Hydroxymethylcytosine-containing and Tryptophandependent Bacteriophages Isolated from City Effluents

Kay, D.; Fildes, Paul

doi:10.1099/00221287-27-1-143

Cited by 21 publications

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References 1 publication

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“…Plasmids expressing proOmpA mutants were transformed into E. coli MC4100rh -[F -, araD139∆(argF-lac)U169, rpsL150 (Str r ), erlA1, flbB5301 deoC1 ptsF25, rbsR, recA, Ox2 r ] or JC6650 [lac, proAB, rpsL, recA1, supF(F′, proAB + , lacI q , lacZ∆M14)], which are OmpAstrains (9). Phages K3 (10) and Ox2 (11) have been described. They were propagated in strain JC6650 using LB broth as described (9).…”

Section: Methodsmentioning

confidence: 99%

Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism

et al. 1999

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Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996−5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS−PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 °C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 Å from the bilayer center. In a narrow temperature range between 30 and 35 °C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40 °C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 Å from the center after refolding was complete. In contrast Trp-7 approached the 10 Å distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four β-hairpins of the eight-stranded β-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four β-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.

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Section: Methodsmentioning

confidence: 99%

Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism

et al. 1999

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“…It is AompC and ompRI and hence does not transcribe ompF, which is highly homologous to ornpC (36). The OmpA-specific phages Ox2 (24,37,38) and K3 (22,37,38), as well as those specific for OmpC and OmpF, TuIb and TuIa (11), respectively, have been described. Cells were grown in L broth (35) at 37°C if not specified otherwise.…”

Section: Methodsmentioning

confidence: 99%

Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF

1990

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Selection was performed for resistance to a phage, Ox2, specific for the Escherichia coli outer membrane protein OmpA, under conditions which excluded recovery of ompA mutants. All mutants analyzed produced normal quantities of OmpA, which was also normally assembled in the outer membrane. They had become essentially resistant to OmpC and OmpF-specific phages and synthesized these outer membrane porins at much reduced rates. The inhibition of synthesis acted at the level of translation. This was due to the presence of lipopolysaccharides (LPS) with defective core oligosaccharides. Cerulenin blocks fatty acid synthesis and therefore that of LPS. It also inhibits synthesis of OmpC and OmpF but not of OmpA (C. Bocquet-Pages, C. Lazdunski, and A. Lazdunski, Eur. J. Biochem. 118:105-111, 1981). In the presence of the antibiotic, OmpA synthesis and membrane incorporation remained unaffected at a time when OmpC and OmpF synthesis had almost ceased. The similarity of these results with those obtained with the mutants suggests that normal porin synthesis is not only interfered with by production of mutant LPS but also requires de novo synthesis of LPS. Since synthesis and assembly of OmpA into the outer membrane was not affected in the mutants or in the presence of cerulenin, association of this protein with LPS appears to occur with outer membrane-located LPS.It remains unknown how proteins of the outer membrane of gram-negative bacteria are sorted to this membrane. We are studying this question with the 325-residue OmpA protein (7) of Escherichia coli (18,19,25,26) and have asked whether non-ompA mutants exist which might no longer incorporate the OmpA protein, and possibly others, into the outer membrane. Toward this end we have performed selections for resistance to an OmpA-specific phage under conditions which exclude the appearance of mutations in the ompA gene. Paradoxically, almost all mutants recovered so far possessed an unaltered OmpA protein, present in the outer membrane, and greatly reduced quantities of the outer membrane porins OmpC and OmpF, which have nothing to do with infection by this phage. Here we show that these mutants produced defective lipopolysaccharides (LPS). These and some other results shed some light on the role of LPS in the membrane assembly of these three proteins. MATERIALS AND METHODSBacterial strains, phages, and growth conditions. The ompA+ derivative of strain UH203 (19) is lac supF recA proA (or proB) rpsL(F' lacIq lacZAMJS proAB+). Its secA derivative, strain UH204, has the same characteristics but, in addition, is leu:: TnJO and secA(Ts5l) (17,44). The construction of a non mutant (49) in strain UH203 ompA+ was started with a precursor of this strain, JC6650 pro his (15). Phage P1 was grown on strain P400 (which is his' non [57]) and used to transduce the former strain to his'; the non allele is cotransduced at a frequency of -30% and was identified by the inability of strains carrying it to give rise to mutants resistant to phage T7,(49). The his+ non strain was made recA by t...

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“…The T-even-type Escherichia coli phage Ox2 (20) uses the outer membrane protein OmpA as a receptor (15,46). Host range mutants of this phage which had switched receptors in consecutive steps have been described.…”

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confidence: 99%

New outer membrane-associated protease of Escherichia coli K-12

1994

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The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced. The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence. Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT. OmpP possessed the same enzymatic activity as OmpT. Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface. Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected. It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity. Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system.The T-even-type Escherichia coli phage Ox2 (20) (Dianova, Hamburg [16]). Strain Y1090 was lysogenized with one of these, Agtll-15. The lysogen was thermoinduced, and the phage was precipitated with polyethylene glycol (57) and then centrifuged through a CsCl step gradient (43). The 4.5-kb EcoRI insert of this phage was cloned into pUC18, yielding pAK1. The ompP gene was present on a 2.2-kb BglII fragment of this insert (see Results), which was cloned into the BamHI site of pUC18, resulting in pAK1-5. DNA sequencing (44) was performed using plasmids which were purified by CsCl density gradient centrifugation. Sequencing was started with a 17-mer sequencing primer and a 24-mer reverse sequencing primer (New England Biolabs). The sequence from bp 1 to 1020 was obtained from subclones by using restriction sites for AccI (bp 1), BglII (bp 50), HindIII (bp 561 and 610), and HinclI (bp 334 and 777; for the positions, see Fig. 4). For further sequencing, new primers annealing within the cloned chromosomal fragment were obtained from Appligene (Heidelberg, Germany [also shown in Fig. 4]). This strategy left a few uncertainties and gaps in the complementary strand upstream from bp 900 and 1400. A site for EclXI starts at bp 1474 (CGGCCG), and an EcoRI site exists in the vector (see Fig. 3). A deletion was obtained by restricting pAK1-5 with the two enzymes, followed by filling-in with the Klenow fragment of DNA polymerase I and religation. The resulting DNA fragment was sequenced by using the reverse sequencing primer, covering the sequence from bp 1450 to 1100. For the generation of two other deletions, pAK1-5 was opened with EclXI, digested for various time intervals with exonuclease III, and made blunt-ended with nuclease SI. After cleavage with EcoRI, the DNA was made blunt ended as above and religated. Two deletions were recovered; one started at bp 1066 and the other started at position 903. Both were sequenced with the reverse primer, providing the sequence from bp 1050 to 650. With these deletions, the sequence was completed for both strands from bp 334 to 1675. Southern blots were probed by ...

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Hydroxymethylcytosine-containing and Tryptophandependent Bacteriophages Isolated from City Effluents

Cited by 21 publications

References 1 publication

Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism

Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism

Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF

New outer membrane-associated protease of Escherichia coli K-12

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