Characterization of TonB Interactions with the FepA Cork Domain and FecA N-terminal Signaling Domain

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11–154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103–239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1–96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676–10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.     

Infectious molecular clones from a simian immunodeficiency virus-infected rapid-progressor (RP) macaque: evidence of differential selection of RP-specific envelope mutations in vitro and in vivo

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of env in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.     

Aberrant Double-Strand Break Repair Resulting in Half Crossovers in Mutants Defective for Rad51 or the DNA Polymerase δ Complex

Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Δ mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Δ and pol3-ct mutants, defective for components of the DNA polymerase δ (Pol δ) complex. The half crossovers formed in Pol δ complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Δ mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol δ complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.DNA double-strand breaks (DSBs) are potentially lethal lesions that can occur spontaneously during normal cell metabolism, by treatment of cells with DNA-damaging agents, or during programmed recombination processes (54). There are two major pathways to repair DSBs: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ involves the religation of the two ends of the broken chromosome and can occur with high fidelity or be accompanied by a gain or loss of nucleotides at the junction (9). Repair of two-ended DSBs by HR generally occurs by gene conversion resulting from a transfer of information from the intact donor duplex to the broken chromosome (Fig. ​(Fig.1).1). HR occurs preferentially during S and G₂ when a sister chromatid is available to template repair (2, 19, 22). Sister-chromatid recombination events are genetically silent, whereas gene conversion between nonsister chromatids associated with an exchange of flanking markers can result in extensive loss of heterozygosity (LOH) or chromosome rearrangements (3, 21). One-ended DSBs that arise by replication fork collapse or by erosion of uncapped telomeres are thought to repair by strand invasion into homologous duplex DNA followed by replication to the end of the chromosome, a process referred to as break-induced replication (BIR) (35). BIR appears to be suppressed at two-ended breaks, presumably because it can lead to extensive LOH if it occurs between homologues or to chromosome translocations when strand invasion initiates within dispersed repeated sequences (5, 28, 31, 50, 52, 55).Open in a separate windowFIG. 1.Models for gene conversion and BIR. After formation of a DSB, the ends are resected to generate 3′ single-strand DNA tails. One end undergoes Rad51-dependent strand invasion to prime DNA synthesis from the invading 3′ end templated by the donor duplex. For gene conversion by the synthesis-dependent strand annealing model, the extended invading end is displaced and can anneal to the other side of the break; completion of repair requires DNA synthesis primed from the noninvading 3′ end. For a one-ended break, or if the other side of the break lacks homology to the donor duplex, DNA synthesis proceeds to the end of the chromosome. Centromeres are shown as solid ovals and a heterozygous marker centromere distal to the site of repair as A/a.The strand invasion step of BIR is assumed to be the same as that for gene conversion based on the requirement for the same HR proteins: Rad51, Rad52, Rad54, Rad55, and Rad57 (10). However, subsequent steps in BIR are less well defined. Recent studies of the fate of the invading end during BIR in diploid strains with polymorphic chromosome III homologues using a plasmid-based assay have shown that following strand invasion, the invading end is capable of dissociating from the initial homologous template. Following dissociation, the displaced end subsequently reinvades into the same or a different chromosome III homologue by a process termed template switching (52). One of the interesting features of the template switching events is that they occur over a region of about 10 kb downstream of the site of strand invasion and do not extend over the entire left arm of chromosome III. There are a number of possible mechanisms that could account for this apparent change in the processivity of BIR. First, it is possible that the strand invasion intermediate is cleaved by a structure-specific nuclease and once the invading strand is covalently joined to one of the template strands, the strand invasion process is irreversible. Recent studies of Schizosaccharomyces pombe have shown an essential role for Mus81, a structure-specific nuclease, in resolution of sister chromatid recombination intermediates during repair of collapsed replication forks (48). Another possibility is that there could be a switch between a translesion DNA polymerase and a highly processive DNA polymerase during BIR. The translesion polymerases in budding yeast, polymerase ζ (Pol ζ) and Pol η, are encoded by REV3-REV7 and RAD30, respectively (34, 40, 43). Deletion of REV3 has been shown to increase the fidelity of DNA synthesis associated with HR but has no effect on the overall frequency of DSB-induced HR (16). Deletion of POLη in chicken DT40 cells reduces the frequency of DSB-induced gene conversion, and human POL η has been shown to extend the invading 3′ end of D-loop intermediates in vitro (23, 36). However, this same preference for Pol η is not found for Saccharomyces cerevisiae. Instead, DNA synthesis during meiotic and mitotic recombination appears to be carried out by Pol δ, one of the three nuclear replicative polymerases, which normally functions with Pol α in Okazaki fragment synthesis (13, 32, 33, 44). Pol ɛ is thought to be the primary leading-strand polymerase (47), but in the absence of the Pol ɛ catalytic domain, Pol δ is presumed to carry out leading-strand synthesis (24). Recent studies by Lydeard et al. (30) have shown a requirement for the lagging-strand polymerases, Pol δ and Pol α, to form the initial primer extension product during BIR, and Pol ɛ is required to complete replication to the end of the chromosome. In contrast, repair of DSBs by gene conversion does not require Pol α, and there appears to be functional redundancy between Pol δ and Pol ɛ (56).To address the roles of Mus81, Pol δ, and Pol η in BIR and in particular template switching, we used the transformation-based BIR assay with diploids with polymorphic chromosome III homologues. Because the transformation assay can only be used with strains with viable mutations of replication factors, we used a null allele of POL32, encoding a nonessential subunit of the Pol δ complex (14), and a point mutation in the gene encoding the essential catalytic subunit, POL3. The pol3-ct allele results in a truncation removing the last four amino acids of the Pol3 protein; the C-terminal region of Pol3 is implicated in interaction with the other essential subunit of the Pol δ complex, Pol31 (15, 49). The interesting feature of the pol3-ct allele is that it decreases the length of gene conversion tracts during mitotic and meiotic recombination, presumably by affecting the processivity of Pol δ, but confers no apparent defect in normal DNA synthesis (32, 33). Because BIR requires more-extensive tracts of DNA synthesis than gene conversion, we expected the pol3-ct mutant to exhibit a BIR defect. We found that in the absence of a fully functional Pol δ complex, chromosome fragment (CF) formation proceeds by a half-crossover mechanism associated with loss of the template chromosome, an event with potentially catastrophic consequences (6, 57). This was also found to occur in rad51 mutants, suggesting nonreciprocal translocations arise by failure to undergo strand invasion or because replication following strand invasion is inefficient. In contrast to rad51 mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion and fully resistant to ionizing radiation, suggesting there is a unique requirement for Pol δ to complete BIR. Consistent with studies of gene conversion in S. cerevisiae (33), we found no role for Pol η in BIR or the process of template switching.     

Contrasting response of seedlings of two tropical species Clusia minor and Clusia multiflora to mycorrhizal inoculation in two soils with different pH

Differences in mycotrophic growth and response to phosphorus (P) fertilization were studied in seedlings of two woody native species: Clusia minor L. and Clusia multiflora H.B.K. from a cloud montane forest of tropical America. Greenhouse investigation was undertaken to determine the relationships between mycorrhizal dependency of host species associated with P utilization and growth in two different soils contrasting in pH (acidic and neutral) and nutrient content. Four treatments were performed: sterilized soil; sterilized soil plus 375 mg/kg of triple superphosphate (TSP); sterilized soil inoculated with Scutellospora fulgida (20 g/pot); and sterilized soil plus S. fulgida and TSP, with 10 replications per treatment for the two species. Results showed that both Clusia species presented high growth response to increasing P availability, which indicates that the root morphology (magnolioid roots) of these species is not a limiting factor for the incorporation of P from soils. Plants inoculated with arbuscular mycorrhizal fungi (AMF) in acidic soil had significantly increased shoot and root biomass, leaf area and height, in comparison to the biomass of P-fertilized plants and nonmycorrhizal plants. In neutral soil, seedlings of C. minor and C. multiflora were negatively affected by inoculation with AMF. In contrast, a significant decrease in growth was observed when inoculated plants were compared with noninoculated plants on neutral soil. Results indicate that an increase in the availability of a limiting nutrient (P) can turn a balanced mutualistic relationship into a less balanced nonmutualistic one.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

DAYLENGTH AND LIGHT RESPONSES IN GROWTH AND FERTILITY OF GLOSSOPHORA KUNTHII (PHAEOPHYTA,DICTYOTALES) FROM PACIFIC SOUTH AMERICA1

Excised ligulae of Glossophora kunthii (C. Ag.) J. Ag. were cultured in photoperiods of 4–24 h and photon fluence rates of 10–75 μmol.m^?2.s^?1. Daylength interacted with irradiance on the growth of the ligulae. Maximal growth of primary ligulae occurred in long-day regimens with high irradiances suggesting an effect of irradiance on photosynthesis and growth. In contrast, growth of secondary ligulae was greatest in short-day regimes. Differences were significant at the highest irradiance tested. Differentiation of tetrasporangia on the ligulae is a short-day photoperiodic response. Daylengths of 8.5 h or less induced a sharp increase in numbers of fertile ligulae and tetrasporangia attaining maturity. Interruptions of the dark period decreased the development of tetrasporangia; the number of interruptions had a cumulative inhibitory effect. Differentiation of reproductive structures was influenced by interactions of photoperiod and irradiance. Maximum numbers of tetrasporangia were formed at short-day regimes and low irradiances; differentiation was completely inhibited at long-day conditions and high irradiance.