Cloning and characterization of the Escherichia coli lit gene, which blocks bacteriophage T4 late gene expression.

Escherichia coli lit mutations inhibit gene expression late in infection by bacteriophage T4. We cloned the lit gene from wild-type E. coli and three independent lit mutants. We present evidence that lit mutations [renamed lit(Con) mutations] cause overproduction of the lit gene product and that overproduction of this product causes the inhibition of gene expression. We also present evidence that the lit gene product is nonessential for E. coli growth, although the gene is common to most E. coli K-12 strains.     

Bacteriophage T4 gol site: sequence analysis and effects of the site on plasmid transformation.

The Escherichia coli lit gene product is required for the multiplication of bacteriophage T4 at temperatures below 34 degrees C. After infection of a lit mutant host, early gene product synthesis is normal, as is T4 DNA replication; however, the late gene products never appear, and early gene product synthesis eventually ceases. Consequently, at late times, there is no protein synthesis of any kind (W. Cooley, K. Sirotkin, R. Green, and L. Snyder, J. Bacteriol. 140:83-91, 1979; W. Champness and L. Snyder, J. Mol. Biol. 155:395-407, 1982), and no phage are produced. We have isolated T4 mutants which can multiply in lit mutant hosts. The responsible T4 mutations (called gol mutations) completely overcome the block to T4 gene expression (Cooley et al., J. Bacteriol. 140:83-91). We have proposed that gol mutations alter a cis-acting regulatory site on T4 DNA rather than a diffusible gene product and that the wild-type form of the gol site (gol+) somehow interferes with gene expression late in infection (Champness and Snyder, J. Mol. Biol. 155:395-409). In this communication, we report the sequence of the gol region of the T4 genome from five different gol mutants. The gol mutations are all single-base-pair transitions within 40 base pairs of DNA. Therefore, the gol site is at least 40 base pairs long. The sequence data confirm that the gol phenotype is not due to an altered protein. We also report that the gol+ site in plasmids prevents transformation of Lit- but not Lit+ E. coli. Thus, the gol site is at least partially active in the absence of the T4 genome.     

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.     

Inversion of the metE-oriC-rpsE chromosome segment in Escherichia coli K12

We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC). The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10). It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene. The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E. coli genetic map and including the rpsE, crp and metE::Tn5 markers. The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E. coli chromosome.     

Identification of cutC and cutF (nlpE) genes involved in copper tolerance in Escherichia coli.

It has been suggested previously that copper transport in Escherichia coli is mediated by the products of at least six genes, cutA, cutB, cutC, cutD, cutE, and cutF. A mutation in one or more of these genes results in an increased copper sensitivity (D. Rouch, J. Camakaris, and B. T. O. Lee, p. 469-477, in D. H. Hamer and D. R. Winge, ed., Metal Ion Homeostasis: Molecular Biology and Chemistry, 1989). Copper-sensitive cutC and cutF mutants were transformed with a genomic library of E. coli, and copper-tolerant transformants were selected. Two distinct clones were identified, each of which partially restores copper tolerance in both the cutC and cutF mutants of E. coli. Subcloning, physical mapping, and sequence analysis have revealed that the cutC gene is located at 42.15 min on the E. coli genome and encodes a cytoplasmic protein of 146 amino acids and that the cutF gene is located at 4.77 min on the E. coli genome and is allelic to the nlpE gene independently identified by Silhavy and coworkers (W. B. Snyder, L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy, J. Bacteriol. 177:4216-4223, 1995). Results from the genetic mapping of the copper-sensitive mutations in the cutF mutant and sequencing of the cutC and cutF (nlpE) alleles from both cutC and cutF mutants indicate that both the cutC and cutF mutants are in fact double mutants altered in these two genes, and mutations in both the genes appear to be required for the copper-sensitive phenotype in each mutant.     

Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts)

The gene pth, encoding peptidyl-tRNA hydrolase (Pth), is essential for protein synthesis and viability of Escherichia coli. Two pth mutants have been studied in depth: a pth(Ts) mutant isolated as temperature sensitive and a pth(rap) mutant selected as nonpermissive for bacteriophage lambda vegetative growth. Here we show that each mutant protein is defective in a different way. The Pth(Ts) protein was very unstable in vivo, both at 43 degrees C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt). Conversely, the mutant Pth(rap) protein had the same stability as Pth(wt), but its specific activity was low. The thermosensitivity of the pth(Ts) mutant, presumably, ensues after Pth(Ts) protein levels are reduced at 43 degrees C. Conditions that increased the cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature. Antibiotic-mediated inhibition of mRNA and protein synthesis, but not of peptidyl-tRNA drop-off, reduced pth(Ts) cell viability even at a permissive temperature. Based on these results, we suggest that Pth(Ts) protein, being unstable in vivo, supports cell viability only if its concentration is maintained above a threshold that allows general protein synthesis.     

The lit gene product which blocks bacteriophage T4 late gene expression is a membrane protein encoded by a cryptic DNA element, e14.

Escherichia coli lit(Con) mutations cause a severe inhibition of gene expression late in infection by bacteriophage T4 owing to the overproduction of one, and possibly two, proteins (C. Kao, E. Gumbs, and L. Snyder, J. Bacteriol. 169:1232-1238, 1987). One or both of these proteins interact, either directly or indirectly, with a short sequence about one-quarter of the way into the major capsid protein gene of T4, and the inhibition occurs when this late gene of the virus is expressed. In this report we show that lit(Con) mutations are up-promoter mutations in the cryptic DNA element e14 and that only one of the proteins, gplit, of about 34 kilodaltons, is required for the inhibition. We have sequenced the lit gene and the surrounding regions. From the sequence, and from cell fractionation studies, we conclude that gplit is an inner membrane protein. Since the assembly of T4 heads is thought to occur on the inner face of the inner membrane, we propose that gplit interferes with a normal regulation which coordinates the synthesis of proteins and the assembly of T4 heads.     

Precise mapping of the rnpB gene encoding the RNA component of RNase P in Escherichia coli K-12.

In Kohara's library derived from Escherichia coli K-12 W3110 (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), multiple copies of chromosomal sequence are found at 68 and at 64 to 65 min (M. Umeda and E. Ohtsubo, J. Mol. Biol. 213:229-237, 1990). We have determined that the rnpB gene (previously mapped at 70 min [B. J. Bachmann, Microbiol. Rev. 54:130-197, 1990]) is located within these segments of repeated sequences as five separate copies, together with tdcA, B, C, and R (mapped at 68 min [Bachmann, 1990]) and six unidentified open reading frames. Since close linkage of rnpB and tdc is found in various strains of E. coli K-12, the rnpB gene should be mapped at 68 min rather than 70 min.     

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.     

Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product.

In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product.     

Localization of the structural gene for threonine dehydrogenase in Escherichia coli.

The threonine dehydrogenase (tdh) gene of Escherichia coli, cloned within the plasmid pDR121, was inactivated in vitro by inserting a segment of DNA carrying the chloramphenicol acetyltransferase (cat) gene. The insertionally inactivated tdh gene was then transferred by homologous recombination into the E. coli chromosome by the procedure of Winans et al. (J. Bacteriol. 161:1219-1221, 1985). Mating experiments, followed by P1-mediated two- and three-point crosses, enabled us to localize tdh near min 81.2. The order with respect to known markers is mtl-cysE-tdh-pyrE.     

Inositol monophosphatase activity from the Escherichia coli suhB gene product.

The suhB gene is located at 55 min on the Escherichia coli chromosome and encodes a protein of 268 amino acids. Mutant alleles of suhB have been isolated as extragenic suppressors for the protein secretion mutation (secY24), the heat shock response mutation (rpoH15), and the DNA synthesis mutation (dnaB121) (K. Shiba, K. Ito, and T. Yura, J. Bacteriol. 160:696-701, 1984; R. Yano, H. Nagai, K. Shiba, and T. Yura, J. Bacteriol. 172:2124-2130, 1990; S. Chang, D. Ng, L. Baird, and C. Georgopoulos, J. Biol. Chem. 266:3654-3660, 1991). These mutant alleles of suhB cause cold-sensitive cell growth, indicating that the suhB gene is essential at low temperatures. Little work has been done, however, to elucidate the role of the product of suhB in a normal cell and the suppression mechanisms of the suhB mutations in the aforementioned mutants. The sequence similarity shared between the suhB gene product and mammalian inositol monophosphatase has prompted us to test the inositol monophosphatase activity of the suhB gene product. We report here that the purified SuhB protein showed inositol monophosphatase activity. The kinetic parameters of SuhB inositol monophosphatase (Km = 0.071 mM; Vmax = 12.3 mumol/min per mg) are similar to those of mammalian inositol monophosphatase. The ssyA3 and suhB2 mutations, which were isolated as extragenic suppressors for secY24 and rpoH15, respectively, had a DNA insertion at the 5' proximal region of the suhB gene, and the amount of SuhB protein within mutant cells decreased. The possible role of suhB in E. coli is discussed.     

The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase.

The essential gene efg, which complements ammonia-dependent growth (adgA) mutations in Rhodobacter capsulatus and is located at 38.1 min on the Escherichia coli chromosome, was found to code for NH3-dependent NAD synthetase. Crude extracts from a strain which overproduces the efg gene product contained up to 400 times more activity than crude extracts from the control strain, and the purified Efg protein possessed-NH3-dependent NAD synthetase activity. Glutamine-dependent NAD synthetase activity was found in crude extracts of E. coli but not in the purified enzyme, suggesting that it may be catalyzed by an additional subunit. An R. capsulatus strain carrying an adgA mutation was found to be deficient in NAD synthetase activity, and activity was restored by complementation with the E. coli gene. In accordance with the nomenclature proposed for Salmonella typhimurium (K. T. Hughes, B. M. Olivera, and J. R. Roth, J. Bacteriol. 170:2113-2120, 1988), the efg and adgA genes should now be designated nadE.     

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.     

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

The existence of a conditional lethal temperature-sensitive mutant affecting peptidyl-tRNA hydrolase in Escherichia coli suggests that this enzyme is essential to cell survival. We report here the isolation of both chromosomal and multicopy suppressors of this mutant in pth, the gene encoding the hydrolase. In one case, the cloned gene responsible for suppression is shown to be lysV, one of three genes encoding the unique lysine acceptor tRNA; 10 other cloned tRNA genes are without effect. Overexpression of lysV leading to a 2- to 3-fold increase in tRNA(Lys) concentration overcomes the shortage of peptidyl-tRNA hydrolase activity in the cell at non-permissive temperature. Conversely, in pth, supN double mutants, where the tRNA(Lys) concentration is reduced due to the conversion of lysV to an ochre suppressor (supN), the thermosensitivity of the initial pth mutant becomes accentuated. Thus, cells carrying both mutations show practically no growth at 39 degrees C, a temperature at which the pth mutant grows almost normally. Growth of the double mutant is restored by the expression of lysV from a plasmid. These results indicate that the limitation of growth in mutants of E.coli deficient in Pth is due to the sequestration of tRNA(Lys) as peptidyl-tRNA. This is consistent with previous observations that this tRNA is particularly prone to premature dissociation from the ribosome.     

Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes.

The synthesis of the Escherichia coli capsular polysaccharide varies with growth medium, temperature of growth, and genetic background. lac fusions to genes necessary for capsule synthesis (cps) demonstrated that these genes are regulated negatively in vivo by the lon gene product. We have now isolated, characterized, and mapped mutations in three new regulatory genes (rcs, for regulator of capsule synthesis) that control expression of these same fusions. rcsA and rcsB are positive regulators of capsule synthesis. rcsA is located at min 43 on the E. coli map, whereas rcsB lies at 47 min. rcsC, a negative regulator of capsule synthesis, is located at min 47, close to rcsB. All three regulatory mutations are unlinked to either the structural genes cpsA-F or lon. Mutations in all three rcs genes are recessive to the wild type. We postulate that lon may regulate capsule synthesis indirectly, by regulating the availability of one of the positive regulators.     

Mapping of noninvasion TnphoA mutations on the Escherichia coli O18:K1:H7 chromosome

Abstract The most virulent newborn meningitis-associated Escherichia coli are of the serotype O18: K1: H7. We previously isolated a large number of E. coli O18:K1:H7 mutants resulting from transposon Tn phoA mutagenesis that fail to invade brain microvascular endothelial cells. We have now determined the locations of 45 independent insertions. Twelve were localized to the 98 min region, containing a 120 kb segment that is characteristic of E. coli O18:K1:H7. Another, the previously described insertion ibe -10::Tn phoA , was localized to the 87 min region, containing a 20 kb segment found in this E. coli . These noninvasion mutations may define new O18:K1:H7 pathogenicity islands carrying genes for penetration of the blood-brain barrier of newborn mammals.     

Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae.