Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.The movement of water across cell membranes has long been thought to occur by free diffusion through the lipid bilayer. However, the discovery of the membrane protein CHIP28 in red blood cells has suggested the involvement of protein channels (29), and it is now well established that transmembrane water permeability is facilitated by aquaporins (AQPs), water channel proteins that are found in bacteria, fungi, plants, and animals (1, 7, 13, 24). AQPs contain six transmembrane α-helices and five connecting loops, and both the N and C termini are located in the cytosol. The monomers assemble into tetrameric complexes, with each monomer forming an individual water channel (11, 14, 24, 33). Apart from the exceptions of AQP11 and AQP12 from mice, as described by K. Ishibashi (15), AQPs have two signature Asn-Pro-Ala motifs, which are located in the second intracellular and the fifth extracellular loops, B and E.While 13 different AQPs have been identified in mammals (16), more than 33 AQP homologues have been discovered in plants (6, 17, 30). Plant AQPs fall into four subclasses: (i) the plasma membrane (PM) intrinsic proteins (PIPs), which are localized in the PM; (ii) the tonoplast intrinsic proteins (TIPs), which are localized in the vacuolar membranes; (iii) the nodulin-26-like intrinsic proteins; and (iv) the small basic intrinsic proteins (24). In Arabidopsis and maize, there are 13 PIPs, which can be divided further into two subfamilies, PIP1 and PIP2 (6, 17).The functions and mechanisms of regulation of plant AQPs have been extensively investigated (7, 13, 18, 24). There have been several reports on the water channel activity (WCA) of specific AQPs and their regulation by protein phosphorylation (3, 4, 8, 12, 18, 25, 32, 33). It has been shown that the WCA of the PIP2 member SoPIP2;1 from spinach is regulated by phosphorylation at two Ser residues (19, 33).The physiologically interesting temperature-dependent opening and closing of tulip (Tulipa gesneriana) petals occur concomitantly with water transport and are regulated by reversible phosphorylation of an undefined PIP (4, 5). Recently, four PIP homologues were isolated from tulip petals, and their WCAs have been analyzed by heterologous expression in Xenopus laevis oocytes (3). It has been shown that the tulip PIP TgPIP2;2 (DDBJ/EMBL/GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB305617","term_id":"196259707","term_text":"AB305617"}}AB305617) is ubiquitously expressed in all organs of the tulip and that TgPIP2;2 is the most likely of the TgPIP homologues to be modulated by the reversible phosphorylation that regulates transcellular water transport and mediates petal opening and closing (3, 4). However, while the members of the PIP2 subfamily are characterized as water channels (6), TgPIP2;1 (DDBJ/EMBL/GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB305616","term_id":"196259705","term_text":"AB305616"}}AB305616) shows no significant WCA in the oocyte expression system (3). There is growing interest in research on AQPs due to their crucial roles in the physiology of plants and animals (1, 16, 21-24, 26-28, 36). The assay of AQP channel activity is usually performed using either a X. laevis oocyte expression system (29) or a stopped-flow light-scattering spectrophotometer (35), both of which are not widely available. Furthermore, the complexity of these methods and requirement of expertise limit their high-throughput applications. In contrast, a Pichia pastoris expression system is simple to use, inexpensive, and feasible and can be used in high-throughput applications. Although a P. pastoris expression system has been shown to assay the WCA of a TIP (9), extensive research is necessary with other AQPs such as PIPs or AQPs present in intragranular membranes to establish whether this assay system can be used to characterize a water channel and study its regulation mechanisms. With this in view, in the study reported herein, TgPIP2;1 and TgPIP2;2 have been heterologously expressed in P. pastoris, and their WCAs have been assayed. The effects of several factors, such as osmolarity, pH, and inhibitors of protein kinases (PKs) and protein phosphatases (PPs), on the WCA of the recombinant P. pastoris have been investigated. Based on the results, we demonstrate that the P. pastoris heterologous expression system can be used to rapidly characterize PIP channels, to monitor the effects of mutations, and to score the effects of inhibitors and abiotic factors.     

The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.Sinorhizobium meliloti and Brucella abortus form chronic intracellular infections within legumes and mammalian hosts, respectively (3, 20), and their BacA proteins play essential roles in these processes (8, 12). The precise function(s) of the BacA proteins has not been resolved, but free-living S. meliloti and B. abortus mutants lacking BacA have increased resistance to the glycopeptide bleomycin (9, 12) and there are ∼50% decreases in their lipid A very-long-chain fatty acid (VLCFA) contents (4, 7). It has also been determined that the increased resistance of an S. meliloti bacA null mutant to bleomycin and a truncated eukaryotic peptide, Bac7_1-16, is independent of its lipid A VLCFA alteration (6, 15). Together, these findings support a model in which BacA could have multiple nonoverlapping functions which lead to lipid A VLCFA modification and peptide uptake. The fact that two symbiotically defective S. meliloti BacA site-directed mutants (Q193G and R389G) (13) show defects in BacA-mediated lipid A VLCFA modification (4) but are still capable of peptide uptake (15) suggests that the S. meliloti lipid A VLCFA modification could play a key role in the symbiosis of this organism with alfalfa.Since the mechanism by which BacA leads to the lipid A VLCFA modification has not been resolved (4), S. meliloti mutants were constructed with mutations in the lpxXL and acpXL genes, which encode a lipid A VLCFA acyl transferase and a VLCFA acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A (5, 23). The S. meliloti lpxXL and acpXL mutants completely lack the lipid A VLCFA modification in their free-living states, but, unlike the S. meliloti bacA null mutant, these mutants can still form a successful symbiosis with alfalfa (5, 8, 23). However, the fact that the S. meliloti acpXL and lpxXL mutants are substantially less competitive in the alfalfa symbiosis than the parent strain (5, 23) indicates that the AcpXL and LpxXL proteins play important roles in at least one of the stages of the alfalfa symbiosis. Although the free-living S. meliloti acpXL and lpxXL mutants completely lack the lipid A VLCFA, they produce different species of lipid A (5). For example, in the absence of AcpXL, S. meliloti is able to modify lipid A with either C16:0 or C18:0 in the position normally modified with the VLCFA in the parent strain lipid A. This process is LpxXL dependent, as it does not occur in either an S. meliloti lpxXL single mutant or an S. meliloti acpXL lpxXL double mutant. In addition, since a Rhizobium leguminosarum acpXL mutant completely lacks the lipid A VLCFA modification in its free-living state but its lipid A is partially modified with the VLCFA to ∼58% of the amount in the parent strain lipid A during passage through peas (25), it is also possible that the S. meliloti acpXL mutant and possibly the S. meliloti lpxXL mutant undergo further lipid A changes during the interaction with alfalfa.In this study, we found that LpxXL and AcpXL play important but distinct roles in S. meliloti bacteroid development during alfalfa symbiosis. Additionally, we demonstrated that there is a minor host-induced AcpXL-independent mechanism by which S. meliloti bacteroid lipopolysaccharide (LPS) can be modified with the VLCFA. In contrast, we found that the LpxXL protein plays an essential role in the modification of S. meliloti bacteroids with VLCFAs.     

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.     

VP23R of Infectious Spleen and Kidney Necrosis Virus Mediates Formation of Virus-Mock Basement Membrane To Provide Attaching Sites for Lymphatic Endothelial Cells

Putative open reading frames (ORFs) encoding laminin-like proteins are found in all members of the genus Megalocytivirus, family Iridoviridae. This is the first study that identified the VP23R protein encoded by ORF23R of the infectious spleen and kidney necrosis virus (ISKNV), a member of these genes of megalocytiviruses. The VP23R mRNA covering the ISKNV genomic coordinates 19547 to 22273 was transcribed ahead of the major capsid protein. Immunofluorescence analysis demonstrated that VP23R was expressed on the plasma membrane of the ISKNV-infected cells and could not be a viral envelope protein. Residues 292 to 576 of VP23R are homologous to the laminin γ1III2-6 fragment, which covers the nidogen-binding site. An immunoprecipitation assay showed that VP23R could interact with nidogen-1, and immunohistochemistry showed that nidogen-1 was localized on the outer membrane of the infected cells. Electron microscopy showed that a virus-mock basement membrane (VMBM) was formed on the surface of the infected cells and a layer of endothelial cells (ECs) was attached to the VMBM. The VMBM contained VP23R and nidogen-1 but not collagen IV. The attached ECs were identified as lymphatic endothelial cells (LECs), which have unique feature of overlapping intercellular junctions and can be stained by immunohistochemistry using an antibody against a specific lymphatic marker, Prox-1. Such infection signs have never been described in viruses. Elucidating the functions of LECs attached to the surface of the infected cells may be useful for studies on the pathogenic mechanisms of megalocytiviruses and may also be important for studies on lymphangiogenesis and basement membrane functions.Basement membrane (BM), a dense and sheetlike structure that is always associated with cells, is a very important specialized form of extracellular matrix (31, 67). BMs mediate tissue compartmentalization and provide structural support to the epithelium, endothelium, peripheral nerve axons, fat cells, and muscle cells, as well as structural and functional foundations of the vasculature (25, 31, 52). BM is also an important regulator of cell behaviors, such as adhesion, migration, proliferation, and differentiation. BMs are highly cross-linked and insoluble materials. They are highly complex and are made up of more than 50 known components (31, 54). Although the molecular composition of BMs is unique in each tissue, their basic structures are similar. Even if many more isoforms exist in different species, the major BM proteins and their receptors are conserved from Caenorhabditis elegans to mammals. BM consists of a layer of laminin polymer, a layer of type IV collagen network, and the nidogen protein, which acts as a cross-linker of these two networks. Other BM components, such as perlecan and fibulin, interact with the laminin polymer and the type IV collagen network to organize a functional BM on the basolateral aspect of the cells (31, 45, 52).The components of BM are able to self-assemble and form a sheetlike structure, and laminin is the key molecule in this process (50). Laminin protein consists of three different chains (α, β, and γ), which comprise a cross-shaped molecular structure with three short amino-terminal arms and a long carboxyl-terminal triple-helical arm (58, 68). The three short arms of this cross-shaped structure can interact with each other in the presence of calcium. Through the binding of globular G domain at the carboxyl-terminal end of the α chain to the cell receptors (e.g., integrins and dystroglycans), laminin self-assembles into polygonal lattices on cell surfaces. This process initiates BM self-assembly (15, 21, 25, 38, 65, 66). To date, 17 laminin isoforms have been observed in different tissues (51). Among them, laminin-1, the crux of early embryonic BM assembly, has been well studied. Laminin-1 consists of α1, β1, and γ1 chains and can interact with nidogen-1 with high affinity through a laminin-type epidermal growth factor-like (LE) module, γ1III4, within the domain III of the γ1 chain (1, 42). The heptapeptide “NIDPNAV” within the γ1III4 motif of laminin-1 is essential for the interaction between laminin-1 and nidogen-1 (41, 46). Blocking the interactions between laminin-1 and nidogen-1 leads to the disruption of BMs. This indicates that the formation of laminin/nidogen complex is essential for BM assembly and stability (30, 61). Nidogen-1, also called entactin-1, is a dumbbell-shaped sulfated 150-kDa glycoprotein consisted of three domains (G1, G2, and G3) (12). By interacting with collagen IV through its G2 domain and binding with laminin γ1 chain through its G3 domain, nidogen-1 bridges the layers of the laminin network and the collagen IV network to construct the fundamental structure of BMs (48). Collagen IV is a triple-helical trimer composed of three α chains. Through the hexamer formation of the carboxyl-terminal globular non-collagenous-1 (NC1) domain of each α chain, two collagen IV proteins assemble into a dimer. Dimers of collagen IV connect with each other via their amino-terminal 7S domains and self-assemble into a network (24, 27, 31, 32). Six kinds of α chains of collagen IV have been identified in mammals. Among them, α1 and α2 chains are the most abundant forms of collagen IV found in all BMs (19, 23). They commonly form a collagen IV molecule with a α1 and α2 ratio of 2:1 (31, 35).Iridoviruses infect invertebrates and poikilothermic vertebrates, including insects, fish, amphibians, and reptiles. These viruses are a group of icosahedral cytoplasmic DNA viruses with circularly permuted and terminally redundant DNA genomes (6, 8, 9, 10, 57, 62). The family Iridoviridae has been subdivided into five genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus, and Megalocystivirus (7). The genus Megalocystivirus, characterized by the ability to cause swelling of the infected cells, is one group of the most harmful viruses to cultured fish (7, 26, 29). Infectious spleen and kidney necrosis virus (ISKNV), the causative agent of a disease that causes high mortality rates in farmed mandarin fish, Siniperca chuatsi, and large-mouth bass, Micropterus salmoides, is regarded as the type species of Megalocystivirus (7). Similar to infection caused by other members of the Megalocystivirus, fish ISKNV infection is characterized by cell hypertrophy in the spleen, kidney, cranial connective tissue, and endocardium (16, 17). Aside from mandarin fish and large-mouth bass, ISKNV-like virus can also be detected in the tissues of more than 60 marine and freshwater fishes (14, 28, 59, 64). The entire genome of ISKNV has been sequenced, and the organization of open reading frames (ORFs) of ISKNV was analyzed by using DNASTAR Omiga 2.0 and Genescan (18). The ISKNV genome is about 110 kbp and contains 125 putative ORFs (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF371960","term_id":"19773610","term_text":"AF371960"}}AF371960).Putative ORFs, encoding viral proteins containing a fragment homologous to laminin and a putative transmembrane fragment, were found in all of the sequenced genomes of the members of Megalocystivirus. These ORFs include ORF23R of ISKNV (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAL98747","term_id":"19773633","term_text":"AAL98747"}}AAL98747), laminin-like protein gene of olive flounder iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAT76907","term_id":"50404491","term_text":"AAT76907"}}AAT76907), ORF2 of sea perch iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAV51313","term_id":"55418187","term_text":"AAV51313"}}AAV51313), predicted laminin-type epidermal growth factor-like protein of large yellow croaker iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ABI32391","term_id":"113200502","term_text":"ABI32391"}}ABI32391), an unknown gene of red sea bream iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAQ07956","term_id":"33324323","term_text":"AAQ07956"}}AAQ07956), ORF2 of rock bream iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAN86692","term_id":"60458808","term_text":"AAN86692"}}AAN86692), and laminin-type epidermal growth factor-like protein of orange-spotted grouper iridovirus (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAX82335","term_id":"62421215","term_text":"AAX82335"}}AAX82335). These putative proteins are highly homologous to each other in amino acid sequence (65 to 99% identity). However, the functions of these proteins have never been identified. This is the first study to identify that the VP23R protein encoded by ORF23R of ISKNV is a plasma membrane-localized viral protein. In addition, we discovered a new function of VP23R in a unique pathological phenomenon of virus infection: the attachment of lymphatic endothelial cells (LECs) to the infected cells. Nidogen-1 assisted VP23R in the construction of a BM-like structure, providing an attachment site for LECs. This unique pathological phenomenon has never been found in viruses and is an attractive direction for studies of pathogenic mechanisms of megalocystiviruses. Moreover, studies on the unique profiles of the virus-mock BM can help us learn more about the functions of BM components and the mechanisms of lymphangiogenesis.     

Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.Bacteria use type IV secretion (T4S) to deliver macromolecules to prokaryotes and eukaryotes (12). Animal and human pathogens deliver proteins to their eukaryotic hosts to affect cellular processes causing disease. The plant-pathogenic bacterium Agrobacterium tumefaciens delivers both proteins and DNA to plants and other eukaryotes. DNA delivered by Agrobacterium directs constitutive synthesis of phytohormones in a transformed plant cell, promoting cancerous growth (56). The Ptl toxin of Bordetella pertussis modifies G proteins by ADP-ribosylation, affecting intracellular cell signaling, and CagA of Helicobacter pylori disrupts epithelial cell polarity by inhibiting PAR1 kinase activity (37, 44, 47). T4S is ancestrally related to bacterial conjugation, a mechanism used by bacteria for interbacterial plasmid transfer, enabling them to acquire novel genes for antibiotic resistance, degradation of organic molecules, toxin production, and other virulence traits (29).The VirD4/VirB family of proteins, found conserved in many alphaproteobacteria, mediates T4S (12). The Ti plasmid-encoded Agrobacterium T4S system requires VirD4 and 11 VirB proteins, VirB1 to VirB11, for efficient DNA transfer (7, 54). The membrane and membrane-associated VirB proteins assemble a macromolecular structure at the cell membrane to promote substrate transfer (12). The octopine Ti plasmid pTiA6NC-encoded VirB6 to VirB11 proteins assemble the T4S apparatus at a cell pole (34, 35, 39). The VirD4 coupling protein targets the VirE2 substrate protein to the cell pole (4). A recent study found that the nopaline Ti plasmid pTiC58 T4S system (T4SS) and its substrates form a helical array around the cell circumference (1). Structural studies using Escherichia coli conjugative plasmid pKM101-encoded VirB homologues showed that TraN (VirB7), TraO (VirB9), and TraF (VirB10) form the core complex and that TraF forms a channel at the outer membrane (11, 23). The Agrobacterium VirB proteins assemble a T-pilus, an appendage composed primarily of VirB2, with VirB5 and VirB7 as its minor constituents (38, 40, 41, 48, 50, 55). VirB3, a homolog of the pilin-like TraL protein encoded in E. coli plasmids, is postulated to function in T-pilus assembly (52). Three ATP-utilizing proteins, VirB4, VirB11, and VirD4, supply energy for substrate translocation (3, 9, 34).The membrane topology of all the VirB proteins, except for VirB3, was determined by analyses of random phoA insertion mutants, targeted phoA fusions, and targeted bla fusions (6, 14, 15, 21, 22, 31, 35, 53). phoA and bla, which encode alkaline phosphatase and β-lactamase, respectively, serve as excellent markers for periplasmic proteins, as they are enzymatically active only when targeted to the cell periplasm (8, 30). Green fluorescent protein (GFP) is an ideal cytoplasmic marker because it fluoresces only when located in the cytoplasm (19, 20). When GFP is targeted to the periplasm through fusion with a membrane-spanning domain (MSD), it fails to fold properly and does not fluoresce.The prevailing view, based on in silico analysis, is that VirB3 is a bitopic membrane protein with a periplasmic C terminus. No phoA-positive insertions in virB3, however, were identified in two random mutagenesis studies of the virB operon (6, 15). The small size of VirB3, a polypeptide of 108 amino acids (aa), could be a contributing factor to the negative findings. Yet several PhoA-positive insertions in two smaller VirB proteins, VirB2 (74-aa mature peptide) and VirB7 (41-aa mature peptide), were successfully obtained in both studies. Therefore, the negative findings may also be indicative of the presence of a small periplasmic domain in VirB3. Biochemical studies showed that the nopaline Ti plasmid pTiC58-encoded VirB3 protein (pTiC58 VirB3) associates with the bacterial outer membrane, while VirB2 associates with both the inner and outer membranes (52). The pTiC58 VirB4 protein is required for localization of VirB3 to the outer membrane (33). VirB4 is also required for VirB3 stability (33, 55). A low level of VirB3 accumulated in a nonpolar pTiC58 virB6 deletion mutant; however, addition of virB6 in trans did not restore the level of the protein, even though it restored tumorigenicity (27). VirB3 participates in the formation of protein complexes with the T-pilus proteins VirB2 and VirB5 (55).Homologues of VirB3 are found in many alphaproteobacteria with a T4SS. While most VirB3 homologues are small proteins, several recently identified homologues are fusions of VirB3 and the immediate downstream protein VirB4 (5, 10, 24). These fusion homologs, which include Actinobacillus MagB03 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAG24434","term_id":"10880920","term_text":"AAG24434"}}AAG24434), Campylobacter CmgB3/4 ({"type":"entrez-protein","attrs":{"text":"EAQ71805","term_id":"87248841","term_text":"EAQ71805"}}EAQ71805), Yersinia pseudotuberculosis TriC ({"type":"entrez-protein","attrs":{"text":"CAF25448","term_id":"51591645","term_text":"CAF25448"}}CAF25448), Citrobacter koseri PilX3-4 ({"type":"entrez-protein","attrs":{"text":"ABV12046","term_id":"157082368","term_text":"ABV12046"}}ABV12046), and Klebsiella pneumoniae PilX3-4 ({"type":"entrez-protein","attrs":{"text":"BAF49490","term_id":"134048868","term_text":"BAF49490"}}BAF49490), have VirB3 at the N terminus and VirB4 at the C terminus. Agrobacterium VirB4 is an integral membrane protein with a cytoplasmic N terminus (14). Its homologues are expected to have a similar topology. The prevailing view that pTi VirB3 has a periplasmic C terminus is inconsistent with the cytoplasmic location of the N terminus of VirB4 in the VirB3-VirB4 fusion protein homologues.In this study, we report the membrane topology of Agrobacterium VirB3 and demonstrate that the C terminus of the protein resides in the cytoplasm. We also demonstrate that VirB3 is an inner membrane protein, not an outer membrane protein as previously reported (52). The octopine Ti plasmid pTiA6NC VirB4 protein does not affect membrane localization of VirB3 but does stabilize VirB3. VirB4, however, is not sufficient for pTiA6NC VirB3 stabilization. Two additional proteins, VirB7 and VirB8, are required for the stabilization of pTiA6NC VirB3.     

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).     

Association of Missense Mutations in Epoxyalkane Coenzyme M Transferase with Adaptation of Mycobacterium sp. Strain JS623 to Growth on Vinyl Chloride

Vinyl chloride (VC) is a toxic groundwater pollutant associated with plastic manufacture and chlorinated solvent use. Aerobic bacteria that grow on VC as a carbon and energy source can evolve in the laboratory from bacteria that grow on ethene, but the genetic changes involved are unknown. We investigated VC adaptation in two variants (JS623-E and JS623-T) of the ethene-oxidizing Mycobacterium strain JS623. Missense mutations in the EtnE gene developed at two positions (W243 and R257) in cultures exposed to VC but not in cultures maintained on ethene. Epoxyalkane-coenzyme M transferase (EaCoMT) activities in cell extracts of JS623-E and JS623-T (150 and 645 nmol/min/mg protein, respectively) were higher than that of wild-type JS623 (74 nmol/min/mg protein), and in both variant cultures epoxyethane no longer accumulated during growth on ethene. The heterologous expression of two variant etnE alleles (W243G [etnE1] and R257L [etnE2]) from strain JS623 in Mycobacterium smegmatis showed that they had 42 to 59% higher activities than the wild type. Recombinant JS623 cultures containing mutant EtnE genes cloned in the vector pMV261 adapted to growth on VC more rapidly than the wild-type JS623 strain, with incubation times of 60 days (wild type), 1 day (pMVetnE1), and 35 days (pMVetnE2). The JS623(pMVetnE) culture did not adapt to VC after more than 60 days of incubation. Adaptation to VC in strain JS623 is consistently associated with two particular missense mutations in the etnE gene that lead to higher EaCoMT activity. This is the first report to pinpoint a genetic change associated with the transition from cometabolic to growth-linked VC oxidation in bacteria.Bacteria that biodegrade pollutants are useful for the cleanup of contaminated sites (i.e., bioremediation) and are interesting as models of evolutionary processes (21, 38, 40). Understanding the molecular genetic and evolutionary basis of biodegradation processes allows improved monitoring and predictions of bacterial activities in situ (39) and promises the development of improved strains and enzymes with increased specific activity (3), increased substrate affinity (16), extended substrate range (3, 16, 21, 37), extended inducer range (30, 31), or constitutive expression (39). Missense mutations in catabolic enzymes or regulatory proteins commonly lead to these changes (43), although other important mechanisms include duplication, deletion, and inversion (38-40).Vinyl chloride (VC) is a common groundwater pollutant (35) and known human carcinogen (24), and it poses a health risk to exposed populations. Although trace amounts (e.g., parts per trillion) of VC have been detected in uncontaminated soil (23), higher concentrations are found only associated with human industry, particularly the manufacture of polyvinylchloride (PVC) plastic and the chlorinated solvents trichloroethene (TCE) and perchloroethene (PCE) (4). Aerobic bacteria that grow on VC as a sole carbon and energy source are diverse, including strains of Mycobacterium (8, 17, 18), Nocardioides (8), Pseudomonas (11, 41, 42), Ochrobactrum (11), and Ralstonia (13, 33). The relative ease of the isolation of VC assimilators from chlorinated ethene-contaminated sites suggests that such bacteria are influential in the natural attenuation of VC, but this interpretation is complicated by the fact that VC-assimilating bacteria are closely related to ethene-assimilating bacteria (8-10, 29) and cannot yet be distinguished from them by molecular tests.The VC and ethene pathway and genes are homologous to some extent with the propene assimilation pathway and genes in Xanthobacter Py2 and Gordonia B-276. The comparison of the genomes of the VC-assimilating Nocardioides JS614 and the propene-assimilating Xanthobacter Py2 indicates that growth on alkenes requires about 20 kb of alkene/epoxide catabolic genes and approximately 7 kb of coenzyme M (CoM) biosynthesis genes. The oxidation of VC and ethene is initiated by an alkene monooxygenase (AkMO; EtnABCD) (8-10, 29), which yields epoxyethane from ethene and chlorooxirane from VC (8, 17). An epoxyalkane-coenzyme M transferase (EaCoMT) enzyme, EtnE, acts upon these reactive, toxic, and mutagenic epoxides (2, 19), converting them to hydroxyalkyl-CoM derivatives. The remainder of the VC/ethene pathway is unclear. The JS614 genome indicates further homology with propene oxidizers, in that a reductase/carboxylase and SDR family dehydrogenase are present, but that other aspects of the VC/ethene pathway gene cluster are unique (e.g., the presence of a semialdehyde dehydrogenase [5] and a disulfide reductase-like gene [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_008697","term_id":"119714007","term_text":"NC_008697"}}NC_008697]).The EtnE enzyme and the homologous XecA enzyme that acts on epoxypropane in Xanthobacter Py2 and Gordonia B-276 (9, 10, 12, 29) are unusual in their requirement for CoM as a cofactor. The C2- and C3-alkene oxidizers are the only Eubacteria known to biosynthesize and require CoM, which is otherwise found only in methanogenic Archaea. The XecA protein of Py2 has been purified and shown to be a Zn-dependent enzyme (1, 14, 26, 44). Based on sequence homology and the presence of the Cys-X-His-X_n-Cys motif (see Fig. S1 in the supplemental material), the EtnE enzymes also are likely to be Zn-dependent enzymes. Heterologous expression systems for XecA and EtnE have been developed (9, 25), but no crystal structures are available yet for EaCoMT from any source.Pure cultures of ethene-assimilating bacteria are capable of spontaneously adapting to growth on VC as a carbon source (22, 42), but the molecular basis of this phenomenon is not clear. This knowledge gap confounds the development of molecular probes specific for VC-assimilating bacteria. Pseudomonas aeruginosa strain DL1 shifted from cometabolism to growth on VC after more than 40 days of incubation (42), while Mycobacterium strains JS622, JS623, JS624, and JS625 took between 55 and 476 days to adapt to VC (22). The VC-adapted phenotype in Mycobacterium strains was not lost after growth in nonselective medium, suggesting a genetic change rather than a physiological adaptation (22).Here, we tested the hypothesis that mutations in the alkene/epoxide catabolic genes are responsible for VC adaptation. This was done by sequencing EtnEABCD genes in fosmid clones from cultures before and after VC adaptation, by sequencing etnE PCR products at different time points during VC adaptation, and by examining the EtnE enzyme activity in VC-adapted strains and recombinant strains carrying evolved etnE alleles.     

Bocavirus Infection Induces Mitochondrion-Mediated Apoptosis and Cell Cycle Arrest at G2/M Phase

Bocavirus is a newly classified genus of the family Parvovirinae. Infection with Bocavirus minute virus of canines (MVC) produces a strong cytopathic effect in permissive Walter Reed/3873D (WRD) canine cells. We have systematically characterized the MVC infection-produced cytopathic effect in WRD cells, namely, the cell death and cell cycle arrest, and carefully examined how MVC infection induces the cytopathic effect. We found that MVC infection induces an apoptotic cell death characterized by Bax translocalization to the mitochondrial outer membrane, disruption of the mitochondrial outer membrane potential, and caspase activation. Moreover, we observed that the activation of caspases occurred only when the MVC genome was replicating, suggesting that replication of the MVC genome induces apoptosis. MVC infection also induced a gradual cell cycle arrest from the S phase in early infection to the G₂/M phase at a later stage, which was confirmed by the upregulation of cyclin B1 and phosphorylation of cdc2. Cell cycle arrest at the G₂/M phase was reproduced by transfection of a nonreplicative NS1 knockout mutant of the MVC infectious clone, as well as by inoculation of UV-irradiated MVC. In contrast with other parvoviruses, only expression of the MVC proteins by transfection did not induce apoptosis or cell cycle arrest. Taken together, our results demonstrate that MVC infection induces a mitochondrion-mediated apoptosis that is dependent on the replication of the viral genome, and the MVC genome per se is able to arrest the cell cycle at the G₂/M phase. Our results may shed light on the molecular pathogenesis of Bocavirus infection in general.The Bocavirus genus is newly classified within the subfamily Parvovirinae of the family Parvoviridae (21). The currently known members of the Bocavirus genus include bovine parvovirus type 1 (BPV1) (17), minute virus of canines (MVC) (57), and the recently identified human bocaviruses (HBoV, HBoV2, and HBoV3) (4, 7, 36).MVC was first recovered from canine fecal samples in 1970 (10). The virus causes respiratory disease with breathing difficulty (14, 32, 49) and enteritis with severe diarrhea (11, 39), which often occurs with coinfection with other viruses (39), spontaneous abortion of fetuses, and death of newborn puppies (14, 29). Pathological lesions in fetuses in experimental infections were found in the lymphoid tissue of the lung and small intestine (14). MVC was isolated and grown in the Walter Reed/3873D (WRD) canine cell line (10), which is derived from a subdermoid cyst of an irradiated male dog (10). The full-length 5.4-kb genome of MVC was recently mapped with palindromic termini (60). Under the control of a single P6 promoter, through the mechanism of alternative splicing and alternative polyadenylation, MVC expresses two nonstructural proteins (NS1 and NP1) and two capsid proteins (VP1 and VP2). Like the NS1 proteins of other parvoviruses, the NS1 of MVC is indispensable for genome replication. The NP1 protein, which is unique to the Bocavirus genus, appears to be critical for optimal viral replication, as the NP1 knockout mutant of MVC suffers from severe impairment of replication (60). A severe cytopathic effect during MVC infection of WRD cells has been documented (10, 60).The HBoV genome has been frequently detected worldwide in respiratory specimens from children under 2 years old with acute respiratory illnesses (2, 34, 55). HBoV is associated with acute expiratory wheezing and pneumonia (3, 34, 55) and is commonly detected in association with other respiratory viruses (34, 55). Further studies are necessary, however, to identify potential associations of HBoV infection with clinical symptoms or disease of acute gastroenteritis (7, 36). The full-length sequence of infectious MVC DNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ214110","term_id":"219665308","term_text":"FJ214110"}}FJ214110) that we have reported shows 52.6% identity to HBoV, while the NS1, NP1, and VP1 proteins are 38.5%, 39.9%, and 43.7% identical to those of HBoV, respectively (60).The cytopathic effect induced during parvovirus infection has been widely documented, e.g., in infections with minute virus of mice (MVM) (13), human parvovirus B19 (B19V) (58), parvovirus H-1 (25, 52), and BPV1 (1). In Bocavirus, cell death during BPV1 infection of embryonic bovine tracheal cells has been shown to be achieved through necrosis, independent of apoptosis (1). B19V-induced cell death of primary erythroid progenitor cells has been shown to be mainly mediated by an apoptotic pathway (58) in which the nonstructural protein 11kDa plays a key role (16). In contrast, the MVM-induced cytopathic effect has been revealed to be mediated by NS1 interference with intracellular casein kinase II (CKII) signaling (22, 44, 45), a nonapoptotic cell death. Oncolytic parvovirus H-1 infections can induce either apoptosis or nonapoptotic cell death, depending on the cell type (25, 40). Therefore, the mechanisms underlying parvovirus infection-induced cell death vary, although NS1 has been widely shown to be involved in both apoptotic and nonapoptotic cell death. The nature of the cytopathic effect during Bocavirus MVC infection has not been studied.Parvovirus replication requires infected cells at the S phase. Infection with parvovirus has been revealed to accompany a cell cycle perturbation that mostly leads to an arrest in the S/G₂ phase or the G₂/M phase during infection (30, 33, 42, 47, 65). MVM NS1 expression induces an accumulation of sensitive cells in the S/G₂ phase (6, 46, 47). Whether MVC infection-induced cell death is accompanied by an alternation of cell cycle progression and whether the viral nonstructural protein is involved in these processes have not been addressed.In this study, we found, in contrast with other members of the family Parvoviridae, expression of both the nonstructural and structural proteins of MVC by transfection did not induce cell death or cell cycle arrest. However, the cytopathic effect induced during MVC infection is a replication-coupled, mitochondrion-mediated and caspase-dependent apoptosis, accompanied with a gradual cell cycle arrest from the S phase to the G₂/M phase, which is facilitated by the MVC genome.