Location of F plasmid transfer operon genes traC and traW and identification of the traW product.

As part of an analysis of the conjugative transfer genes associated with the expression of F pili by plasmid F, we have investigated the physical location of the traC and traW genes. We found that plasmid clones carrying a 2.95-kilobase EcoRI-EcoRV F transfer operon fragment were able to complement transfer of F lac traC mutants and expressed an approximately 92,000-dalton product that comigrates with TraC. We also found that traW-complementing activity was expressed from plasmids carrying a 900-base-pair SmaI-HincII fragment. The traW product was identified as an approximately 23,000-dalton protein. The two different F DNA fragments that expressed traC and traW activities do not overlap. Our data indicate that the traC gene is located in a more-tra operon promoter-proximal position than suggested on earlier maps and that traW is distal to traC. These results resolve a long-standing question concerning the relationship of traW to traC. The clones we have constructed are expected to be useful in elucidating the role of proteins TraC and TraW in F-pilus assembly.     

A traC mutant that retains sensitivity to f1 bacteriophage but lacks F pili.

An F lac pro mutant which was temperature sensitive for infection by the filamentous bacteriophage f1 but resistant to the F-specific icosahedral RNA phage f2 was isolated. Cells carrying the F' mutation failed to elaborate F pili at all temperatures. Mutant cells were able to pair with recipient cells during bacterial conjugation, but transfer of conjugal DNA occurred at a greatly reduced frequency. Complementation analyses showed the F' mutation to be in the traC gene. When a plasmid carrying traC was introduced into hosts harboring the F' mutation, phage sensitivity, the ability to elaborate F pili, and conjugation efficiency were restored. The mutation was named traC1044. The F lac pro traC1044 mutant appears to be unique among traC mutants in retaining host sensitivity to the filamentous phage f1 in the absence of expression of extended F pili. Phage f1 attachment sites appeared to be present at the cell surface in traC1044 mutants. The reduced accessibility of these sites may account for the reduced efficiency of phage f1 infection of traC1044 hosts, although the possibility that a defect was present in the receptor site itself was not eliminated. Membranes of hosts carrying the F' mutation contained a full complement of mature F-pilin subunits, so the product of traC is presumably required for pilus assembly but not for pilin processing. This, together with the deficiency in conjugal DNA transfer, suggests that traC may be part of a membrane-spanning tra protein complex responsible for pilus assembly and disassembly and conjugal DNA transmission.     

Analysis of transfer genes and gene products within the traB-traC region of the Escherichia coli fertility factor, F.

A series of plasmids that carry overlapping segments of F DNA encoding the genes in the traB-traC interval was constructed, and a restriction enzyme map of the region was derived. Plasmids carrying deletions that had been introduced at an HpaI site within this interval were also isolated. The ability of these plasmids to complement transfer of F lac plasmids carrying mutations in traB, traV, and traW, and traC was analyzed. The protein products of the plasmids were labeled in UV-irradiated cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These analyses showed that the product of traV is a polypeptide that migrates with an apparent molecular weight of 21,000. It was not detected when [35S]methionine was used to label plasmid products, but was readily detected in 14C-amino acid labeling experiments. A 21,500-dalton product appeared to stem from the region assigned to traP. A 9,000-dalton product was found to stem from a locus, named traR, that is located between traV and traC. No traW activity could be detected from the region of tra DNA examined. Our data also indicated that traC is located in a more promoter-proximal position than suggested on earlier maps. The plasmids constructed are expected to be useful in studies designed to identify the specific functions of the traB, -P, -V, -R, and -C products.     

Conjugal transfer system of the IncN plasmid pKM101.

The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically. Its organization differed significantly from that of the F plasmid. The tra genes are located in three regions, each between 3 and 4 kilobases in length. All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus. The plasmid's origin of transfer was localized to a 1.2-kilobase region at an extreme end of the transfer region. Using two different methods, we have identified 11 complementation groups required for transfer. One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids. One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused.     

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F.

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.     

Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response.

The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Emr) or Tn916 (Tcr) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF- orfY-sea-1 with all but repB and traA oriented in the same (left-to-right) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.     

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.     

Deletion Map of the Escherichia coli K-12 Sex Factor F: the Order of Eleven Transfer Cistrons

A series of Hfr deletion mutants was isolated. These mutants contain deletions which extend from a lambda prophage into an Flac which is integrated into the gal operon. Transfer-deficient deletion mutants were found to fall into four different phenotypic groups when tested for male- and female-specific phage resistance. Conjugational and transductional complementation tests with Flac point mutants deficient in transfer (tra(-)) were performed, and the order of 11 tra cistrons was determined. The tra genes are all located between an F gene for the inhibition of female-specific phages and the transposed lac operon originally carried by the Flac. The order of genes in the Hfr studied was established to be: proC... phi(II) (R)... traJ traA traE traK traB traC traF traH traG traD traI...lac...attlambda...bio.     

Localization of TraC, a protein involved in assembly of the F conjugative pilus.

TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure. Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified. When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies. Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm. However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction. Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1. These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region. This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.     

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI.

The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.     

Structure, expression and regulation of the murine 4F2 heavy chain.

The murine 4F2 molecule is a 125 kilodalton disulfide-linked heterodimeric cell-surface glycoprotein which has been shown to be involved in the processes of cellular activation and proliferation (1). To elucidate the structure, expression, and regulation of the 4F2 molecule, a murine 4F2 heavy chain (4F2HC) cDNA has been isolated and structurally characterized. The murine 4F2HC is a 526 amino acid (aa) type II membrane glycoprotein which is composed of a 75 aa N-terminal intracytoplasmic region, a single hydrophobic putative transmembrane domain, and a 428 aa C-terminal extracellular domain. Comparison with the human 4F2HC cDNA reveals the highest degree of sequence identity within the transmembrane and intracytoplasmic domains. Northern blot analyses have demonstrated that the 4F2HC gene is expressed at relatively high levels in adult testis, lung, brain, kidney, and spleen, and at significantly lower levels in adult liver and cardiac and skeletal muscle. Studies designed to elucidate the pattern of regulation of the murine 4F2HC gene have demonstrated that it is induced during the process of cell activation, but is subsequently expressed at constant levels throughout the cell cycle in exponentially growing cells.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.     

Human mitochondrial ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling factor 6

Coupling factor 6 (F6) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lambda gt10 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3'-untranslated sequence. The remainder of the cDNA sequence coded for a mature human F6 protein of 76 aa. The deduced primary aa sequence showed 81% homology to that of bovine F6, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.     

Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1

The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes. The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation. The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753. The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.     

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.     

Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro

Although p48 is the most conserved subunit of mammalian DNA polymerase alpha-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.     

The Capsid Gene of Feline Calicivirus Contains Linear B-Cell Epitopes in both Variable and Conserved Regions

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.     

Characterization of two monoclonal antibodies, 38F10 and 44D11,against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor(F_0) and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies(mAbs) against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus(HearNPV)(Ha F) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10,recognizes amino acid(aa) 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process.     

Purification and characterization of six fibrinolytic serine-proteases from earthworm Lumbricus rubellus

The six lumbrokinase fractions (F1 to F6) with fibrinolytic activities were purified from earthworm Lumbricus rubellus lysates using the procedures of autolysis, ammonium sulfate fractionation, and column chromatography. The proteolytic activities on the casein substrate of the six iso-enzymes ranged from 11.3 to 167.5 unit/mg with the rank activity orders of F2 > F1 > F5 > F6 > F3 > F4. The fibrinolytic activities of the six fractions on the fibrin plates ranged from 20.8 to 207.2 unit/mg with rank orders of F6 > F2 > F5 > F3 > F1 > F4. The molecular weights of each iso-enzyme, as estimated by SDS-PAGE, were 24.6 (F1), 26.8 (F2), 28.2 (F3), 25.4 (F4), 33.1 (F5), and 33.0 kDa (F6), respectively. The plasminogen was activated into plasmin by the enzymes. The optimal temperature of the six iso-enzymes was 50 degrees C, and the optimal pH ranged from pH 4-12. The four iso-enzymes (F1-F4) were completely inhibited by PMSF. The two enzymes (F5 and F6) were completely inhibited by aprotinin, TLCK, TPCK, SBTI, LBTI, and leupeptin. The N-terminal amino acid (aa) sequences of the first 20 to 22 residues of each fraction had high homology. All six iso-enzymes had identical aa residues 2-3 and 13-15. The N-terminal 21-22 aa sequences of the F2, F3, and F4 iso-enzymes were almost the same. The N-terminal aa sequences of F5 and F6 were identical.     

Pheromone-regulated expression of sex pheromone plasmid pAD1-encoded aggregation substance depends on at least six upstream genes and a cis-acting, orientation-dependent factor

Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an alpha-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated alpha-amylase activity, a total of at least six genes, traB, traC, traA, traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of traC); only traC can relieve traB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation of traE1, orfY, or orf1, respectively, results in a total loss of alpha-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering traA, P(0), and traE1 without disrupting any gene or control element switches off amyL or asa1 expression, indicating the involvement of a cis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed.