High-resolution visualization by field emission scanning electron microscopy of Enterococcus faecalis surface proteins encoded by the pheromone-inducible conjugative plasmid pCF10.

Enterococcus faecalis can acquire antibiotic resistance and virulence genes by transfer of pheromone-inducible conjugative plasmids such as pCF10, which encodes tetracycline resistance. Two pCF10-encoded cell surface proteins, Sec10 and Asc10, have been previously shown to play an important role in the transfer of this plasmid. We used high-resolution, field emission scanning electron microscopy to visualize these proteins on the surfaces of a series of isogenic strains of E. faecalis. Immunogold labeling, using both 6- and 12-nm colloidal gold, unambiguously demonstrated the expression and distribution of Sec10 and Asc10 on the surface of the E. faecalis cells. On unlabeled E. faecalis cells which expressed either Sec10 or Asc10, the former appeared to be more readily detected. Immunogold labeling of E. faecalis cells expressing both Asc10 and Sec10 clearly demonstrated the abundance and intermixing of both proteins on the cell surface except at septal regions. Sec10 was observed to be distributed over the cell surface. At regions of cell-cell contact, fine strands representing Asc10 were observed directly attaching adjacent cells to one another.     

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.     

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.     

Cloning and expression of genes encoding pheromone-inducible antigens of Enterococcus (Streptococcus) faecalis.

Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the Tra150 and Tra130 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing Tn5 or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tra130 and Tra150.     

Role of the pheromone-inducible surface protein Asc10 in mating aggregate formation and conjugal transfer of the Enterococcus faecalis plasmid pCF10.

The high transfer frequency of pheromone-inducible conjugative plasmids of Enterococcus faecalis in liquid culture is due in part to the formation of mating aggregates. These aggregates result from the interaction of two surface components, aggregation substance (AS), which is plasmid encoded, and the chromosomally encoded binding substance (BS). In the accompanying paper (S.-M. Kao, S. B. Olmsted, A. S. Viksnins, J.C. Gallo, G. M. Dunny, J. Bacteriol, 173:7650-7664, 1991), the sequence of the prgB gene encoding the AS molecule (Asc10) produced by pheromone-induced cells carrying plasmid pCF10 is presented. Here we report the results of genetic and immunological experiments which define the role of Asc10 in aggregation and plasmid transfer. These data indicate expression of AS on the surface of an E. faecalis cell and its binding to BS expressed on a second cell are required for the formation of a mating pair and the efficient transfer of pCF10 in liquid matings. However, the orientation of the receptors was not critical for transfer; ie., AS expressed on recipient cells could facilitate plasmid transfer via binding to BS on the donor. Our results suggest that additional (as yet unidentified) products are involved in forming the channel that ultimately serves to transfer the DNA, with AS-BS binding serving primarily to generate the initial attachment between cells. The putative prgC gene product, identified by DNA sequencing (data presented in the accompanying paper), could be involved in transfer events occurring subsequent to aggregation.     

Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.     

Characterization of a rat tRNA gene cluster containing the genes for tRNAAsp, tRNAGly and tRNAGlu, and pseudogenes.

The putative genes for tRNAGAUAsp(C), tRNAGGAGly(G) and tRNAGAGGlu are in a cluster on the rat chromosome and are present exclusively in a 3.3 kb region cleaved with a restriction endonuclease EcoRI. The cluster reiterates about 10 times on the haploid DNA. Four lambda clones each containing an independent repeating unit were isolated from a rat gene library. The studies on the cloned DNA revealed that the length of the repeating unit including the 3.3 kb EcoRI fragment was at least 13.5 kb. Nucleotide sequence analysis of the 3.3 kb DNA in the isolated clones showed sequence variations among the repeating units and incomplete genes for tRNAGly and tRNAGlu within the clusters.     

The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S. cerevisiae. Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6-Sec8-Sec15 complex, indicating that these genes may encode some of the remaining components of this complex. To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co-immunoprecipitation studies. Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6-Sec8-Sec15 complex for peptide microsequencing. The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components. In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70. Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst.     

Cloning and analysis of structural and regulatory pectate lyase genes of Erwinia chrysanthemi ENA49

Erwinia chrysanthemi ENA49 structural and regulatory ptl genes, coding for pectate lyase (Ptl) were cloned in Escherichia coli cells. Phage vector lambda L47.1 and phasmid vector lambda pMYF131 were used for constructing libraries of BamHI and EcoRI fragments, respectively, of Er. chrysanthemi chromosomal DNA. Among the 1,100 hybrid clones containing BamHI Er. chrysanthemi DNA fragments and 11,000 hybrid clones containing EcoRI fragments, six and 45 clones, respectively, were identified as having pectolytic activity. Two different structural genes, designated ptlA and ptlB, have been subcloned on multi-copy plasmids. Genes ptlA and ptlB are located side by side on the chromosome of Er. chrysanthemi and transcribe in the same direction. Each of the genes has its own promoter. Southern-blot hybridization analysis showed that the cloned ptl genes shared practically no homology and each of the genes was represented by a single copy on the Er. chrysanthemi chromosome. Other ptl genes capable of expression in E. coli cells were not found in the gene libraries. Negative regulation of the ptlA gene expression by a cloned gene called ptlR was shown. To screen the gene library for the ptlR gene, a specific genetic system was devised. The genes studied are located within an EcoRI chromosomal DNA fragment of 7.3 kb in the order: ptlA-ptlB-ptlR.     

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.     

Localization of a chromosomal mutation affecting expression of extracellular lipase in Staphylococcus aureus.

We describe a Tn551 chromosomal insertion in Staphylococcus aureus S6C that results in sharply reduced expression of extracellular lipase. With Tn917 as a probe, the insertion in the original mutant (KSI905) was localized to a 12.6-kb EcoRI DNA fragment. The 12.6-kb fragment was cloned and used as a probe to identify a 26-kb EcoRI fragment containing the Tn551 insertion site in the S6C parent strain. Restriction endonuclease analysis of the 12.6- and 26-kb EcoRI fragments confirmed that the Tn551 insertion in KSI905 was accompanied by a deletion of 18.7 kb of chromosomal DNA. Tn551 was transduced from KSI905 back into the S6C parent strain. All transductants exhibited the same lipase-negative (Lip-) phenotype and contained the same mutation with respect to both the insertion and the 18.7-kb deletion. The inability to produce lipase was not caused by disruption of the lipase structural gene, since all Lip- mutants carried intact copies of geh. Moreover, the Tn551 insertion was localized to a region of the staphylococcal chromosome at least 650 kb from geh. Taken together, these results suggest that the Tn551 insertion occurred in a region of the chromosome encoding a trans-active element required for the expression of extracellular lipase. A 20-bp oligonucleotide corresponding to a sequence within the region encoding RNA II near the Tn551 insertion site in ISP546 (H.L. Peng, R.P. Novick, B. Kreiswirth, J. Kornblum, and P. Schlievert, J. Bacteriol. 170:4365-4372, 1988) and a 1.75-kb DNA fragment representing the region encoding RNA III were used as gene probes to show that the Tn551 insertion did not occur in the agr locus. We conclude that the genetic element functions independently of agr or as an unrecognized part of that regulatory system.     

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.     

Cloning of genes for naphthalene metabolism in Pseudomonas putida.

Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate. Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus. Deletion plasmids provide a restriction map of both operons. Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression.     

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.     

The 35- and 36-kDa beta subunits of GTP-binding regulatory proteins are products of separate genes

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the alpha, beta, and gamma subunits of G proteins. Recently two cDNA clones encoding beta subunits, beta 1 and beta 2, were isolated from bovine and human cDNA libraries. We report here that the beta 2 gene encodes the 35-kilodalton (kDa) component of the beta 35/beta 36 subunit of G proteins and that the beta 1 gene encodes the 36-kilodalton component. The in vitro translation product of the beta 2 cDNA co-migrates with the 35-kDa beta subunit (beta 35), while the in vitro product of the beta 1 cDNA co-migrates with the 36-kDa beta subunit (beta 36) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic beta 2 peptides bind specifically to the beta 35 component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two beta subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution.     

Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins, The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein presumably forming a copper-processing system. A consensus sequence for an Anr- or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN-binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N2-fixing ability.