PubMed Health⌕ Search

Biomedical subjects

E B Goldberg

Publications and source records attributed to E B Goldberg.

At least 19 recordsLinked to original sources
PubMed2000

Bacteriophage T4 self-assembly: in vitro reconstitution of recombinant gp2 into infectious phage.

T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in the recBCD(+) host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2(+) phage infection (H(+)) with tails from a gp2(+) or gp2(-) phage infection (T(+) or T(-)), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H(-)) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV(+) cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2(+) phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.

Bacteriophage T4↗
PubMed2000

Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length.

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

Bacteriophage T4↗
PubMed1999

Bacteriophage T4 gp2 interferes with cell viability and with bacteriophage lambda Red recombination.

The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.

Bacteriophage T4↗
PubMed1996

The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs.

The sequenced members of a novel family of small, hydrophobic, bacterial multidrug-resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic alpha-helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.

Amino Acid Sequence↗
PubMed1994

Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump.

Multidrug transport system in proteoliposomes was reconstituted using the highly purified membrane transport protein responsible for bacterial multidrug resistance. This protein (named Smr, for staphylococcal multidrug resistance) consists of 107 amino acid residues and displays four putative transmembrane domains. The Smr protein was tagged with a FLAG epitope, and the modified protein was expressed, purified, characterized, and reconstituted into proteoliposomes. With this in vitro experimental system, it has been demonstrated that a highly purified multidrug resistance protein functions as a drug pump, which transports methyltriphenylphosphonium actively against a 10(3)-fold concentration gradient. Delta mu H+ was shown to be a driving force, and an electrogenic drug/proton antiport was suggested as the molecular mechanism of the drug transport. Of the 2 Glu residues in putative extramembrane loops of the Smr polypeptide chain, Glu-24 was shown to be involved in determining the specificity of drug resistance. Replacement of both of these Glu residues with Asp produced active Smr. In contrast, Smr was unable to protect cells from multiple drugs when a Glu-13-->Asp-13 replacement was made. We suggest that Glu-13, a unique acidic residue located in the hydrophobic domain of Smr, is directly involved in the drug/proton antiport.

Amino Acid Sequence↗
PubMed1994

The NhaB Na+/H+ antiporter is essential for intracellular pH regulation under alkaline conditions in Escherichia coli.

We isolated a mutant of Escherichia coli which was defective in an Na+/H+ antiporter and grew poorly under alkaline conditions [Ishikawa, T., Hama, H., Tsuda, T., and Tsuchiya, T. (1987) J. Biol. Chem. 262, 7443-7446]. Later, it was concluded that the defective Na+/H+ antiporter in the mutant was the NhaB system, and the nhaB gene was mapped to 25.6 min on the E. coli chromosome [Thelen, P., Tsuchiya, T., and Goldberg, E.B. (1991) J. Bacteriol. 173, 6553-6557]. We found that the NhaB-defective cells cannot grow in a high pH medium. Furthermore, intracellular pH in the mutant cells was almost the same as extracellular pH between 7.9 and 9.1, that is, intracellular pH was not regulated at this pH range. On the other hand, intracellular pH of the wild-type cells was maintained at about 7.6 when the extracellular pH was between 7.6 and 8.5. Thus, the NhaB Na+/H+ antiporter is essential for the regulation of intracellular pH under alkaline conditions in E. coli. Introduction of nhaA gene into the mutant cells increased Na+/H+ antiporter activity, but did not restore the defective growth and defective intracellular pH regulation under alkaline conditions.

Bacterial Proteins↗
PubMed1992

A staphylococcal multidrug resistance gene product is a member of a new protein family.

The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.

Amino Acid Sequence↗
PubMed1992

Properties of recombinant cells capable of growing on serine without NhaB Na+/H+ antiporter in Escherichia coli.

Escherichia coli HIT-1 has a mutation in the Na+/H+ antiporter gene, nhaB (P. Thelen, T. Tsuchiya, and E. B. Goldberg, J. Bacteriol. 173:6553-6557, 1991). This strain is not able to utilize serine as a carbon source (T. Ishikawa, H. Hama, M. Tsuda, and T. Tsuchiya, J. Biol. Chem. 262:7443-7446, 1987), because an active NhaB is required to maintain the electrochemical potential of Na+, which drives serine transport via the Na+/serine carrier, the major transport system for serine. We isolated recombinant cells from a cross between strains HIT-1 and Hfr, and these cells were able to grow on serine even though the NhaB Na+/H+ antiporter of the recombinant cells was still defective. We found that the activity of the H+/serine cotransport system, one of the minor serine transport systems in E. coli, was elevated in the recombinant cells. H+/serine cotransport activity was induced by leucine in the recombinant cells more strongly than in strain HIT-1. A kinetic analysis showed that the Vmax, but not the Km, of the transport system was much higher in the recombinant cells than in strain HIT-1 cells.

Biological Transport↗
PubMed1991

Characterization and mapping of a major Na+/H+ antiporter gene of Escherichia coli.

Using in vivo assays, we show that the Na+/H+ antiporter activity of the Escherichia coli mutant HIT-1 is reduced dramatically compared with activity in wild-type cells. An isogenic nhaA (formerly antA) deletion strain, however, is not significantly different from wild type in this respect. We call the locus affecting Na+/H+ antiporter activity of the HIT-1 mutant nhaB. The nhaB activity exhibits no pH dependence in the range between 7.0 and 8.5, whereas that of the nhaA gene increases considerably at pH levels above 8.0. Mutants with defects in nhaB grow normally on agar media containing 0.5 M NaCl, but nhaA mutants are sensitive to 0.5 M NaCl. We have mapped the nhaB mutation of HIT-1 to 25.6 min on the E. coli map. It is unlinked to the nhaA region, which is located at about 0.5 min. Since a cell with a mutation in nhaB alone is essentially Na+/H+ antiporter negative up to pH 8.0, we conclude that nhaB is required for the major Na+/H+ antiporter activity in the usual physiological pH range.

Carrier Proteins↗
PubMed1989

Cloning and identification of bacteriophage T4 gene 2 product gp2 and action of gp2 on infecting DNA in vivo.

We sequenced bacteriophage T4 genes 2 and 3 and the putative C-terminal portion of gene 50. They were found to have appropriate open reading frames directed counterclockwise on the T4 map. Mutations in genes 2 and 64 were shown to be in the same open reading frame, which we now call gene 2. This gene codes for a protein of 27,068 daltons. The open reading frame corresponding to gene 3 codes for a protein of 20,634 daltons. Appropriate bands on polyacrylamide gels were identified at 30 and 20 kilodaltons, respectively. We found that the product of the cloned gene 2 can protect T4 DNA double-stranded ends from exonuclease V action.

Amino Acid Sequence↗
PubMed1987

Characterization of a Na+/H+ antiporter gene of Escherichia coli.

A mutant of Escherichia coli with increased Na+/H+ antiport activity was isolated and found by other workers to harbor two mutations [Niiya, S., Yamasaki, K., Wilson, T.H. & Tsuchiya, T. (1982) J. Biol. Chem. 257, 8902-8906]. The mutation that leads to increased Na+/H+ antiport (antup) has now been separated and mapped. antup maps in the vicinity of 0.5 min on the E. coli map, and the presence of this mutation alone in an isogenic pair raises antiport activity (Vmax) by approximately 4-fold. We also characterized cells hearing plasmids containing fragments of a 15-kilobase-pair segment of DNA between carA and dnaJ. A wild-type gene located within 2 kilobase pairs counterclockwise of rpsT increases the Na+/H+ antiport activity when present in multiple copies.

Carrier Proteins↗
PubMed1982

Physical mapping and cloning of bacteriophage T4 anti-restriction endonuclease gene.

We have proposed that the ability of T4 to produce non-glucosylated progeny after a single cycle of growth on a galU rglA rglB+ mutant of Escherichia coli is due to the initiation of the rglB+ function by a phage-coded, anti-restriction endonuclease protein. Based on this hypothesis, we screened T4 deletion mutants for failure to give a burst in this host. The absence of an arn gene in phage mutants lacking the 55.5- to 58.4-kilobase region is verified by their inability to protect secondary infecting non-glucosylated phage from rglB-controlled cleavage. A functional arn gene was cloned on plasmid pBR325, and the 0.8-kilobase insert DNA was shown to be homologous to the DNA missing in the arn deletion phage.

Cloning, Molecular↗
PubMed1980

Restriction in vivo. V. Introduction of SOS functions in Escherichia coli by restricted T4 phage DNA, and alleviation of restriction by SOS functions.

Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degradation of restricted nonglycosylated T4 DNA by exonuclease V delayed cell division and induced filament formation and mutagenesis in lexA+ but not in lexA- cells. Delay of cell division was also dependent upon recA and recBC functions. Such degradation of DNA also dramatically increased mutagenesis in tif- Sfi- cells at 42 degrees. The synthesis of recA protein continued in the restricting host after infection by the monglucosylated T4 phage, but enhanced synthesis is not induced to the extent seen in SOS induced tif- cells grown at 42 degrees. We also found that restriction of nonglycosylated T4 was alleviated in UV irradiated cells. The UV induced alleviation of rgl and rK restriction depended upon post irradiation protein synthesis and was not observed in recA, lexA or recBC mutants.

DNA Repair↗
PubMed1980

Region-specific recombination in phage T4. II. Structure of the recombinants.

In this paper, we present results of crosses designed to elucidate the structure of recombinants in the tail-fiber region of bacteriophage T4, in which a glucosylation-dependent recombinations mechanism is operative, and the cause of the "special" recombination in glycosylated crosses is discussed. We present evidence that, when phage are nonglycosylated, recombination in the tail-fiber region proceeds via long heteroduplex overlaps. Mismatched bases within such regions (in nonglycosylated phage) are repaired efficiently (as contrasted to those of glucosylated phage), but asymmetrically; that is, there may be an equal probability of resolving the mismatch to mutant or wild type.

Capsid↗