DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

Multiple Interaction Domains in FtsL,a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.The division of an Escherichia coli cell into two daughter cells requires a complex of proteins, the divisome, to coordinate the constriction of the three layers of the Gram-negative cell envelope. In E. coli, there are 10 proteins known to be essential for cell division; in the absence of any one of these proteins, cells continue to elongate and to replicate and segregate their chromosomes but fail to divide (29). Numerous additional nonessential proteins have been identified that localize to midcell and assist in cell division (7-9, 20, 25, 34, 56, 59).A localization dependency pathway has been determined for the 10 essential division proteins (FtsZ→FtsA/ZipA→FtsK→FtsQ→FtsL/FtsB→FtsW→FtsI→FtsN), suggesting that the divisome assembles in a hierarchical manner (29). Based on this pathway, a given protein depends on the presence of all upstream proteins (to the left) for its localization and that protein is then required for the localization of the downstream division proteins (to the right). While the localization dependency pathway of cell division proteins suggests that a sequence of interactions is necessary for divisome formation, recent work using a variety of techniques reveals that a more complex web of interactions among these proteins is necessary for a functionally stable complex (6, 10, 19, 23, 24, 30-32, 40). While numerous interactions have been identified between division proteins, further work is needed to define which domains are involved and which interactions are necessary for assembly of the divisome.One subcomplex of the divisome, composed of the bitopic membrane proteins FtsB, FtsL, and FtsQ, appears to be the bridge between the predominantly cytoplasmic cell division proteins and the predominantly periplasmic cell division proteins (10). FtsB, FtsL, and FtsQ share a similar topology: short amino-terminal cytoplasmic domains and larger carboxy-terminal periplasmic domains. This tripartite complex can be divided further into a subcomplex of FtsB and FtsL, which forms in the absence of FtsQ and interacts with the downstream division proteins FtsW and FtsI in the absence of FtsQ (30). The presence of an FtsB/FtsL/FtsQ subcomplex appears to be evolutionarily conserved, as there is evidence that the homologs of FtsB, FtsL, and FtsQ in the Gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae also assemble into complexes (18, 52, 55).The assembly of the FtsB/FtsL/FtsQ complex is important for the stabilization and localization of one or more of its component proteins in both E. coli and B. subtilis (11, 16, 18, 33). In E. coli, FtsB and FtsL are codependent for their stabilization and for localization to midcell, while FtsQ does not require either FtsB or FtsL for its stabilization or localization to midcell (11, 33). Both FtsL and FtsB require FtsQ for localization to midcell, and in the absence of FtsQ the levels of full-length FtsB are significantly reduced (11, 33). The observed reduction in full-length FtsB levels that occurs in the absence of FtsQ or FtsL results from the degradation of the FtsB C terminus (33). However, the C-terminally degraded FtsB generated upon depletion of FtsQ can still interact with and stabilize FtsL (33).While a portion of the FtsB C terminus is dispensable for interaction with FtsL and for the recruitment of the downstream division proteins FtsW and FtsI, it is required for interaction with FtsQ (33). Correspondingly, the FtsQ C terminus also appears to be important for interaction with FtsB and FtsL (32, 61). The interaction between FtsB and FtsL appears to be mediated by the predicted coiled-coil motifs within the periplasmic domains of the two proteins, although only the membrane-proximal half of the FtsB coiled coil is necessary for interaction with FtsL (10, 32, 33). Additionally, the transmembrane domains of FtsB and FtsL are important for their interaction with each other, while the cytoplasmic domain of FtsL is not necessary for interaction with FtsB, which has only a short 3-amino-acid cytoplasmic domain (10, 33).In this study, we focused on the interaction domains of FtsL. We find that, as with FtsB, the C terminus of FtsL is required for the interaction of FtsQ with the FtsB/FtsL subcomplex while the cytoplasmic domain of FtsL is involved in recruitment of the downstream division proteins. Finally, we provide a comprehensive analysis of the presence of FtsB, FtsL, and FtsQ homologs among bacteria and find that the proteins of this complex are likely more widely distributed among bacteria than was previously thought.     

Characterization of the FtsZ-Interacting Septal Proteins SepF and Ftn6 in the Spherical-Celled Cyanobacterium Synechocystis Strain PCC 6803

Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure at midcell establishes the location of the nascent division sites in prokaryotes. However, it is not yet known how the assembly and contraction of the Z ring are regulated, especially in cyanobacteria, the environmentally crucial organisms for which only one FtsZ partner protein, ZipN, has been described so far. Here, we characterized SepF and Ftn6, two novel septal proteins, in the spherical-celled strain Synechocystis PCC 6803. Both proteins were found to be indispensable to Synechocystis sp. strain PCC 6803. The depletion of both SepF and Ftn6 resulted in delayed cytokinesis and the generation of giant cells but did not prevent FtsZ polymerization, as shown by the visualization of green fluorescent protein (GFP)-tagged FtsZ polymers. These GFP-tagged Z-ring-like structures often appeared to be abnormal, because these reporter cells respond to the depletion of either SepF or Ftn6 with an increased abundance of total, natural, and GFP-tagged FtsZ proteins. In agreement with their septal localization, we found that both SepF and Ftn6 interact physically with FtsZ. Finally, we showed that SepF, but not Ftn6, stimulates the formation and/or stability of FtsZ polymers in vitro.Binary fission of a mother cell to form two daughter cells is a widely conserved cell proliferation mechanism. In nearly all bacteria, cell division is initiated by the polymerization into a ring-like structure at midcell of the tubulin homolog GTPase protein FtsZ, which is also found in some archae, as well as in plastids and some mitochondria (for reviews, see references 7, 21, and 33). The Z-ring is subsequently used as a scaffold for recruitment of downstream factors that execute the synthesis of the division septum. The assembly of this complex, also referred to as the divisome, has been thoroughly investigated in studies of the rod-shaped model organisms Escherichia coli and Bacillus subtilis) (for reviews, see references 3, 4, 7, 9, 11, 19, and 21). In E. coli, more than 10 different proteins are required for the progression and completion of cell division. They are designated Fts proteins because their depletion leads to filamentation of the bacteria, and they are recruited to the division site in the following sequential order: FtsZ→FtsA/ZipA/ZapB→FtsK→FtsQ and FtsL/FtsB→FtsW→FtsI and FtsN.The stability of the FtsZ protofilaments is thought to be important for assembly of the septal Z ring. Four FtsZ-interacting proteins have been shown to promote FtsZ polymerization and/or Z-ring stabilization, namely, ZapA and ZipA (found only in gammaproteobacteria), FtsA (an actin-like protein), and SepF (not found in gammaproteobacteria) (10, 31). Both FtsA and ZipA assemble at the Z-ring early and participate in its anchorage to the inner face of the cytoplasmic membrane of the cell. They also participate in the recruitment of the downstream cytokinetic factor FtsK. Subsequently, the recruitment of FtsQ and the FtsB/FtsL complex allow the progressive assembly of downstream factors (FtsW, FtsI, and FtsN) involved in synthesis of the septal cell wall (7).By contrast, the negative regulatory proteins MinCDE, DivIVA, EzrA, SulA, and Noc operate in the destabilization and positioning of the Z-ring at midcell (7, 21, 30), sometimes through a direct interaction with FtsZ (SulA, MinC, and ErzA).Little is known concerning cell division in cyanobacteria, in spite of their crucial importance to the biosphere (5, 27, 34) and their interest for biotechnologists (1, 6, 32). Cyanobacteria are also attractive because many species (such as E. coli and B. subtilis) exhibit a cylindrical morphology with a well-defined middle, whereas many others have a spherical shape (29) and thus possess an infinite number of potential division planes at the point of greatest cell diameter. Furthermore, as the progenitor of the chloroplasts (8), cyanobacteria can be of help for deciphering the stromal chloroplastic division machinery (33). Interestingly, several cell division factors occurring in E. coli and B. subtilis have been shown (FtsZ, MinCDE, and SulA) or proposed (FtsE, FtsI, FtsQ, and FtsW) to be conserved in cyanobacteria (23, 26) and chloroplasts (which lack MinC) (33). In contrast, ftsA, ftsB, zipA, ftsK, ftsL, ftsN, and zapA have not been detected in cyanobacteria.So far, cyanobacterial cytokinesis has mainly been investigated using the two unicellular species Synechococcus sp. strain PCC 7942 (rod shaped; hereafter S. elongatus) and Synechocystis sp. strain PCC 6803 (spherical-celled; hereafter Synechocystis sp.) and the filamentous strain Anabaena PCC 7120, all of which possess a fully sequenced genome (http://genome.kazusa.or.jp/cyanobase/) that is easily manipulated (16). Both FtsZ and ZipN/Ftn2/Arc6, a protein occurring only in cyanobacteria (ZipN [alternative name, Ftn2]) and plant chloroplasts (Arc6), were found to be crucial for cytokinesis (17, 23, 26) and to physically interact with each other (20, 23). We also reported that the MinCDE system participates in determining the correct positioning of the septal Z ring at midcell (23). In addition, it has recently been shown in studies of Synechococcus sp. that inactivation of both the cdv2 gene (an orthologue of the gene encoding B.subtilis sepF) and the ftn6 gene (present in only some cyanobacteria) promotes filamentation, though their role in cell division has yet to be characterized (16, 26).In a continuous effort to characterize the divisome machine of Synechocystis sp., we have used a combination of in vivo and in vitro techniques for thorough analysis of the SepF and Ftn6 proteins. We report here that both SepF and Ftn6 are crucial cytokinetic proteins that localize at the division site at midcell and whose depletion leads to the formation of giant cells that remain spherical. In agreement with their septal localization, both SepF and Ftn6 were found to interact physically with FtsZ; also, SepF, but not Ftn6, was found to stimulate the formation and/or stability of FtsZ polymers.     

Changes in the Min Oscillation Pattern before and after Cell Birth

The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.Rod-shaped bacteria, such as Escherichia coli, divide by binary fission and thus assemble their cell division apparatus (the divisome) at the cell midpoint. Tubulin-like FtsZ is the major cytoskeletal protein of the divisome (17) and assembles into a polymeric ring on the inner surface of the cytoplasmic membrane (the Z ring). Assembly and eventual contraction of the Z ring are crucial for divisome function, and thus it is not surprising that many regulatory factors control FtsZ assembly (25). Notably, two negatively acting spatial regulatory systems, the Min system and nucleoid occlusion, ensure that the Z ring is located properly at the cell midpoint (18). Whereas a major component of the nucleoid occlusion system can be deleted with no major effects on cell division (2), inactivation of the Min system causes cells to divide either at midcell or aberrantly at cell poles (27). The result of polar cell division is the formation of chromosome-free minicells.The Min system consists of three proteins, MinC, MinD, and MinE (7). MinC has two separate domains, each of which binds to FtsZ and promotes disassembly of FtsZ polymers and polymer bundles (6, 29, 30). MinC also binds to MinD, an ATPase with a carboxy-terminal amphipathic helix that binds to the membrane only when the protein is bound to ATP (11, 12). MinD also forms polymers (31). Finally, MinE is a small protein that binds to MinD and stimulates hydrolysis of its bound ATP in the presence of membranes. By doing so, MinE helps to dislodge MinD from the membrane, although MinE itself can bind to the membrane (10). The result is that MinD and MinE form zones that oscillate from one cell pole to the other, with an oscillation period of seconds to minutes, depending on a number of factors, including temperature (9, 23, 24, 34). In typical cells, MinD spends most of its time bound to the membrane at a cell pole, forming a U-shaped zone, and its transit to the opposite pole is rapid compared to its dwell time (23). MinE typically forms a ring at the edge of the MinD zone (22, 24). The direction of the oscillation is determined strongly by cell geometry (5, 35). Other factors, such as membrane phospholipid composition, also influence MinD oscillation; MinD-ATP preferentially binds anionic phospholipids, such as cardiolipin, which is enriched at cell poles (15, 21, 32).Because MinC binds to MinD, MinC oscillates in concert with MinD and therefore is present at the cell poles for longer times than anywhere else in the cell (13, 22). This sets up a gradient of MinC, with the average smallest amount of MinC at midcell at any one time. The current model is that Z rings are most likely to assemble at the trough of the MinC gradient and are discouraged from assembling at cell poles at the peak of the gradient (14). This is supported by the observation that nonring FtsZ itself oscillates from pole to pole, presumably being chased back and forth by the alternating zones of high MinC concentration (33).However, recent work in Bacillus subtilis has shed new light on the possible function of MinC on the Z ring and the divisome. B. subtilis lacks MinE and thus relies on a static MinC gradient. This is set up by the recruitment of MinC and MinD (MinCD) to the Z ring during formation of the division septum (19, 20). This seems paradoxical, as the presence of MinCD at the Z ring is predicted to destabilize it. However, in B. subtilis, Z rings containing MinCD remain functional. Therefore, MinCD seems to have an important role in preventing the immediate reassembly of Z rings at developing cell poles next to a recently used ring (4, 8).This recruitment of MinCD to the Z ring of B. subtilis prompted us to examine in more detail Min oscillations in E. coli cells undergoing septation. We hypothesized that MinCD might bind to the Z ring at later stages of septation, perhaps helping the Z ring to function by stimulation of FtsZ disassembly. Previous results with green fluorescent protein (GFP)-MinC suggested that MinC could transiently localize to the Z ring during septation (13). Consequently, we tested if MinD, the driving force of the oscillation, could also localize to the Z ring and if this localization was dependent on MinC. We also hypothesized that a more central localization of MinCD during the time of septum formation might explain how Min proteins are partitioned equitably to both daughter cells.     

Adenine Nucleotide-dependent Regulation of Assembly of Bacterial Tubulin-like FtsZ by a Hypermorph of Bacterial Actin-like FtsA

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.Bacterial cell division requires a large number of proteins that colocalize to form a putative protein machine at the cell membrane (1). This machine, sometimes called the divisome, recruits enzymes to synthesize the septum cell wall and to initiate and coordinate the invagination of the cytoplasmic membrane (and in Gram-negative bacteria, the outer membrane). The most widely conserved and key protein for this process is FtsZ, a homolog of tubulin that forms a ring structure called the Z ring, which marks the site of septum formation (2, 3). Like tubulin, FtsZ assembles into filaments with GTP but does not form microtubules (4). The precise assembly state and conformation of these FtsZ filaments at the division ring is not clear, although recent electron tomography work suggests that the FtsZ ring consists of multiple short filaments tethered to the membrane at discrete junctures (5), which may represent points along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One of these, ZipA, is not well conserved but is an essential protein in E. coli. ZipA binds to the C-terminal tail of FtsZ (6–8), and purified ZipA promotes bundling of FtsZ filaments in vitro (9, 10). The other, FtsA, is also essential in E. coli and is more widely conserved among bacterial species. FtsA is a member of the HSP70/actin superfamily (11, 12), and like ZipA, it interacts with the C-terminal tail of FtsZ (7, 13–15). FtsA can self-associate (16, 17) and bind ATP (12, 18), but reports of ATPase activity vary, with Bacillus subtilis FtsA having high activity (19) and Streptococcus pneumoniae FtsA exhibiting no detectable activity (20). There are no reports of any other in vitro activities of FtsA, including effects on FtsZ assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has a number of key activities in the cell. It is required for recruitment of a number of divisome proteins (21, 22) and helps to tether the Z ring to the membrane via a C-terminal membrane-targeting sequence (23). FtsA, like ZipA and other divisome proteins, is necessary to activate the contraction of the Z ring (24, 25). In E. coli, the FtsA:FtsZ ratio is crucial for proper cell division, with either too high or too low a ratio inhibiting septum formation (26, 27). This ratio is roughly 1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell (28), which works out to concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in the protein can result in significant enhancement of divisome activity. For example, the R286W mutation of FtsA, also called FtsA*, can substitute for the native FtsA and divide the cell. However, this mutant FtsA causes E. coli cells to divide at less than 80% of their normal length (29) and allows efficient division of E. coli cells in the absence of ZipA (30), indicating that it has gain-of-function activity. FtsA* and other hypermorphic mutations such as E124A and I143L can also increase division activity in cells lacking other essential divisome components (31–33). The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule, allowing cell division to occur at higher ratios than with WT² FtsA. This may be because the altered FtsA proteins self-associate more readily than WT FtsA, which may cause different changes in FtsZ assembly state as compared with WT FtsA (17, 34).In this study, we use an in vitro system with purified FtsZ and a purified tagged version of FtsA* to elucidate the role of FtsA in activating constriction of the Z ring in vivo. We show that FtsA*, at physiological concentrations in the presence of ATP or ADP, has significant effects on the assembly of FtsZ filaments.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24, 26, 34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34, 38, 54).Type IV pili are typically composed of major pilins, the primary structural components of the pilus, and several minor pilin-like proteins that play important roles in pilus assembly or function (15, 17, 46). Pilin precursor proteins are transported across the cytoplasmic membrane via the Sec translocation pathway (7, 20). Most Sec substrates contain either a class I or a class II signal peptide that is cleaved at a recognition site that lies subsequent to the hydrophobic portion of the signal peptide (18, 43). However, the precursors of type IV pilins contain class III signal peptides, which are processed at recognition sites that precede the hydrophobic domain by a prepilin-specific peptidase (SPase III) (38, 43, 45). Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/PibD) (3, 8, 10, 11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38, 44, 53, 54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11, 26, 44, 54).Interestingly, most sequenced archaeal genomes also contain diverse sets of genes that encode type IV pilin-like proteins with little or no homology to archaeal flagellins (3, 39, 52). While often coregulated with pilB and pilC homologs, these genes are never found in clusters containing the motility-specific flaCDEFG and flaH homologs; however, the proteins they encode do contain class III signal peptides (52). Several of these proteins have been shown to be processed by an SPase III (4, 52). Moreover, in Sulfolobus solfataricus and Methanococcus maripaludis, some of these archaeal type IV pilin-like proteins were confirmed to form surface filaments that are distinct from the flagella (21, 22, 56). These findings strongly suggest that the genes encode subunits of pilus-like surface structures that are involved in functions other than swimming motility.In bacteria, type IV pili are multifunctional filamentous protein complexes that, in addition to facilitating twitching motility, mediate adherence to abiotic surfaces and make close intercellular associations possible (15, 17, 46). For instance, mating between Escherichia coli in liquid medium has been shown to require type IV pili (often referred to as thin sex pili), which bring cells into close proximity (29, 30, 57). Recent work has shown that the S. solfataricus pilus, Ups, is required not only for efficient adhesion to surfaces of these crenarchaeal cells but also for UV-induced aggregation (21, 22, 58). Frols et al. postulate that autoaggregation is required for DNA exchange under these highly mutagenic conditions (22). Halobacterium salinarum has also been shown to form Ca²⁺-induced aggregates (27, 28). Furthermore, conjugation has been observed in H. volcanii, which likely requires that cells be held in close proximity for a sustained period to allow time for the cells to construct the cytoplasmic bridges that facilitate DNA transfer between them (35).To determine the roles played by haloarchaeal flagella and other putative type IV pilus-like structures in swimming and surface motility, surface adhesion, autoaggregation, and conjugation, we constructed and characterized two mutant strains of H. volcanii, one lacking the genes that encode the flagellins and the other lacking pibD. Our analyses indicate that although this archaeon was previously thought to be nonmotile (14, 36), wild-type (wt) H. volcanii can swim in a flagellum-dependent manner. Consistent with the involvement of PibD in processing flagellins, the peptidase mutant is nonmotile. Unlike nonhalophilic archaea, however, the flagellum mutant can adhere to glass as effectively as the wild type. Conversely, the ΔpibD strain fails to adhere to glass surfaces, strongly suggesting that in H. volcanii surface adhesion involves nonflagellar, type IV pilus-like structures.     

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.African swine fever virus (ASFV) (61, 72) is the only known DNA-containing arbovirus and the sole member of the Asfarviridae family (24). Infection by this virus of its natural hosts, the wild swine warthogs and bushpigs and the argasid ticks of the genus Ornithodoros, results in a mild disease, often asymptomatic, with low viremia titers, that in many cases develops into a persistent infection (3, 43, 71). In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which the only available methods of disease control are the quarantine of the affected area and the elimination of the infected animals (51).The ASFV genome is a lineal molecule of double-stranded DNA of 170 to 190 kbp in length with convalently closed ends and terminal inverted repeats. The genome encodes more than 150 open reading frames, half of which lack any known or predictable function (16, 75).The virus particle, with an overall icosahedral shape and an average diameter of 200 nm (11), is organized in several concentric layers (6, 11, 15) containing more than 50 structural proteins (29). Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7, 34) on top of which the icosahedral capsid is assembled (26, 27, 31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11). Both forms of the virus, intracellular and extracellular, are infective (8).The assembly of ASFV particles occurs in the cytoplasm of the infected cell, in viral factories located close to the cell nucleus (6, 13, 49). ASFV factories possess several characteristics similar to those of the cellular aggresomes (35), which are accumulations of aggregates of cellular proteins that form perinuclear inclusions (44).Current models propose that ASFV assembly begins with the modification of endoplasmic reticulum (ER) membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. These ER-derived viral membranes represent the precursors of the inner viral envelope and are the first morphological evidence of viral assembly (7, 60). ASFV viral membrane precursors evolve into icosahedral intermediates and icosahedral particles by the progressive assembly of the outer capsid layer at the convex face of the precursor membranes (5, 26, 27, 31) through an ATP- and calcium-dependent process (19). At the same time, the core shell is formed underneath the concave face of the viral envelope, and the viral DNA and nucleoproteins are packaged and condensed to form the innermost electron-dense nucleoid (6, 9, 12, 69). However, the assembly of the capsid and the internal envelope appears to be largely independent of the components of the core of the particle, since the absence of the viral polyprotein pp220 during assembly produces empty virus-like particles that do not contain the core (9).Comparative genome analysis suggests that ASFV shares a common origin with the members of the proposed nucleocytoplasmic large DNA viruses (NCLDVs) (40, 41). The reconstructed phylogeny of NCLDVs as well as the similitude in the structures and organizations of the genomes indicates that ASFV is more closely related to poxviruses than to other members of the NCLDVs. A consensus about the origin and nature of the envelope of the immature form of vaccinia virus (VV), the prototypical poxvirus, seems to be emerging (10, 17, 20, 54). VV assembly starts with the appearance of crescent-shaped structures within specialized regions of the cytoplasm also known as viral factories (21, 23). The crescent membranes originate from preexisting membranes derived from some specialized compartment of the ER (32, 37, 52, 53, 67), and an operative pathway from the ER to the crescent membrane has recently been described (38, 39). VV crescents apparently grow in length while maintaining the same curvature until they become closed circles, spheres in three dimensions, called immature virions (IV) (22). The uniform curvature is produced by a honeycomb lattice of protein D13L (36, 70), which attaches rapidly to the membranes so that nascent viral membranes always appear to be coated over their entirety. The D13L protein is evolutionarily related to the capsid proteins of the other members of the NCLDV group, including ASFV, but lacks the C-terminal jelly roll motif (40). This structural difference is probably related to the fact that poxviruses are the only member of this group without an icosahedral capsid; instead, the spherical D13L coat acts as a scaffold during the IV stage but is discarded in subsequent steps of morphogenesis (10, 28, 46, 66). Thus, although crescents in VV and precursors of the inner envelope in ASFV are the first morphogenetic stages discernible in the viral factories of these viruses, they seem to be different in nature. Crescents are covered by the D13L protein and are more akin to the icosahedral intermediates of ASFV assembly, whereas ASFV viral membrane precursors are more similar to the naked membranes seen when VV morphogenesis is arrested by rifampin treatment (33, 47, 48, 50) or when the expression of the D13L and A17L proteins are repressed during infection with lethal conditional VV viruses (45, 55, 56, 68, 74, 76).Although available evidence strongly supports the reticular origin of the ASFV inner envelope (7, 60), the mechanism of acquisition remains unknown, and the number of membranes present in the inner envelope is controversial. The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7, 59, 60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope. In the case of VV, the coat of the D13L protein has been suggested to play a key role in the stabilization of the single membrane structure of the crescent (10, 17, 36), but the ASFV capsid protein p72 is not a component of the viral membrane precursors. The identification and functional characterization of the proteins involved in the generation of these structures are essential for the understanding of the mechanisms involved in these early stages of viral assembly. For this reason, we are focusing our interest on the study of abundant structural membrane proteins that reside at the inner envelope of the viral particle. We have shown previously that one of these proteins, p54, is essential for the recruitment of ER membranes to the viral factory (59). Repression of protein p54 expression has a profound impact on virus production and leads to an early arrest in virion morphogenesis, resulting in the virtual absence of membranes in the viral factory.Protein p17, encoded by the late gene D117L in the BA71V strain, is an abundant structural protein (60, 65). Its sequence, which is highly conserved among ASFV isolates (16), does not show any significant similarity with the sequences present in the databases. Protein p17 is an integral membrane protein (18) that is predicted to insert in membranes with a Singer type I topology and has been localized in the envelope precursors as well as in both intracellular and extracellular mature particles (60), suggesting that it resides at the internal envelope, the only membranous structure of the intracellular particles.In this work, we analyze the role of protein p17 in viral assembly by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent lethal conditional virus. The data presented indicate that protein p17 is essential for viral morphogenesis. The repression of this protein appears to block assembly at the level of viral precursor membranes, resulting in their accumulation at the viral factory.From the electron microscopy analysis of serial sections of viral factories at very early times during morphogenesis, we present experimental evidence that suggests that, during assembly, viral precursor membranes and core material organize into large helicoidal intermediates from which icosahedral particles emerge. The possible role of these structures during ASFV morphogenesis is discussed.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli

Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of β-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM⁻ cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.Cytokinesis in Escherichia coli and other gram-negative bacteria proceeds via the coordinated constriction of their envelope layers (outer membrane, inner membrane, and peptidoglycan [PG]) (12, 13, 34, 89). This coordination is achieved by a multi-protein division machine referred to as the septal ring or divisome (20). Assembly of the septal ring begins with the polymerization of the bacterial tubulin protein, FtsZ, into a ring structure just underneath the inner membrane at the prospective site of cell division (8). Once formed, this so-called Z-ring facilitates the recruitment of a number of essential and nonessential division proteins to the division site for the assembly of the trans-envelope divisome organelle (20).A major function of the cytokinetic machinery is to promote the synthesis of the PG layer that will eventually fortify the new poles of the developing daughter cells. PG is a polysaccharide polymer composed of repeating units of N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) linked by a β-1,4-glycosidic bond (46). Attached to the MurNAc sugar is a short peptide that is used to form cross-links between adjacent polysaccharide strands (46). Such cross-links allow for the construction of a cell-shaped PG meshwork that surrounds the cell membrane and protects it from osmotic rupture.A new wave of zonal PG synthesis is initiated at the division site during each cell cycle (23, 25, 72, 77, 91). Several of the major PG synthases called penicillin-binding proteins are components of the divisome organelle and play important roles in the synthesis of PG during division (7, 21, 62, 67, 73, 74, 80, 81, 88, 90). The septal PG layer produced by these and perhaps other components of the divisome is thought to be initially shared by the daughter cells (46). In gram-positive bacteria, this septal PG layer is typically split some time after the daughter cells have been compartmentalized by membrane fusion (11). In gram-negative bacteria, however, the septal PG layer is split shortly after it is formed to allow constriction of the outer membrane to closely follow that of the inner (cytoplasmic) membrane (12, 13, 34, 89). This gives rise to the characteristic constricted appearance of predivisional cells of E. coli and its relatives.PG hydrolysis is required to promote septal PG splitting and eventual daughter cell separation (87). E. coli, like many bacteria, encodes a vast array of factors with known or predicted PG hydrolase activity (at least 30 genes and 11 different protein families) (29, 31, 87). In most cases, the loss of individual PG hydrolase factors has little effect on growth and division, suggesting that there is significant functional overlap between the various hydrolases (87). This dearth of phenotypic information has consequently made it difficult to understand the physiological roles of PG hydrolases and identify the subset of these factors needed for septal PG splitting. An approach that has helped overcome this limitation in E. coli, however, has been the systematic deletion of all members of a particular PG hydrolase family from the genome (22, 44, 45, 63). Thus far, of all the families of PG hydrolases encoded by E. coli, the factors that play the predominant role in cell separation appear to be the LytC-type N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC (44, 45, 69). Loss of all three of these amidases results in a severe defect in cell separation and the formation of extremely long cell chains. This chaining phenotype can be exacerbated by the loss of members of other classes of PG hydrolases like the lytic transglycosylases or d,d-endopeptidases (44, 68). However, relative to strains defective for the amidases, mutants lacking multiple lytic transglycosylases or d,d-endopeptidases alone do not display significant chaining phenotypes in E. coli. These PG hydrolases therefore appear to be playing more of an ancillary role in cell separation.The LytM (lysostaphin/peptidase M23)-domain containing factors (referred to as LytM factors for convenience) are a widely distributed class of putative PG hydrolases that have been poorly characterized with regard to their role in PG biogenesis in E. coli and other bacteria (31). The most well-studied members of this family of factors, LytM and lysostaphin, are metallo-endopeptidases that cleave the pentaglycine cross-bridges found in staphylococcal PG (9, 30, 64). Based on this activity, other LytM factors are also likely to be PG hydrolases but with altered cleavage specificity because pentaglycine cross-bridges are only found among the staphylococci (75). Indeed, the LytM protein, gp13, from the Bacillus subtilis phage Φ29 was recently shown to be a d,d-endopeptidase that cleaves the meso-diaminopimelic acid-d-Ala cross-links of B. subtilis PG (17).E. coli encodes four factors with identifiable LytM-domains: EnvC, NlpD, YgeR, and YebA (29) (Fig. ​(Fig.1).1). Of the four, only EnvC has been studied in appreciable detail. EnvC⁻ mutants have a mild cell separation (chaining) defect when grown in medium containing salt and a severe division defect when grown at high temperatures in medium lacking salt (5, 42, 48, 71). In addition, purified EnvC protein was found to possess PG hydrolase activity using a gel-based zymogram assay, and an EnvC-green fluorescent protein (GFP) fusion exported to the periplasm via the Tat system was shown to be recruited to the division site (5). In all, these results support a model in which EnvC is targeted to the division site to participate directly in septal PG splitting and daughter cell separation.Open in a separate windowFIG. 1.Predicted domain structure of the E. coli LytM factors. Shown is a diagram depicting the predicted domain architecture of the four E. coli factors with identifiable LytM domains. Abbreviations: LytM, LytM domain; LysM, LysM PG-binding domain (29); CC, coiled coil; T, transmembrane domain; SS, signal sequence; SS*, lipoprotein signal sequence. The UniProtKB/Swiss-Prot accession numbers are as follows: EnvC (P37690), NlpD (P0ADA3), YebA (P0AFS9), and YgeR (Q46798).In the present study, we investigated the physiological role(s) of the entire set of E. coli LytM factors by generating mutant strains lacking all possible combinations of them. We found that, like the amidases, LytM factors play a critical role in daughter cell separation. Furthermore, studies of their subcellular localization revealed that NlpD is recruited to the division site along with EnvC, indicating that both of these LytM factors are likely to be participating directly in the septal PG splitting process. We also discovered that mutants lacking multiple LytM factors lyse more slowly and display an altered morphological response relative to wild-type (WT) cells when they are treated with ampicillin. This finding suggests that in addition to cell separation, LytM proteins play a role in the lytic mechanism of β-lactam antibiotics.     

Characterization of the Synechocystis Strain PCC 6803 Penicillin-Binding Proteins and Cytokinetic Proteins FtsQ and FtsW and Their Network of Interactions with ZipN

Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.The peptidoglycan layer (PG) of bacterial cell wall is a major determinant of cell shape, and the target of our best antibiotics. It is built from long glycan strands of repeating disaccharides cross-linked by short peptides (38). The resultant meshwork structure forms a strong and elastic exoskeleton essential for maintaining shape and withstanding intracellular pressure. Cell morphogenesis and division have been essentially studied in the rod-shaped organisms Escherichia coli and Bacillus subtilis, which divide through a single medial plane (8, 10, 21, 23). These organisms have two modes of cell wall synthesis: one involved in cell elongation and the second operating in septation (2). Each mode of synthesis is ensured by specific protein complexes involving factors implicated in the last step of PG synthesis (2). The complete assembly of PG requires a glycosyl transferase that polymerizes the glycan strands and a transpeptidase that cross-links them via their peptide side chains (35). Both activities are catalyzed by penicillin-binding proteins (PBPs), which can be divided into three classes: class A and class B high-molecular-weight (HMW) PBPs and class C low-molecular-weight (LMW) PBPs (35).Class A PBPs exhibit both transglycosylase and transpeptidase activities. In E. coli, they seem to be nonspecialized (2), as they operate in the synthesis of both cylindrical wall (cell elongation) and septal PG (cytokinesis). In B. subtilis, PBP1 (class A) is partially localized to septal sites and its depletion leads to cell division defects (31).Class B PBPs, which comprise two proteins in most bacteria, are monofunctional transpeptidases (35), each involved in longitudinal and septal growth of cell wall, respectively (36). In E. coli, this protein, PBP3, is also termed FtsI, because it belongs to the Fts group of cell division factors whose depletion leads to the filamentation phenotype (11). These at least 10 Fts proteins are recruited to the division site at mid-cell in the following sequential order: FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL/FtsB, FtsW, FtsI, and FtsN (11). The cytoplasmic protein FtsZ is the first recruited to the division site, where it polymerizes in a ring-like structure (1), which serves as a scaffold for the recruitment of the other Fts proteins and has been proposed to drive the division process (6). Together the Fts proteins form a complex machine coordinating nucleoid segregation, membrane constriction, septal PG synthesis, and possibly membrane fusion.Unlike the other PBPs, class C PBPs do not operate in PG synthesis but rather in maturation or recycling of PG during cell septation (35). They are subdivided into four types. Class C type 5 PBP removes the terminal d-alanine residue from pentapeptide side-chains (dd-carboxypeptidase activity). Types 4 and 7 are able to cleave the peptide cross-links (endopeptidase activity). Finally, type AmpH, which does not have a defined enzymatic activity, is believed to play a role in the normal course of PG synthesis, remodeling or recycling (for a review, see reference 35).In contrast to rod-shaped bacteria, less is known concerning PG synthesis, morphogenesis, and cytokinesis, and their relationships, in spherical-celled bacteria, even though a wealth of them have a strong impact on the environment and/or human health. Furthermore, unlike rod-shaped bacteria spherical-celled bacteria possess an infinite number of potential division planes at the point of greater cell diameter, and they divide through alternative perpendicular planes (26, 36, 37, 39). The spherical cells of Staphylococcus aureus seem to insert new PG strands only at the septum, and accordingly the unique class A PBP localizes at the septum during cell division (36). In contrast, the rugby-ball-shaped cells of Streptococcus pneumoniae synthesize cell wall at both the septum and the neighboring region called “equatorial rings” (36). Accordingly, class A PBP2a and PBP1a were found to operate in elongation and septation, respectively (29).In cyanobacteria, which are crucial to the biosphere in using solar energy to renew the oxygenic atmosphere and which make up the biomass for the food chain (7, 30, 40), cell division is currently investigated in two unicellular models with different morphologies: the rod-shaped Synechococcus elongatus strain PCC 7942 (19, 28) and the spherical-celled Synechocystis strain PCC 6803 (26), which both possess a small fully sequenced genome (http://genome.kazusa.or.jp/cyanobase/) that is easily manipulable (18). In both organisms FtsZ and ZipN/Arc6, a protein occurring only in cyanobacteria (ZipN) and plant chloroplasts (Arc6), were found to be crucial for cytokinesis (19, 26, 28) and to physically interact with each other (25, 26). Also, interestingly, recent studies of cell division in the filamentous cyanobacterium Anabaena (Nostoc) strain PCC 7120, showed that this process is connected with the differentiation of heterocysts, the cells dedicated to nitrogen fixation (34).In a continuous effort to study the cell division machine of the unicellular spherical cyanobacterium Synechocystis, we have presently characterized its eight presumptive PBPs (22) that define three classes and the putative cytokinetic proteins FtsQ and FtsW, as well as their network of interactions between each other and ZipN. Both FtsI and FtsQ were found to be key players in cell division in interacting with ZipN and class A PBPs. Consequently, ZipN in interacting with FtsZ (26), FtsI, and FtQ, like the FtsA protein of E. coli, could play a role similar to FtsA, which is absent in cyanobacteria and chloroplasts.     

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

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