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E Yagil

Publications and source records attributed to E Yagil.

At least 19 recordsLinked to original sources
PubMed1996

A second site-specific recombination event in the lambdoid bacteriophage HK022.

An in vitro site-specific recombination reaction of the lambdoid phage HK022 has revealed two supercoiled products that proved to be Holliday intermediates. One of them is the Holliday intermediate which has resulted from an attP x attB reaction. The other is an intermediate which has resulted from a recombination reaction between attP and the attL site of the product from the first reaction. The preferential attL x attP over attR x attP reaction was confirmed in vitro and in vivo by challenging attP sites with attL and attR sites. The biased attP x attL over attP x attR reaction in phage HK022 is discussed.

Bacteriophage lambda↗
PubMed1995

Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases.

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.

Amino Acid Sequence↗
PubMed1995

Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases.

The Integrases of bacteriophages lambda and HK022 promote recombination between DNA molecules that carry attachment sites. The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected mutants of lambda integrase with increased ability to recombine HK022 sites. This selection yielded eleven different amino acid substitutions at eight different positions. Three of the positions belong to a larger set that were identified as important for the lambda/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding lambda residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels. Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the lambda/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine lambda sites. However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of lambda sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase. The first three substitutions act principally to prevent recombination of lambda sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity.

Amino Acid Sequence↗
PubMed1995

Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi.

Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp). This induction is relA independent but depends on the spoT gene product. A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT. We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon.

Alkaline Phosphatase↗
PubMed1994

Position and direction of strand exchange in bacteriophage HK022 integration.

The positions of the endonucleolytic cleavages promoted by the integrase protein (Int) of coliphage HK022 within its attB site were determined. The protein catalyses a staggered cut, which defines an overlap sequence of 7 bp within the core site. The overlap region is at the center of symmetry of a palindromic sequence which appears in all four putative att core binding sites for Int. We confirm that the order of strand exchange is similar to that in phage lambda.

Attachment Sites, Microbiological↗
PubMed1994

Lambda integrase cleaves DNA in cis.

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.

Bacteriophage lambda↗
PubMed1989

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity.

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.

Amino Acid Sequence↗
PubMed1985

The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli.

The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding capacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The KD values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the KD value was found to be 9-31 times higher than the values obtained for the wild type and the phoT mutant. The apparent Km values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. Vmax of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.

Bacterial Proteins↗
PubMed1984

A new locus in the phosphate specific transport (PST) region of Escherichia coli.

PhoS64 is a mutation in the Phosphate Specific Transport (PST) region on the E. coli chromosome which lacks the periplasmic phosphate binding protein. In contrast to other phoS mutations (which have the same phenotype) it complements the mutations in phoT and pstB. A detailed genetic map of the PST region constructed by three point transductional crosses has revealed that phoS64 is located distally from other phoS mutations. The genetic order obtained was phoS64-phoU35-pstB401-phoT-phoS-ilvC. The data indicate that phoS64 belongs to a different complementation unit in the PST region not known hitherto. We propose to name it phoV.

Bacterial Proteins↗
PubMed1980

Chi mutation in a transposon and the orientation-dependence of Chi phenotype.

Chi, an element that stimulates recombination via the E. coli RecBC pathway, can arise by spontaneous mutation in the transposon Tn5. When in phage lambda in one orientation, the mutant transposon confers Chi+ phenotype (large plaque and a high rate of exchange near the transposon). In the other orientation, however, the transposon does not confer Chi+ phenotype. The mobility of the transposon allows us to show that the Chi+ orientation of the mutant Tn5 is the same at different locations in lambda. These include a site near gene J, one in gam at 69, one to the right of gam at 73 and several to the right of R between 95.7 and 99.5. To the right of R, the mutant transposon could be found in only one orientation, that which confers Chi+ phenotype. We speculate that the other orientation of Tn5 in that locale is lethal to lambda. The orientation-dependence of Chi+ phenotype also revealed that Tn5 flip-flops in lambda.

Bacteriophage lambda↗
PubMed1977

Arsenate-resistant alkaline phosphatase-constitutive mutants of Escherichia coli.

When arsenate-resistant mutants are selected approximately 50 per cent of them are also consitutive for the synthesis of alkaline phosphatase and the Pi-binding protein. Some of these mutants are linked to ilv (phoS- or phoT-), other are linked to proC (phoR-). One of the mutant strains linked to ilv lost the Pi-binding protein (the phoS gene product). Resistance to arsenate, constitutivty for alkaline phosphatase synthesis and loss of the Pi-binding protein occurred pleiotropically by the same phoS- mutation.

Alkaline Phosphatase↗
PubMed1977

Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane.

Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity. The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration. The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source. The Vmax is not altered. Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane. Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain. One cryptic mutant (3--41), however, has altered sensitivity to phage T4. By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained. These mutants are no longer cryptic. We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide.

Adenosine Monophosphate↗