Roles of internal cysteines in the function,localization, and reactivity of the TraV outer membrane lipoprotein encoded by the F plasmid

We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein. Each was mutated to a serine separately and together to yield three mutant traV genes: traV(C10S), traV(C18S), and traV(C10S/C18S). All three cysteine mutations complemented a traV mutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of the traV(C18S) and, especially, traV(C10S/C18S) mutant strains were significantly less than those of the traV(+) and traV(C10S) strains. Thus, unlike the Agrobacterium tumefaciens T-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity. By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane. However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraV(C18S) mutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraV(C10S) or TraV(C10S/C18S) proteins. Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation. Finally, whereas the TraV(C10S) and TraV(C18S) proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraV(C10S/C18S) protein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions. Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.     

TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.     

The TraK accessory factor activates substrate transfer through the pKM101 type IV secretion system independently of its role in relaxosome assembly

A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin-of-transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4-like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon-Helix-Helix (RHH) family of DNA-binding proteins, to transfer of DNA and protein substrates through the pKM101-encoded T4SS. Using a combination of cross-linking/affinity pull-downs and two-hybrid assays, we determined that TraK self-associates as a probable tetramer and also forms heteromeric contacts with pKM101-encoded TraI relaxase, VirD4-like TraJ receptor, and VirB11-like and VirB4-like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ–TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the Tra_pKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.     

The effect of tra mutations on the synthesis of the F-pilin membrane polypeptide

Summary We had previously demonstrated that several F specific polypeptide bands could be detected in the membranes of Flac, but not F^- strains of Escherichia coli K 12, (Moore et al. 1981). One of these polypeptides co-migrated with F-pilin protein on polyacrylamide gels. We have now analyzed ³⁵[S]methionine labelled membrane preparations from a series of strains containing Flac tra mutant plasmids. The F-pilin polypeptide was absent from preparations of strains containing all traA mutants tested, confirming the importance of the traA gene in F-pilin biosynthesis. A polypeptide which migrated in the F-pilin position was still present, however, in membranes prepared from Flac strains carrying mutations in traL, traE, traK, traB, traV, traW, traC, traU, traF, traH or traG despite the inability of these mutants to elaborate F-pili filaments. Thus, all of these gene products may be concerned with F-pilus assembly and outgrowth rather than biosynthesis of the F-pilin subunit. The polar mutation tra-4 did, however, prevent the appearance of pilin polypeptide, indicating that at least one unidentified gene in the region between traE and traG must also be required in F-pilin biosynthesis.Our analysis also permitted the identification of a 100,000 dalton membrane protein as the product of traG. The appearance of an F specific 12,000 dalton protein was prevented by traD amber mutants. As expected, traJ mutants prevented the expression of all the tra operon products detected except the product of traT. The traT product band was reduced only to 50–60% of its normal intensity.     

The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP-binding motifs and is localized to the cytoplasmic membrane

The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type-A and two type-B NTP-binding motifs. Site-directed mutagenesis revealed that the type-A motif and one of the type-B motifs, 109 amino acid residues downstream of the type-A motif, were essential for both plasmid transfer and Cma. The second type-B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino-terminal c-Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c-Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter- and intramycelial transfer processes.     

TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin.

Conjugative transfer of the self-transmissible IncP plasmid RP4 requires the product of the RP4 traK gene. By using the phage T7 expression system, the traK gene product was efficiently overproduced and purified to near homogeneity. traK encodes a basic protein (pI = 10.7) of 14.6 kDa that, as shown by DNA fragment retention assay, interacts exclusively with its cognate transfer origin. The apparent equilibrium constant K(app) for the complex of TraK and oriT-DNA was estimated to be 4 nM. Footprinting experiments using DNase I or hydroxyl radicals indicate that several TraK molecules interact specifically with an intrinsically bent region of oriT, covering a range of almost 200 base pairs. The TraK target sequence maps in the leading region adjacent to the relaxation nick site and recognition sequences involved in relaxosome formation but does not overlap them. Specific interactions between TraK and the DNA occur only on one side of the double helix. Electron microscopy of TraK-oriT complexes demonstrates that binding of TraK to its recognition region apparently shrinks the length of the target DNA, suggesting that the nucleic acid becomes wrapped around a core of TraK molecules. Formation of this structure could be favored by the presence of the sequence-directed bend in the TraK recognition region.     

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.     

Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [(3)H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.     

Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12

Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.     

A two-step two-hybrid system to identify functionally significant protein-protein interactions

The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system. Its purpose is to identify proteins that interact with a relatively small, defined, functionally significant domain of a protein of interest. In this method, a first round of screening is performed to identify proteins that interact with bait comprised of the wild type protein. Next, each of the prey identified in this first round is tested for its ability to interact with functionally impaired, mutant bait. Any proteins that interact with the wild type bait, but not the mutant bait, are candidate effectors or regulators of the protein of interest.     

The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein.

The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane. The virB operon of A. tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence. Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus. Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins. VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion. Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels. A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes. The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.     

Genetic Analysis of Transfer by the Escherichia coli Sex Factor F, Using P1 Transductional Complementation

P1 transduction has been used to perform a complementation analysis of a series of transfer-deficient mutants of Flac. The results define ten cistrons and are consistent with the results of a conjugational analysis presented in an accompanying report. Both sets of results are summarized here. Between them, they define eleven cistrons, traA through traK, necessary for conjugational deoxyribonucleic acid (DNA) transfer. Mutants in traI and traD and some in traG still make F-pili, although traD mutants are resistant to f2 phage; their products may be involved in conjugational DNA metabolism. Other mutants in traG and all mutants in the remaining eight cistrons do not make F-pili. One of these, traJ, may be a control cistron, and the others may specify a biosynthetic pathway responsible for synthesis and modification of the F-pilin subunit protein and its assembly into the F-pilus.     

Analysis of the transfer region of the Streptomyces plasmid SCP2*

plJ903, a bifunctional derivative of the 31.4 kb low-copy number, conjugative Streptomyces plasmid SCP², was mutagenized in Streptomyces lividans using Tn4560. Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated. The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb. The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.     

Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background

Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP(S210A) protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP(S210A) was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP(S210A) also fractionated. These results suggested that the DegP(S210A)-mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.     

人FAM92A1基因诱饵表达质粒的构建与鉴定

目的 构建人FAM92A1基因（hFAM92A1）的诱饵表达质粒pGBKT7-hFAM92A1并检测其蛋白表达、毒性和自激活作用.方法 PCR扩增hFAM92A1的基因编码序列并克隆入诱饵表达载体pGBKT7中,酶切和测序鉴定后,转化到酵母AHl09细胞中,Western印迹检测诱饵蛋白表达情况,同时检测诱饵蛋白的毒性和自激活作用.结果 成功构建FAM92A1基因的诱饵表达质粒pGBKT7-hFAM92A1,测序结果正确.Western印迹实验证实酵母细胞高表达诱饵蛋白hFAM92A1,诱饵蛋白没有自激活作用.结论 构建的诱饵表达质粒pGBKT7-hFAM92A1可用于下一步酵母双杂交系统实验,为进一步研究hFAM92A1功能奠定了基础.     

Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein.

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.     

Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system

Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 106 clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.     

A novel approach for the identification of protein-protein interaction with integral membrane proteins

Protein-protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein-protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein-protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein-protein interactions with high specificity and selectivity, where other methods fail.