Cyclic-di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa: the pilY1 Gene and Its Impact on Surface-Associated Behaviors

The intracellular signaling molecule cyclic-di-GMP (c-di-GMP) has been shown to influence surface-associated behaviors of Pseudomonas aeruginosa, including biofilm formation and swarming motility. Previously, we reported a role for the bifA gene in the inverse regulation of biofilm formation and swarming motility. The bifA gene encodes a c-di-GMP-degrading phosphodiesterase (PDE), and the ΔbifA mutant exhibits increased cellular pools of c-di-GMP, forms hyperbiofilms, and is unable to swarm. In this study, we isolated suppressors of the ΔbifA swarming defect. Strains with mutations in the pilY1 gene, but not in the pilin subunit pilA gene, show robust suppression of the swarming defect of the ΔbifA mutant, as well as its hyperbiofilm phenotype. Despite the ability of the pilY1 mutation to suppress all the c-di-GMP-related phenotypes, the global pools of c-di-GMP are not detectably altered in the ΔbifA ΔpilY1 mutant relative to the ΔbifA single mutant. We also show that enhanced expression of the pilY1 gene inhibits swarming motility, and we identify residues in the putative VWA domain of PilY1 that are important for this phenotype. Furthermore, swarming repression by PilY1 specifically requires the diguanylate cyclase (DGC) SadC, and epistasis analysis indicates that PilY1 functions upstream of SadC. Our data indicate that PilY1 participates in multiple surface behaviors of P. aeruginosa, and we propose that PilY1 may act via regulation of SadC DGC activity but independently of altering global c-di-GMP levels.Pseudomonas aeruginosa forms surface-attached communities known as biofilms, and this microbe is also capable of surface-associated motility, including twitching and swarming. The mechanism by which cells regulate and coordinate these various surface-associated behaviors, or how these microbes transition from one surface behavior to another, has yet to be elucidated. Given that P. aeruginosa is capable of such diverse surface-associated lifestyles, this Gram-negative organism serves as a useful model to address questions regarding the regulation of surface-associated behaviors.Recent studies indicate that biofilm formation and swarming motility by P. aeruginosa are inversely regulated via a common pathway (12, 27, 37). Important factors that influence early biofilm formation by P. aeruginosa strain PA14 include control of flagellar motility and the robust production of the Pel exopolysaccharide (EPS). Swarming occurs when cells move across a hydrated, viscous semisolid surface, and like biofilm formation, flagellar function is important for this surface-associated motility. Additionally, swarming requires production of rhamnolipid surfactant acting as a surface-wetting agent (25, 58). In contrast to biofilm formation, swarming motility is enhanced in strains which are defective for the production of Pel EPS (12).The inverse regulation of biofilm formation and swarming motility is reminiscent of the regulation of sessile and motile behaviors that occurs in a wide range of bacterial species via the intracellular signaling molecule cyclic-di-GMP (c-di-GMP) (17, 24, 50, 51, 56). High levels of this signaling molecule promote sessile behaviors and inhibit motility, whereas low levels of c-di-GMP favor motile behaviors (8, 9, 22, 56). Recently, we reported that the BifA phosphodiesterase, which catalyzes the breakdown of c-di-GMP, inversely regulates biofilm formation and swarming motility (27). In addition, Merritt et al. reported that SadC, a diguanylate cyclase (DGC) which synthesizes c-di-GMP, participates with BifA to modulate cellular c-di-GMP levels and thus regulate biofilm formation and swarming motility (37).Consistent with a role for BifA as a c-di-GMP phosphodiesterase, ΔbifA mutants exhibit increased cellular pools of c-di-GMP relative to the wild type (WT) (27). Phenotypically, ΔbifA mutants form hyperbiofilms and are unable to swarm. The hyperbiofilm phenotype of the ΔbifA mutant results largely from increased synthesis of the pel-derived polysaccharide; that is, the ΔbifAΔpel double mutant shows a marked decrease in biofilm formation compared to the ΔbifA mutant (27). Interestingly, elevated Pel polysaccharide production alone is not sufficient to explain the swarming defect of the ΔbifA mutant, as the ΔbifAΔpel double mutant recovers only minimal swarming ability (27). These data indicate that high levels of c-di-GMP inhibit swarming motility in a largely Pel-independent manner.To better understand how elevated c-di-GMP levels in the cell inhibit swarming motility, we exploited the swarming defect of the ΔbifA mutant, and using a genetic screen, we identified suppressors in the ΔbifA background that restored the ability to swarm. Here we report a role for the PilY1 protein in repression of swarming motility in the ΔbifA mutant background. Our data are consistent with a model in which PilY1 functions upstream of the c-di-GMP diguanylate cyclase SadC to regulate swarming motility by P. aeruginosa.     

The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms in Vibrio fischeri

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the ΔbinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.Bacterial biofilms play important roles in the environment and in interactions with eukaryotic hosts (for reviews, see references 17 and 32). Exopolysaccharides are a major component of biofilms (23), and many bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Salmonella spp., have the ability to produce multiple different exopolysaccharides (23). For some of these bacteria, it has been demonstrated that the different polysaccharides contribute to biofilm formation in different settings. For example, several strains of Salmonella require an exopolysaccharide called O-antigen capsule to form biofilms on human gallstones but not to form biofilms on glass or plastic (11). Conversely, the exopolysaccharides cellulose and colanic acid are required for optimal biofilm formation by Salmonella spp. on glass and plastic but are not required for biofilm formation on human gallstones (11, 35). For some bacteria, a particular exopolysaccharide promotes attachment to one surface but seems to interfere with attachment to other surfaces. One example of this is E. coli O157:H7, which requires the exopolysaccharides poly-β-1,6-N-acetylglucosamine (PGA), colanic acid, and cellulose for optimal binding to alfalfa sprouts and plastic (27). In contrast, these polysaccharides are not required for binding by E. coli O157:H7 cells to human intestinal epithelial (Caco-2) cells and binding was actually enhanced in cellulose and PGA mutants, suggesting that while these polysaccharides are important for attachment to sprouts and plastic, they interfere with attachment to Caco-2 cells (27).The marine bacterium Vibrio fischeri is known to produce at least two different exopolysaccharides that play roles in biofilm formation, the symbiosis polysaccharide (Syp) and cellulose (12, 59, 60). The Syp polysaccharide is critical for the formation of a biofilm-like aggregate at the initiation of symbiosis with the Hawaiian bobtail squid Euprymna scolopes (59). The natural condition(s) under which V. fischeri cells use cellulose in biofilm formation is not yet known. However, in other bacteria, cellulose contributes to the ability to attach to a variety of surfaces, including plant roots, other plant cells, mammalian epithelial cells, glass, and plastic (27, 28, 31, 35, 37, 45).Although V. fischeri biofilm formation appears to be important for interaction with its symbiotic host and is likely also to be important in the marine environment outside the host, wild-type V. fischeri cells do not produce substantial biofilms under a variety of standard laboratory conditions. V. fischeri''s ability to form biofilms in culture, however, is greatly enhanced when the syp biosynthetic gene cluster (Fig. ​(Fig.1)1) is induced by overexpression of the response regulator SypG, the sensor kinase RscS, or the sensor kinase SypF (12, 59, 60).Open in a separate windowFIG. 1.The binA gene and its predicted protein. (A) The binA gene (VFA1038) is located downstream from and oriented in the same direction as the syp gene locus. Individual genes are indicated by block arrows, and the four known or putative promoters within the syp locus are indicated by line arrows. (B) The BinA protein (630 amino acids [aa]) is predicted to have three domains, GAF (∼Q20 to L151), GGDEF (∼H205 to A338), and EAL (∼L374 to D611), as indicated. Only the EAL domain is well conserved.In culture, V. fischeri also forms biofilms when cellulose production is induced. Cellulose contributes to the biofilms formed when either SypF or the putative response regulator VpsR is overexpressed (12). Additionally, overexpression of the diguanylate cyclase MifA induces biofilm formation and increases binding to two dyes that are cellulose indicators, Congo red and calcofluor (34, 37, 54).The product of diguanylate cyclase activity, cyclic diguanylate (c-di-GMP), is an intracellular signaling molecule that plays an important role in regulating biofilm formation and motility (reviewed in references 10, 22, 38, 39, 48, and 56). C-di-GMP is produced from two GTP molecules by diguanylate cyclases with conserved GGDEF domains and is depleted by hydrolysis to linear pGpG by phosphodiesterases (PDEs) with EAL or HD-GYP domains. There is evidence of increased cellulose production in response to increased c-di-GMP levels in many bacteria (36, 41). In general, high levels of c-di-GMP enhance biofilm formation and low levels of c-di-GMP enhance motility (39).Here we report that V. fischeri biofilm formation also increases in the absence of BinA, a GGDEF/EAL domain protein encoded by a gene that is adjacent to the syp cluster. We also report the discovery of genes involved in biofilm formation by binA mutants and the identification of amino acids that are critical to BinA activity. Finally, we suggest a mechanism by which the production of two different polysaccharides may be coordinated by BinA.     

Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.Cryptococcus neoformans is a fungal pathogen that is ubiquitous in the environment and enters the body via the inhalation of airborne particles. The C. neoformans cell is surrounded by a layer of polysaccharide that consists predominantly of glucuronoxylomannan (GXM), which forms a protective capsule around the microbe. The capsule has been shown to be essential for virulence in murine models of infection (5-7) and, thus, is considered a key virulence factor. C. neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immune systems, and is a significant problem in AIDS patients (21, 31). The most common manifestation of cryptococcosis is meningoencephalitis.Biofilms are communities of microbes that are attached to surfaces and held together by an extracellular matrix, often consisting predominantly of polysaccharides (8, 10). A great deal is known about bacterial biofilms (3, 9, 24, 30), but fungal biofilm formation is much less studied. Candida albicans is known to synthesize biofilms (11, 28, 29), as is C. neoformans. Biofilm-like structures consisting of innumerable cryptococcal cells encased in a polysaccharide matrix have been reported in human cases of cryptococcosis (32). Biofilm formation confers upon the microbe the capacity for drug resistance, and microbial cells in biofilms are less susceptible to host defense mechanisms (2, 4, 9, 12). In this regard, cells within C. neoformans biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19).Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic infection (10, 13, 15, 27). C. neoformans biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). C. neoformans can form biofilms on the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32).The polysaccharide capsule of C. neoformans is essential for biofilm formation (18), and biofilm formation involves the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in solution could inhibit biofilm formation through a process that presumably involves interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of C. neoformans cells on cryptococcal biofilm formation has not been explored. In this paper, we report that the monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize C. neoformans to surfaces, a process that promotes biofilm formation. Interestingly, we identified planktonic variant C. neoformans cells that appeared to escape from the biofilm, but whose functions are not known. The results provide new insights on biofilm formation.     

Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.Yeast sporulation is a developmental process that involves multiple, sequential events that need to be tightly coordinated (59, 68). In the fission yeast Schizosaccharomyces pombe, when cells of opposite mating type (h⁺ and h⁻) are mixed and shifted to conditions of nitrogen starvation, cell fusion and karyogamy occur to form a diploid zygote, which then undergoes premeiotic DNA replication, the two meiotic divisions, formation of the spore envelopes (comprising the plasma membrane and a specialized cell wall), and maturation of the spores (74, 81). At the onset of meiosis II, precursors of the spore envelopes, the forespore membranes (FSMs), are formed by the fusion of vesicles at the cytoplasmic surface of each spindle-pole body (SPB) and then extend to engulf the four nuclear lobes (the nuclear envelope does not break down during meiosis), thus capturing the haploid nuclei, along with associated cytoplasm and organelles, to form the nascent spores (55, 68, 81). How the FSMs recognize and interact with the nuclear envelope, extend in a properly oriented manner, and close to form uniformly sized spherical spores is not understood, and study of this model system should also help to elucidate the more general question of how membranes obtain their shapes in vivo.It has been shown that both the SPB and the vesicle trafficking system play important roles in the formation and development of the FSM and of its counterpart in the budding yeast Saccharomyces cerevisiae, the prospore membrane (PSM). In S. pombe, the SPB changes its shape from a compact dot to a crescent at metaphase of meiosis II (26, 29), and its outer plaque acquires meiosis-specific components such as Spo2, Spo13, and Spo15 (30, 57, 68). This modified outer plaque is required for the initiation of FSM assembly. In S. cerevisiae, it is well established that various secretory (SEC) gene products are required for PSM formation (58, 59). Similarly, proteins presumably involved in the docking and/or fusion of post-Golgi vesicles and organelles in S. pombe, such as the syntaxin-1A Psy1, the SNAP-25 homologue Sec9, and the Rab7 GTPase homologue Ypt7, are also required for proper FSM extension (34, 53, 54). Consistent with this hypothesis, Psy1 disappears from the plasma membrane upon exit from meiosis I and reappears in the nascent FSM.Phosphoinositide-mediated membrane trafficking also contributes to the development of the FSM. Pik3/Vps34 is a phosphatidylinositol 3-kinase whose product is phosphatidylinositol 3-phosphate [PtdIns(3)P] (35, 72). S. pombe cells lacking this protein exhibit defects in various steps of FSM formation, such as aberrant starting positions for extension, disoriented extension and/or failure of closure, and the formation of spore-like bodies near, rather than surrounding, the nuclei, suggesting that Pik3 plays multiple roles during sporulation (61). The targets of PtdIns(3)P during sporulation appear to include two sorting nexins, Vps5 and Vps17, and the FYVE domain-containing protein Sst4/Vps27. vps5Δ and vps17Δ mutant cells share some of the phenotypes of pik3Δ cells (38). sst4Δ cells also share some of the phenotypes of pik3Δ cells but are distinct from vps5Δ and vps17Δ cells, consistent with the hypothesis that Pik3 has multiple roles during sporulation (62).Membrane trafficking processes alone do not seem sufficient to explain how the FSMs and PSMs extend around and engulf the nuclei, suggesting that some other mechanism(s) must regulate and orient FSM/PSM extension. The observation that the FSM is attached to the SPB until formation of the immature spore is complete (68) suggests that the SPB may regulate FSM extension. In addition, the leading edge of the S. cerevisiae PSM is coated with a complex of proteins (the LEPs) that appear to be involved in PSM extension (51, 59). S. pombe Meu14 also localizes to the leading edge of the FSM, and deletion of meu14 causes aberrant FSM formation in addition to a failure in SPB modification (60). However, it has remained unclear whether the SPB- and LEP-based mechanisms are sufficient to account for the formation of closed FSMs and PSMs of proper size and position (relative to the nuclear envelope), and evidence from S. cerevisiae has suggested that the septin proteins may also be involved.The septins are a conserved family of GTP-binding proteins that were first identified in S. cerevisiae by analysis of the cytokinesis-defective cdc3, cdc10, cdc11, and cdc12 mutants (41). Cdc3, Cdc10, Cdc11, and Cdc12 are related to each other in sequence and form an oligomeric complex that localizes to a ring in close apposition to the plasma membrane at the mother-bud neck in vegetative cells (12, 20, 25, 41, 47, 77). The septin ring appears to be filamentous in vivo (12), and indeed, the septins from both yeast (11, 20) and metazoans (31, 36, 69) can form filaments in vitro. The yeast septin ring appears to form a scaffold for the localization and organization of a wide variety of other proteins (8, 22), and it forms a diffusion barrier that constrains movement of membrane proteins through the neck region (7, 8, 73). In metazoan cells, the septins are involved in cytokinesis but are also implicated in a variety of other cellular processes, such as vesicular transport, organization of the actin and microtubule cytoskeletons, and oncogenesis (27, 70).In S. cerevisiae, a fifth septin (Shs1) is also expressed in vegetative cells, but the remaining two septin genes, SPR3 and SPR28, are expressed at detectable levels only during sporulation (15, 17). In addition, at least some of the vegetatively expressed septins are also present in sporulating cells (17, 48), and one of them (Cdc10) is expressed at much higher levels there than in vegetative cells (32). The septins present during sporulation are associated with the PSM (15, 17, 48, 51), and their normal organization there depends on the Gip1-Glc7 protein phosphatase complex (71). However, it has been difficult to gain insight into the precise roles of the septins during sporulation in S. cerevisiae (59), because some septins are essential for viability during vegetative growth, and the viable mutants have only mild phenotypes during sporulation (15, 17), possibly because of functional redundancy among the multiple septins.S. pombe seemed likely to provide a better opportunity for investigating the role of septins during spore formation. There are seven septin genes (spn1⁺ to spn7⁺) in this organism (23, 41, 63). Four of these genes (spn1⁺ to spn4⁺) are expressed in vegetative cells, and their products form a hetero-oligomeric complex that assembles during cytokinesis into a ring at the division site (2, 3, 10, 76, 79). The septin ring is important for proper targeting of endoglucanases to the division site (44), and septin mutants show a corresponding delay in cell separation (10, 41, 44, 76). However, even the spn1Δ spn2Δ spn3Δ spn4Δ quadruple mutant is viable and grows nearly as rapidly as the wild type (our unpublished results), a circumstance that greatly facilitates studies of the septins'' role during sporulation.spn5⁺, spn6⁺, and spn7⁺ are expressed at detectable levels only during sporulation (1, 45, 78; our unpublished results), and spn2⁺, like its orthologue CDC10 (see above), is strongly induced (45), but the roles of the S. pombe septins in sporulation have not previously been investigated. In this study, we show that the septins are important for the orientation of FSM extension, suggesting that the septins may have a more general role in dynamic membrane organization and shape determination.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Roles of ExoI and SbcCD Nucleases in “Reckless” DNA Degradation in recA Mutants of Escherichia coli

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this “reckless” DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.The RecA protein plays a central role in homologous recombination and recombinational DNA repair in Escherichia coli, as well as in other bacterial species. It catalyzes the key stages of the recombination process—homologous pairing and DNA strand exchange. Cells carrying null mutations in the recA gene are completely deficient for homologous recombination and are extremely sensitive to DNA-damaging agents (for a review, see references 21, 24, and 25). Populations of recA null mutants contain a large proportion (50 to 60%) of nonviable cells, reflecting the inability of these mutants to repair spontaneously occurring DNA damage (31). Also, exponentially growing recA cells display pronounced spontaneous DNA degradation that presumably starts at the sites of DNA damage and that depends on RecBCD nuclease (ExoV) activity (5, 48). This phenotype of recA cells is aggravated after DNA-damaging treatment, such as UV irradiation (48).According to the present data, the majority of RecA-catalyzed DNA transactions in E. coli start with binding of the RecA protein onto single-stranded DNA (ssDNA) substrates. This binding is mediated by the RecBCD and/or RecFOR protein, which helps RecA to overcome hindrance imposed by the SSB protein during competition for the DNA substrate. The RecBCD and RecFOR proteins begin RecA polymerization on ssDNA, giving rise to a nucleoprotein filament that is indispensable for further recombination reactions (3, 33; reviewed in reference 44).The RecBCD enzyme is crucial for initiation of recombinational processes at double-stranded DNA (dsDNA) ends (or breaks [DSBs]) in wild-type E. coli (a set of reactions known as the RecBCD pathway) (9, 43, 44). Upon recognizing a blunt or nearly blunt dsDNA end and binding to it, RecBCD acts as a combination of powerful helicase and nuclease, thus unwinding and simultaneously degrading both strands of the DNA duplex. After encountering a specific octanucleotide sequence designated Chi, the strong 3′-5′ nuclease activity of the enzyme is attenuated and a weaker 5′-3′ nuclease activity is upregulated (1). This Chi-dependent modification allows RecBCD to create a long 3′ ssDNA tail and to direct the loading of RecA protein onto it (2, 3). In vivo data suggest that this transition of RecBCD from a nuclease to a recombinase mode of action requires the presence of the RecA protein, suggesting that the two proteins might interact (27).In wild-type E. coli cells, the RecFOR protein complex works predominantly on DNA gaps, which may arise in chromosomes due to replication forks passing over the noncoding lesions (e.g., UV-induced pyrimidine dimers) or may be present in replication forks stalled at different obstacles in DNA (44). On the other hand, the RecFOR complex has an important role in recBC sbcBC(D) mutant cells, replacing the RecA-loading activity of RecBCD during recombination reactions starting from dsDNA ends. Recombination reactions mediated by RecFOR proteins are termed the RecF (or RecFOR) pathway (44).Cells mutated in the recB and/or recC gene exhibit strong deficiency in conjugational and transductional recombination, as well as in the repair of DSBs (8, 21). These defects can be rectified by extragenic sbcB and sbcC(D) suppressor mutations that inactivate two nucleases, thus enabling full efficiency of the RecF pathway on dsDNA ends (21, 44). The sbcB gene (also designated xonA) encodes exonuclease I (ExoI), the enzyme that digests ssDNA in the 3′-5′ direction (23). The sbcC and sbcD genes encode subunits of the SbcCD nuclease, which acts both as an endonuclease that cleaves hairpin structures and as an exonuclease that degrades linear dsDNA molecules (10, 11). Inactivation of either of the two subunits leads to the loss of SbcCD enzyme activity (18).The exact mechanism of activation of the RecF pathway by sbc mutations is not completely understood. A plausible explanation is that inactivation of ExoI and SbcCD nucleases is necessary to prevent the degradation of recombinogenic 3′ DNA ends created in a RecBCD-independent manner (8, 23, 38, 45, 46). It was recently shown that the sbcB15 mutant allele (encoding a protein without nucleolytic activity) (37) is a better suppressor of the RecBCD⁻ phenotype than an sbcB deletion (50), suggesting that some nonnucleolytic activity of ExoI may also contribute to the efficiency of the RecF pathway (46, 50).ExoI and SbcCD are usually viewed as enzymes with inhibitory roles in recombination due to their deleterious actions on the RecF pathway. However, some results suggest that these enzymes could also have stimulatory roles in recombination reactions proceeding on the RecBCD pathway. Genetic experiments with UV-irradiated E. coli cells indicated that ExoI and SbcCD might be involved in blunting radiation-induced DNA ends prior to RecBC(D) action (38, 45, 46). Such a role of ExoI and SbcCD seems to be particularly critical in recD recF mutants, in which the majority of DSB repair depends on the RecBC enzyme (38). It was also suggested that the blunting roles of the two nucleases may be required during conjugational recombination (16, 46).In this work, we studied the effects of sbcB (xonA) and sbcD mutations on DNA degradation occurring spontaneously in exponentially growing recA mutant cells, as well as on DNA degradation induced in recA mutants by UV irradiation. We have demonstrated that in both cases DNA degradation is strongly reduced in recA mutants that carry in addition a combination of xonA and sbcD null mutations. The results described in this paper suggest that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells.     

Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the template-dependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science.Several model systems for biochemical study of viral RNA-dependent RNA synthesis exist (4, 19, 20, 25, 37, 42). Well-characterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47-49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60).RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed conformation, even in the absence of template, in contrast to DNA-dependent RNA polymerases, which transition from open to closed complexes upon template recognition (13). The closed form of the phage φ6 RdRp has been proposed to allow specific recognition of the single-stranded viral RNA (7). The template channel formed by the closed structure, however, is too narrow to accommodate the partially duplexed RNA that forms during RNA synthesis, and hence, the closed conformation needs to undergo significant rearrangements in the ternary complex. Biswal et al. (3) have captured an X-ray crystallographic structure of a partially open conformation of the HCV RdRp. Bovine viral diarrhea virus (BVDV) RdRp was also shown to exist in a partially open conformation (11). Ranjith-Kumar and Kao (49) demonstrated that the HCV RdRp could initiate RNA synthesis from a circular RNA template, and thus, the threading of a single-stranded RNA into the template channel is not required for de novo-initiated RNA synthesis. Altogether, these results raise the possibility that the HCV RdRp can undergo rearrangements from the closed conformation seen in the crystal structure prior to de novo initiation.A secondary structure that extends from the finger to the thumb subdomains, named the Δ1 loop, has been proposed to serve as a gate to cover the template channel and regulate the switch from de novo initiation to elongation (5, 10). Mutations that affect the interaction between the Δ1 loop and the mostly hydrophobic residues that it contacts have resulted in polymerases that are defective for de novo initiation but can bind to partially duplexed RNA and can extend from the 3′ terminus of an RNA primer (10).Two general models for RNA synthesis by the HCV RdRp can be proposed (Fig. ​(Fig.1).1). The first posits that the HCV RdRp functions as a monomer at least during de novo initiation because the closed template channel is needed for specific recognition of the template (5, 7, 10). It was presumed that the Δ1 loop and thumb domain interaction in the HCV RdRp is stable and mutations that disrupted this interaction would render the enzyme catalytically inactive (5, 24). However, a deletion of five residues in the tip of the Δ1 loop did not prevent RNA synthesis from a primed template by the polymerase (10). Furthermore, a genotype 2a RdRp was crystallized in a form with altered interaction between the Δ1 loop and thumb domain in comparison to the 1b RdRp (3). Interestingly, a low-affinity GTP binding site exists on the thumb domain close to the base of the Δ1 loop binding pocket. GTP binding at this site has been proposed to stabilize the Δ1 loop and thumb domain interactions, favoring the closed monomer model (6). A second model is based on the reports that HCV RdRp can oligomerize and that oligomerization increases its activity (12, 16, 46, 54). The dimer could be active due to either the second subunit increasing the stability of the Δ1 loop and thumb interactions in the first subunit to increase de novo initiation or the two subunits forming a common template-binding domain (Fig. ​(Fig.1).1). Here we have attempted to determine whether monomers or oligomers of the HCV RdRp can better perform de novo initiation using biochemical and biophysical analyses.Open in a separate windowFIG. 1.Models for RNA synthesis by the HCV RdRp. The monomer model is based on the central tenet that intramolecular interactions within an RdRp molecule regulate the modes of RNA synthesis. The curved arrow represents the possible orientation of the template RNA. The oligomer model is an adaptation from the dimer model of the norovirus RdRp (18). T, P, and F represent the thumb, palm, and finger domains, respectively, in different shades of gray, and the thick black line connecting the thumb and finger domains represents the Δ1 loop.     

Conjugative Plasmid Transfer and Adhesion Dynamics in an Escherichia coli Biofilm

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.Bacterial biofilms are complex communities of bacterial cells living in close association with a surface (17). Bacterial cells in these protected environments are often resistant to multiple factors, including antimicrobials, changes in the pH, oxygen radicals, and host immune defenses (19, 38). Biofilm formation is a property of many bacterial species, and a range of molecular mechanisms that facilitate this process have been described (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form a biofilm is dependent on the production of adhesins on the bacterial cell surface. In Escherichia coli, biofilm formation is enhanced by the production of certain types of fimbriae (e.g., type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g., autotransporter proteins such as antigen 43, AidA, TibA, EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).The close proximity of bacterial cells in biofilms creates an environment conducive for the exchange of genetic material. Indeed, plasmid-mediated conjugation in monospecific and mixed E. coli biofilms has been demonstrated (6, 18, 24, 31). The F plasmid represents the best-characterized conjugative system for biofilm formation by E. coli. The F pilus mediates adhesion to abiotic surfaces and stabilizes the biofilm structure through cell-cell interactions (16, 30). Many other conjugative plasmids also contribute directly to biofilm formation upon derepression of the conjugative function (16).One example of a conjugative system employed by gram-negative Enterobacteriaceae is the type 4 secretion (T4S) system. The T4S system is a multisubunit structure that spans the cell envelope and contains a secretion channel often linked to a pilus or other surface filament or protein (8). The Agrobacterium tumefaciens VirB-VirD4 system is the archetypical T4S system and is encoded by 11 genes in the virB operon and one gene (virD4) in the virD operon (7, 8). Genes with strong homology to genes in the virB operon have also been identified on other conjugative plasmids. For example, the pilX1 to pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to virB11 genes are highly conserved at the nucleotide level (28).We recently described identification and characterization of the mrk genes encoding type 3 fimbriae in a uropathogenic strain of E. coli isolated from a patient with a nosocomial catheter-associated urinary tract infection (CAUTI) (29). The mrk genes were located on a conjugative plasmid (pMAS2027) and were strongly associated with biofilm formation. In this study we determined the entire sequence of plasmid pMAS2027 and revealed the presence of conjugative transfer genes homologous to the pilX1 to pilX11 genes of E. coli R6K (in addition to the mrk genes). We show here that biofilm formation is driven primarily by type 3 fimbriae and that the T4S apparatus is unable to mediate biofilm growth in the absence of the mrk genes. Finally, we demonstrate that conjugative transfer of pMAS2027 within a mixed biofilm confers biofilm formation properties on recipient cells due to acquisition of the type 3 fimbria-encoding mrk genes.     

Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.The serovars of Salmonella enterica are diverse, infect a broad array of hosts, and cause significant morbidity and mortality in impoverished and industrialized nations worldwide. S. enterica serovar Typhi is the etiologic agent of typhoid fever, a severe illness characterized by sustained bacteremia and a delayed onset of symptoms that afflicts approximately 20 million people each year (14, 19). Serovar Typhi can establish a chronic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment.There is a strong correlation between gallbladder abnormalities, particularly gallstones, and development of the asymptomatic Salmonella carrier state (47). Antibiotic regimens are typically ineffective in carriers with gallstones (47), and these patients have an 8.47-fold-higher risk of developing hepatobiliary carcinomas (28, 46, 91). Elimination of chronic infections usually requires gallbladder removal (47), but surgical intervention is cost-prohibitive in developing countries where serovar Typhi is prevalent. Thus, understanding the progression of infection to the carrier state and developing alternative treatment options are of critical importance to human health.The formation of biofilms on gallstones has been hypothesized to facilitate enhanced colonization of and persistence in the gallbladder. Over the past 2 decades, bacterial biofilms have been increasingly implicated as burdens for food and public safety worldwide, and they are broadly defined as heterogeneous communities of microorganisms that adhere to each other and to inert or live surfaces (17, 22, 67, 89, 102). A sessile environment provides selective advantages in natural, medical, and industrial ecosystems for diverse species of commensal and pathogenic bacteria, including Streptococcus mutans (40, 92, 104), Staphylococcus aureus (15, 35, 100), Escherichia coli (21, 74), Vibrio cholerae (39, 52, 107), and Pseudomonas aeruginosa (23, 58, 73, 105). Bacterial biofilms are increasingly associated with many chronic infections in humans and exhibit heightened resistance to commonly administered antibiotics and to engulfment by professional phagocytes (54, 55, 59). The bacterial gene expression profiles for planktonic and biofilm phenotypes differ (42, 90), and the changes are likely regulated by external stimuli, including nutrient availability, the presence of antimicrobials, and the composition of the binding substrate.Biofilm formation occurs in sequential, highly ordered stages and begins with attachment of free-swimming, planktonic bacteria to a surface. Subsequent biofilm maturation is characterized by the production of a self-initiated extracellular matrix (ECM) composed of nucleic acid, proteins, or exopolysaccharides (EPS) that encase the community of microorganisms. Planktonic cells are continuously shed from the sessile, matrix-bound population, which can result in reattachment and fortification of the biofilm or systemic infection and release of the organism into the environment. Shedding of serovar Typhi by asymptomatic carriers can contaminate food and water and account for much of the person-to-person transmission in underdeveloped countries.Our laboratory has previously reported that bile is required for formation of mature biofilms with characteristic EPS production by S. enterica serovars Typhimurium, Enteritidis, and Typhi on human gallstones and cholesterol-coated Eppendorf tubes (18, 78). Cholesterol is the primary constituent of human cholesterol gallstones, and use of cholesterol-coated tubes creates an in vitro uniform surface that mimics human gallstones (18). It was also demonstrated that Salmonella biofilms that formed on different surfaces had unique phenotypes and required expression of specific EPS (18, 77), yet the factors mediating Salmonella binding to gallstones and cholesterol-coated surfaces during the initiation of biofilm formation remain unknown. Here, we show that the presence of serovar Typhimurium flagella promotes binding specifically to cholesterol in the early stages of biofilm development and that the FliC subunit is a critical component. Bound salmonellae expressing intact flagella provided a scaffold for other cells to bind to during later stages of biofilm growth. Elucidation of key mechanisms that mediate adherence to cholesterol during Salmonella bile-induced biofilm formation on gallstone surfaces promises to reveal novel drug targets for alleviating biofilm formation in chronic cases.     

Role of Extracellular DNA during Biofilm Formation by Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in L. monocytogenes extracellular DNA (eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41 L. monocytogenes strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither Listeria genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically N-acetylglucosamine (NAG), interacted with the DNA in a manner which restored adhesion. If a short DNA fragment (less than approximately 500 bp long) was added to an eDNA-free culture prior to addition of genomic or salmon sperm DNA, adhesion was prevented, indicating that high-molecular-weight DNA is required for adhesion and that the number of attachment sites on the cell surface can be saturated.The food-borne pathogen Listeria monocytogenes is known to persist in food processing plants (28, 48), and it has been reported that some strains of this species are capable of forming biofilms (2, 16). The mechanisms of biofilm formation have not been elucidated, but this process seems to depend on factors such as temperature and inducing compounds (14). One inducing compound is NaCl (22), but ethanol, isopropanol (14), quorum sensing (36), and an increasing temperature (8, 14, 38) also seem to enhance attachment and biofilm formation, whereas an acidic pH reduces adhesion (17, 38, 43). Furthermore, at 30°C flagellum-based motility seems to be a specific determinant for the initial adhesion (23, 42) and biofilm formation (23); however, it has recently been reported that in time nonflagellated mutants can produce hyperbiofilms (42).Since bacteria adhering to surfaces, both in biofilms and as single cells, exhibit increased resistance to sanitizers and antimicrobial agents (10, 41), examining the essential steps in adhesion and biofilm formation is important in order to develop new and improved sanitation processes.Extracellular DNA (eDNA) is a ubiquitous component of the organic matter pool in soil, marine, and freshwater habitats (26), but it is also found in environments as diverse as tissue cultures and the blood of mammals (11, 25). The presence of eDNA in the matrix of multicellular structures has recently been reported to influence the initial attachment and/or biofilm structure of Pseudomonas (1, 47), Streptococcus (29), and Staphylococcus (21, 33, 34) species.The prevalence of eDNA in nature appears to be associated with both lysis of cells and active secretion. The concentrations of eDNA released can be up to 2 μg g⁻¹ soil (30) and up to 0.5 g (m²)⁻¹ in the top few centimeters of deep-sea sediment (where more than 90% of the DNA is extracellular) (5). In the deep sea eDNA plays a key role in the ecosystem, functioning as a nitrogen and phosphorus reservoir (5). At present, there are different theories concerning both the function and the release of eDNA in multicellular structures. The presence of eDNA could be a result of either cell lysis (33, 34) or vesicle release (47), whereas active transport is a more speculative explanation. The role of eDNA in biofilm structure has not been revealed yet, but various functions, including a role as a structural component, an energy and nutrition source, or a gene pool for horizontal gene transfer (HGT) in naturally competent bacteria, can be envisaged.Until now there have been no studies of L. monocytogenes eDNA as a possible matrix component in relation to adhesion and biofilm development. In this study, we determined for the first time the presence of L. monocytogenes eDNA, its origin, and its role as a matrix component for both single-cell adhesion and biofilm formation using static assays, as well as flow cell systems. Furthermore, we showed that an additional component is necessary for eDNA-mediated adhesion.     

Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.The intracellular Gram-negative bacterium Coxiella burnetii is the causative agent of the zoonosis Q fever. Inside the host cell, C. burnetii thrives in a unique parasitophorous vacuole (PV) that is considered “phagolysosome-like” due to its moderate acidity (pH ∼5), the presence of active hydrolytic enzymes, and labeling with lysosomal markers (14, 21). Proteins secreted by a specialized Dot/Icm type IV secretion system (T4SS) are thought to modify the PV to support pathogen replication (21). The C. burnetii PV promiscuously fuses with endosomes and lysosomes; however, it does not appear to intercept Golgi body-derived vesicles or to closely associate with the endoplasmic reticulum (ER) (4, 12).The C. burnetii PV membrane is structurally strong and contains lipid raft proteins such as flotillin, characteristics that likely reflect its high cholesterol content (13). Cholesterol is a critical component of cellular membranes, where it provides structural stability and platforms for signaling proteins. Cholesterol is also a precursor for a variety of signaling molecules (8). Intracellular pathogens exploit cholesterol as sources of energy (6, 19) and membrane lipid (7) and interact with cholesterol to manipulate host cell trafficking (10, 25). Indeed, we have previously shown that pharmacological inhibition of host cell cholesterol biosynthesis or uptake blocks C. burnetii PV formation and growth (13), suggesting a critical role for sterols in C. burnetii pathogenesis.Cholesterol synthesis occurs in the ER through a complex series of enzymatic reactions, with the final and required step being the reduction of the carbon-24 bond by a Δ24 sterol reductase (Fig. ​(Fig.1).1). Mutations in the human Δ24 sterol reductase DHCR24 result in desmosterolosis, where the absence of cholesterol results in severe developmental and neurological problems (24). Interestingly, analysis of the C. burnetii genome revealed two genes encoding putative sterol reductases: CBU1158 and CBU1206 (3, 20). Here, we utilize heterologous expression in Saccharomyces cerevisiae to demonstrate that CBU1206 is an active Δ24 sterol reductase.Open in a separate windowFIG. 1.Schematic showing reduction of carbon-24 double bonds by Δ24 sterol reductases. In mammalian cells, the final step in cholesterol synthesis is reduction of the C24 bond in desmosterol by DHCR24, a Δ24 sterol reductase. Ergosterol is the final sterol in yeast, with the Erg4 Δ24 sterol reductase catalyzing the reduction of ergosta-5,7,22,24(28)-tetraen-3β-ol.