 (20.0)
B11 (0.1)
C132471362776572 (8.0)
D11 (0.1)
E11 (0.1)
F123 (0.3)
G11 (0.1)
H1314133221122 (2.4)
I1113 (0.3)
J11 (0.1)
K112 (0.2)
L11 (0.1)
Total no. of variants (%)b17 (21.3)24 (30.0)15 (37.5)18 (22.5)18 (25.0)22 (30.6)2 (25.0)20 (25.0)34 (42.5)35 (87.5)21 (26.3)27 (37.5)17 (26.6)19 (29.2)289 (31.9)
Open in a separate windowaPFGE profile of inoculated strain Sakai-215.bTotal numbers of isolates having PFGE profiles A to L.Kudva et al. (16) demonstrated that the difference in PFGE profiles between EHEC O157:H7 strains was due to distinct insertions or deletions that contained XbaI sites rather than to single-nucleotide polymorphisms in the XbaI sites themselves. To identify the locations of insertions or deletions in the genome of the EHEC O157:H7 isolates recovered from experimentally infected cattle, whole-genome PCR scanning (WGP scanning) was performed as described previously (19). Briefly, 549 pairs of PCR primers were used to amplify 549 segments covering the whole chromosome of EHEC O157:H7 strain RIMD 0509952, with overlaps of a certain length at every segment end. The inoculated strain (strain Sakai-215) and the strain whose genome was sequenced (RIMD 0509952) were isolated from the same outbreak in Japan in 1996 (23) and had same PFGE profile after XbaI digestion. All primer sequences are available at http://genome.gen-info.osaka-u.ac.jp/bacteria/o157/pcrscan.html. PCR were performed using genomic DNA as the template and long accurate PCR (LA-PCR) kits. The cycling conditions for the LA-PCR included an initial incubation at 96°C for 100 s, followed by 30 cycles of 96°C for 20 s and 69°C for 10 min.Prior to the WGP scanning of the isolates, we scanned an approximately 1.2-Mb region covered by 116 segments (71/72 to 146/147) of the EHEC O157:H7 genome using 24 strains, including inoculated strain Sakai-215, 4 isolates with the same PFGE profile as the inoculated strain, 3 isolates with PFGE profile A, 3 isolates with PFGE profile C, 2 isolates with PFGE profile F, 2 isolates with PFGE profile H, 2 isolates with PFGE profile I, and one isolate each with PFGE profiles B, D, E, G, J, K, and L. The main purpose of this preliminary scanning was to determine the extent of variation in the data for isolates having the same PFGE profiles.As shown in Fig. Fig.2,2, we successfully amplified products that were the expected sizes for 103 of the 116 segments for the 24 strains tested. No amplification in a segment was observed for the 24 strains. Polymorphism (expected amplification was observed in some but not all strains) was observed in 12 segments. Eleven of the 12 polymorphic segments consisted of two different sequentially unamplified regions. An IS629 insertion was also observed in a polymorphic segment in one strain. In other words, variation in the data for isolates with the same PFGE profile was not observed except for the isolates having the same PFGE profile as the inoculated strain. Hence, we performed WGP scanning using one isolate with each of the selected PFGE profiles.Open in a separate windowFIG. 2.Summary of the results of PCR scanning analysis of part of the EHEC O157:H7 genome using 24 strains recovered from experimentally infected cattle. The line at the top indicates data for the inoculated strain. The positions of Sp5 and Sp6 are indicated above the data lines. Segments showing polymorphism (expected amplification was observed in some strains but not in all strains) are indicated below the data lines. In, inoculated strain.The results of WGP scanning of the seven isolates with different PFGE profiles in addition to inoculated strain Sakai-215 are summarized in Fig. Fig.3.3. We successfully amplified products of the expected sizes for 530 of 549 segments for the eight strains tested. No amplification was observed for any of the eight strains for three segments (133.2/133.3, 164.4/164.5, and 164.5/164.6). Polymorphism was observed in 16 segments. Fourteen of the 16 polymorphic segments were located in four different regions.Open in a separate windowFIG. 3.Summary of the results of WGP scanning analysis of the EHEC O157:H7 isolates recovered from experimentally infected cattle and the inoculated strain. The positions of Sp5 and Sp13 are indicated above the data lines. Segments showing polymorphism (expected amplification was observed in some strains but not in all strains) are indicated below the data lines. In, inoculated strain.The 110/110.1-to-110.5/111 region in PFGE profile I, the 122/122.1-to-122.4/123 region in PFGE profile K, and the 199/199.1-to-199.2/200 region in PFGE profiles B, C, and G corresponded to prophages Sp5, Sp6, and Sp13, respectively. The 283/284-to-285/286 region in PFGE profile E and the 448/448.1-to-448.1/448.2 region in PFGE profile B corresponded to nonprophage regions on the chromosome. The sizes of the deletion sites of nonprophage regions 283/284 to 285/286 and 448/448.1 to 448.1/448.2 were 17 kb and 9.5 kb, respectively. We synthesized new primer pairs upstream and downstream of these five regions and performed LA-PCR (data not shown). The results of the sequencing analysis of the products indicated that the three prophage genomes were cured at their integration sites (Fig. 4A to C). It is not clear from this study whether deletion of the three prophages represented phage excisions or simple deletions. We identified short direct CCGCCA and GC repeats at both ends of the 17-kb and 9.5-kb deletion sites, respectively, compared with the sequence data for the Sakai-215 strain, although the deleted regions included one side of the direct repeats (Fig. 5D and E).Open in a separate windowFIG. 4.Schematic diagrams showing the relationships between deletions of chromosomal regions and changes in the sizes of restricted fragments. (A) The 467-kb restricted fragment of PFGE profile I was generated by deletion of prophage Sp5 located in the 530-kb fragments of the inoculated strain. (B) The 759-kb restricted fragment of PFGE profile K was generated by deletion of Sp6 located in the adjacent 530-kb and 278-kb fragments of the inoculated strain. (C) The 291-kb restricted fragment of PFGE profiles C, G, and H was generated by deletion of prophage Sp13 located in the adjacent 255-kb and 55-kb fragments of the inoculated strain. (D) The 188-kb restricted fragment of PFGE profile E was generated by deletion of the 17-kb chromosomal region in the 205-kb fragment of the inoculated strain. (E) The 334-kb restricted fragment of PFGE profile B was generated by deletion of the 9.5-kb region located in the adjacent 343-kb and 6.2-kb fragments of the inoculated strain.Open in a separate windowFIG. 5.Comparison of the PFGE profiles of the EHEC O157:H7 isolates recovered from experimentally infected cattle and the inoculated strain. Lane M, λ ladder used as a size marker; lane 1, inoculated strain; lanes 2 to 13, isolates with PFGE profiles A to L, respectively.The deleted 17-kb region contains 16 open reading frames, including formate hydrogenase-related genes (4), mutS (21), and rpoS (8), suggesting that the strain with PFGE profile E is more susceptible to environmental stresses than the inoculated strain. In fact, the isolate with PFGE profile E was more susceptible to low-pH, high-temperature, and high-osmolarity conditions or to the presence of deoxycholate in vitro than the other isolates obtained in this study (data not shown). The fact that this isolate was obtained 4 days after inoculation from calf 1 and could not be detected after that time suggested that the isolate with PFGE profile E could not survive in the intestine of the calf due to the loss of genes related to stress resistance. The deleted 9.5-kb region contains nine open reading frames whose functions are unknown. The strain with this deletion was isolated 1 day after inoculation from calf 3 and could not be detected after that time.Sp5 is one of the prophages in EHEC O157:H7 RIMD 0509952 carrying the stx2 gene. Deletion of this prophage affected the PFGE profile of inoculated strain Sakai-215. The loss of a 530-kb fragment and the gain of a 467-kb fragment due to deletion of the 63-kb prophage Sp5 were identified in PFGE profile I (Fig. (Fig.4A4A and and55).Sp6 is one of the lambda-like phages and has a single XbaI site in its genome. The loss of 530-kb and 278-kb fragments and the gain of a 759-kb fragment due to deletion of this phage were identified in PFGE profile K (Fig. (Fig.4B4B and and55).Sp13 is one of the P2-like phages that have a single XbaI site in the genome. The loss of 255-kb and 55-kb fragments and the gain of a 291-kb fragment due to deletion of this prophage were identified in PFGE profiles C, G, and H (Fig. (Fig.4C4C and and5).5). The same changes in PFGE profile B were not observed, although we found a sequentially unamplified region in which Sp13 was located in the genomes of isolates with PFGE profile B (Fig. (Fig.3).3). We detected part of the Sp13 sequence by Southern blot analysis; however, this part of the sequence was not detected in isolates with PFGE profiles C, G, and H (data not shown). One possible explanation for this phenomenon is that deletion of part of the Sp13 sequence included deletion of primer annealing sites. However, the details of mutation in this region for the isolates with PFGE profile B are not clear.Deletion of the two nonprophage regions also affected the PFGE profiles. The loss of a 205-kb fragment and the gain of a 188-kb fragment due to deletion of a 17-kb region were identified in PFGE profile E (Fig. (Fig.4D4D and and5).5). The loss of a 343-kb fragment and the gain of a 334-kb fragment due to deletion of a 9.5-kb region were identified in PFGE profile B (Fig. (Fig.4E4E and and55).Two single unamplified segments were both observed in the strain with PFGE profile F (106.3/106.4 and 204.2/204.3). We could not amplify these regions using additional primer pairs (data not shown). Insertion of DNA or large-scale inversion might have occurred in these regions. The other unamplified segments all corresponded to deletion of chromosomal regions. Recombination successfully occurred and cured three prophages and two other chromosomal regions. These data suggest that the changes in PFGE profiles after passage through the intestinal tract of cattle are generated in part by deletion of chromosomal regions. Obviously, deletion of five chromosomal regions does not explain the other changes in the PFGE profiles, including profiles A, D, F, J, and L. The genetic events behind such changes are not clear.Prior to drawing a conclusion, we need to consider the use of nalidixic acid, a potent inducer of bacteriophage induction (24), for selection of the isolates. In addition, most of the EHEC O157:H7 isolates obtained on day 8 postinoculation and later were isolated from enrichment cultures (Fig. (Fig.1).1). The possibility that the culturing process itself affected the deletion events affecting the PFGE profiles cannot be ruled out. Taken together, the results suggest that deletions can cause a single strain to mutate into several variants while it is passing through the gastrointestinal tract of a host, provided that the culture technique used does not contribute to this process. Hence, this study may explain why EHEC O157:H7 isolates with various PFGE profiles can be isolated from a single animal. What causes the deletion mutations and why the PFGE profiles show such patterns after passage through cattle are subjects for future studies.  相似文献   

13.
14.
Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (14-16, 31, 34, 45). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (18-20, 43) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ∼80% of HIV-infected heterosexuals and in ∼60% of HIV-infected men who have sex with men (MSM) (1, 13, 18, 24). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (6, 11-13, 17, 25, 28, 30, 35, 38, 42, 47-49) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (15, 36).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (2, 3, 29, 42) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (26, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from single or multiple transmitted/founder viruses could be identified following intravenous inoculation and that the rectal mucosal barrier to infection was 2,000- to 20,000-fold greater than with intravenous inoculation (19). However, we also recognized potentially important differences between virus transmission in Indian rhesus macaques and virus transmission in humans that could complicate an IDU acquisition study. For example, in the SIV macaque model, the virus inocula can be well characterized genetically and the route and timing of virus exposure in relation to plasma sampling precisely defined, whereas in IDUs, the virus inoculum is generally undefined and the timing of virus infection only approximated based on clinical history and seroconversion testing (8). In addition, IDUs may have additional routes of potential virus acquisition due to concomitant sexual activity. Finally, there is a paucity of IDU cohorts for whom incident infection is monitored sufficiently frequently and clinical samples are collected often enough to allow for the identification and enumeration of transmitted/founder viruses. To address these special challenges, we proposed a pilot study of 10 IDU subjects designed to determine with 95% confidence if the proportion of multivariant transmissions in IDUs was more than 2-fold greater than the 20% frequency established for heterosexual transmission (1, 13, 18, 24). A secondary objective of the study was to determine whether the range in numbers of transmitted/founder viruses in IDUs exceeded the 1-to-6 range observed in heterosexuals (1, 13, 18, 24). To ensure comparability among the studies, we employed SGA-direct amplicon sequencing approaches, statistical methods, and power calculations identical to those that we had used previously to enumerate transmitted/founder viruses in heterosexual and MSM cohorts (1, 13, 18, 20, 24).We first surveyed investigators representing acute-infection cohorts in the United States, Canada, Russia, and China; only one cohort—the Montreal Primary HIV Infection Cohort (41)—had IDU clinical samples and clinical data available for study. The Montreal cohort of subjects with acute and early-stage HIV-1 infection was established in 1996 and recruits subjects from both academic and private medical centers throughout the city. Injection drug use is an important contributing factor to Montreal''s HIV burden, with IDUs comprising approximately 20% of the city''s AIDS cases and 35% of the cohort (21, 40, 41). A large proportion of Montreal''s IDUs use injection cocaine, with 50 to 69% of subjects reporting cocaine as their injection drug of choice (4, 5, 9, 22, 23).Subjects with documented serological evidence of recent HIV-1 infection and a concurrent history of injection drug use were selected for study. These individuals had few or no reported risk factors for sexual HIV-1 acquisition. Clinical history and laboratory tests of HIV-1 viremia and antibody seroconversion were used to determine the Fiebig clinical stage (8) and to estimate the date of infection (Table (Table1).1). One subject was determined to be in Fiebig stage III, one subject was in Fiebig stage IV, five subjects were in Fiebig stage V, and three subjects were in Fiebig stage VI. We performed SGA-direct amplicon sequencing on stored plasma samples and obtained a total of 391 3′ half-genomes (median, 25 per subject; range, 19 to 167). Nine of these sequences contained large deletions or were G-to-A hypermutated and were excluded from subsequent analysis. Sequences were aligned, visually inspected using the Highlighter tool (www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), and analyzed by neighbor-joining (NJ) phylogenetic-tree construction. A composite NJ tree of full-length gp160 env sequences from all 10 subjects (Fig. (Fig.1A)1A) revealed distinct patient-specific monophyletic lineages, each with high bootstrap support and separated from the others by a mean genetic distance of 10.79% (median, 11.29%; range, 3.00 to 13.42%). Maximum within-patient env gene diversity ranged from 0.23% to 3.34% (Table (Table1).1). Four subjects displayed distinctly lower within-patient maximum env diversities (0.23 to 0.49%) than the other six subjects (1.48% to 3.34%). The lower maximum env diversities in the former group are consistent with infection either by a single virus or by multiple closely related viruses, while the higher diversities can be explained only by transmission of more than one virus based on empirical observations (1, 13, 18, 24) and mathematical modeling (18, 20).Open in a separate windowFIG. 1.NJ trees and Highlighter plots of HIV-1 gp160 env sequences. (A) Composite tree of 382 gp160 env sequences from all study subjects. The numerals at the nodes indicate bootstrap values for which statistical support exceeded 70%. (B) Subject ACT54869022 sequences suggest productive infection by a single virus (V1). (C) Subject HDNDRPI032 sequences suggest productive infection by as many as three viruses. (D) Subject HDNDRPI001 sequences suggest productive infection by at least five viruses with extensive interlineage recombination. Sequences are color coded to indicate viral progeny from distinct transmitted/founder viruses. Recombinant virus sequences are depicted in black. Methods for SGA, sequencing, model analysis, Highlighter plotting, and identification of transmitted/founder virus lineages are described elsewhere (18, 20, 24, 44). The horizontal scale bars represent genetic distance. nt, nucleotide.

TABLE 1.

Subject demographics and HIV-1 envelope analysis results
Subject identifierAge (yr)SexaFiebig stageEstimated no. of days postinfectionbCD4 countPlasma viral load (log)No. of SGA ampliconsDiversity of env genes (%)c
No. of transmitted/ founder viruses
MeanInterquartile rangeMaximumdModel predictionePhylogenetic estimatef
HDNDRPI03447MIII292407.881631.070.553.34>116
HDNDRPI02918FIV484404.34290.160.150.4911
HTM38524MV624065.37220.120.080.2711
CQLDR0342MV66NDg5.01210.080.080.2311
HDNDRPI00136MV286905.94250.900.631.91>15
HTM31939MV685204.43250.770.461.54>13
HDNDRPI03237MV731,0403.53191.482.993.34>13
ACTDM58020839MVI933874.53301.170.972.64>13
ACT5486902228MVI687233.43270.070.040.2411
PSL02446MVI823404.46210.820.631.57>13
Open in a separate windowaM, male; F, female.bNumbers of days postinfection were estimated on the basis of serological markers, clinical symptoms, or a history of a high-risk behavior leading to virus exposure.cDiversity measurements determined by PAUP* analysis.dThe model prediction of the maximum achievable env diversity 100 days after transmission is 0.60% (95% CI, 0.54 to 0.68%). Diversity values exceeding this range imply transmission and productive infection by more than one virus. Diversity values less than 0.54% can be explained by transmission of one virus or of multiple closely related viruses (18).eModel described in Keele et al. (18).fMinimum estimate of transmitted/founder viruses.gND, not determined.An example of productive clinical infection by a single virus is shown in phylogenetic tree and Highlighter plots from subject ACT54869022 (Fig. (Fig.1B).1B). A similar phylogenetic pattern of single-variant transmission was found in 4 of 10 IDU subjects (Table (Table1).1). Examples of multivariant transmission are shown for subject HDNDRPI032, for whom there was evidence of infection by 3 transmitted/founder viruses (Fig. (Fig.1C)1C) and for subject HDNDRPI001, for whom there was evidence of infection by at least 5 transmitted/founder viruses (Fig. (Fig.1D).1D). One IDU subject, HDNDRPI034, had evidence of multivariant transmission to an extent not previously seen in any of 225 subjects who acquired their infection by mucosal routes (1, 13, 18, 24) or in any of 13 IDUs, as recently reported by Masharsky and colleagues (29). We greatly extended the depth of our analysis in this subject to include 163 3′ half-genome sequences in order to increase the sensitivity of detection of low-frequency viral variants. Power calculations indicated that a sample size of 163 sequences gave us a >95% probability of sampling minor variants comprising as little as 2% of the virus population. By this approach, we found evidence of productive infection by at least 16 genetically distinct viruses (Fig. (Fig.2).2). Fourteen of these could be identified unambiguously based on the presence of discrete low-diversity viral lineages, each consisting of between 2 and 48 sequences. Two additional unique viral sequences with long branch lengths (3F8 and G10) exhibited diversity that was sufficiently great to indicate a distinct transmission event as opposed to divergence from other transmitted/founder lineages (see the legend to Fig. Fig.2).2). It is possible that still other unique sequences from this subject also represented transmitted/founder viruses, but we could not demonstrate this formally. We also could not determine if all 16 (or more) transmission events resulted from a single intravenous inoculation or from a series of inoculations separated by hours or days; however, it is likely that all transmitted viruses in this subject resulted from exposure to plasma from a single infected individual, since the maximum env diversity was only 3.34% (Fig. (Fig.1A).1A). It is also likely that transmission occurred within a brief window of time, since the period from transmission to the end of Fiebig stage III is typically only about 25 days (95% CI, 22 to 37 days) (18, 20) and the diversity observed in all transmitted/founder viral lineages in subject HDNDRPI034 was exceedingly low, consistent with model predictions for subjects with very recent infections (18, 20).Open in a separate windowFIG. 2.NJ tree and Highlighter plot of HIV-1 3′ half-genome sequences from subject HDNDRPI034. Sequences emanating from 16 transmitted/founder viruses are color coded. Fourteen transmitted/founder viral lineages comprised of 2 or more identical or nearly identical sequences could be readily distinguished from recombinant sequences (depicted in black), which invariably appeared as unique sequences containing interspersed segments shared with other transmitted/founder virus lineages. The two sequences with the longest branch lengths (3F8 and G10) were interpreted to represent rare progeny of discrete transmitted/founder viruses because their unique polymorphisms far exceeded the maximum diversity estimated to occur in the first 30 days of infection (0.22%; CI, 0.15 to 0.31%) (18) and far exceeded the diversity observed within the other transmitted/founder virus lineages. The horizontal scale bar represents genetic distance.Lastly, we compared the multiplicity of HIV-1 transmission in the Montreal IDU subjects with that of non-IDU subjects for whom identical SGA methods had been employed. In this combined-cohort analysis, we found the frequency of multiple-variant transmission in heterosexuals to be 19% (34 of 175) and in MSM 38% (19 of 50) (Table (Table2)2) (24). The current study was powered to detect a >2-fold difference in multivariant transmission between IDUs and heterosexual subjects; in fact, we observed a 3-fold-higher frequency of multiple-variant transmission in Montreal IDUs (6 of 10 subjects [60%]) than in heterosexuals (odds ratio, 6.14; 95% CI, 1.37 to 31.27; Fisher exact test, P = 0.008) and a 1.5-fold-higher frequency in Montreal IDUs than in MSM (odds ratio, 2.41; 95% CI, 0.50 to 13.20; P = 0.294, not significant). In addition, we found that the range of numbers of transmitted/founder viruses was greater in IDUs (range, 1 to 16 viruses; median, 3) than in either heterosexuals (range, 1 to 6 viruses; median, 1) or MSM (range, 1 to 10 viruses; median, 1). The finding of larger numbers of transmitted/founder viruses in IDUs was not simply the result of more intensive sampling, since the numbers of sequences analyzed in all studies were comparable. Moreover, it is notable that in studies reported elsewhere, we sampled as many as 239 sequences by SGA or as many as 500,000 sequences by 454 pyrosequencing from four acutely infected MSM subjects and in each case found evidence of productive clinical infection by only a single virus (24; W. Fischer, B. Keele, G. Shaw, and B. Korber, unpublished). These results thus suggest that IDUs may be infected by more viruses and by a greater range of viruses than is the case following mucosal transmission. On this count, our findings differ from those reported by Masharsky and coworkers for an IDU cohort from St. Petersburg, Russia (29). Their study found a low frequency of multiple virus transmissions (31%), not significantly different from that of acutely infected heterosexuals, and a low number of transmitted/founder viruses (range, 1 to 3 viruses; median, 1). Because the SGA methods employed in both studies were identical, the numbers of sequences analyzed per subject were comparable (median of 25 sequences in Montreal versus 33 in St. Petersburg), and because the discriminating power of the SGA-direct sequencing method was sufficient to distinguish transmitted/founder viruses differing by as few as 3 nucleotides, or <0.1% of nucleotides (Fig. (Fig.2,2, compare lineages V4 and V5), it is unlikely that differences in the genetic diversity of HIV-1 in the two IDU populations explain the differences in findings between the two studies. Instead, we suspect that the explanation lies in the small cohort sizes (10 versus 13 subjects) and the particular risk behaviors of the IDUs in each cohort. The Russian cohort is heavily weighted toward heroine use, whereas the Montreal cohort is weighted toward injection cocaine use, the latter being associated with more frequent drug administration and the attendant infection risks of needle sharing (4).

TABLE 2.

Multiplicity of HIV-1 infection in IDU, heterosexual, and MSM subjects
CohortReferenceVirus subtypeTotal no. of subjectsSingle-variant transmission
Multiple-variant transmission
P valueOdds ratio95% CIMedianRange
No. of subjects% of totalNo. of subjects% of total
HeterosexualsKeele et al. (18)B796582.301417.7011-4
Abrahams et al. (1)C695478.301521.7011-5
Haaland et al. (13)A or C272281.50518.5011-6
Total17514180.603419.400.008a6.141.37-31.2711-6
MSMKeele et al. (18)B221359.10940.9011-6
Li et al. (24)B281864.301035.7011-10
Total503162.001938.000.294b2.410.50-13.2011-10
IDUsBarB10440.00660.0031-16
Open in a separate windowaFisher''s exact test of multiple-variant transmission in heterosexuals versus in IDUs.bFisher''s exact test of multiple-variant transmission in MSM versus in IDUs.The results from the present study indicate that transmission of HIV-1 to IDUs can be associated with a high frequency of multiple-variant transmission and a broad range in the numbers of transmitted viruses. This wide variation in the multiplicity of HIV-1 infection in IDUs is likely due to the absence of a mucosal barrier to virus transmission (12, 19) and differences in the virus inocula (27, 29, 32, 39). The findings substantiate concerns raised in recent HIV-1 vaccine efficacy trials that different vaccine candidates may be more efficacious in preventing infection by some exposure routes than by others (7, 10, 33, 37). They further suggest that biological comparisons of molecularly cloned transmitted/founder viruses responsible for vaginal, rectal, penile, and intravenous infection could facilitate a mechanistic understanding of HIV-1 transmission and vaccine prevention (24, 44).  相似文献   

15.
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Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.Cellulases catalyze the breakdown of cellulose into simple sugars that can be fermented to ethanol. The large amount of natural cellulose available is an exciting potential source of fuels and chemicals. However, the detailed molecular mechanisms of crystalline cellulose degradation by glycoside hydrolases are still not well understood and their low efficiency is a major barrier to cellulosic ethanol production.Thermobifida fusca is a filamentous soil bacterium that grows at 50°C in defined medium and can utilize cellulose as its sole carbon source. It is a major degrader of plant cell walls in heated organic materials such as compost piles and rotting hay and produces a set of enzymes that includes six different cellulases, three xylanases, a xyloglucanase, and two CBM33 binding proteins (12). Among them are three endocellulases, Cel9B, Cel6A, and Cel5A (7, 8), two exocellulases, Cel48A and Cel6B (6, 19), and a processive endocellulase, Cel9A (5, 7).T. fusca Cel9A-90 (Uniprot P26221 and YP_290232) is a multidomain enzyme consisting of a family 9 catalytic domain (CD) rigidly attached by a short linker to a family 3c cellulose binding module (CBM3c), followed by a fibronectin III-like domain and a family 2 CBM (CBM2). Cel9A-68 consists of the family 9 CD and CBM3c. The crystal structure of this species (Fig. (Fig.1)1) was determined by X-ray crystallography at 1.9 Å resolution (Protein Data Bank [PDB] code 4tf4) (15). Previous work has shown that E424 is the catalytic acid and D58 is the catalytic base (11, 20). H125 and Y206 were shown to play an important role in activity by forming a hydrogen bonding network with D58, an important supporting residue, D55, and Glc(−1)O1. Several enzymes with amino acid changes in subsites Glc(−1) to Glc(−4) had less than 20% activity on bacterial cellulose (BC) and markedly reduced processivity. It was proposed that these modifications disturb the coordination between product release and the subsequent binding of a cellulose chain into subsites Glc(−1) to Glc(−4) (11). Another variant enzyme with a deletion of a group of amino acids forming a block at the end of the catalytic cleft, Cel9A-68 Δ(T245-L251)R252K (DEL), showed slightly improved filter paper (FP) activity and binding to BC (20).Open in a separate windowFIG. 1.Crystal structure of Cel9A-68 (PDB code 4tf4) showing the locations of the variant residues, catalytic acid E424, catalytic base D58, hydrogen bonding network residues D55, H125, and Y206, and six glucose residues, Glc(−4) to Glc(+2). Part of the linker is visible in dark blue.The CBM3c domain is critical for hydrolysis and processivity. Cel9A-51, an enzyme with the family 9 CD and the linker but without CBM3c, had low activity on carboxymethyl cellulose (CMC), BC, and swollen cellulose (SWC) and showed no processivity (4). The role of CBM3c was investigated by mutagenesis, and one modified enzyme, R557A/E559A, had impaired activity on all of these substrates but normal binding and processivity (11). Variants with changes at five other CBM3c residues were found to slightly lower the activity of the modified enzymes, while Cel9A-68 enzymes containing either F476A, D513A, or I514H were found to have slightly increased binding and processivity (11) (see Table Table1).1). In the present work, CBM3c has been investigated more extensively to identify residues involved in substrate binding and processivity, understand the role of CBM3c more clearly, and study the coordination between the CD and CBM3c. An additional goal was to combine amino acid variants showing increased crystalline cellulose activity to see if this further increased activity. Finally, we have investigated whether the changes that improved the activity of Cel9A-68 also enhanced the activity of intact Cel9A-90.

TABLE 1.

Activities of Cel9A-68 CBM3c variant enzymes and CD variant enzymes used to create the double variants
EnzymeActivity (% of wild type) on:
% Processivity% BC bindingReference
CMCSWCBCFPa
Wild type10010010010010015This work
R378K9891103931392011
DELb981011011289620
F476A97105791001452111
D513A1001151211071192011
I514H104911121041102311
Y520A1087833a79871411
R557A1039860a9390This work
E559A869030a7094This work
R557A+E559A907515a751061511
Q561A1035651a7874This work
R563A977052a931292011
Open in a separate windowaThe target percent digestion could not be reached; activity was calculated using 1.5 μM enzyme.bDEL refers to deletion of T245 to L251 and R252K.  相似文献   

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