High-affinity arabinose transport mutants of Escherichia coli: isolation and gene location.

The gene araF, the product of which is the L-arabinose-binding protein--a component of the high-affinity L-arabinose transport system, was located on the Escherichia coli linkage map at 45 min. We established this location using bacteriophage P2 eductates and bacteriophage P1 cotransduction frequencies with the adjacent genetic loci, his (histidine biosynthesis) and mgl (methylgalactoside transport). In addition, we isolated a number of mutants that phenotypically exhibited altered high-affinity L-arabinose transport capacities. At least two of these mutations were located in the araF gene, as binding protein purified from these strains exhibited altered in vitro arabinose-binding properties.     

Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.     

Null mutation in the stringent starvation protein of Escherichia coli disrupts lytic development of bacteriophage P1.

As initial steps toward understanding the regulation and function of the stringent starvation protein (SSP) of Escherichia coli, we have isolated the ssp gene (encoding SSP), defined the operon in which ssp is found, and created insertion-deletion mutations of the ssp gene in recBC, sbc and recD strains by linear DNA transformation. During attempts to move the insertion-deletion structure to other strains by P1 transduction, we found that P1 was unable to form plaques on hosts lacking an intact ssp gene. The delta ssp mutation, however, did not affect transduction of the delta ssp strains and mutant strains were able to support lysogenic P1. When P1 lytic growth was induced, an increase in P1 DNA was detected without lysis or plaque formation. Examination of proteins synthesized in the delta ssp host during induction revealed the absence of P1 late gene products. Also, the apparent continued synthesis of early gene products during late time points was observed in the delta ssp host. The results reported here suggest that the defect in P1 lytic growth brought about by the absence of SSP occurs at the point at which bacteriophage P1 shifts from early to late gene expression. We also report the results of experiments on stable RNA synthesis following amino acid (aa) starvation induced by serine hydroxamate, and experiments on stable RNA synthesis following resupplementation of a limiting aa. These experiments show that SSP is not involved in stable RNA synthesis. Additionally, complementation studies have shown that ssp is identical to the previously described pog gene of E. coli.     

Sensitivity and Resistance to Spectinomycin in Escherichia coli

Inhibition of growth and division of Escherichia coli by spectinomycin is reversible, and the kinetics of its interference with deoxyribonucleic and ribonucleic acid synthesis may be interpreted as secondary effects of inhibition of protein synthesis on the ribosome. Spontaneous mutations to spectinomycin resistance occur in E. coli K-12 at a rate of about 2 x 10(-10). Resistance is transducible with a discrete lag in phenotypic expression, and the kinetics of its development is about the same as that for streptomycin resistance. All spectinomycin-resistant mutants tested contain resistant ribosomes, and all map in a locus (spc) counterclockwise to and 70% cotransducible with the classical str locus. Differences in the residual drug sensitivity of various spectinomycin-resistant mutants, and of their ribosomes, indicate the existence of more than one phenotypic class of resistance.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

Deletions in bacteriophage P2. Circularity of the genetic map and its orientation relative to the DNA denaturation map

Several types of viable chromosomal deletions of bacteriophage P2 were isolated. One type gives the immunity insensitive phenotype and may extend to the genes for the immunity repressor (C) and for integrative recombination (int). Two other types delete genes (old and fun) known to be active in the lysogenic state. For such deletion mutants the relationship between particle density and DNA length was established. The deletions were located in respect to previously mapped genes and the results were compared with electron microscopical studies (by Inman and collaborators) of the P2 chromosome. It is concluded that the best representation of the genetic map of P2 is circular. The cohesive ends of the linear P2 DNA molecule are most likely formed between genes old and Q. Except for the neighborhood of gene old, the previously published, linear genetic map of P2 (Lindahl) is colinear with the melting map of the P2 chromosome (Inman). Preliminary evidence for some specific recombination event often accompanying integrative recombination between phage chromosomes is presented.     

Escherichia coli mutants completely deficient in adenosylmethionine decarboxylase and in spermidine biosynthesis.

Mutants of Escherichia coli deficient in adenosylmethionine decarboxylase, an enzyme in the biosynthetic pathway for spermidine, were isolated after mutagenesis of E. coli K 12 with N-methyl-N-nitro-N-nitrosoguanidine or with the bacteriophage Mu. The mutated gene, designated speD, is at 2.7 min on the E. coli chromosome map. In several of the mutants resulting from Mu insertion both adenosylmethionine decarboxylase activity and spermidine were undetectable. The absence of spermidine from speD strains proves the essential role of adenosylmethionine decarboxylase in the biosynthetic pathway for spermidine. Despite the complete absence of spermidine, these mutants grew at 75% of the wild type rate.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA.

Escherichia coli mia strains were shown to lack delta 2-isopentenylpyrophosphate transferase activity, the first step in the synthesis of the 2-methylthio derivative of 6-(delta 2-isopentenyl) adenosine (ms2i6A). A double mutant, rpsL (Smp) miaA, was streptomycin dependent. The wild-type miaA gene was cloned by selecting for lambda recombinant bacteriophage which eliminated the streptomycin-dependent phenotype and was subsequently recloned into plasmid vectors. The cloned miaA gene restored the ms2i6A modification to tRNA. The miaA gene mapped to 95 min on the E. coli map, and we propose the order mutL-miaA-hflA-purA.     

A new gene of Escherichia coli K-12 whose product participates in T4 bacteriophage late gene expression: interaction of lit with the T4-induced polynucleotide 5''-kinase 3''-phosphatase.

We isolated five Escherichia coli mutants deficient in their ability to support the late (replication-coupled) gene expression of T4 bacteriophage at 30 degrees C. These mutants, which we call Lit mutants, define at least one novel gene at 25 min on the E. coli map. They were selected in an attempt to obtain mutants which restrict the growth of T4 mutants deficient in polynucleotide 5'-kinase 3'-phosphatase but not that of wild-type T4 at 37 degrees C. Some of the mutants do have these phenotypes under some conditions. Studies of the block in T4 development in some of the E. coli mutants suggest that Lit mutants are affected in a gene product involved in the metabolism of deoxyribonucleic acid nicks or single-strand gaps. None of the Lit mutants is deficient in the major, bacterial, 3'-phosphatase activity in crude extracts.     

Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.     

Amber mutants of bacteriophage T4D: their isolation and genetic characterization

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome.     

Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells.

The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.     

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Restriction insensitivity in bacteriophage T5 I. Genetic characterization of mutants sensitive to EcoRI restriction.

Unmodified bacteriophage T5 is able to grow normally on bacterial hosts carrying three different Escherichia coli restriction systems, EcoK, EcoPI, and EcoRI. Under the same conditions, the plating efficiency of bacteriophage gamma is less than 10(-9). At least in the case of EcoRI, this lack of in vivo restriction is not due to lack of restriction sites on the T5 DNA molecule. These observations suggest that bacteriophage T5 specifies one or more restriction protection systems. Mutants (ris) of T5 have been isolated which confer sensitivity to EcoRI restriction but not to EcoK or EcoPI. The mutations are located in the pre-early region of the genetic map but are too far apart to be alleles of a single gene. Complementation studies show that the ris mutants can be helped to grow on the EcoRI-restricting host by coinfection with T5+. This result provides evidence for a restriction protection function but does not necessarily show that the ris mutants are defective in such a system.     

A novel pleiotropic mutation in Escherichia coli K12 which affects transduction, transformation and rates of mutation.

A mutant strain of Escherichia coli K12, R2721, has been shown to differ from its parent strain, S491, in four associated phenotypic characters as a result of a single mutation. This strain did not give recombinants with DNA transduced by bacteriophage PI or bacteriophage Mu, nor transformats after exposure to R factor DNA: lysates of bacteriophage PI grown on this strain did not appear to contain any transducing particles when tested on normal recipients. Moreover, the reversion rates, both spontaneous and ultraviolet-induced, for two auxotrophic markers were reduced. The frequency of revertants was at least two orders of magnitude lower in cultures of R2721 than in cultures of S491I. Many of the rare revertants for one or other of the auxotrophic markers were found to have regained normal reversion frequencies for the other marker and for the capacity to be transduced. In all other respects, recombination in R2721 appeared normal, the frequency of chromosomal mobilization by and F' factor was unaffected and normal yields of recombinants were obtained from matings with Hfr strains. The only circumstance in which transduction of R2721 was observed was when the capacity to ferment galactose was selected and PI had been grown on a strain carrying lambdadgal when, presumably, integration was effected by the phage-coded gene products. The mutation has been located on the E. coli chromosone map between tonA and pro and has been given the symbol tdi (transduction inhibition). Double mutants, (tdi recA) and (tdi recB), have been isolated and show no unexpected properties.