Role of Plant Residues in Determining Temporal Patterns of the Activity,Size, and Structure of Nitrate Reducer Communities in Soil

The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.Modern agricultural practices include a return of plant residues to soil, as this is considered sustainable to the environment. It is now recognized that the conversion of native land into cultivated systems leads to carbon losses, which can be up to 20 to 40% (17). Postharvest plant residues therefore represent an important source of carbon, helping to replenish soil organic matter that decomposes as a result of cultivation. Decomposing plant residues are also a source of nutrients, such as nitrogen, with reduced nitrate leaching compared to mineral fertilizers, which is beneficial for water quality (3). In addition, leaving the plant residue on the soil surface limits water losses by evaporation and prevents soil erosion by wind or water (15).The biochemical composition of plant residues is one of the most important factors influencing their decomposition in soil (14, 28, 29, 51). Indeed, Manzoni et al. (28), using a data set of 2,800 observations, showed previously that the patterns of decomposition were regulated by the initial residue stoichiometry. Several other factors such as climatic conditions, soil type, or localization of the residue in the soil (incorporated or on the soil surface) were also reported previously to influence decomposition (2, 24, 29, 44). Microorganisms are the major decomposers of organic matter in soil, and therefore, the diversity and activity of the microbial community during plant residue decomposition has received much attention (6, 23, 26, 27, 35). It was shown previously that the biochemical composition of plant residues influences microbial respiration (8) and microbial community structure (7, 37). The recent development of carbon-labeling approaches has furthered our knowledge of the microorganisms that actively assimilate the carbon derived from various plant residues (10, 31). However, most of those studies focused on microorganisms involved in C mineralization, and in contrast, very little is known about the effect of plant residue decomposition on the microbial communities involved in biochemical cycles other than the carbon cycle. Thus, despite the influence of plant residues on nitrogen cycling (1, 4, 5, 16, 20), studies assessing the effect of the presence and composition of plant residues on the ecology of microbial communities involved in nitrogen cycling are rare (21, 32, 36).The dissimilatory reduction of nitrate into nitrite is the first step in the processes of denitrification and the dissimilatory reduction of nitrate to ammonium (33, 41). The reduction of nitrate by denitrification leads to losses of nitrogen, which is often a limiting nutrient for plant growth in agriculture. Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membrane-bound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9, 53). Nitrate reducers can harbor either Nar, Nap, or both (40, 47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42). Because of this high level of diversity of heterotrophs sharing the ability to produce energy from nitrate reduction, nitrate reducers are an excellent model system to investigate the response of the N-cycling community to plant residue addition.The aim of this work was to determine how the incorporation of plant residues with contrasting biochemical compositions into soil affects the nitrate-reducing community. For this purpose, we monitored the dynamics of the potential activity, size, and structure of the nitrate-reducing community after the addition of wheat, rape, or alfalfa residues to soil in a field experiment. As the nature and availability of the substrate change during residue decomposition (38, 39, 48), the influence of the incorporation of different plant residues on the nitrate-reducing community was investigated at several sampling times for 11 months.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

Probing the Spatial Organization of Measles Virus Fusion Complexes

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.Paramyxoviruses infect cells through fusion of the viral envelope with target cell membranes. For all members of the Paramyxovirinae subfamily, this involves the concerted action of two envelope glycoproteins, the fusion (F) and attachment (H, HN, or G, depending on the Paramyxovirinae genus) proteins. Both proteins feature short lumenal tails, a single transmembrane domain, and large ectodomains. The F protein, in type I orientation, forms homotrimers, while homodimers or homotetramers have been suggested as functional units for attachment proteins of different Paramyxovirinae subfamily members (7, 14, 28, 41, 49, 50, 66). For entry, upon receptor binding, the attachment protein is considered to initiate a series of conformational rearrangements in the metastable prefusion F protein (15, 77), which ultimately brings together transmembrane domains and fusion peptides and, thus, donor and target membranes (3, 32, 45, 53, 80).Multiple studies have demonstrated that specific interactions between compatible F and attachment proteins of paramyxovirinae are imperative for the formation of functional fusion complexes (6, 29, 36, 42, 43, 56, 75). However, the molecular nature of these interactions and the spatial organization of functional glycoprotein hetero-oligomers remain largely unknown. Individual ectodomain and partial ectodomain crystal structures have been obtained for different paramyxovirus F (13, 76, 77) and attachment (8, 14, 17, 28, 35, 79) proteins, respectively. For F, a stabilized human parainfluenza virus type 5 (HPIV5) ectodomain that is believed to represent a prefusion conformation folds into a globular head structure that is attached to the transmembrane domains through a helical stalk consisting of the membrane-proximal heptad repeat B (HR-B) domains (77). For the attachment protein, a globular head that harbors the receptor binding sites is considered to be connected to the transmembrane region through extended stalk domains (34, 78). Crystal structures of isolated head domains have been solved for several paramyxovirus attachment proteins, including measles virus (MeV) H, and reveal the six-blade propeller fold typical of sialidase structures (8, 14, 17, 28, 79). However, morbilliviruses recognize proteinaceous receptors (for MeV, the regulator of complement activation [CD46] and/or signaling lymphocytic activation molecule [SLAM], depending on the virus strain) (21, 40, 46, 51, 64, 65). X-ray data do not extend to the stalk domains, but circular dichroism analysis (78) and structure predictions (36, 78) support an α-helical coiled-coil configuration of the stalk.The nature of individual residues that engage in specific intermolecular interactions between glycoproteins of paramyxovirinae prior to refolding has been studied most extensively for the attachment protein. The stalk domains of several paramyxovirus HN proteins have been implicated in mediating specificity for their homotypic F proteins (18, 20, 43, 63, 70, 72). We have found that this extends to MeV and canine distemper virus H and, thus, to paramyxovirinae recognizing proteinaceous receptors (36), supporting the general hypothesis that F-interacting residues may reside in the stalk region of the attachment protein (30, 78).Considerably less information concerning the nature of F microdomains that mediate attachment protein specificity is available. Among the few exceptions are peptides derived from Newcastle disease virus (NDV) and Sendai virus F HR-B domains, which interact with soluble variants of the respective HN proteins in vitro (25, 67). Multiple domains have been suggested to mediate specificity of HPIV2 F for its HN (69). However, a conclusive N-glycan shielding study (43) and structural information (77, 78) argue against direct contacts between NDV F HR-B domains and HN in native glycoprotein complexes. Thus, the role of individual HPIV2 F residues in HN binding is unclear (25, 43).Building on the observation that MeV H is able to engage in productive heterotypic interactions with F proteins derived from some but not all isolates of closely related canine distemper virus, we have recently identified residues in morbillivirus F (MeV F residue 121) and H (H stalk residues 110 to 114) that interdependently contribute to physical MeV glycoprotein interaction and F triggering for fusion (36). While these residues could mediate reciprocal glycoprotein specificity through long-range effects, molecular modeling of the MeV H stalk in an α-helical conformation has posited F residue 121 at the same level above the viral envelope as H residues 110 to 114, making direct contacts structurally conceivable (36). This spatial organization of functional fusion complexes furthermore provides a comprehensive explanation for previous demonstrations of a specific role for attachment protein stalk domains of paramyxovirinae in functional and physical interactions with F (18, 43, 63, 70, 72). However, this “staggered-head” model mandates positioning the globular head of the attachment protein above the prefusion F trimer (36), as opposed to a suggested “parallel-head” alignment of the glycoproteins (31, 47). The latter is mostly based on transmission electron microscopy micrographs of viral particles apparently showing glycoprotein spikes of equal length (33). Unfortunately, these images lack the resolution for an identification of the molecular nature of the spikes (attachment or F protein) or the distinguishing between densely packaged H and F head domains of different heights and laterally aligned head domains. Indeed, a recent single-particle reconstruction based on cryo-electron microscopy images of HPIV5 particles revealed that defined spikes correspond to F in a postfusion conformation, which was interpreted as a product of possible premature F refolding (38). These two-dimensional images of heavy-metal-stained particles did not reveal F spikes in a prefusion conformation. Rather, a dense surface layer was considered to correspond to prefusion glycoprotein hetero-oligomers (38). In addition to further-advanced image reconstructions, biochemical assessment of alternative docking modes is imperative for the elucidation of the organization of functional fusion complexes of paramyxovirinae.In this study, we subjected central predictions of the hypothetical alignment models to experimental analysis. By employing carbohydrate shielding, directed mutagenesis, and variation of the length of the H stalk domain, we examined the proximity of different regions of the H stalk to F, probed a role of individual residues around the previously identified H stalk section from positions 110 to 114 in the formation of functional fusion complexes, tested the effect of varying the length of the H stalk membrane proximal and membrane distal to this section, and explored the general possibility of whether specific contacts between the prefusion F and H head domains are required for F triggering. Experimental data were interpreted in the light of a working model of MeV glycoprotein hetero-oligomers prior to receptor binding.     

Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts

Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W₃₀-GLW₅₁-C₅₄-C₆₄. Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.Hepatitis C virus (HCV) is a major human pathogen that affects approximately 3% of the global population, leading to cirrhosis and hepatocellular carcinoma in chronically infected individuals (5, 23, 42). Hepatocytes are the major target cells of HCV (11), and entry follows a complex cascade of interactions with several cellular factors (6, 8, 12, 17). Infectious viral particles are associated with lipoproteins and initially attach to target cells via glycosaminoglycans and the low-density lipoprotein receptor (1, 7, 31). These interactions are followed by direct binding of the E2 envelope glycoprotein to the scavenger receptor class B type I (SR-B1) and then to the CD81 tetraspanin (14, 15, 33, 36). Early studies showed that CD81 and SR-B1 were necessary but not sufficient for HCV entry, and claudin-1 was discovered to be a requisite HCV entry cofactor that appears to act at a very late stage of the process (18).Claudin-1 is a member of the claudin protein family that participates in the formation of tight junctions between adjacent cells (25, 30, 37). Tight junctions regulate the paracellular transport of solutes, water, and ions and also generate apical-basal cell polarity (25, 37). In the liver, the apical surfaces of hepatocytes form bile canaliculi, whereas the basolateral surfaces face the underside of the endothelial layer that lines liver sinusoids. Claudin-1 is highly expressed in tight junctions formed by liver hepatocytes as well as on all hepatoma cell lines that are permissive to HCV entry (18, 24, 28). Importantly, nonhepatic cell lines that are engineered to express claudin-1 become permissive to HCV entry (18). Claudin-6 and -9 are two other members of the human claudin family that enable HCV entry into nonpermissive cells (28, 43).The precise role of claudin-1 in HCV entry remains to be determined. A direct interaction between claudins and HCV particles or soluble E2 envelope glycoprotein has not been demonstrated (18; T. Dragic, unpublished data). It is possible that claudin-1 interacts with HCV entry receptors SR-B1 or CD81, thereby modulating their ability to bind to E2. Alternatively, claudin-1 may ferry the receptor-virus complex to fusion-permissive intracellular compartments. Recent studies show that claudin-1 colocalizes with the CD81 tetraspanin at the cell surface of permissive cell lines (22, 34, 41). With respect to nonpermissive cells, one group observed that claudin-1 was predominantly intracellular (41), whereas another reported associations of claudin-1 and CD81 at the cell surface, similar to what is observed in permissive cells (22).Claudins comprise four transmembrane domains along with two extracellular loops and two cytoplasmic domains (19, 20, 25, 30, 37). The first extracellular loop (ECL1) participates in pore formation and influences paracellular charge selectivity (25, 37). It has been shown that the ECL1 of claudin-1 is required for HCV entry (18). All human claudins comprise a highly conserved motif, W₃₀-GLW₅₁-C₅₄-C₆₄, in the crown of ECL1 (25, 37). The exact function of this domain is unknown, and we hypothesized that it is important for HCV entry. The second extracellular loop is required for the holding function and oligomerization of the protein (25). Claudin-1 also comprises various signaling domains and a PDZ binding motif in the intracellular C terminus that binds ZO-1, another major component of tight junctions (30, 32, 37). We further hypothesized that some of these domains may play a role in HCV entry.To understand the role of claudin-1 in HCV infection, we developed a mutagenesis strategy targeting the putative sites for internalization, glycosylation, palmitoylation, and phosphorylation. The functionality of these domains has been described by others (4, 16, 25, 35, 37, 40). We also mutagenized charged and bulky residues in ECL1, including all six residues within the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄. None of the intracellular domains were found to affect HCV entry. However, we identified seven residues in ECL1 that are critical for entry mediated by envelope glycoproteins derived from several HCV subtypes, including all six residues of the conserved motif. These mutants were still expressed at the cell surface and able to form lateral homophilic interactions within the plasma membrane as well as to engage in lateral interactions with CD81. In contrast, they no longer engaged in homophilic trans interactions at cell-cell contacts. We conclude that the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄ of claudin-1 is important for HCV entry into target cells and participates in the formation of cell-cell contacts.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

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.     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

Residue Histidine 669 Is Essential for the Catalytic Activity of Bacillus anthracis Lethal Factor

The lethal factor (LF) of Bacillus anthracis is a Zn²⁺-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.Anthrax is a zoonotic disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis (24). Because infections are highly fatal, the organisms are easily produced, and the spores spread easily, B. anthracis has been used as a bioweapon in biological war and biological terrorism (38). If inhaled, the spores are phagocytosed by alveolar macrophages, where they germinate to produce vegetative bacteria (10, 24). The vegetative bacteria further release anthrax toxins, which inhibit the innate and adaptive immune responses of the hosts. This enables the capsulated bacteria to escape the lymph node defense barrier to reach the blood system, causing bacteremia and toxemia, which can rapidly kill the hosts (24, 26). The great threat posed by anthrax to the public is not only due to the highly lethal rate of inhaled anthrax, but also is due to the social panic caused by the lethality. Therefore, efficient ways to defend against anthrax infection and spreading are greatly needed. This mostly depends on a full understanding of the mechanisms of anthrax infection and toxicities.Anthrax toxins are the dominant virulence factors of Bacillus anthracis (6, 33, 37). They consist of three proteins: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa). The 83-kDa PA (PA₈₃) directly binds to cellular membrane receptors and was cleaved to an active fragment of 63-kDa PA (PA₆₃) by cellular proteases of the furin family or by serum proteases. The receptor-bound portion of PA₆₃ self-assembles into either ring-shaped heptamers, which bind to three molecules of LF and/or EF, resulting in (PA₆₃)₇(LF/EF)₃ (21), or octamers which bind up to four molecules of these moieties, resulting in (PA₆₃)₈(LF/EF)₄ complexes (16, 17). The catalytic partners (EF and/or LF) are subsequently transported across the membrane to the cell cytosol (24, 27). EF is a Ca²⁺- and calmodulin-dependent adenylate cyclase that, together with PA, forms edema toxin. EF causes a rapid increase in intracellular cyclic AMP (cAMP) levels in host cells and alters the elaborate balance of intracellular signaling pathways (20, 23). LF is a Zn²⁺-dependent protease that, together with PA, forms lethal toxin (LT). It is a dominant virulence factor and the major cause of death for the B. anthracis-infected animals (1, 29, 30). LF specifically cleaves the N-terminal domain of mitogen-activated protein kinase kinases (MAPKKs) (11, 35). Because the N-terminal domain of MAPKKs is essential for the interaction between MAPKKs and MAPKs, the cleavage of this domain impairs the activation of MAPKs (8, 11, 15) and leads to the inhibition of three major cellular signaling pathways—the ERK (extracellular signal-regulated kinase), p38, and JNK (c-Jun N-terminal kinase) pathways (29, 31)—and thus induces the lysis of the host cells in an unknown mechanism.The crystal structure of LF with the N-terminal domain of MEK2 has been reported (28). LF has 776 amino acids and comprises four different domains. Domain I (residues 1 to 254) is a PA-binding domain which delivers the remaining domains of the LF to the cell cytoplasm (3). The interface among domains II, III, and IV creates long, deep, 40-Å-long catalytic grooves into which the N terminus of MEK fits and forms an active site complex (28). Domain IV is central to catalytic activities of LF, containing two zinc-binding motifs (residues 686 to 690 and residues E735 to E739) and bound to a single Zn ion (18). However, which residues of LF are critical for efficient catalytic activities and execute the substrate cleavage remains unclear.Histidine is the only naturally occurring amino acid to contain an imidazole residue as a side chain. The catalytic activity of histidine mostly depends on the special features of the imidazole residue. The logarithm of the proton dissociation constant of imidazolyl in the histidine residue is about 6.5; thus, under the physiological condition, it tends to form hydrogen bonds and shares donor and acceptor properties that can take part in either nucleophilic or base catalysis. The speed of the imidazole residue to give or accept protons is very fast, with a half-life of less than 10 s. So in the process of natural selection, histidine was chosen as the catalytic structure, indicating that it plays an important role in the catalysis process of enzymes (9, 12, 14). There are 21 histidines in LF, with 9 of them in LF domain I and 12 of them in domains II, III, and IV. The histidine residues important to LF activities in domain I have been identified (2, 22). The other 12 histidine residues in the remaining three domains include His-277, His-280, and His-424 in domain II; His-309 in domain III; and His-588, His-645, His-654, His-669, His-686, His-690, His-745, and His-749 in domain IV (28). His-686 and His-690 in domain IV were demonstrated to form a zinc binding site constituting a thermolysin-like zinc metalloprotease motif, HEXXH (18). The activities of the remaining 10 histidine residues in domains II, III, and IV have not been explored yet. In this study, we replaced these 10 histidine residues separately with alanine residues by site-directed mutagenesis. By the cytotoxicity assay of all these mutants, the H669A mutant was found to lose cell toxicity completely. Further assay revealed that residue His-669 was involved in neither zinc stabilization nor PA binding but participated in the substrate proteolytic activity of LF.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.Nitrilases (EC 3.5.5.1) are hydrolytic enzymes found in many bacteria, fungi, and plants which convert nitriles to the corresponding carboxylic acids and ammonia. They are members of the CN hydrolase (or nitrilase) superfamily of enzymes, which also encompasses other enzymes which attack C-N bonds, such as aliphatic amidases, carbamoylases, and N-acyltransferases (1). Nitrilases possess a catalytic triad which is composed of a cysteine, a glutamate, and a lysine residue and form during the catalytic cycle a covalent adduct between the cysteine residue and the carbon atom of the nitrile group (11, 12, 29). Nitriles are important intermediates in chemical industry, and several processes which utilize the chemo-, regio-, or enantioselectivity of nitrilases for the production of commercially interesting products have been investigated (13, 16, 17, 18, 22, 26, 27, 33, 34). There is also growing biotechnological interest in nitrilases because they form (as other members of the so-called nitrilase superfamily) spiral quaternary structures which can be studied by electron microscopy and which might be useful as templates in nanotechnology (30, 31).We are currently investigating a nitrilase from Pseudomonas fluorescens EBC191 which converts various substituted phenylacetonitriles [e.g., 2-phenylpropionitrile (2-PPN), mandelonitrile (2-hydroxyphenylacetonitrile), or phenylglycinonitrile (2-aminophenylacetonitrile)] and also aliphatic 2-acetoxynitriles with moderate enantioselectivities into the corresponding α-substituted carboxylic acids. This enzyme forms with certain nitriles also significant amounts of the corresponding amides as side products (3, 5, 8, 15, 19, 24). The enzyme has recently been studied intensively in order to analyze the molecular basis for the substrate specificity, reaction specificity, and enantiospecificity of nitrilases (9, 10). In the course of these investigations, the effects of various carboxy-terminal mutations and mutations in close proximity to the catalytic active cysteine residue were analyzed. These experiments demonstrated that deletions of 47 to 67 amino acids (aa) from the carboxy terminus of the nitrilase resulted in variant forms that demonstrated increased amide formation and an increased formation of the (R)-acids (9). In addition, it was demonstrated that the size of the amino acid residue in direct proximity to the catalytic active cysteine residue (toward the C terminus) is determinative of the enantioselectivity of acid formation. Thus, it was found that only enzyme variants with large amino acid residues at this position showed a high degree of enantioselectivity for the formation of (R)-mandelic acid from racemic mandelonitrile (10). In the present study, we investigated a set of enzyme variants that carried mutations located in the amino-terminal part of the enzyme (in relation to the catalytic active cysteine residue). Thus, several mutations that resulted in changes in the enantioselectivity of the reactions and increased formation of amides were identified.     

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).     

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

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.     

Identity,Diversity, and Molecular Phylogeny of the Endophytic Mycobiota in the Roots of Rare Wild Rice (Oryza granulate) from a Nature Reserve in Yunnan,China

Rice (Oryza sativa L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root endophytic mycobiota residing in roots of O. granulate by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia macrospinosa, and the Ceratobasidium/Rhizoctonia complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions.The majority of terrestrial plant roots are intimately associated with mycorrhizal fungi, and many aspects of the ecological roles played by these mycorrhizal fungi are well understood. In recent years, however, endophytic fungi have been gaining increasing interest. There is accumulating evidence that plant roots usually harbor mycorrhizal as well as endophytic fungi (29, 30, 34, 39, 52, 63). Dark septate endophytes (DSEs), which are characterized by dark pigmented hyphae and sclerotium-like structures, are believed to represent primary nonmycorrhizal root-inhabiting fungi (23). In some cases, DSEs are even more frequent than mycorrhizal fungi (68).Endophytic fungi have frequently been reported to be associated with crop plants, including wheat (Triticum aestivum), wild barley (Hordeum brevisubulatum and Hordeum bogdanii), soya bean (Glycine max), and maize (Zea mays) (6, 9, 11, 13, 21, 26, 27, 33, 36, 67). Some of the endophytic fungi in these crops conferred resistance of the plant to insect or fungal pathogens (55).Domesticated from the wild grass Oryza rufipogon 10,000 to 14,000 years ago, rice is today the main staple for more than 3 billion people (i.e., half of the world''s population). Its consumption exceeds 100 kg per capita annually in many Asian countries, and it is the principal food for most of the world''s poorest people, particularly in Asia. The association of arbuscular mycorrhizal fungi and endophytic bacteria with rice plants has been well documented (15, 32, 35, 44, 53, 56, 60). Less, however, is known about its fungal endophytes. Fungal endophytes have been detected in cultivated rice (Oryza sativa L.) (12, 14, 37, 61, 70), and antagonistic or plant growth-stimulating properties have been claimed for some of these isolates. For example, endophytic Fusarium spp. from cultivated rice roots proved to be effective in biocontrol of a root-knot nematode (28). The occurrence of mycorrhizal and endophytic fungi in a variety of rice cultivars has also recently been reported (63).Nondomesticated, wild plant species may live in symbiosis with a unique and rich mycoflora that may have been lost during breeding of the cultivars used in agriculture (20, 59). The purpose of this research was to characterize the endophytic fungal community of the roots of rare (nearly extinct) wild rice (Oryza granulate) from a nature reserve in Yunnan, China. Our results showed that arbuscular mycorrhizal fungi were apparently absent from wild rice roots. This finding was confirmed by standard root staining techniques and molecular detection using the arbuscular mycorrhizal (AM)-specific primer pairs (69). The characterization of root endophytes in wild rice as reported in this study will improve our knowledge concerning the ecology and evolution of mutualistic plant-fungus interactions.