Internal structure and dynamics of isolated Escherichia coli nucleoids assessed by fluorescence correlation spectroscopy

The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from approximately 50 kbp to approximately 3 kbp).     

Characterization of a nalidixic-acid-resistant mutant of Escherichia coli as a strict aerobe

An Escherichia coli mutant, C18, which plates at an efficiency of 5.0 x 10(-4) under anaerobic condition, was isolated among spontaneous nalidixic-acid-resistant mutants. This strict aerobic mutation was mapped by P1 cotransduction with a gyrA linked transposon Tn10 and found to be at the gyrA gene. A low degree of superhelicity of pBR322 DNA isolated from C18 was demonstrated by agarose gel electrophoresis with various concentrations of ethidium bromide. The superhelical density of pBR322 isolated from C18 was 80% of the value of pBR322 isolated from wild-type bacteria cultured under aerobic condition, and 50% cultured under anaerobic condition. These results lead us to conclude that a certain mutation of the gyrA gene causes a decrease in DNA superhelicity and prevents anaerobic growth.     

Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase.

We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step.     

Functional Interactions between the DNA Ligase of ESCHERICHIA COLI and Components of the DNA Metabolic Apparatus of T4 Bacteriophage

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.     

A superhelical spiral in the Escherichia coli DNA gyrase A C-terminal domain imparts unidirectional supercoiling bias

DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped beta-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.     

Molecular cloning and expression in E. coli of a yeast gene coding for beta-galactosidase.

The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose. We have isolated the gene that codes for this enzyme using recombinant DNA techniques. K. lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase. ligase. A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA. Three lac+ transformants containing recombinant plasmids were selected. Two of the plasmids (pK15 and pK17) contain four Eco R1-K. lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons. The other plasmid (pK16) lacks the smallest fragment. E. coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K. lactis beta-galactosidase and distinctly different from E. coli beta-galactosidase. DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K. lactis but not to E. coli DNA.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.     

Genetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.

The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive locus seems to be a gene, or genes, unique to CTr5X or to be an altered form of a nonessential gene, since deleting the locus seems to cause loss of the phenotypes. In addition to restricting RNA ligase- and polynucleotide kinase-deficient T4, the locus also restricts bacteriophages lambda and T4 with cytosine DNA. When lambda or T4 with cytosine DNA infect strains with the prr locus, the phage DNA is injected, but phage genes are not expressed and the host cells survive. These phenotypes are unlike anything yet described for a phage-host interaction.     

In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.

Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate.     

Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.     

Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities

The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.     

Existence of two D-alanine:D-alanine ligases in Escherichia coli: cloning and sequencing of the ddlA gene and purification and characterization of the DdlA and DdlB enzymes

Two distinct genes encoding D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) activity in Escherichia coli have been cloned by complementation of E. coli strain ST640(lambda 112) deficient in D-Ala-D-Ala ligase activity with a lambda library of E. coli DNA. One of the two genes, designated as ddlB, is identical with the ddl gene already sequenced [Robinson, A.C., Kenan, D.L., Sweeney, J., & Donachie, W.D. (1986) J. Bacteriol. 167, 809-817]. We describe the subcloning and DNA sequencing of the other gene, designated as ddlA on the basis of similarities with the Salmonella typhimurium ddlA gene [Daub, E., Zawadzke, L.E., Botstein, D., & Walsh, C.T. (1988) Biochemistry 27, 3701-3708]. The predicted amino acid sequence of the E. coli DdlA enzyme shows 90% homology with the S. typhimurium DdlA sequence. The ddlB gene was subcloned by use of the polymerase chain reaction into an expression vector containing an optimized ribosome binding site, which expressed the DdlB enzyme to greater than 50% soluble cell protein. Both DdlA and DdlB enzymes were purified to greater than 90% homogeneity and characterized kinetically.     

DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression.

We show that several interacting environmental factors influence the topology of intracellular DNA. Negative supercoiling of DNA in vivo is increased by anaerobic growth and is also influenced by growth phase. The tonB promoter of Escherichia coli and Salmonella typhimurium was found to be highly sensitive to changes in DNA supercoiling. Expression was increased by novobiocin, an inhibitor of DNA gyrase, and was decreased by factors which increase DNA superhelicity. Expression of the plasmid-encoded tonB gene was enhanced by gamma delta insertions in cis in a distance- and orientation-independent fashion. Both the res site and the TnpR protein of gamma delta, which is known to function as a type I topoisomerase, were required for this activation. tonB expression increased during the growth cycle and was reduced by anaerobiosis. There was excellent correlation between tonB expression from a plasmid and the level of supercoiling of that plasmid under a wide range of conditions. The chromosomal tonB gene was regulated in a manner identical to that of the plasmid-encoded gene. Thus, the physiological regulation of tonB expression in response to anaerobiosis and growth phase appears to be mediated by environmentally induced changes in DNA superhelicity.