Mutant Trp repressors with new DNA-binding specificities.

Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of the Escherichia coli trpR (tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes. Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensus trp operator. Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities. These new specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair. The Trp repressor does not use the first two amino acids of its "recognition alpha-helix," Ile79 and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites.

Bacterial motility: secretory secrets of gliding bacteria.

Many bacteria glide over surfaces without the aid of flagella. Gliding is still somewhat mysterious, but recent studies show that it involves specialized secretory systems that assemble membrane-associated filaments, and the recognition of extracellular components that trigger movement via transmembrane transducers.

Primary structure of the immI immunity region of bacteriophage P22.

The DNA sequence of the immI immunity region of bacteriophage P22 has been determined. This region includes the ant gene, which encodes the P22 antirepressor protein, and the mnt and arc genes, which encode proteins that negatively regulate antirepressor synthesis. We have purified antirepressor protein and selected tryptic peptides of antirepressor, and have determined the amino terminal sequences and amino acid composition of these molecules. These data, in combination with the DNA sequence, locate the ant gene and define the complete amino acid sequence of antirepressor (300 residues). The mnt and arc genes have been located by sequencing the mnt-am343 and arc-amH1605 mutations. The Mnt and Arc proteins are predicted to be small, basic polypeptides that are homologous in amino acid sequence. The Mnt protein also shows significant sequence homology with the lambda Cro protein. The arc and ant genes are transcribed rightward from the Pant promoter, while mnt is transcribed leftward from a promotor that may overlap Pant. The Mnt protein apparently acts by binding to an operator site located immediately adjacent to the startpoint of Pant transcription.

Structural and regulatory divergence among site-specific recombination genes of lambdoid phage.

The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini.

Phage lambda Cro protein and cI repressor use two different patterns of specific protein-DNA interactions to achieve sequence specificity in vivo.

By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.

Mutant lambda repressors with increased operator affinities reveal new, specific protein-DNA contacts.

The binding specificities of four mutant lambda cI repressor proteins with increased affinities for operator DNA were examined. Two mutant repressors (Glu34----Lys and Glu83----Lys) have the same specificity of binding as wild-type repressor, whereas two (Gly48----Ser and Gly48----Asn) have new binding specificities. The Gly48----Asn mutant repressor recognizes lambda operators with changes at base pair 3 with a different order of affinity than wild-type repressor, suggesting that the side chain of Asn48 makes additional specific DNA contacts at or near this base pair. When paired with a change that disrupts the specific interaction of the amino-terminal arm of lambda repressor with DNA (Lys4----Gln), one change that increases the affinity of repressor (Gly48----Ser) suppresses the binding defect of the Lys4----Gln repressor, resulting in a double mutant repressor with a new binding specificity different than that of both its parents and of wild type. These results lend strong support to the model of direct recognition of the lambda operator by lambda repressor proposed from the crystal structure of the repressor/operator complex.

DNA sequence determinants of lambda repressor binding in vivo.

The critical operator determinants for lambda repressor recognition have been defined by analyzing the binding of wild-type repressor to a set of mutant operators in vivo. Base pair substitutions at six positions within the lambda operator half-site impair binding severely, and define these base pairs as critical for operator function. One mutant operator binds repressor better than the consensus operator, and is a superoperator. The model proposed by M. Lewis in 1983 for the binding of lambda repressor to its operator accurately predicts the observed operator requirements for binding in vivo, with several minor exceptions. The order of affinities of the six natural lambda operators has also been determined.

Mutations that improve the ant promoter of Salmonella phage P22.

Mutations that increase the activity of the promoter for the antirepressor gene of phage P22 were isolated by pseudoreversion of four severe promoter-down mutations. The sequence changes in these pseudorevertants include single base pair substitutions, single base pair deletions, tandem double base pair deletions and multisite mutations. The single base pair substitutions change nonconsensus base pairs to consensus base pairs at positions -14 and -8. The other mutations provide support for the idea that the length of the spacer region between the conserved -35 and -10 hexamers is an important determinant of promoter strength. Deletions of one or two base pairs in the spacer region apparently activate an alternate -10 hexamer by shifting it from a spacing of 19 base pairs to a spacing of 18 or 17 base pairs, respectively.

Mutant tryptophan aporepressors with altered specificities of corepressor recognition.

The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.

Spontaneous mutations occur near dam recognition sites in a dam- Escherichia coli host.

The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.

Mutations that improve the binding of yeast FLP recombinase to its substrate.

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

General selection for specific DNA-binding activities.

We present a general strategy for the selection of bacterial clones that express DNA-binding activities corresponding to particular DNA recognition sites. The selection uses a "challenge phage" vector, P22 Kn9 arc-amH1605, into which is substituted a synthetic DNA-binding site for a site that controls transcription of the P22 antirepressor (ant) gene. Constitutive synthesis of antirepressor channels a challenge phage into lytic development and efficiently kills an infected host, unless the substituted site is bound by a specific protein; in this case, the challenge phage prefers lysogenic development, and the host survives and acquires an antibiotic-resistance phenotype. Infections with challenge phages carrying the E. coli Lac operator, phage lambda OL1 operator, or synthetic, "idealized" E. coli Trp and Tn10 Tet operators select clones that express each of the corresponding binding activities. The use of challenge phage vectors may be extended to select clones that express eukaryotic DNA-binding activities.

Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages.

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.

Direct recognition of the trp operator by the trp holorepressor--a review.

Two different X-ray co-crystal structures of the Escherichia coli trp holorepressor complexed with DNA suggest that the TrpR protein recognizes specific DNA sequences primarily with a network of water-mediated H-bonds. However, the more recent nuclear magnetic resonance (NMR) solution structures of the holorepressor-operator complex show no long-lived, ordered water molecules at the protein-DNA interface and place amino acids in intimate contact with nucleotide bases. Both genetic and biochemical studies support a model in which the trp repressor recognizes specific DNA sequences by a direct mechanism, as seen in the NMR solution structures, not by the 'indirect readout' mechanism initially proposed on the basis of X-ray studies.

Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.

Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues. Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt). Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro. Whereas three of these eight mutant repressors are unstable in E. coli, all are made at similar steady-state levels in Salmonella typhimurium. Three of the eight mutant holorepressors are lethal when overproduced in S. typhimurium, because they confer an induced auxotrophy. Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA. These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work.

Automated kinetic assay of beta-galactosidase activity.

An automated kinetic assay for beta-galactosidase activity in Escherichia coli was developed to permit the measurement of many independent samples simultaneously. Bacteria are grown, lysed from without (by adsorption of a high multiplicity of bacteriophage T4) and assayed in microtiter plates with 96 wells. Absorbance data are collected and analyzed by computer. The growth and lysis procedure, apparatus and software used in this assay can be used for other spectrophotometric enzyme assays.

New genes in the left arm of the bacteriophage phi 80 chromosome.

We describe the isolation and partial genetic characterization of 247 amber (suppressor-sensitive) mutants of temperate bacteriophage phi 80 of Escherichia coli. Of these 247 mutants, the mutations of 201 mapped to the left arm of the phi 809 chromosome and the mutations of 39 mapped to the right arm of the genome. Complementation tests among these and previously described left arm mutants defined five additional genes in the left arm of the chromosome. The positions of these genes are consistent with the hypothesis that four of them represent functions essential for phi 80 tail assembly and one represents a capsid assembly function, probably the major coat protein. The identification of these genes brings the phi 80 genome into closer correspondence with the organization of the phage lambda genome. Two- and three-factor crosses performed between mutants with defects in each of the previously identified genes and mutants with defects in the five new genes allowed us to construct a consistent, roughly additive recombination map of the left arm of the bacteriophage phi 80 genome.