Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Two Novel Membrane Proteins,TcpD and TcpE,Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.     

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.Conjugation systems are important contributors to the dissemination of antibiotic resistance determinants and virulence factors. Extensive analysis of conjugative plasmids from gram-negative bacteria has led to the elucidation of a general mechanism of conjugative transfer (10, 22). In this process, the transferred DNA is processed by components of a relaxosome complex. Specifically, the DNA is nicked at the origin of transfer (oriT) by a relaxase, which remains covalently coupled to the transferred DNA strand. The single-stranded DNA complex then interacts with the coupling protein, a DNA-dependent ATPase that provides the energy to actively pump the DNA through the mating pair formation (Mpf) complex into the recipient cell (36). The coupling protein interacts with both DNA processing proteins and components of the Mpf complex (1, 4, 12, 35, 38). These interactions have been demonstrated using bacterial and yeast two-hybrid approaches as well as gel filtration, pull-down, and coimmunoprecipitation studies.The mechanism of conjugative transfer has yet to be precisely determined for conjugative plasmids from gram-positive bacteria although bioinformatics analysis has identified similar gene arrangements and conservation of gene sequences within the transfer regions encoded on conjugative plasmids identified from strains of streptococcal, staphylococcal, enterococcal, and lactococcal origin (15). It was proposed that gram-positive and gram-negative conjugation systems utilize a similar transfer mechanism (15).In the anaerobic pathogen Clostridium perfringens conjugative plasmids have been shown to encode antibiotic resistance genes or extracellular toxins (3, 8, 9, 18). Although the contribution of conjugation to disease dissemination has not been systematically evaluated, it has been proposed that transfer of the C. perfringens enterotoxin plasmid pCPF4969 to normal flora isolates of C. perfringens may contribute to the severity of disease caused by non-food-borne isolates of C. perfringens (9).The prototype conjugative plasmid in C. perfringens is the 47-kb tetracycline resistance plasmid, pCW3. The complete sequence of pCW3 has been determined, and its unique replication protein and conjugation locus have been identified (8). Bioinformatics analysis of this C. perfringens tcp conjugation locus identified several proteins with limited similarity to proteins encoded within the transfer region of the conjugative transposon, Tn916 (8). The role of the tcp locus in the transfer of pCW3 has been confirmed by isolation of independent tcpA, tcpF, and tcpH mutants and subsequent complementation studies (8, 29). Since the region that encompasses the tcp locus is conserved in all conjugative plasmids from C. perfringens (2, 3, 8, 9, 18, 27) and since divergent tcpA homologues can complement a pCW3tcpA mutant (29), it appears that the conjugative transfer of both antibiotic resistance and toxin plasmids from this bacterium utilizes a common but poorly understood mechanism. Note that the C. perfringens tcp conjugation locus is different from the transfer regions of conjugative plasmids from other gram-positive bacteria.We have recently shown that the essential conjugation protein TcpH, a putative membrane-associated Mpf complex component, is localized to the poles of C. perfringens cells, as is another essential conjugation protein, TcpF (37). TcpH has also been shown to interact with itself and with the pCW3-encoded TcpC protein (37). In this study we have focused on the essential conjugation protein TcpA. Since TcpA encodes an FtsK/SpoIIIE domain found in DNA translocases (8), it is proposed that TcpA is involved in the movement of DNA during conjugative transfer, fulfilling a role equivalent to that of coupling proteins in other conjugation systems. Like such proteins, TcpA encodes two N-terminal transmembrane domains (TMDs) and a C-terminal cytoplasmic region that contains three motifs predicted to be involved in ATP binding and hydrolysis (8). Our previous studies revealed that the conserved motifs, motif I (Walker A box), motif II (Walker B box), and motif III (RAAG box), are essential for the function of TcpA. The C-terminal 61 amino acids (aa), though not essential for TcpA function, were shown to be important for efficient transfer of pCW3, as were the putative TMDs (29).To further investigate pCW3 transfer and the role of TcpA in this process, we have used bacterial two-hybrid analysis to examine protein-protein interactions involving TcpA. Using this system, interactions were observed between TcpA and itself, TcpC, TcpG, and TcpH. In addition, TcpC and TcpG were also found to self-interact. By combining these data with other data generated in this laboratory (37), we have constructed a model for the conjugative transfer of pCW3.     

Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed. Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms. The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin. Each plasmid contained the pUC18-derived multiple cloning site and the lacZ′ gene which enabled direct screening for recombinants in E. coli . These plasmids should prove invaluable for the genetic manipulation of C. perfringens.