Search PubMed⌕ Search

Biomedical subjects

D Bramhill

Publications and source records attributed to D Bramhill.

18 recordsLinked to original sources
PubMed1998

Bacterial SOS checkpoint protein SulA inhibits polymerization of purified FtsZ cell division protein.

Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.

Bacterial Proteins↗
PubMed1997

Bacterial cell division.

Bacteria usually divide by building a central septum across the middle of the cell. This review focuses on recent results indicating that the tubulin-like FtsZ protein plays a central role in cytokinesis as a major component of a contractile cytoskeleton. Assembly of this cytoskeletal element abutting the membrane is a key point for regulation. The characterization of FtsZ homologues in Mycoplasmas, Archaea, and chloroplasts implies that the constriction mechanism is conserved and that FtsZ can constrict in the absence of peptidoglycan synthesis. In most Eubacteria, the internal cytoskeleton must also regulate synthesis of septal peptidoglycan. The Escherichia coli septum-specific penicillin-binding protein 3 (PBP3) forms a complex with other enzymes involved in murein metabolism, suggesting a centrally located transmembrane complex capable of splicing multiple new strands of peptidoglycan into the cell wall. Important questions remain about the spatial and temporal control of bacterial division.

Bacteria↗
PubMed1996

Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.

The bacterial cell division protein FtsZ is a homolog of tubulin, but it has not been determined whether FtsZ polymers are structurally related to the microtubule lattice. In the present study, we have obtained high-resolution electron micrographs of two FtsZ polymers that show remarkable similarity to tubulin polymers. The first is a two-dimensional sheet of protofilaments with a lattice very similar to that of the microtubule wall. The second is a miniring, consisting of a single protofilament in a sharply curved, planar conformation. FtsZ minirings are very similar to tubulin rings that are formed upon disassembly of microtubules but are about half the diameter. This suggests that the curved conformation occurs at every FtsZ subunit, but in tubulin rings the conformation occurs at either beta- or alpha-tubulin subunits but not both. We conclude that the functional polymer of FtsZ in bacterial cell division is a long thin sheet of protofilaments. There is sufficient FtsZ in Escherichia coli to form a protofilament that encircles the cell 20 times. The similarity of polymers formed by FtsZ and tubulin implies that the protofilament sheet is an ancient cytoskeletal system, originally functioning in bacterial cell division and later modified to make microtubules.

Animals↗
PubMed1996

PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence.

Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.

Animals↗
PubMed1996

A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model.

Although many detection methods have been used to determine Helicobacter colonization in small animal models, the sensitivity and specificity of these detection methods are limited. To improve the Helicobacter felis conventional mouse model for accurate evaluation of therapeutic regimens, we developed a PCR for detection of, and a competitive PCR for quantitation of, H. felis in viral antibody-free (VAF) mice. The PCR was based on the H. felis 16S rRNA gene. An internal control DNA was used for competitive quantitation of the PCR. VAF conventional Swiss-Webster mice were infected with an H. felis culture by oral gavage. At various times after H. felis challenge and therapy, stomach mucosa was collected and evaluated by PCR. PCR detected approximately 50 to 100 H. felis cells per mouse stomach and showed no cross-reaction with other bacteria commonly found in mouse stomachs. Colonization of H. felis in the mouse stomach was confirmed by culture isolation from germfree mice and histological examination of VAF mice. Response to therapy in this H. felis model correlated well with results seen in human clinical trials with H. pylori. A model utilizing PCR detection which may be useful for discovering new antibiotics and/or vaccines against Helicobacter ulcer disease has been developed.

Animals↗
PubMed1995

The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene.

Alleles↗
PubMed1995

The dnaA gene of Rhizobium meliloti lies within an unusual gene arrangement.

Rhizobium meliloti exists either as a free-living soil organism or as a differentiated endosymbiont bacteroid form within the nodules of its host plant, alfalfa (Medicago sativa), where it fixes atmospheric N2. Differentiation is accompanied by major changes in DNA replication and cell division. In addition, R. meliloti harbors three unique large circular chromosome-like elements whose replication coordination may be complex. As part of a study of DNA replication control in R. meliloti, we isolated a dnaA homolog. The deduced open reading frame predicts a protein of 57 kDa that is 36% identical to the DnaA protein of Escherichia coli, and the predicted protein was confirmed by immunoblot analysis. In a comparison with the other known DnaA proteins, this protein showed the highest similarity to that of Caulobacter crescentus and was divergent in some domains that are highly conserved in other unrelated species. The dnaA genes of a diverse group of bacteria are adjacent to a common set of genes. Surprisingly, analysis of the DNA sequence flanking dnaA revealed none of these genes, except for an rpsT homolog, also found upstream of dnaA in C. crescentus. Instead, upstream of rpsT lie homologs of fpg, encoding a DNA glycosylase, and fadB1, encoding an enoyl-coenzyme A hydratase with a strikingly high (53 to 55%) level of predicted amino acid identity to two mammalian mitochondrial homologs. Downstream of dnaA, there are two open reading frames that are probably expressed but are not highly similar to any genes in the databases. These results show that R. meliloti dnaA is located within a novel gene arrangement.

Amino Acid Sequence↗
PubMed1994

GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules.

The FtsZ protein is a GTPase that is essential for cell division in Escherichia coli. During cytokinesis, FtsZ localizes to a ring at the leading edge of septum synthesis. We report the GTP-dependent polymerization of purified FtsZ measured by sedimentation and light scattering. Electron microscopy of polymerized FtsZ revealed structures including tubules 14-20 nm in diameter with longitudinal arrays of protofilaments. FtsZ depolymerized upon removal of GTP and repolymerized after subsequent GTP addition. Mutant FtsZ84 protein polymerized inefficiently, suggesting that polymerization is important for the cellular role of FtsZ in division. The possibility that tubules of FtsZ protein form a cytoskeleton involved in septum synthesis is consistent with our data.

Bacterial Proteins↗
PubMed1994

Open complex formation by DnaA initiation protein at the Escherichia coli chromosomal origin requires the 13-mers precisely spaced relative to the 9-mers.

The 245 bp chromosomal origin, oriC, of Escherichia coli contains two iterated motifs. Three 13-mers tandemly repeated at one end of the origin and four 9-mers in a nearby segment of oriC are highly conserved in enteric bacteria, as is the distance separating these two sequence clusters. Mutant origins were constructed with altered spacing of the 9-mers relative to the 13-mers. Loss or addition of even a single base drastically reduced replication, both in vivo and in vitro. Spacing mutant origins bound effectively to DnaA protein but failed to support efficient open complex formation. These results suggest that interaction with the 9-mers positions at least one subunit of DnaA to recognize directly the nearest 13-mer for DNA melting.

Bacterial Proteins↗
PubMed1988

A model for initiation at origins of DNA replication.

Many prokaryotic origins resemble E. coli oriC in possessing essential AT-rich sequences, tandemly repeated. The role of these repeats may be in the initial opening of the duplex by the initiator protein, as has been found for the 13-mers in oriC and is implied for the 11-mers of the lambda origin. Regulatory influences on the effective action of the initiator protein of E. coli (dnaA protein) include transcriptional activation of the origin, nucleotide binding and membrane attachment of the protein, and interactions leading to the introduction of helicases to start replication forks.

Adenosine Triphosphate↗
PubMed1988

Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome.

Complexes previously identified in the reconstitution of stages in the initiation of replication of plasmids (oriC) bearing the origin of the Escherichia coli chromosome have been examined further. These are: (i) an ATP complex of dnaA protein, (ii) an initial complex of ATP.dnaA protein with oriC DNA, (iii) an open complex in which a portion of the oriC duplex has been opened by dnaA protein action, (iv) a prepriming complex of the open complex with dnaB, dnaC, and HU proteins, and (v) a complex with a small bubble opened at oriC by dnaB helicase action and by coating with single strand-binding protein (SSB). Helicase and gyrase actions can enlarge the bubble; coupling to priming and replication propagates bidirectional fork movement. Formation and stability of these complexes are profoundly affected by ATP, Mg2+, and temperature, as well as the levels of the participating proteins, including HU and SSB. As examples, the open complex is stable to isolation at a temperature near 38 degrees C but not at 24 degrees C; the prepriming complex requires an elevated temperature and high ATP levels for its formation, but is maintained at a low temperature and is destabilized by Mg2+. These successive steps, subject to a variety of controls, are designed to open the supercoiled duplex for priming and bidirectional replication.

Adenosine Triphosphate↗
PubMed1988

Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome.

Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This "open complex" requires ATP and 38 degrees C for optimum formation and stability. The subsequent dnaC-dependent entry of dnaB helicase to form a "prepriming complex" stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication.

Adenine Nucleotides↗
PubMed1987

ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome.

ATP is bound to dnaA protein with high affinity (KD = 0.03 microM) and hydrolyzed slowly to ADP in the presence of DNA. ADP is also bound tightly to dnaA protein and exchanges with ATP very slowly. The ATP form is active in replication; the ADP form is not. A unique conformation of oriC, formed in an early initiation stage, depends on dnaA protein being in the ATP form. The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP. Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATP gamma S can replace ATP, the ATP function appears to be allosteric. The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle.

Adenosine Triphosphate↗
PubMed1985

Initiation of replication of the Escherichia coli chromosomal origin reconstituted with purified enzymes.

A mixture of purified proteins has replaced a crude enzyme fraction capable of efficient replication of oriC-containing plasmids. The reconstituted enzyme system contains proteins which provide initiation, replication, and specificity functions required for dnaA-dependent replication specific for an oriC template. Replication can be separated into successive stages of RNA synthesis and DNA replication. Isolation of an intermediate no longer requiring RNA polymerase action requires the presence of dnaA protein, DNA gyrase, dnaB protein and dnaC protein. Intermediate formation likely involves binding of dnaA protein to a 9-bp sequence present 4 times as inverted repeats within the chromosomal origin sequence.

Bacterial Proteins↗
PubMed1983

The E. coli uvrD gene product is DNA helicase II.

We have shown that the uvrD gene product, previously identified in maxicell extracts as a 73 kilodalton protein, copurifies with single stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities. This protein is specifically precipitated from maxicell extracts by antibodies raised against DNA helicase II. In order to facilitate purification of the UvrD protein we have subcloned the uvrD gene into a plasmid vector in which its transcription is under the control of the phage lambda leftward promoter. Using cells harbouring this recombinant plasmid as a source of elevated levels of the UvrD protein we have purified this protein to homogeneity by a simple, rapid procedure. The purified protein has single stranded DNA-dependent ATPase activity and ATP-dependent DNA helicase activity, and both activities are specifically inactivated by antibodies raised against DNA helicase II. We conclude that DNA helicase II is the uvrD gene product.

Adenosine Triphosphatases↗
PubMed1982

Cloning of the uvrD gene of E. coli and identification of the product.

The uvrD gene has been cloned from Escherichia coli chromosomal DNA into phage lambda, cosmid, and low-copy-number plasmid vectors. Comparison of the proteins encoded by the cloned fragments with those encoded by fragments in which the uvrD gene is inactivated by transposon insertion or by deletion shows that the uvrD gene product is a protein of Mr = 73000.

Bacteriophage lambda↗