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Anneke Funk Katherine Truong Tomoko Nagasaki Shessy Torres Nadia Floden Ezequiel Balmori Melian Judy Edmonds Hongping Dong Pei-Yong Shi Alexander A. Khromykh 《Journal of virology》2010,84(21):11407-11417
Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3′ untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3′ UTR. Mutations or deletions of various secondary structures in the 3′ UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNVKUN) 3′ UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.Arthropod-borne flaviviruses such as West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) cause major outbreaks of potentially fatal disease and affect over 50 million people every year. The highly pathogenic North American strain of WNV (WNVNY99) has already claimed more than 1,000 lives with over 27,000 cases reported since its emergence in New York in 1999 and has raised global public health concerns (9). In contrast, the closely related Australian strain of WNV, WNVKUN, is highly attenuated and does not cause overt disease in humans and animals (11). WNVKUN has been used extensively as a model virus to study flavivirus replication and flavivirus-host interactions (13, 14, 16-19, 26, 38, 39).The ∼11-kb positive-stranded, capped WNV genomic RNA (gRNA) lacks a poly(A) tail and consists of 5′ and 3′ untranslated regions (UTRs) flanking one open reading frame, which encodes the viral proteins required for the viral life cycle (6, 15, 38, 39). Flavivirus UTRs are involved in translation and initiation of RNA replication and likely determine genome packaging (13, 14, 16, 21, 30, 39-41). Both the 5′ UTR (∼100 nucleotides [nt] in size) and the 3′ UTR (from ∼400 to 700 nucleotides) can form secondary and tertiary structures which are highly conserved among mosquito-borne flaviviruses (1, 8, 10, 14, 29, 32, 34). More specifically, the WNVKUN 3′ UTR consists of several conserved regions and secondary structures (Fig. (Fig.1A)1A) which were previously predicted or shown to exist in various flaviviruses by computational and chemical analyses, respectively (4, 10, 25, 26, 29-32). The 5′ end of the 3′ UTR starts with an AU-rich region which can form stem-loop structure I (SL-I) followed by SL-II, which we previously showed to be vitally important for subgenomic flavivirus RNA (sfRNA) production (26; see also below). SL-II is followed by a short, repeated conserved hairpin (RCS3) and SL-III (26). Further downstream of SL-III are the SL-IV and CS3 structures, which are remarkably similar to the preceding SL-II-RCS3 structure (26, 29). Further downstream of the SL-IV-CS3 structure are dumbbells 1 and 2 (DB1 and DB2, respectively) followed by a short SL and the 3′ SL (25, 26).Open in a separate windowFIG. 1.(A) Model of the WNVKUN 3′ UTR RNA structure. Highlighted in bold are the secondary structures investigated here. Dashed lines indicate putative PKs. The two sites of the putative PK interactions are shown in open boxes. sfRNA1, -2, -3, and -4 start sites are indicated by arrows. (R)CS, (repeated) conserved sequence; DB, dumbbell structure; PK, pseudoknot; SL, stem-loop. (B) Structural model of PK1 in SL-II with disruptive mutations. Nucleotide numbering is from the end of the 3′ UTR. The sfRNA1 start is indicated by an arrow. Nucleotides forming PK1 are on a gray background, and mutated nucleotides are white on a black background. (C) Sequences mutated in the different constructs. Nucleotides in the wt PK sequences used for mutations are bold and underlined. Introduced mutations are shown under the corresponding nucleotides in the wt sequence.The described structures have been investigated in some detail for their requirement in RNA replication and translation. Generally, a progressive negative effect on viral growth was shown with progressive deletions into the 3′-proximal region of the JEV 3′ UTR (41). However, only a relatively short region of the JEV 3′ UTR, consisting of the 3′-terminal 193 nt, was shown to be absolutely essential for gRNA replication (41). The minimal region for DENV replication was reported to be even shorter (23). Extensive analysis has shown that the most 3′-terminal, essential regions of the 3′ UTR include the cyclization sequence and 3′ SL, which are required for efficient RNA replication (2, 14, 16, 23, 35). As we showed, deletion of SL-II or SL-I did not overtly affect WNVKUN replication (26). However, deletion of CS2, RCS2, CS3, or RCS3 in WNV replicon RNA significantly reduced RNA replication but not translation (20), indicating that these elements facilitate but are not essential for RNA replication. In addition, it was shown that deletion of DB1 or DB2 resulted in a viable mutant virus that was reduced in growth efficiency, while deletion of both DB structures resulted in a nonviable mutant (23).In addition to the above-mentioned secondary stem-loop structures, computational and chemical analysis of the flavivirus 3′ UTR suggested the presence of 5 pseudoknot (PK) interactions (Fig. (Fig.1A)1A) (25, 26, 32). A PK is a structure formed upon base pairing of a single-stranded region of RNA in the loop of a hairpin to a stretch of complementary nucleotides elsewhere in the RNA chain (Fig. (Fig.1B).1B). These structures are referred to as hairpin type (H-type) PKs (3), and they usually stabilize secondary RNA structures. Typically, the final tertiary structure does not significantly alter the preformed secondary structure (5). In general, PK interactions have been shown to be important in biological processes such as initiation and/or elongation of translation, initiation of gRNA replication, and ribosomal frameshifting for a number of different viruses, including flaviviruses (reviewed in references 3 and 22). The first PK in the WNV 3′ UTR was predicted to form in SL-II, followed by a similar PK in SL-IV (26) (PK1 and PK2 in Fig. Fig.1A).1A). For the DENV, yellow fever virus (YFV), and JEV subgroup of flaviviruses, two PKs further downstream were predicted to form between DB1 and DB2 and corresponding single-stranded RNA regions located further downstream (25) (PK3 and PK4 in Fig. Fig.1A).1A). The formation of these structures is supported by covariations in the WNV RNAs. In addition, a PK was proposed to form between a short SL and the 3′ SL at the 3′ terminus of the viral genome (32) (PK5 in Fig. Fig.1A1A).Importantly, in addition to its role in viral replication and translation, we have shown that the WNVKUN 3′ UTR is important for the production of a small noncoding RNA fragment designated sfRNA (26). This short RNA fragment of ∼0.5 kb is derived from the 3′ UTR of the gRNA and exclusively produced by the members of the Flavivirus genus of the Flaviviridae family, where it is required for efficient viral replication, cytopathicity, and pathogenicity (26). Our studies suggested that sfRNA is a product of incomplete degradation of the gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, resulting from XRN1 stalling on the rigid secondary/tertiary structures located at the beginning of the 3′ UTR (26). XRN1 is an exoribonuclease which usually degrades mRNA from the 5′ to the 3′ end as part of cellular mRNA decay and turnover (33), and it was shown previously that XRN1 can be stalled by SL structures (28). Mutations or deletions of WNV 3′ UTR secondary structures resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3) (26). In particular, SL-II (Fig. (Fig.1A)1A) was shown to be important for sfRNA1 production; deletion of this structure either alone or in conjunction with other structures located downstream of SL-II abolished sfRNA1 production, leading to the production of the smaller RNA fragments sfRNA2 and sfRNA3.Here, we extended our investigation and studied the importance of several predicted 3′ UTR secondary structures and PK interactions for the production of sfRNA. To further understand the generation mechanism of sfRNA and its requirements, we deleted or mutated a number of RNA structures in the WNVKUN 3′ UTR and investigated the size and amount of sfRNA generated from these mutant RNAs. The results show that not only SLs but also PK interactions play a vital role in stabilizing the 3′ UTR RNA and preventing complete degradation of viral gRNA to produce nuclease-resistant sfRNA, which is required for efficient virus replication and cytopathicity in cells and virulence in mice. 相似文献
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Deusdedith R. Mbanzibwa Yanping Tian Settumba B. Mukasa Jari P. T. Valkonen 《Journal of virology》2009,83(13):6934-6940
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Is Single-Strand Conformation Polymorphism Analysis of the Full 5′ Untranslated Region an Adequate Approach To Study Hepatitis C Virus Quasispecies Distribution?
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Jorge Vera-Otarola María Inés Barría Ursula León Pilar Carvallo Alejandro Soza Marcelo López-Lastra 《Journal of virology》2009,83(17):9018-9021
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Gareth A. Cromie 《Journal of bacteriology》2009,191(16):5076-5084
RecBCD and AddAB are bacterial enzymes that share similar helicase and nuclease activities and initiate repair of DNA double-strand breaks by homologous recombination. Examination of the phylogenetic distribution of AddAB and RecBCD revealed that one or the other complex is present in most sequenced bacteria. In addition, horizontal gene transfer (HGT) events involving addAB and recBCD appear to be common, with the genes encoding one complex frequently replacing those encoding the other. HGT may also explain the unexpected identification of archaeal addAB genes. More than 85% of addAB and recBCD genes are clustered on the genome, suggesting operon structures. A few organisms, including the Mycobacteria, encode multiple copies of these complexes of either the same or mixed classes. The possibility that the enzymatic activities of the AddAB and RecBCD enzymes promote their horizontal transfer is discussed, and the distribution of AddAB/RecBCD is compared to that of the RecU/RuvC resolvases. Finally, it appears that two sequence motifs, the Walker A box involved in ATP binding and an iron-sulfur-cysteine cluster, are present only in subsets of AddB proteins, suggesting the existence of mechanistically distinct classes of AddB.Homologous recombination is central to the repair of DNA damaged by single-strand (SS) and double-strand (DS) breaks and gaps. Such discontinuities can occur after exposure to exogenous agents such as ionizing radiation but also when DNA replication forks break and as intermediates in DNA repair processes. Homologous recombination can also rearrange genetic information, making it important in processes such as phase variation of bacterial outer membrane proteins (for an example, see reference 2).Homologous recombination is highly conserved among viruses, bacteria, archaea, and eukaryotes. There are three recognizable stages (for reviews, see references 25 and 35). In the first stage, presynapsis, the DNA substrate is processed to give a SS region coated with strand exchange protein(s). In bacteria, this protein is RecA. In the second stage, synapsis, the protein-DNA complex pairs with its complementary homologous DNA target, displacing the other strand at the target site and forming a joint molecule. In the final stage, postsynapsis, DNA replication fills in any gaps in the joint molecule, which is then resolved to give recombinant DNA products.In bacteria, there appear to be two different presynaptic pathways that use either the related AddAB or RecBCD holoenzymes (Fig. (Fig.1)1) or, alternatively, the RecFOR proteins. RecFOR proteins appear to operate on SS gaps to ensure RecA is loaded there, whereas RecBCD and AddAB act on DNA DS breaks (DSBs), processing them to yield SS 3′ DNA ends coated with RecA (3, 43).Open in a separate windowFIG. 1.Structure of the RecBCD and AddAB proteins. The RecB, RecD, and AddA proteins include a helicase domain (solid green) with the canonical six-helicase motifs of helicase superfamily I. The most N-terminal of these motifs is the Walker A box (orange). The RecC and AddB proteins include an inactivated helicase domain (striped green). The RecB, AddA, and AddB proteins additionally possess similar short nuclease domains toward their C termini (red). Some, but not all, AddB proteins possess an N-terminal Walker A and/or a mostly C-terminal iron-sulfur motif made up of four cysteines (blue).The RecBCD complex has most thoroughly been studied in Escherichia coli, a member of the Gamma class of the phylum Proteobacteria. RecB and RecD are superfamily I helicases, and RecB is also a nuclease. After the RecBCD complex loads onto a DS DNA end, the RecB and RecD helicases separate the strands, with the slower RecB helicase traveling on one strand in the 3′-to-5′ direction and the faster RecD helicase traveling on the other strand from 5′ to 3′ (38). During translocation, E. coli RecBCD recognizes a specific DNA sequence, Chi (5′-GCTGGTGG-3′), perhaps via the RecC protein, which appears to be an inactivated helicase (Fig. (Fig.1)1) (32, 34). Chi is a recombination hot spot that switches the complex to a recombinogenic mode, producing a 3′ SS end at the Chi site via the nuclease activity of RecB (for reviews, see references 25 and 35). In addition to producing a 3′ SS “tail” needed for recombination, the RecBCD complex actively loads the RecA strand exchange protein onto this DNA (5). In E. coli, the RecBCD complex is responsible for essentially all recombinational repair of DSBs.Bacillus subtilis and some other bacteria lack RecBCD but possess a different complex with many of the same activities. This complex is composed of the AddA and AddB proteins (22). Like RecB, AddA is a superfamily I helicase and a nuclease (18, 21), organized with the same domain structure as RecB (Fig. (Fig.1).1). Similar to RecC, AddB appears to be an inactivated helicase, but it also possesses a nuclease domain similar to those of RecB and AddA (Fig. (Fig.1)1) (2, 11, 43). Like RecBCD, AddAB is an ATP-dependent helicase that acts as a nuclease in conjunction with its helicase activity, with the AddA nuclease acting on the 3′-end strand and the AddB nuclease on the 5′-end strand (43). Also like RecBCD, AddAB recognizes Chi-like control sequences, for instance, 5′-GCGCGTG-3′ in Lactococcus lactis and 5′-AGCGG-3′ in B. subtilis (7, 12). The AddAB complex is required for recombinational repair of DSBs (1, 2, 44), but it is not known if, like RecBCD, it actively loads RecA.Both RecBCD and AddAB are important for bacterial pathogenicity. addAB mutations reduce the ability of Helicobacter pylori to colonize mouse stomachs, and recBC mutants of Salmonella enterica serovar Typhimurium are severely compromised for infection and killing (2, 8).Using a strict set of criteria, Rocha et al. (30) examined the distribution across bacteria of the RecBCD and AddAB enzymes, along with other recombination and DNA repair proteins. They determined that the AddAB proteins are ubiquitous in some taxa and the RecBCD proteins in others, while yet other taxa have examples of both complexes in different species. In addition, they classified several species as lacking both the AddAB and RecBCD complexes. Recently, addAB genes from Epsilonproteobacteria were identified (2, 27, 40). Previously, all examined members of this proteobacterial class had been classified by Rocha et al. as lacking every component of the RecBCD and AddAB systems. This finding suggests that the stringent criteria used by Rocha et al. (30) might have led them to miss other examples of AddAB and RecBCD from sequenced bacteria. On this basis, and to gain a fuller understanding of the importance of the AddAB and RecBCD enzymes, their phylogenetic distribution was reexamined. 相似文献
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Brian H. Lower Ruchirej Yongsunthon Liang Shi Linda Wildling Hermann J. Gruber Nicholas S. Wigginton Catherine L. Reardon Grigoriy E. Pinchuk Timothy C. Droubay Jean-Fran?ois Boily Steven K. Lower 《Applied and environmental microbiology》2009,75(9):2931-2935
Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe2O3), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacterium that is well known for its ability to use a variety of anaerobic terminal electron acceptors (TEAs), including solid-phase iron oxide minerals, such as goethite and hematite (8, 10). Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2, 8, 10, 15, 16, 20, 21, 23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.In the present study, we used atomic force microscopy (AFM) to probe the surface of live S. oneidensis MR-1 cells, using AFM tips that were functionalized with cytochrome-specific polyclonal antibodies (i.e., anti-OmcA or anti-MtrC). This technique, termed antibody recognition force microscopy (Ig-RFM), detects binding events that occur between antibodies (e.g., anti-OmcA) on an AFM tip and antigens (e.g., OmcA) that are exposed on a cell surface. While this is a relatively new technique, Ig-RFM has been used to map the nanoscale spatial location of single molecules in complex biological structures under physiological conditions (5, 9, 11, 13).Anti-MtrC or anti-OmcA molecules were covalently coupled to silicon nitride (Si3N4) cantilevers (Veeco or Olympus) via a flexible, heterofunctional polyethylene glycol (PEG) linker molecule. The PEG linker consists of an NHS (N-hydroxysuccinimide) group at one end and an aldehyde group at the other end (i.e., NHS-PEG-aldehyde). AFM tips were functionalized with amine groups, using ethanolamine (6, 7). The active NHS ester of the NHS-PEG-aldehyde linker molecule was then used to form a covalent linkage between PEG-aldehyde and the amine groups on the AFM tips (6, 7). Next, anti-MtrC or anti-OmcA molecules were covalently tethered to these tips via the linker molecule''s aldehyde group. This was accomplished by incubating the tips with antibody (0.2 mg/ml) and NaCNBH3 as described previously (7). The cantilevers were purchased from Veeco and had spring constant values between 0.06 and 0.07 N/m, as determined by the thermal method of Hutter and Bechhoefer (12).Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24, 28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Briefly, 2.5 μg of purified OmcA or MtrC (23) was resolved by sodium dodecyl sulfate-PAGE or native PAGE, transferred to a polyvinylidene difluoride membrane, incubated with either anti-OmcA or anti-MtrC, and then visualized using the Amersham ECL Plus Western blotting detection kit. Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed cross-reactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).To determine if MtrC or OmcA was expressed on the external surface of live bacteria when Fe(III) served as the TEA, Ig-RFM was conducted on wild-type versus ΔomcA ΔmtrC double mutant cells. For these experiments, bacteria were cultivated anaerobically with Fe(III), in the form of Fe(III) chelated to nitrilotriacetic acid (NTA), serving as the TEA (19, 23). Growth conditions have been described elsewhere (3, 15) and were based on previous studies (3, 15, 16, 18) that suggest that S. oneidensis MR-1 targets OmcA and MtrC to the cell surface when Fe(III) serves as the TEA.An Asylum Research MFP-3D-BIO AFM or a Digital Instruments Bioscope AFM (16, 17) was used for these experiments. The z-piezoelectric scanners were calibrated as described previously (17). Cells were deposited on a hydrophobic glass coverslip and immersed in imaging buffer (i.e., phosphate-buffered saline [pH 7.4]). The hydrophobic glass coverslips were made as described previously (17) using a self-assembling silane compound called octadecyltrichlorosilane (OTS; Sigma-Aldrich). S. oneidensis MR-1 cells readily adsorbed onto OTS glass coverslips and remained attached to the coverslips during the entire experiment. No lateral cell movement was observed during the experiment, consistent with previous studies that used OTS glass to immobilize bacteria (15, 17, 18, 27).The AFM tip was brought into contact with the surface of a bacterium, and the antibody-functionalized tip was repeatedly brought into and out of contact with the sample, “fishing” for a binding reaction with cytochrome molecules that were exposed on the external cell surface. Binding events were observed upon separating anti-OmcA- or anti-MtrC-functionalized tips from wild-type S. oneidensis MR-1 cells (Fig. (Fig.1).1). For the wild-type cells, we observed both nonspecific and specific interactions (Fig. (Fig.11).Open in a separate windowFIG. 1.Retraction force curves for anti-MtrC-functionalized tips (A) and anti-OmcA-functionalized tips (B) that are being pulled away from the surface of living ΔomcA ΔmtrC double mutant (gray dotted line) or wild-type (solid black line) S. oneidensis MR-1. These bacteria were adsorbed onto OTS glass coverslips. (C) Retraction curves exhibiting nonspecific binding, specific binding, or no binding between the AFM tip and the cell surface.The distinction between “specific” and “nonspecific” adhesion is made by observing the change in slope of the force curve during the retraction process (26). During specific binding (Fig. (Fig.1C),1C), the cantilever is initially relaxed as it is pulled away from the sample. Upon further retraction, the ligand-receptor complex becomes stretched and unravels, resulting in a nonlinear force profile as noted in references 26 and 16. On the other hand, nonspecific adhesion (Fig. (Fig.1C)1C) maintains the same slope during the retraction process because only the cantilever flexes (26).Figure Figure22 summarizes the frequency or probability of observing a binding event for both anti-OmcA and anti-MtrC tips. Each bar in Fig. Fig.22 represents one experiment in which 500 to 1,000 force curves were collected between one AFM tip and two to four live bacterial cells. This figure does not make a distinction between specific and nonspecific binding. It simply shows the frequency of observing an attractive interaction as the antibody-functionalized tip was pulled away from the surface of S. oneidensis MR-1. Binding events occurred with roughly the same frequency when wild-type S. oneidensis MR-1 cells were probed with anti-MtrC-functionalized tips as when they were probed with anti-OmcA-functionalized tips (Fig. (Fig.22).Open in a separate windowFIG. 2.Histograms showing the frequency of observing a binding event for anti-MtrC-functionalized (blue) or anti-OmcA-functionalized (red) AFM tips on live wild-type S. oneidensis MR-1 (solid bars) or ΔomcA ΔmtrC double mutant (diagonally hatched bars) cells. The downward arrows designate injection of free antibody into the imaging buffer. The solid gray bars correspond to results obtained with unbaited AFM tips.A number of control experiments were performed to verify the detection of OmcA and MtrC on the surface of wild-type S. oneidensis MR-1. First, 0.1 μM of free anti-OmcA (or anti-MtrC) was added to the imaging fluid to block binding between the antibody-functionalized AFM tip and surface-exposed cytochromes (11, 16). This decreased the adhesion that was observed between the antibody-functionalized tip and the cell surface (Fig. (Fig.22).Second, we performed force measurements on ΔomcA ΔmtrC double mutant S. oneidensis MR-1 cells. This mutant is deficient in both OmcA and MtrC (19, 23, 24) but produces other proteins native to the outer surface of S. oneidensis MR-1. The resulting force spectra showed a noticeable reduction in binding events for the ΔomcA ΔmtrC double mutant cells (Fig. (Fig.2).2). The binding events that were observed for the double mutant were only nonspecific in nature (Fig. (Fig.1).1). This indicates that the antibodies on the tip do not participate in specific interactions with other proteins on the surface of S. oneidensis MR-1 cells.As a final control experiment, force measurements were conducted on wild-type S. oneidensis MR-1 cells, using Si3N4 tips conjugated with the PEG linker but not functionalized with polyclonal antibody (unbaited tips). Like the results with the double mutant, the unbaited tips were largely unreactive with the surface of the bacteria (Fig. (Fig.2).2). Those binding events that were observed were nonspecific in nature. Taken together, these results demonstrate that the antibody-coated tips have a specific reactivity with OmcA and MtrC molecules. Furthermore, these force measurements show that MtrC and OmcA are present on the external cell surface when Fe(III) serves as the TEA.To map the distribution of cytochromes on living cells, Ig-RFM was conducted on living S. oneidensis MR-1 cells that were growing on a hematite (α-Fe2O3) thin film. The conditions for these experiments were as follows. A hematite film was grown on a 10-mm by 10-mm by 1-mm oxide substrate via oxygen plasma-assisted molecular beam epitaxy (14, 16). The cells were grown anaerobically to mid-log phase with Fe(III)-NTA serving as the TEA. Cells were deposited onto the hematite thin film along with anaerobic growth medium that lacked Fe(III)-NTA. The cells were allowed to attach to the hematite surface (without drying) overnight in an anaerobic chamber. The following day, the liquid was carefully removed and immediately replaced with fresh anaerobic solution (pH 7.4). Ig-RFM was performed on the cells by raster scanning an antibody-functionalized AFM tip across the sample surface, thereby creating an affinity map (1). Force curves were collected for a 32-by-32 array. The raw pixilated force-volume data were deconvoluted using a regularized filter algorithm. The total time to acquire a complete image was approximately 20 min.As noted above, attractive interactions between an antibody tip and cell resulted in relatively short-range, nonspecific and longer-range, specific adhesive forces (Fig. (Fig.1C).1C). To distinguish between these two interactions, we integrated each force curve beginning at >20 nm and ending at the full retraction of the piezoelectric motor (∼1,800 nm). This integration procedure quantifies the work of binding, measured in joules, between the antibody tip and a particular position on the sample. While this integration procedure does not totally exclude nonspecific binding, it does select for those events associated primarily with specific antibody-antigen binding. Figure Figure33 is the antibody-cytochrome recognition images for MtrC and OmcA. The corresponding height (or topography) images of the bacterial cells are also shown in Fig. Fig.33.Open in a separate windowFIG. 3.Ig-RFM of live S. oneidensis MR-1 cells deposited on a hematite (α-Fe2O3) thin film. Height image (A) and corresponding Ig-RFM image (B) for a bare unfunctionalized Si3N4 tip. Height and corresponding Ig-RFM image for a tip functionalized with anti-MtrC (C and D) or anti-OmcA (E and F). Each panel contains a thin white oval showing the approximate location of the bacterium on the hematite surface. A color-coded scale bar is shown on the right (height in micrometers [μm], and the work required to separate the tip from the surface in attojoules [aJ]).OmcA molecules were concentrated at the boundary between the bacterial cell and hematite surface (Fig. 3E and F). MtrC molecules were also detected at the edge of a cell (Fig. 3C and D). Some MtrC, unlike OmcA, was observed on the cell surface distal from the point of contact with the mineral (Fig. 3C and D). Both OmcA and MtrC were also present in an extracellular polymeric substance (EPS) on the hematite surface (Fig. 3D and F), which is consistent with previous results showing MtrC and OmcA in an EPS produced by cells under anaerobic conditions (19, 24). This discovery is interesting in light of the research by Rosso et al. (22) and Bose et al. (4), who found that Shewanella can implement a nonlocal electron transfer strategy to reduce the surface of hematite at locations distant from the point of cell attachment. Rosso et al. (22) proposed that the bacteria utilize unknown extracellular factors to access the most energetically favorable regions of the Fe(III) oxide surface. The Ig-AFM results (Fig. (Fig.3)3) suggest the possibility that MtrC and/or OmcA are the “unknown extracellular factors” that are synthesized by Shewanella to reduce crystalline Fe(III) oxides at points distal from the cell. Additional experiments showing reductive dissolution features coinciding with the extracellular location of MtrC and/or OmcA would need to be performed to test this hypothesis.It is important to note that these affinity maps were collected on only a few cells because it so challenging to produce large numbers of quality images. Future work should be conducted on a population of cells. Until this time, these affinity maps can be used to provide a crude, lowest-order estimate of the number of cytochromes on the outer surface of living S. oneidensis MR-1. For example, there were 236 force curves collected on the bacterium shown in Fig. Fig.3D.3D. Thirty-eight of these curves exhibited a distinct, sawtooth-shaped, antibody-antigen binding event. In other words, MtrC molecules were detected in one out of every six force curves (16%) that were collected on the cell surface.This probability can be compared to other independent studies that estimated the density and size of MtrC and OmcA molecules from S. oneidensis MR-1. Lower et al. (16) estimated that S. oneidensis has 4 × 1015 to 7 × 1015 cytochromes per square meter by comparing AFM measurements for whole cells to force curves on purified MtrC and OmcA molecules. Wigginton et al. (25) used scanning tunneling microscopy to determine that the diameter of an individual cytochrome is 5 to 8 nm. These values can be used to create a simple, geometric, close-packing arrangement of MtrC or OmcA molecules on a surface. Using this approach, cytochromes could occupy 8 to 34% of the cell surface.This estimate is consistent with the observed number of putative MtrC molecules shown in Fig. Fig.3D.3D. Therefore, it appears that these affinity maps can be used as a lowest-order estimate for the number of cytochromes on S. oneidensis MR-1 even though we do not know a priori the exact configuration of the antibody tip (e.g., the concentration of antibody on the tip, the exact shape of the tip, the binding epitopes within the antibody).In summary, the data presented here show that S. oneidensis MR-1 localizes OmcA and MtrC molecules to the exterior cell surface, including an EPS, when Fe(III) is the TEA. Here, the cytochromes presumably serve as terminal reductases that catalyze the reduction of Fe(III) through direct contact with the extracellular iron-oxide mineral. 相似文献
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Changming Zhao Tingting Huang Wenqing Chen Zixin Deng 《Applied and environmental microbiology》2010,76(21):7343-7347
Polyoxins consist of 14 structurally variable components which differentiate at three branch sites of the carbon skeleton. Open reading frame (ORF) SAV_4805 of Streptomyces avermitilis, showing similarity to thymine-7-hydroxylase, was proved to enhance the diversity of polyoxins at the C-5 site of the 1-(5′-amino-5′-deoxy-β-d-allofuranuronosyl) pyrimidine moiety.The antifungal nucleoside antibiotic polyoxins synthesized naturally by Streptomyces cacaoi subsp. asoensis (S. cacaoi here) consist of a mixture of at least 14 different compounds called polyoxins A to N (Fig. (Fig.1)1) (6, 13, 25). Most of the polyoxins have a common nucleoside skeleton that is attached with variable side groups at three different places (polyoxins C, I, and N have a modified skeleton). Polyoxins are grouped into four different classes according to the identity of the side group R1 attached at C-5 of the 1-(5′-amino-5′-deoxy-β-d-allofuranuronosyl) pyrimidine moiety. Different classes of polyoxins differ markedly in their activity spectra against plant pathogenic fungi (1, 10, 20).Open in a separate windowFIG. 1.Chemical structure of polyoxin complex. Class is determined by R1. The × indicates a different skeleton that does not have this particular residue.It was deduced that the nucleoside moiety originated from the condensation of uridine with phosphoenolpyruvate (PEP) to generate octosyl acid as the intermediate (7, 12, 14). Then, a subsequent oxidative elimination of the two terminal carbons would create the nucleoside moiety (12). The detailed biosynthetic pathway of nucleoside moiety of polyoxins remains obscure. Our previous work showed that heterologous expression of the polyoxin biosynthetic gene cluster pol in Streptomyces lividans TK24 produces only thymine-derived polyoxin H (class III) (5). This prompted us to investigate the origin of a gene(s) related to the structural variation of polyoxins at the R1 site, which could be located outside the polyoxin biosynthetic gene cluster in the native producer.The pyrimidine rings of polyoxins correspond one to one to the intermediates of the thymidine salvage pathway, which was characterized only for some fungi (see Fig. S1 in the supplemental material) (22). In the prime pathway of nucleotide metabolism, dUMP could be converted to dTMP under the catalysis of thymidylate synthase (TS) with tetrahydrofolic acid as a methyl donor while dTMP could not be converted back to dUMP reversibly (4, 17). In the 1970s, Shaffer et al. separated two enzymes, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and isoorotate decarboxylase (IDCase), from fungal sources which showed potential ability to convert thymine to uracil (22). THase is a trifunctional oxygenase and catalyzes three enzymatic reactions: thymine to 5-hydroxymethyluracil, 5-hydroxymethyluracil to 5-formyluracil, and 5-formyluracil to uracil-5-carboxylic acid (also designated isoorotate) (18, 26). IDCase catalyzes the subsequent decarboxylation reaction, and uracil-5-carboxylic acid is converted to uracil in the end (21). This process is catalyzed by THase, and IDCase is the core step of the thymidine salvage pathway in fungi.BLAST analysis results with the amino acid sequence of THase from Rhodotorula glutinis and IDCase from Neurospora crassa as queries in the 25 sequenced Streptomyces strains (5 finished and 20 in process) of the genome database (from NCBI GenBank, accessed 24 July 2010) showed that the THase and IDCase gene homologs are distributed extensively in Streptomyces, such as open reading frame (ORF) SAV_4805 (sharing 27% identity and 45% similarity with the THase gene) from S. avermitilis strain MA4680 and SCO_6305 (sharing 25% identity and 44% similarity with the IDCase gene) from S. coelicolor strain A3(2) (2, 11, 23). The results indicated a potential thymidine salvage pathway in Streptomyces, and the salvage pathway provides alternative nucleoside monophosphates as precursors for polyoxin biosynthesis. 相似文献
16.
Toshana L. Foster Tamara Belyaeva Nicola J. Stonehouse Arwen R. Pearson Mark Harris 《Journal of virology》2010,84(18):9267-9277
The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.Hepatitis C virus (HCV) is a human pathogen which chronically infects nearly 3% of the world''s population (36, 37). Persistent infection, in 80% of cases, leads to chronic hepatitis which can progress to liver cirrhosis and, in the worst cases, hepatocellular carcinoma (37). Current therapies lack specificity and efficacy due largely to an incomplete understanding of the complex molecular mechanisms of virus infectivity, RNA replication, and assembly (4, 36). HCV is a member of the Flaviviridae family of enveloped viruses (30), with a positive-sense RNA genome of ∼9.6 kb consisting of a single open reading frame (ORF) that encodes 10 structural and nonstructural viral proteins (3, 16, 25). Cap-independent translation of the ORF (29) yields a large polyprotein of approximately 3,000 amino acid residues that is cleaved co- and posttranslationally by host and viral proteases into 10 mature virus proteins; these cleavage products are ordered from the amino to the carboxy terminus as follows: core (C), envelope proteins 1 and 2 (E1 and E2), p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (3, 16, 25). At the flanking ends of the genome are two highly conserved untranslated regions (UTRs). The 5′ UTR is highly structured and consists of the internal ribosome entry site (IRES), which is important for the initiation of cap-independent translation of the polyprotein (29). The 3′ UTR consists of a short genotype-specific variable region, a tract of variable length comprising solely pyrimidine residues (predominantly U), and a conserved 98-nucleotide sequence, known as the X region, containing three stem-loops (13, 23) (Fig. (Fig.1A).1A). The 3′ UTR is the initiation site for the synthesis of the negative-strand RNA during viral replication (13) and is involved in translational regulation.Open in a separate windowFIG. 1.The HCV 3′ UTR RNA. (A) The positive-strand 3′ UTR consists of three distinct regions, i.e., a short genotype-specific variable region, a polypyrimidine tract [poly(U/UC)] of variable length, and a conserved 98-nucleotide sequence known as the X region containing three stable stem-loops. The predicted structure of the genotype 1b 3′ UTR is shown. (B) Left panel, the integrities of in vitro-transcribed radiolabeled full-length 3′ UTR RNAs of genotypes 1b (nucleotides 9375 to 9595) and 2a (nucleotides 9443 to 9678) and the poly(U/UC) (nucleotides 9406 to 9497) and X region (nucleotides 9498 to 9595) of genotype 1b are shown on denaturing polyacrylamide gels. Right panel, the integrities of in vitro-transcribed radiolabeled RNAs comprising the 3′-terminal NS5B-coding region plus the 3′ UTR RNAs of genotypes 1b (nucleotides 9136 to 9595) and 2a (nucleotides 9204 to 9678) (KL-3′ UTR) are shown on denaturing polyacrylamide gels.HCV RNA replication occurs on membranous structures derived from the endoplasmic reticulum (ER) in a complex that includes host cell factors as well as viral nonstructural proteins, including NS5B, the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome in vivo and in vitro (2, 25, 30). Initiation of the synthesis of the negative-strand RNA is thought to occur upon recognition and specific binding of the NS5B polymerase to the 3′ UTR of the genomic RNA (2, 16, 26). This replication activity and template specificity of NS5B in vivo are dependent, however, on the presence of the other nonstructural proteins, such as the proteases NS2 and NS3, which are required for polyprotein processing and helicase activity, and the multifunctional protein NS5A (16).NS5A is a proline-rich phosphoprotein that is absolutely required for viral replication and is also involved in virus particle assembly (9, 10, 20, 22, 35). Its specific function in the latter process is, however, still unknown. NS5A is membrane associated due to the presence of an N-terminal amphipathic helix that serves as a membrane anchor allowing association with ER-derived membranes (Fig. (Fig.2)2) (24, 27). The cytoplasmic portion of NS5A is organized into three domains that are separated by low-complexity sequences (Fig. (Fig.2A)2A) (20). The X-ray crystal structure of domain I has revealed that it is a zinc binding domain which forms a homodimer with contacts at the N-terminal ends of the molecules; the resultant large, basic groove at the dimeric interface has been proposed to be involved in RNA binding during viral replication (17, 33). NS5A has also been shown to interact with uridylate and guanylate-rich RNA and to bind to the 3′ ends of the HCV positive- and negative-strand RNAs (8). These observations suggest that NS5A may specifically interact with the large U/G stretches in the IRES of the 5′ UTR, implying a role in HCV translation and genome multiplication, while its interactions with the polypyrimidine tract of the 3′ UTR suggest that NS5A may affect the efficiency of RNA synthesis by NS5B (8, 28, 32). The reported interactions with both flanking regions of the HCV genome imply that NS5A may play a role in the switch between translation and replication that must occur during the viral life cycle (8).Open in a separate windowFIG. 2.Domain structure and expression of HCV NS5A. (A) Schematic diagram of the functional domains of NS5A and design of the constructs used in the study (genotype 1b NS5A protein numbering). The N-terminal amphipathic helix of NS5A (black box) is responsible for the interaction of NS5A with membranes. NS5A is organized into three domains that are separated by low-complexity sequences, indicated by black boxes. The NS5A constructs used all lacked the N-terminal amphipathic helix and were designed to include an N-terminal Strep tag and a C-terminal hexahistidine tag. (B and C) SDS-PAGE and Western blot analysis of the NS5A(ΔAH) and NS5A domain constructs purified by nickel affinity and Streptactin tag affinity chromatography. Coomassie brilliant blue-stained gels and Western blots (WB) using anti-NS5A antibodies for NS5A proteins of genotype 1b strain J4 (B) and genotype 2a strain JFH-1 (C) are shown.Among HCV genotypes, domains II and III are less well conserved than domain I (34). By mutational analysis, domain II, along with domain I, has been attributed to the replicase activity of NS5A (12). Contrastingly, domain III has been shown to be dispensable for RNA replication, and large heterologous insertions and deletions in this region can be tolerated, maintaining RNA replication (34). It has been shown, however, that these insertions and deletions within domain III do have an impact on virus particle assembly, highlighting the critical role of domain III NS5A in the viral life cycle (1, 10). Recent nuclear magnetic resonance (NMR) studies of domains II and III of NS5A revealed that they both adopt a natively unfolded state (6, 14, 15). The high degree of disorder and flexibility observed in these domains may contribute to the promiscuity of NS5A, which has been shown to interact with a variety of biological partners essential for NS5A function and virus persistence (11, 18, 19, 21, 31). In addition, regions within domains I and II of NS5A interact with NS5B, stimulating the in vitro activity of the polymerase and supporting the hypothesis that NS5A has a role in the modulation of RNA replication (28, 32).In this study, we have investigated in detail the RNA binding properties of NS5A. We have mapped the RNA binding regions of NS5A using bacterially expressed deletion constructs of NS5A and have assayed their binding affinity for HCV positive-strand 3′ UTR RNA. In addition, we provide evidence that the RNA binding activity of NS5A is specific and that NS5A interacts preferentially with the polypyrimidine region of the 3′ UTR. 相似文献
17.
18.
Regulation of vif mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5′ Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G
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Dibyakanti Mandal Colin M. Exline Zehua Feng C. Martin Stoltzfus 《Journal of virology》2009,83(12):6067-6078
The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5′ splice site (5′ss) D1, to the first splice acceptor, 3′ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5′ss D2, which is 50 nucleotides downstream of 3′ss A1; a GGGG silencer motif proximal to 5′ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5′ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3′ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5′ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5′ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.Human immunodeficiency virus type 1 (HIV-1) Vif is a 23-kDa basic protein (4, 9) that is incorporated into virus particles during productive infection (8-10). Replication of HIV-1 in some T-cell lines is dependent on the expression of a functional Vif protein. Replication of Vif-deleted HIV-1 is restricted in these cells, which are termed nonpermissive, because of the presence of several host deaminases, the most important of which for HIV-1 replication is APOBEC3G (A3G) (25, 26). Human A3G is a single-stranded DNA deaminase that inhibits the replication of HIV-1 as well as other types of retroviruses and retrotransposons (5, 12, 17, 25, 32). HIV-1 Vif forms a complex with A3G and other cellular proteins to promote A3G ubiquitination, resulting in proteasomal degradation of A3G (1, 11, 14, 18, 26). Vif-deleted HIV-1 produced in the presence of A3G packages increased levels of A3G compared to those found in the wild type (WT) and has reduced infectivity in nonpermissive T-cell lines. This reduced infectivity in the absence of Vif has been correlated with the dC-to-dU hypermutation of newly synthesized minus-strand viral DNA by A3G (6, 13, 31, 32). However, other studies have shown that A3G is also able to restrict virus replication without hypermutating viral DNA (7, 19).It has previously been shown that the expression of Vif in infected cells is maintained at a relatively low level compared to levels of the other HIV-1 accessory proteins. One mechanism to explain this phenomenon is that Vif is degraded more rapidly than other accessory proteins by the proteasome (3). Another mechanism is that a relatively low level of vif mRNA is produced by alternative splicing (22). Alternative splicing of HIV-1 RNA results in the production of approximately 40 different mRNA species, which include three different mRNA size classes: 1.8-kb, completely spliced RNAs; 4-kb, incompletely spliced RNAs; and 9-kb, unspliced RNAs (Fig. (Fig.1A).1A). The 4-kb mRNA class encodes Vif, Vpr, Tat, Vpu, and Env, and the completely spliced, 1.8-kb mRNA class encodes Tat, Rev, and Nef. Unspliced viral RNA is both packaged into virions as genomic RNA and used as mRNA for Gag and Gag-Pol proteins (2, 27). As shown in Fig. Fig.1A,1A, four different 5′ splice donor sites (5′ss) and eight different 3′ splice acceptor sites (3′ss), which are highly conserved among group M HIV-1 strains, are used to produce alternatively spliced HIV-1 mRNAs at different levels in infected cells (22). The efficiencies with which these 5′ss and 3′ss are used are dependent on the presence of suboptimal cis splicing elements within the 5′ss and 3′ss themselves and more-distant elements, which include exonic splicing silencers, an intronic splicing silencer, and exonic splicing enhancers (ESE) (2, 15, 27).Open in a separate windowFIG. 1.HIV-1 splicing pattern and elements regulating vif mRNA splicing. (A) The conserved 5′ss (D1 to D4) and 3′ss (A1 to A7) located within the 9-kb HIV-1 genome are shown. Completely and incompletely spliced HIV-1 mRNAs (∼4 kb and ∼1.8 kb) are shown as open boxes. Spliced mRNAs are denoted by the translated open reading frame and by the exon content. The incompletely spliced mRNAs, denoted with an I, are differentiated from completely spliced mRNAs by inclusion of the intron between 5′ss D4 and 3′ss A7. Either one or both of the noncoding exons 2 and 3 (shown as gray-shaded exons) can be differentially included within all 1.8- and 4.0-kb mRNA species, with the exception of vif mRNA (1.2I) and vpr mRNA, which can include only exon 2 (1.[2].3I). LTR, long terminal repeat. (B) Three elements regulating vif mRNA splicing are shown: positively acting enhancer ESEVif, the 5′ss D2 (underlined), and a negatively acting G4 silencer motif. The locations of noncoding exon 2 and the start site for Vif protein synthesis are also shown. (C) HIV-1 5′ss D2-down mutants used in this study are shown. Sequences were aligned and compared with that of the consensus metazoan 5′ss. The sequence of the ESEVif mutant used in this study is also aligned and compared with the WT sequence. nt, nucleotides.HIV-1 Vif is translated from a low-abundance, incompletely spliced mRNA resulting from splicing of HIV-1 RNA between the major splice donor site (5′ss D1) and 3′ss A1. We have demonstrated that vif mRNA splicing is tightly regulated by the presence of multiple regulatory elements (Fig. (Fig.1B).1B). These include a highly conserved suboptimal 5′ss (5′ss D2) 50 nucleotides downstream from 3′ss A1, an SRp75-dependent ESE (ESEVif), and a GGGG silencer element proximal to 5′ss D2 (2). Mutations within the relatively weak 5′ss D2 that increase its homology to a consensus 5′ss result in increased inclusion of the noncoding 50-nucleotide exon defined by 3′ss A1 and 5′ss D2 (exon 2), increased single-spliced vif mRNA levels and Vif expression, and an excessive splicing phenotype in which virion production is reduced to 10 to 25% of that of the WT (16). Conversely, mutations that decrease the homology of 5′ss D2 to a consensus 5′ss inhibit splicing at 3′ss A1 and exon 2 inclusion into both incompletely and completely spliced HIV-1 mRNAs as well as decreased levels of vif mRNA. Virus production, however, is not significantly affected. Mutation of ESEVif resulted in a similar phenotype. We have shown previously that increased or decreased exon 2 inclusion into spliced mRNA does not affect the stability or expression of viral mRNAs (15). Based on these results, we hypothesized that the conserved suboptimal 5′ss D2, which together with 3′ss A1 defines exon 2, and ESEVif are necessary to maintain optimal levels of Gag and Gag-Pol required for HIV-1 replication while producing sufficient Vif to overcome the cellular restriction factor A3G (2). To further test this hypothesis, we examined a panel of HIV-1 mutants producing reduced levels of Vif under permissive and nonpermissive conditions. We also investigated the long-term replication capabilities of these mutant viruses in both permissive and nonpermissive A3G-expressing T-cell lines. Mutant viruses demonstrated increasing sensitivity to A3G, which is inversely proportional to their levels of Vif expression. Our results suggest that the reason 5′ss D2 and ESEVif exist in the HIV-1 genome is to regulate the levels of vif mRNA and Vif protein in infected cells. 相似文献
19.
Wolfram Lorenzen Michael W. Ring Gertrud Schw?r Helge B. Bode 《Journal of bacteriology》2009,191(18):5849-5853
It was recently shown that Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.Myxobacteria are unique among the prokaryotes as (i) they can form highly complex fruiting bodies under starvation conditions, even up to microscopic tree-like structures (28); (ii) they can move on solid surfaces using different motility mechanisms (16); (iii) they produce some of the most cytotoxic secondary metabolites, with epothilone already in clinical use against cancer (2, 3); and (iv) they harbor the largest prokaryotic genomes found so far (15, 27). The large genome might be directly related to their complex life-style and the diverse secondary (3) and primary (9) metabolisms. Already in 2002 we found that myxobacteria are able to produce isovaleryl coenzyme A (IV-CoA) and compounds derived thereof via a new pathway that branches from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is the central intermediate of the well-known mevalonate-dependent isoprenoid biosynthesis (Fig. (Fig.1)1) (22, 23). Usually IV-CoA is derived from leucine degradation via the branched-chain keto acid dehydrogenase (BKD) complex (24), which is also the preferred pathway to IV-CoA in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca (Fig. (Fig.2A).2A). However, in bkd mutants, where no or only residual leucine degradation is possible (30), the alternative pathway is induced (Fig. (Fig.2B),2B), presumably to ensure the production of iso-fatty acids (iso-FAs) (5). A possible reason for this alternative pathway is the importance of IV-CoA-derived compounds in the complex myxobacterial life cycle, which is the starvation-induced formation of fruiting bodies in which the cells differentiate into myxospores. We showed that this pathway is induced during fruiting body formation in M. xanthus when leucine is limited. Under these conditions, this pathway might be more important for protein synthesis than for lipid remodeling, as lipids are present in excess during development due to the surface reduction from vegetative rods to round myxospores as described previously (29). Examples of IV-CoA-derived compounds are the unusual iso-branched ether lipids, which are almost exclusively produced in the developing myxospores. They might serve as structural lipids and signaling compounds during fruiting body formation (26).Open in a separate windowFIG. 1.Biosynthesis of IV-CoA and compounds derived thereof and biosynthesis of isoprenoids in M. xanthus. Broken arrows indicate multistep reactions; supplementation (double-lined arrows) with MVL and IVA can be used to complement selected mutants.Open in a separate windowFIG. 2.Short representations of proposed metabolic fluxes through the IV-CoA/isoprenoid network. Broken arrows indicate no metabolic flux. (A) DK1622 (wild type); (B) DK5643 (Δbkd); (C) DK5624 (Δbkd mvaS::kan); (D) HB002 (Δbkd liuC::kan); (E) HB002 with 1 mM IVA; (F) HB002 with 1 mM MVL. Ac-CoA, acetyl-CoA; MVA, mevalonic acid.In M. xanthus, we could recently identify candidate genes involved in the alternative pathway from HMG-CoA to IV-CoA. We also described the genes required for the degradation pathway of leucine and subsequently also those involved in the transformation of IV-CoA to HMG-CoA (4). In myxobacteria leucine is an important precursor for isoprenoid biosynthesis, as was already shown elsewhere for the biosynthesis of steroids (7) and prenylated secondary metabolites like aurachin (22) or leupyrrins (6), as well as volatiles like geosmin or germacradienol in M. xanthus and S. aurantiaca (11, 13). The interconnection of iso-FAs and isoprenoid biosynthesis made it difficult to assign functions to these compound classes during fruiting body formation in M. xanthus because it cannot be excluded that reduced leucine degradation also impairs isoprenoid biosynthesis. A mutant strain of M. xanthus that was blocked in the degradation of leucine and the alternative pathway had a deletion in the bkd locus as well as a plasmid insertion in the mvaS gene encoding the HMG-CoA synthase (strain DK5624). This double mutation severely affected isoprenoid biosynthesis (5), and cultures of DK5624 must be supplemented with mevalonolactone (MVL; the cyclized form of mevalonic acid) in order to enable growth (Fig. (Fig.2C).2C). Since we have identified the genes involved in IV-CoA biosynthesis and the mevalonate pathway (4), we can now start to identify differences between strains that show deficiencies in iso-FAs and strains that show deficiencies in isoprenoids via simple analysis of the FA profile and analysis of the myxobacterial development of selected mutants.All mutants used in this study (HB002 [Δbkd liuC::kan], HB015 [Δbkd MXAN_4265::kan], DK5624 [Δbkd mvaS::kan], HB019 [Δbkd mvaS::kan mvaS+], and HB020 [Δbkd MXAN_4265::kan mvaS+]) have been published previously (4), and FA analysis as well as myxobacterial fruiting body formation has also been described previously (26).M. xanthus HB002 (Δbkd liuC) shows only residual amounts of iso-FAs, as both leucine degradation and the alternative pathway to IV-CoA are blocked (Fig. (Fig.2D)2D) and its capability to form fruiting bodies is strongly reduced (Fig. (Fig.3).3). The residual amount of iso-FAs results from a second BKD activity in M. xanthus that has been identified by residual leucine incorporation as well as by residual enzymatic activity in bkd mutants (23, 30). This second BKD activity might be a side activity of the pyruvate dehydrogenase or a related chemical oxidative decarboxylation, as no second bkd locus could be identified in the genome (unpublished results). Moreover, growth of HB002 is not MVL dependent because the block in the alternative pathway does not affect isoprenoid biosynthesis, as liuC encodes a dehydratase/hydratase that is involved in the conversion of HMG-CoA to 3-methylglutaconyl-CoA and vice versa (4). As expected, the FA profile (4) as well as the developmental phenotype (data not shown) can be complemented (Fig. (Fig.2E)2E) by the addition of isovaleric acid (IVA), the free acid of IV-CoA, indicating the importance of iso-branched compounds for development in M. xanthus. Unexpectedly, addition of MVL (Fig. (Fig.2F)2F) also partially restored fruiting body formation without restoring the FA profile (Fig. (Fig.3).3). Similarly, M. xanthus HB015 (Δbkd MXAN_4265::kan) can produce only traces of iso-FAs, as both pathways to IV-CoA are blocked. MXAN_4265 encodes a protein with similarity to a glutaconyl-CoA transferase subunit, but from our previous results, we postulated it to be involved in the alternative pathway to IV-CoA (Fig. (Fig.1)1) (4). The respective mutant shows a severely impaired developmental phenotype, which can be complemented not only by the addition of IVA (not shown) but also by the addition of MVL (Fig. (Fig.3).3). Again, no change in the FA profile was observed after the addition of MVL. However, a plasmid insertion into MXAN_4265 has a polar effect on mvaS, which is the last gene in this five-gene operon and which is crucial for HMG-CoA formation from acetoacetyl-CoA and acetyl-CoA. Therefore, we assume that both pathways to HMG-CoA are blocked in HB015: no HMG-CoA can be made from acetyl-CoA and hardly any can be made via leucine degradation. In order to prove this hypothesis, we complemented HB015 with an additional copy of mvaS under the constitutive T7A1 promoter as described previously, using the plasmid pCK4267exp (4). The resulting strain, HB020 (Δbkd MXAN_4265::kan mvaS+), showed a restored developmental phenotype but still produced only trace amounts of iso-FAs.Open in a separate windowFIG. 3.Fruiting body formation on TPM agar in selected mutants at 24, 48, and 72 h after starvation. Numbers refer to the relative amounts (in percentages) of the most abundant iso-FA, iso-15:0, which is indicative of iso-FAs in general. Strains were DK1622 (wild type), HB002 (Δbkd liuC::kan), HB015 (Δbkd MXAN_4265::kan), DK5624 (Δbkd mvaS::kan), HB019 (Δbkd mvaS::kan mvaS+), and HB020 (Δbkd MXAN_4265::kan mvaS+). DK5624 was grown with 0.3 mM MVL prior to starvation, and the cells were washed and plated on TPM with or without 1 mM of MVL.The data from HB002, HB015, and HB020 indicate an important function of the mevalonate-dependent isoprenoid pathway for fruiting body formation in M. xanthus. Therefore, MVL addition can at least partially complement the developmental phenotype of DK5624, which cannot form fruiting bodies without MVL (Fig. (Fig.3).3). However, genetic complementation with mvaS in HB019 resulted in the expected complementation of the fruiting body formation and the FA profile (Fig. (Fig.3,3, bottom row).Leucine is one of the most abundant proteinogenic amino acids. It is also an essential amino acid for M. xanthus (8), which has a predatory life-style (1), as it lives on other bacteria and fungi that contain a lot of leucine. Moreover, leucine is very efficiently incorporated into isoprenoids like geosmin and aurachin (10, 22). Thus, one can conclude that in fact leucine degradation is the major pathway for HMG-CoA biosynthesis instead of the usual formation via acetoacetyl-CoA and acetyl-CoA by the HMG-CoA synthase MvaS as indicated in Fig. Fig.2A.2A. No difference in growth was observed between culture with and culture without MVL for HB002 (Δbkd liuC::kan) and HB015 (Δbkd MXAN_4265::kan) in rich medium (data not shown), probably due to the complete MvaS activity (in HB002) or residual BKD activity (in HB002 and HB015), resulting in all precursors for the mevalonate-dependent isoprenoid biosynthesis still being present in excess under these conditions. However, under starvation conditions a small reduction in HMG-CoA biosynthesis caused by completely blocked leucine degradation (as in HB002 due to the mutation in liuC [Fig. [Fig.2D])2D]) or reduced leucine degradation and a mutation in mvaS (as in HB015) might each result in a reduced isoprenoid level, which can be complemented at least partially by the addition of MVL. This would also explain the difference in the developmental phenotypes of HB002 and HB015, with the phenotype being more severe in HB002 (Fig. (Fig.3).3). The fact that complementation with IVA is in all cases more efficient than that with MVL can be explained by the role of the already-mentioned isolipids. They can be produced only after IVA addition, which also complements the (developmental) phenotype of some of these mutants (26).As isoprenoids represent probably the most diverse class of natural products (14), it is very hard to predict which particular isoprenoids might be responsible for the observed effects. Several isoprenoids (7, 11-13), prenylated secondary metabolites (6, 22), and carotenoids (18-21) are known from myxobacteria in general, and a major volatile compound from M. xanthus is the terpenoid geosmin (13). In order to test whether geosmin might be required for fruiting body formation, we constructed a plasmid insertion mutant in MXAN_6247, which is involved in the cyclization of farnesyl diphosphate to geosmin, following published procedures (4, 5). The resulting strain, HB022, showed the expected loss in geosmin production but no developmental phenotype (data not shown).Additionally, it cannot be excluded that prenylated proteins, sugars, or quinones from the respiratory chain are important for fruiting body formation. Moreover, stigmolone has been described as a pheromone involved in fruiting body formation in S. aurantiaca (25). Although its biosynthesis has not been elucidated yet, stigmolone could be an isoprenoid as well, which is deducible from the two iso-branched residues within its chemical structure (17). Nevertheless, the importance of isoprenoids for M. xanthus is evident from the data presented, and clearly more work is needed to identify the compound(s) involved. 相似文献
20.
Judith Becker Corinna Klopprogge Hartwig Schr?der Christoph Wittmann 《Applied and environmental microbiology》2009,75(24):7866-7869
In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.With an annual market volume of more than 1,000,000 tons, lysine is one of the dominating products in biotechnology. In recent years, rational metabolic engineering has emerged as a powerful tool for lysine production (16, 18, 22). Hereby, different target enzymes and pathways in the central metabolism could be identified and successfully modified to create superior production strains (1, 2, 5, 8, 10, 17-20). The tricarboxylic acid (TCA) cycle has not been rationally engineered so far, despite its major role in Corynebacterium glutamicum (6). From metabolic flux studies, however, it seems that the TCA cycle might offer a great potential for optimization (Fig. (Fig.1),1), which is also predicted from in silico pathway analysis (13, 22). Experimental evidence comes from studies with Brevibacterium flavum exhibiting increased lysine production due to an induced bottleneck toward the TCA cycle (21). In the present work, we performed TCA cycle engineering by downregulation of isocitrate dehydrogenase (ICD). ICD is the highest expressed TCA cycle enzyme in C. glutamicum (7). Downregulation was achieved by start codon exchange, controlling ICD expression on the level of translation.Open in a separate windowFIG. 1.Stoichiometric correlation of lysine yield (%), biomass yield (g/mol) and TCA cycle flux (%; entry flux through citrate synthase) determined by 13C metabolic flux analysis achieved by paraboloid fitting of the data set (parameters were determined with Y0 = 105.1, a = −1.27, b = 0.35, c = −9.35 × 10−3, d = −11.16 × 10−3). The data displayed represent values from 18 independent experiments with different C. glutamicum strains taken from previous studies (1-3, 11, 12, 15, 23). 相似文献